effect of nonfiber carbohydrates on product yield...

EFFECT OF NONFIBER CARBOHYDRATES ON PRODUCT YIELD AND FIBER

DIGESTION IN FERMENTATIONS WITH MIXED RUMINAL MICROBES

By

LUCIA HOLTSHAUSEN

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2004

Copyright 2004

by

Lucia Holtshausen

This dissertation is dedicated to Heidi Bissell for her friendship during the last three and a half years. It was her support, endless patience and encouragement that carried me through my PhD program.

iv

ACKNOWLEDGMENTS

I would like to express my appreciation to all the people who made valuable

contributions throughout my PhD program. First, I would like to thank Dr. Mary Beth

Hall, chair of my supervisory committee, for helping me further develop as a scientist and

for her encouragement. I would also like to thank the other members of my committee:

Dr. Ramon Littell for his assistance with the statistical analysis, Dr. Adegbola Adesogan

for acting as assistant chair on short notice and for always being available to answer even

the most trivial of questions, Dr. Christian Cruywagen for continued academic and moral

support and for always taking a keen interest in my academic future, and the late Dr. Bill

Kunkle for providing valuable practical insight at the onset of my program. Next I would

like to thank all the people who helped at various stages with the in vitro studies and

laboratory analyses: Alexandra Amorocho, Heidi Bissell, Jocelyn Croci, Faith Cullens,

Bruno Amaral, Ashley Hughes, Celeste Kearney, Colleen Larson, Sergei Sennikov and

Tina Sheedy. Without their help I would not have been able to do the studies on such a

large scale. I would like to thank Dr. Paul Weimer and Christine Odt for the analysis of

organic acids and their patience in answering my questions, Dr. Glen Broderick for

amino acid and ammonia nitrogen analyses, and Hangxin Hou from US Sugar in

Clewiston (Florida) for sugar analysis. I would also like to thank Sabrina Robinson for

various administrative favors throughout my PhD program. Last but not least I would

like to thank the Liquid Feed Committee of the American Feed Industry Association and

the Florida Milk Check-Off Program for funding this research.

v

TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iv

LIST OF TABLES........................................................................................................... viii

LIST OF FIGURES ........................................................................................................... xi

LIST OF ABBREVIATIONS........................................................................................ xxiv

ABSTRACT.....................................................................................................................xxv

CHAPTER

1 REVIEW OF LITERATURE: MICROBIAL FERMENTATION OF NON-NEUTRAL DETERGENT FIBER CARBOHYDRATES, AND HOW IT MAY RELATE TO ANIMAL PERFORMANCE .................................................................1

Introduction...................................................................................................................1 Chemical Structure and degradation of NFC Sources..................................................2

Sucrose ..................................................................................................................2 Starch.....................................................................................................................3 Pectic Substances...................................................................................................5

NFCs and Microbial Fermentation Products ................................................................5 Organic Acids........................................................................................................6

Total volatile fatty acids .................................................................................6 Acetic acid......................................................................................................7 Propionic acid.................................................................................................9 Butyric acid ..................................................................................................11 Branched chain volatile fatty acids ..............................................................12 Lactic acid ....................................................................................................13

Microbial Mass....................................................................................................14 Microbial composition .................................................................................14 Microbial protein yield.................................................................................15 Microbial α-glucan .......................................................................................17

Other Factors Which Influence Microbial Product Yield...........................................18 Nitrogen Source...................................................................................................18 Fermentation pH..................................................................................................21

NFCs and Ruminal pH, Fiber Digestion and Animal Performance ...........................22 Ruminal pH and Fiber Digestion.........................................................................22 Animal Performance............................................................................................24

vi

Dry Matter Intake ................................................................................................25 Milk Production and Composition ......................................................................25

2 EFFECT OF PH ON MICROBIAL YIELD AND NEUTRAL DETERGENT FIBER DIGESTION FROM IN VITRO FERMENTATIONS OF SUCROSE AND ISOLATED NEUTRAL DETERGENT RESIDUE...................................................34

Introduction.................................................................................................................34 Materials and Methods ...............................................................................................35

Substrates.............................................................................................................35 Medium and reducing solution............................................................................35 Fermentation........................................................................................................36 Sample Handling and Subsequent Analyses .......................................................37 Statistical Analysis ..............................................................................................39

Results and Discussion ...............................................................................................41 Residual Sucrose .................................................................................................41 Microbial Glycogen.............................................................................................43 Fermentation pH..................................................................................................44 Organic Acids......................................................................................................45 Protein Degradation Products..............................................................................47 Neutral Detergent Fiber Digestion ......................................................................48 Microbial Crude Protein Yield and Efficiency....................................................49

Conclusions.................................................................................................................50

3 EFFECT OF NITROGEN SOURCE ON MICROBIAL YIELD AND NEUTRAL DETERGENT FIBER DIGESTION FROM IN VITRO FERMENTATIONS OF SUCROSE AND ISOLATED NEUTRAL DETERGENT RESIDUE......................58


Substrates.............................................................................................................59 Medium................................................................................................................60 Fermentation........................................................................................................60 Sample Handling and Subsequent Analyses .......................................................61 Statistical Analysis ..............................................................................................64

Results and Discussion ...............................................................................................66 Residual Substrate ...............................................................................................66 Microbial Glycogen.............................................................................................67 Fermentation pH..................................................................................................67 Organic Acids......................................................................................................68 Protein Degradation Products..............................................................................69 Neutral Detergent Fiber Digestion ......................................................................70 Microbial Crude Protein Yield and Efficiency....................................................71

Conclusions.................................................................................................................72

vii

4 MICROBIAL PRODUCT YIELD AND NEUTRAL DETERGENT FIBER DIGESTION FROM IN VITRO FERMENTATIONS WITH SUCROSE, STARCH AND PECTIN IN COMBINATION WITH ISOLATED BERMUDAGRASS NEUTRAL DETERGENT RESIDUE .......................................................................83


Substrates and Treatments...................................................................................84 Fermentation........................................................................................................86 Sample Handling and Subsequent Analyses .......................................................88 Statistical Analysis ..............................................................................................91

Treatment mean comparisons and temporal pattern descriptions ................91 Comparisons of maxima, minima and 24 h data..........................................92 Comparisons of NFC mixtures.....................................................................94

Results and Discussion ...............................................................................................95 Residual Sugars (Sucrose, Glucose, Fructose) ....................................................95 Microbial Glycogen Yield...................................................................................96 Fermentation pH..................................................................................................97 Organic Acids......................................................................................................99 Neutral Detergent Fiber Digestion ....................................................................104 Microbial Crude Protein Yield ..........................................................................106

Conclusions...............................................................................................................107

5 CONCLUSIONS ......................................................................................................121

APPENDIX

A ADDITIONAL FIGURES FOR CHAPTER 2.........................................................124

B ADDITIONAL FIGURES FOR CHAPTER 3.........................................................126

C FIGURES FOR CHAPTER 4...................................................................................128

D ADDITIONAL TABLE FOR CHAPTER 4.............................................................166

E CHAPTER 2 RAW DATA.......................................................................................168

F CHAPTER 3 RAW DATA.......................................................................................179

G CHAPTER 4 RAW DATA.......................................................................................193

LIST OF REFERENCES.................................................................................................281

BIOGRAPHICAL SKETCH ...........................................................................................293

viii

LIST OF TABLES

Table page 1-1 The effects of NFC source on ruminal or fermentation pH and organic acid profile.28

1-2 Effects of NFC source on dry matter intake, milk production and milk composition.31

2-1 Type and number of fermentation tubes per medium for each sampling hour, indicating the substrate and analysis for which tubes were reserved in an 24 h in vitro fermentation of sucrose and iNDF...................................................................51

2-2 Residual glucose, fructose, unhydrolyzed sucrose, monosaccharide sucrose equivalent (glucose+fructose) and sucrose equivalent at 0, 4 and 8 h, and averaged for 24 h in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue with initial medium pH of 6.8 or 5.6. ..........................................51

3-1 Type and number of fermentation tubes per medium for one sampling hour, indicating the substrate and analysis for which tubes were reserved in a 16 h in vitro fermentation of sucrose and isolated neutral detergent fiber...........................73

3-2 Residual glucose, fructose, sucrose, monosaccharide sucrose equivalent (glucose + fructose) and sucrose equivalent at 0, 4 and 8 h, and averaged for 16 h in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue with different sources of nitrogen in media......................................................................74

3-3 Organic acid concentrations (least squares means) at 16 h (corrected for blank fermentations) for in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue with different sources of nitrogen in media .....................75

3-4 Maximum microbial crude protein (MCP) yield (hour of maximum) and efficiency of MCP yield at the point of maximum MCP yield for in vitro fermentations of iNDF and of SuNDF with different source of nitrogen in media. Values are least squares means...........................................................................................................75

4-1 Layout of treatments and substrate amounts for a series of three 24 h in vitro fermentations (performed in duplicate) of a mixed batch culture. .........................108

4-2 Residual glucose, fructose, unhydrolyzed sucrose and sucrose equivalent (mg) at 0 and 4 h for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means. ..................109

ix

4-3 Maximum microbial glycogen (GLY) yield (mg), hour of maximum yield and temporal patterns for 24 h in vitro fermentations of iNDF, NFC sources (sucrose and pectin), and combinations of NFCs. Values are least squares means. ...........110

4-4 Main effects and regression coefficients for maximum microbial glycogen (GLY) yield (mg) for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations. .........................................................................................................111

4-5 Fermentation pH (mean, minimum, hour of minimum and temporal pattern) for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means. ......................................112

4-6 Main effects and regression coefficients for minimum fermentation pH for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations...113

4-7 Volatile fatty acid concentrations at 24 h and maximum lactate concentrations (hour of maximum indicated) for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means. ...............................................................................................................114

4-8 Main effects and regression coefficients for maximum organic acid concentrations for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations...115

4-9 Residual NDFOM at 24 h and temporal patterns for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means. .............................................................................117

4-10 Main effects and regression coefficients for residual NDFOM at 24 h for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations. ..........................118

4-11 Microbial crude protein (MCP) yield (mean, maximum, hour of maximum and temporal pattern) for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means. .119

4-12 Main effects and regression coefficients for maximum microbial crude protein (MCP) yield for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations. .........................................................................................................120

D-1 Temporal patterns of organic acid concentrations for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. .....167

x

E-1 Data used for statistical analysis in evaluating the effect of pH on microbial yield and neutral detergen fiber digestion from in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue....................................................168

F-1 Data used for statistical analysis in evaluating the effect of nitrogen source on microbial yield and neutral detergen fiber digestion from in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue. ...............................179

G-1 Data used for statistical analysis in evaluating the effect on microbial yield and neutral detergen fiber digestion from in vitro fermentations of different sources (sucrose, starch and pectin), amounts (0, 40, 80 and 120 mg nominal hexose equivalents) and combinations (sucrose+starch, starch+pectin and sucrose+pectin) of NFCs. ...............................................................................................................193

xi

LIST OF FIGURES

Figure page 2-1 Microbial glycogen yield (LSmeans ± standard error) for 24 h in vitro

fermentations of SuNDF with initial medium pH of 6.8 (■) or 5.6 (▲). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ....................................52

2-2 Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (∆, □) and SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or □) or 5.6 (▲ or ∆). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF...........................................................................................................52

2-3 Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. .....................................................................................................53

2-4 Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ..................................................................53

2-5 Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. .....................................................................................................54

2-6 Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ..................................................................54

2-7 Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. .....................................................................................................55

xii

2-8 Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ..................................................................55

2-9 Ammonia nitrogen concentration (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ..................................................................56

2-10 Total free amino acid concentration (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ..................................................................56

2-11 Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of (iNDF; ∆, □) and SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or □) or 5.6 (▲ or ∆). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose + iNDF.........................................................................................................57

2-12 Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with an initial medium pH of 6.8 (■) or 5.6 (▲). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. .................................57

3-1 Microbial glycogen yield (least squares means ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ....................76

3-2 Fermentation pH (least squares means ± standard error) for 16 h in vitro fermentations of iNDF (□, ∆, ○) and SuNDF (■, ▲, ●) with media containing nitrogen in the form of non-protein nitrogen + true protein (■ or □), true protein only (▲ or ∆) or non-protein nitrogen only (● or ○). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF. ........................76

3-3 Total volatile fatty acid concentrations (LSmeans ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ....................77

3-4 Acetate concentrations (LSmeans ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ....................................77

xiii

3-5 Propionate concentrations (LSmeans ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ....................78

3-6 Butyrate concentrations (LSmeans ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ....................................78

3-7 Lactate concentrations (LSmeans ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ....................................79

3-8 Ammonia nitrogen concentration (LSmeans ± standard error) for 16 h in vitro fermentations with no substrate (□, ∆, ○) or SuNDF as the substrate (■,▲,●) and media containing nitrogen in the form of non-protein nitrogen + true protein (□, ■), true protein only (∆, ▲) or non-protein nitrogen only (○, ●). SuNDF = sucrose+isolated bermudagrass neutral detergent residue. ......................................79

3-9 Total free amino acid concentration (LSmeans ± standard error) for 16 h in vitro fermentations with no substrate (□, ∆, ○) or SuNDF as the substrate (■,▲,●) and media containing nitrogen in the form of non-protein nitrogen + true protein (□, ■), true protein only (∆, ▲) or non-protein nitrogen only (○, ●). SuNDF = sucrose+isolated bermudagrass neutral detergent residue. ......................................80

3-10 Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 16 h in vitro fermentation with no substrate (□, ∆, ○) or SuNDF as the substrate (■,▲,●) and media containing nitrogen in the form of non-protein nitrogen + true protein (□, ■), true protein only (∆, ▲) or non-protein nitrogen only (○, ●). SuNDF = sucrose+isolated bermudagrass neutral detergent residue. ......................80

3-11 Residual NDFOM for 16 h in vitro fermentations of iNDF (A; □, ∆, ○) and SuNDF (B; ■, ▲, ●) with media containing nitrogen in the form of non-protein nitrogen + true protein (□, ■), true protein only (∆, ▲) or non-protein nitrogen only (○, ●). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF.81

3-12 Microbial crude protein yield for 16 h in vitro fermentations of iNDF (□, ∆, ○) and SuNDF (■, ▲, ●) with media containing nitrogen in the form of non-protein nitrogen + true protein (■ or □), true protein only (▲ or ∆) or non-protein nitrogen only (● or ○). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF...........................................................................................................82

A-1 Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with initial medium pH, before addition of reducing solution or inoculum, of 6.8 (■) or 5.6 (▲). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ...................................................................................................124

xiv

A-2 Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with initial medium pH, before addition of reducing solution or inoculum, of 6.8 (■) or 5.6 (▲). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ................................................................125

A-3 Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with initial medium pH, before addition of reducing solution or inoculum, of 6.8 (■) or 5.6 (▲). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ...................................................................................................125

B-1 Residual glucose content (LSmeans ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ..................................126

B-2 Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ..................127

B-3 Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. ..................................127

C-1 Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......128

C-2 Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......129

C-3 Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......129

C-4 Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................130

xv

C-5 Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................130

C-6 Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................131

C-7 Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................131

C-8 Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................132

C-9 Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................132

C-10 Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..................................................133

C-11 Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................133

C-12 Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..................................................134

xvi

C-13 Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......134

C-14 Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......135

C-15 Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......135

C-16 Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................136

C-17 Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................136

C-18 Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................137

C-19 Microbial glycogen yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................137

C-20 Microbial glycogen yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................138

C-21 Microbial glycogen yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..................................................138

xvii

C-22 Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......139

C-23 Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......139

C-24 Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......140

C-25 Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................................................140

C-26 Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................................................141

C-27 Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................141

C-28 Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......142

C-29 Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......142

C-30 Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......143

C-31 Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................143

xviii

C-32 Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................144

C-33 Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................144

C-34 Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................145

C-35 Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................145

C-36 Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................146

C-37 Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..................................................146

C-38 Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................147

C-39 Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..................................................147

xix

C-40 Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......148

C-41 Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......148

C-42 Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......149

C-43 Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................149

C-44 Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................150

C-45 Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................150

C-46 Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................151

C-47 Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................151

C-48 Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................152

xx

C-49 Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..................................................152

C-50 Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................153

C-51 Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..................................................153

C-52 Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...................................................................................................154

C-53 Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...................................................................................................154

C-54 Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...................................................................................................155

C-55 Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ....................................155

C-56 Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ....................................156

xxi

C-57 Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ....................................156

C-58 Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......157

C-59 Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......157

C-60 Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......158

C-61 Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................158

C-62 Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................159

C-63 Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................159

C-64 Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......160

C-65 Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......160

C-66 Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......161

xxii

C-67 Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................................................161

C-68 Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................................................162

C-69 Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................162

C-70 Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................163

C-71 Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................163

C-72 Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. ...............................................................................................................164

C-73 Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..................................................164

C-74 Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..............................................................165

xxiii

C-75 Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate. ..................................................165

xxiv

LIST OF ABBREVIATIONS

ADF acid detergent fiber ApH acidic pH medium B non-protein nitrogen+ true protein medium BCVFA branched chain volatile fatty acid BW body weight C true protein only medium CP crude protein DM dry matter DMI dry matter intake GLY microbial glycogen iNDF isolated neutral detergent residue MCP microbial crude protein MCPeff microbial crude protein efficiency N Nitrogen NDF neutral detergent fiber NDFCP neutral detergent fiber crude protein NDFOM neutral detergent organic matter NFC non-neutral detergent fiber carbohydrate NH3-N ammonia nitrogen NpH neutral pH medium NPN non-protein nitrogen NRC National Research Council NSC non-structural carbohydrate OM organic matter OMD organic matter digested OMI organic matter intake Pe Pectin RDP rumen degradable protein St Starch Su Sucrose SuNDF sucrose + isolated neutral detergent residue TCA trichloroacetic acid U non-protein nitrogen only medium VFA volatile fatty acid

xxv

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

EFFECT OF NONFIBER CARBOHYDRATES ON PRODUCT YIELD AND FIBER DIGESTION IN FERMENTATIONS WITH MIXED RUMINAL MICROBES

By

Lucia Holtshausen

December 2004

Chair: Mary Beth Hall Major Department: Animal Sciences

Effects of nonfiber carbohydrate (NFC) supplementation on fiber digestion and

fermentation product yields were examined in three in vitro fermentation studies. Studies

1 and 2 respectively examined the effects of medium pH (5.6 vs. 6.7) and nitrogen source

(non-protein nitrogen (U) vs. true protein (C) vs. mixture (B)) on fermentation of isolated

neutral detergent residue (iNDF) with or without sucrose (Su). Study 3 examined the

effect of supplementing iNDF with starch, sucrose, pectin or their combinations.

Anaerobic fermentations of 24 h (Studies 1 and 3) and 16 h (Study 2) were performed in

batch culture with Goering and Van Soest medium and rumen inoculum. Fermentation

samples were analyzed for residual substrate, pH, iNDF digestion, and microbial

fermentation products.

In Study 1, maximum microbial crude protein (MCP) and glycogen (GLY) yields

for Su+iNDF were greater at pH 6.7 (MCP:19.4 mg; GLY:6.0 mg) than those at pH 5.7

(MCP:11.1 mg; GLY:3.5mg). At pH 6.7, 24 h iNDF digestion was greater for Su+iNDF

xxvi

(42.4%) than for iNDF (26.4%) and the reverse was true at pH 5.6 (Su+iNDF: 2.2%;

iNDF: 7.8%).

In Study 2, maximum MCP yields from fermentations of Su+NDF in media

containing C (15.7 mg), and B (14.3 mg) were greater than those containing U (8.05 mg).

At 16 h, iNDF digestion for Su+iNDF was lower for U vs. B and C, with no difference

among treatments for iNDF. Maximum GLY was similar among nitrogen treatments.

In Study 3, sucrose decreased pH more than NFC combinations (sucrose+starch,

starch+pectin, sucrose+pectin) followed by pectin, starch and iNDF (6.83, 6.87, 6.89,

7.03 and 7.13, respectively). Residual iNDF was increased by sucrose (60.6%), not

affected by starch (61.5%), and decreased by pectin (65.7%) and NFC combinations

(63.6 %) compared to iNDF fermented alone (61.8%). Maximum MCP yield was

greatest for NFC combinations followed by pectin, starch, sucrose and iNDF fermented

alone (22.4, 18.6, 16.6, 14.8 and 5.12 mg, respectively).

The various types of NFCs as well as pH and nitrogen source altered the yield of

microbial products and extent of fiber digestion. Treatment of NFCs as a uniform entity

in ruminant nutrition is not warranted.

1

CHAPTER 1 REVIEW OF LITERATURE: MICROBIAL FERMENTATION OF NON-NEUTRAL

DETERGENT FIBER CARBOHYDRATES, AND HOW IT MAY RELATE TO ANIMAL PERFORMANCE

Introduction

In order to meet the nutritional needs of high producing beef and dairy cattle, it is

necessary to supplement diets with energy and protein rich feeds. Grain and byproduct

feeds, such as molasses, have been used to increase the energy density of ruminant diets

(Huntington, 1997; Lykos et al., 1997). Other byproduct feeds, such as soybean hulls

(Royes et al., 2001), almond hulls (Grasser et al., 1995), sugar beet pulp (Mansfield et al.,

1994) and citrus pulp (Ammerman et al., 1963; Van Horn et al., 1975; Fegeros et al.,

1995; Leiva et al., 2000; Arthington et al., 2002), have been used as substitutes for grains.

These byproduct feeds have lower starch contents and higher neutral detergent soluble

fiber (NDSF) contents (compared to grain feeds), and may have high sugar contents

(Hall, 1998). Starch, sugars (mono- and oligosaccharides) and NDSF (non-starch, non-

neutral detergent fiber polysaccharides) are three of the major components of the

carbohydrate fraction of feeds referred to as non-neutral detergent fiber carbohydrates

(NFCs).

The NFC fraction of feedstuffs is estimated from the following calculation: NFC =

100 – crude protein – ether extract – ash – neutral detergent fiber + neutral detergent

insoluble crude protein (National Research Council [NRC], 2001). The terms non-

structural carbohydrates (NSCs) and NFCs have at times been used interchangeably for

the fraction derived by this calculation. However, NSCs refer to cell contents, and

2

include organic acids (which are not carbohydrates), mono- and oligosaccharides, starch

and fructans, whilst NFCs also include soluble fiber: pectic substances, β-glucans and

galactans (Van Soest et al., 1991). Therefore, NFCs include structural and non-structural

carbohydrates, as well as fibrous and non-fibrous carbohydrates (Hall, 1998).

From a nutritional standpoint, some of the carbohydrates in plants are also

designated as dietary fiber. This term refers to the non-starch polysaccharides that are

not digestible by mammalian enzymes. Dietary fiber encompasses both NDSF and

neutral detergent fiber (NDF), thus, including both cell wall carbohydrates and some cell

contents. Cellulose, hemicelluloses, lignin, pectic substances, β-glucans, fructans and

galactans are all dietary fiber. Pectic substances, β-glucans, fructans and galactans are

soluble in neutral detergent solution, and thus are included in the NFC fraction.

The NRC (2001) applies a total digestible nutrient content of 98% to the NFC

fraction and it can therefore play a major role in the nutrient supply to the animal. The

rest of this discussion will focus on sucrose, starch and pectic substances (more

specifically, pectin), as three of the major components of NFCs.

Chemical Structure and degradation of NFC Sources

When trying to understand or predict animal responses to supplementation of NFC

sources it is necessary to keep in mind that this carbohydrate fraction is by no means a

homogenous entity, chemically or nutritionally. Some of the fundamental differences of

the individual components are found in their chemical structure.

Sucrose

Sucrose is the primary vehicle for energy transport in plants and the majority of

plants convert sucrose into polymeric forms for storage (Van Soest, 1994). Sucrose, and

its constituent monosaccharides glucose and fructose, are the predominant saccharides of

3

the mono- and oligosaccharide component of NFC and are found in byproduct feeds such

as molasses (Kunkle et al., 2000), sugar beet pulp (Hall, 2002) and citrus pulp (Ben-

Ghedalia et al., 1989). Sucrose is a disaccharide consisting of single glucose and fructose

monomers linked through an α-1→2 linkage. It is considered to be 100% degradable in

the rumen (Sniffen et al., 1992) and is reported to be fermented at a rate as high as

300%/h (Sniffen et al., 1983). Sucrose is hydrolyzed to glucose and fructose by the

enzyme sucrase (Van Soest, 1994). Ruminal bacteria that ferment sucrose include

Streptococcus bovis, Lachnospira multiparus, Lactobacillus ruminis, Lactobacillus

vitulinis, Clostridium longisporum, Eubacterium cellulosolvens, and some strains of

Eubacterium ruminantium, Butyrivibrio fibrisolvens, Ruminococcus albus, Ruminococcus

flavefaciens, Megaspaera elsdenii, Prevotella spp., Selenomonas ruminantium and

Succinivibrio dextrinosolvens (Stewart et al., 1997).

Starch

Starch is a polymer of glucose molecules and is the major storage carbohydrate in

most cereal grains. It consists of amylose, a predominantly linear α-(1→4) linked

polymer, and amylopectin, an α-(1→4) linked polymer with α-(1→6) linked branches,

which can be present in various ratios. Amylopectin comprises 70 to 80% of most cereal

starches and amylose 20 to 30% (Rooney and Pflugfelder, 1986). The proportions of

these two polysaccharides appear to affect the digestion characteristics of starch. There

are contradictions in the literature as to the ease of hydrolysis of amylose as compared to

amylopectin. Rooney and Pflugfelder (1986) described starch granules as having

crystalline and amorphous areas, with amylopectin comprising the majority of the

crystalline region and amylose that of the amorphous region. According to Rooney and

Pflugfelder (1986) amylase attack starts in the amorphous region and hydrolysis of the

4

crystalline areas occurs more slowly. However, the author also stated that the

digestibility of starch is inversely proportional to the amylose content. According to Piva

and Masoero (1996) amylose is slowly degraded in the rumen, whereas amylopectin is

more rapidly degraded. The reason for the conflicting statements may be because

amylose and amylopectin both contain crystalline areas and the crystalline area of

amylose has greater crystal strength (Van Soest, 1994). Therefore, hydrolysis may start

in the amorphous region of amylose, exposing the amylopectin for hydrolysis, while the

crystalline region of amylose hydrolyzes more slowly. Nonetheless, starch fermentation

in the rumen is considered to be extensive, but can vary from 40 to 90% (NRC, 2001)

depending on factors such as structure (amylose/amylopectin ratio; (Piva and Masoero,

1996), plant source (Rooney and Pflugfelder, 1986) and processing or physical form

(Baldwin and Allison, 1983).

Starch can be degraded by ruminal microbial enzymes as well as enzymes in the

small intestine of the animal. A series of enzymes are required to degrade amylose and

amylopectin to glucose in both the rumen and small intestine. These include randomly

acting endo-α-amylases releasing maltodextrins from amylose and β-amylases removing

maltose units from the non-reducing end of the chain. Approximately 50% of

amylopectin can be degraded by β-amylase to maltose. The residue is hydrolyzed by

glucoamylase (cleaving α-(1→4) linkages), and α-dextrin-6-glucanohydrolase and

isoamylase (cleaving at the α-(1→6) linked branch points). Maltose and maltodextrins

are degraded to glucose by α-glucosidase (Chesson and Forsberg, 1997). There are

numerous amylolytic ruminal bacteria, which include Ruminobacter amylophilus,

5

Prevotella ruminicola, Succinimonas amylolytica, S. bovis, S. ruminantium, B.

fibrisolvens, E. ruminantium and Clostridium spp. (Cotta, 1988).

Pectic Substances

Pectic substances are found in the middle lamella and other cell wall layers (Van

Soest, 1994), but are not covalently linked to the lignified portions. They are almost

completely digested (90-100%) in the rumen (Nocek and Tamminga, 1991; NRC, 2001).

Pectic substances are a family of complex molecules that contain a great variety of

monomers and potential branch-points. The pectin backbone consists of galacturonic

acid monomers linked via α-(1→4) linkages, and rhamnose inserts. With the addition of

neutral sugar side chains, made up largely of arabinose and galactose, bound to the

rhamnose inserts, these complex molecules are referred to as pectic substances (Jarvis,

1984). Various degrees of methoxylation (Jarvis, 1984) and acetylation (Marounek and

Dušková, 1999) of the galacturonic acid backbone are possible. The acid groups in the

backbone can also be associated with calcium ions (Van Soest, 1994).

Animals do not have the enzymes to digest pectin, but microorganisms in the

rumen do (McDonald et al., 1995). At least two enzymes, a methylesterase and

polygalacturonidase, are required for pectin hydrolysis (Baldwin and Allison, 1983).

Pectin-utilizing bacteria include some of the prominent ruminal populations such as

Fibrobacter succinogenes, P. ruminicola, B. fibrisolvens, S. bovis and Lachnospira

multiparus (Czerkawski and Breckenridge, 1969; Gradel and Dehority, 1972; Baldwin

and Allison, 1983).

NFCs and Microbial Fermentation Products

Microbial fermentation products play an important part in the nutrient supply to the

ruminant animal and thus also in animal performance. Major products of microbial

6

fermentation of carbohydrates include organic acids, which are absorbed through the

ruminal wall and serve as a source of energy for the animal, and microbial mass, which

provides potentially metabolizable nutrients in the form of protein, glycogen, and lipids

to the animal when they pass to the small intestine. The NFC fraction is considered a

good source of readily available energy for microbial growth (Ariza et al., 2001). It has

been suggested that microbial growth is directly proportional to the rate of carbohydrate

degradation (Russell et al., 1992). As documented in the Cornell Net Carbohydrate and

Protein System model, simple sugars are considered to have a fast degradation rate, and

starch and pectin an intermediate rate (Sniffen et al., 1992). This would imply that

supplementation with sugars would yield more microbial mass and other microbial

fermentation products compared to starch and pectin. There is evidence, both in vitro and

in vivo, of differences in fermentation characteristics among individual NFC components

(Table 1-1). Sometimes these results have not been consistent with the notion that

individual NFC components with faster degradation rates result in greater microbial yield

(Hall and Herejk, 2001). It is important to understand how individual NFC components

differ in their effect on microbial product yield and efficiency of yield to help predict and

explain some of the variation seen in animal performance when supplementing with

NFCs. Differences among NFC components regarding microbial fermentation may also

imply that the complement of NFCs in a particular feedstuff is important when predicting

animal response.

Organic Acids

Total volatile fatty acids

Total volatile fatty acid (VFA) production is generally similar among different

NFC sources (Table 1-1) both in vitro (Mansfield et al., 1994; Ariza et al., 2001) and in

7

vivo (Ben-Ghedalia et al., 1989; Khalili and Huhtanen, 1991a; Chamberlain et al., 1993;

Moloney et al., 1994; O'Mara et al., 1997a; Leiva et al., 2000; Sannes et al., 2002;

Voelker and Allen, 2003c). However, Bach et al. (1999) reported an increase (P > 0.05)

in total VFA concentration for cracked corn compared with beet pulp and molasses in a

continuous culture study. In contrast, total VFA concentration increased (P = 0.01) or

tended to increase (P = 0.07) in lactating dairy cows fed a total mixed ration (TMR)

containing dried citrus pulp and high moisture ear corn in a 50:50 ratio compared to cows

receiving a TMR containing high moisture ear corn or cracked shelled corn, respectively

(Broderick et al., 2002b). The varying results in these studies may be due to the fact that

the combinations of NFCs that were compared differed, which may imply that the effect

on VFA production from supplementation with individual NFC sources may not be

additive.

Acetate, propionate and butyrate are the major VFAs included in the total VFA

concentration. Despite giving relatively similar total VFA yields there may be

differences in the relative proportions of individual VFAs from different NFC sources.

Acetic acid

Acetate (acetic acid) is a lipogenic nutrient, a precursor of fatty acid synthesis and

ultimately of milk fat synthesis in the mammary gland (Van Soest, 1994). Starch and

sucrose (Sutton, 1979; Khalili and Huhtanen, 1991a; Chamberlain et al., 1993; Moloney

et al., 1994; Heldt et al., 1999) have been associated with relative decreases in ruminal

acetate concentration, whereas pectin had either no effect (Van Vuuren et al., 1993; Leiva

et al., 2000) or increased (Broderick et al., 2002b; Voelker and Allen, 2003c) acetate in

the rumen (Table 1-1). In vitro fermentations of different carbohydrates showed a greater

8

acetate production from pectin compared to starch and sucrose (P < 0.05), which did not

differ from each other (P > 0.05; [Strobel and Russell, 1986]).

The effect of sugars on the ruminal molar proportion of acetate in vivo may depend

on the amount of sucrose or glucose included in the diet (Table 1-1). Sucrose

supplementation at 10% of silage DM intake did not affect ruminal acetate molar

proportion for sheep compared to those on the silage control diet (P > 0.05; [Charmely et

al., 1991]). When sucrose was substituted for corn at 3.2% of diet DM in a diet for

lactating dairy cows, acetate production was also not affected (P = 0.15; [Sannes et al.,

2002]). Dextrose (glucose) at 5.6% of diet DM did not affect ruminal acetate proportions

in heifers compared to a medium concentrate diet containing 39.7% ground barley, or the

control diet containing 10% ground barley (P > 0.05; [Piwonka et al., 1994]). However,

when starch or sucrose was supplemented at 200g/d (approximately 5% of daily diet DM)

to a grass silage diet, ruminal acetate proportions for sheep on the starch-supplemented

diet did not differ compared to those on the control diet, whereas sheep on the sucrose-

supplemented diet had decreased ruminal acetate proportions (P < 0.05; [Chamberlain et

al., 1993]). When cane molasses, a source of sugars, was fed to steers at 61% of DM

intake, ruminal acetate proportions was decreased compared to steers fed a diet with the

same amount of barley, a starch source (P < 0.01; [Moloney et al., 1994]).

Pectin is reported to ferment primarily to acetate (Czerkawski and Breckenridge,

1969; Marounek et al., 1985). When citrus pectin was fermented in cultures of B.

fibrisolvens 787 and P. ruminicola AR29, 73.7 and 57.3% of metabolite carbon was

captured in acetate, respectively (Marounek and Dušková, 1999). Citrus pulp can contain

25.2 to 43.7% neutral detergent-soluble fiber (Hall, 2002), of which pectin is a major

9

component. Several continuous culture studies reported an increase in acetate proportion

for fermentations of beet pulp and citrus pulp compared to corn (Table 1-1), whether it

was included at the same concentration as corn (P < 0.05; [Bach et al., 1999]) and P =

0.03; [Ariza et al., 2001]) or replaced a portion of the corn (P < 0.05; [Mansfield et al.,

1994]). This effect was also seen in vivo when increasing concentrations of citrus pulp

substituted for high moisture corn in lactating dairy cow diets resulted in a linear increase

(P < 0.01) in the ruminal acetate proportion (Voelker and Allen, 2003c). Ben-Ghedalia et

al. (1989) also reported increased ruminal acetate proportions for cannulated rams fed

dried citrus pulp compared to those fed barley (P < 0.05). It would appear that

fermentation of pectin in general increases the molar proportion of acetate compared to

fermentation of sugars and starch, while sugars often decrease the acetate proportion

when compared to starch.

Propionic acid

Propionate (propionic acid) is a precursor for glucose synthesis in the liver and thus

important for the glucogenic energy supply to the ruminant. In vitro fermentation with

mixed ruminal bacteria yielded similar propionate concentrations from starch and sucrose

(P > 0.05; [Strobel and Russell, 1986]), Table 1-1). The effect of sugars compared to

starch on ruminal propionate proportion varies among in vivo studies. In some in vivo

studies ruminal molar proportions of propionate did not differ between sugars and starch,

whether small amounts (5.6% dextrose; Piwonka et al., 1994) or larger amounts (61%

molasses; Moloney et al., 1994) of sugar were added to the diet. In contrast, ruminal

propionate molar proportions in sheep on a starch-supplemented diet was similar to that

of sheep fed the control ryegrass silage diet (P > 0.05), whereas ruminal propionate

proportions increased (P < 0.05) for sheep fed a sucrose supplementation (Chamberlain et

10

al., 1993). In another contrasting study, ruminal molar proportions of propionate tended

to increase with starch supplementation (P = 0.11) compared to supplementation of

sugars (sucrose, glucose and fructose) when a low amount of ruminally degradable

protein (RDP; 0.031% BW/d) was supplemented to steers, and increased (P < 0.01)

ruminal propionate proportions when a higher amount (0.122% BW/d) of RDP was

supplemented (Heldt et al., 1999). It may be that other components of the diet such as

protein alter the yield of propionate from NFCs.

Pectin yielded less (P < 0.05) propionate compared to starch and sucrose when

fermented in vitro with mixed ruminal bacteria (Strobel and Russell, 1986, Table 1-1).

Citrus pectin fermented by a P. ruminicola AR29 in vitro culture yielded a small amount

of propionate (3.2 mmol/L), while no propionate production was detected in the culture

with B. fibrisolvens 787 (Marounek and Dušková, 1999). Corn additions increased

propionate molar proportions in continuous culture fermentations when compared to

similar amounts of citrus pulp (P = 0.02; [Ariza et al., 2001] and beet pulp (P < 0.05;

[Bach et al., 1999]). Broderick et al. (2002b) also reported higher ruminal propionate

proportions in lactating dairy cows fed high moisture ear corn (P < 0.01) and cracked

shelled corn (P = 0.04) compared to cows fed a diet in which citrus pulp substituted for

50% of high moisture ear corn. However, when beet pulp was substituted for 50% of the

corn in a continuous culture study no difference was found for the molar proportion of

propionate (P > 0.05, [Mansfield et al., 1994]). Other researchers also reported no effect

on ruminal propionate proportions in lactating dairy cows when replacing beet pulp for

ground corn (O'Mara et al., 1997a) and replacing dried citrus pulp for corn hominy

(Leiva et al., 2000). The varied propionate response when feeding citrus and beet pulp

11

could be a result of the variation in composition of these feedstuffs, especially in the ratio

of sugars to neutral detergent soluble fiber. It would appear that pectin yields less

propionate than sugars and starch, with no clear difference between the latter two NFCs.

Butyric acid

Butyrate (butyric acid) supplies energy to the animal, mainly to the heart and

skeletal muscle, in the form of β-hydroxybutyrate (a ketone body; McDonald et al.,

1995). It is lipogenic and can be used for the production of fat. Ruminally-produced

butyrate is converted to β-hydroxybutyrate in the ruminal epithelial cells, and is

considered more effective than propionate or acetate in enhancing development of

ruminal papillae (Van Soest, 1994). Overall, it would appear that sucrose yields more

butyrate than other NFCs (Table 1-1). In vitro fermentations with mixed ruminal

microorganisms yielded more butyrate from sucrose compared to starch (P < 0.05),

which in turn yielded more butyrate than pectin (P < 0.05; [Strobel and Russell, 1986]).

Several in vivo studies also reported increased butyrate production from sucrose

compared to starch. Ruminal butyrate proportions in cannulated steers increased with

sugar (sucrose, glucose and fructose) supplementation compared to supplementation with

starch (Heldt et al., 1999). Khalili and Huhtanen (1991a) reported greater ruminal molar

proportions of butyrate for bulls consuming a sucrose-supplemented diet compared to a

grass silage and barley-based diet. Steers fed a molasses-based diet also had increased

ruminal butyrate proportions compared to those fed a barley-based diet (Moloney et al.,

1994).

Studies that have evaluated the fermentation of pectin or feeds that are reported to

contain substantial amounts of pectin have shown differences among microorganisms in

the yield of butyrate. Pectin fermentation in a B. fibrisolvens 787 culture yielded a small

12

amount of butyrate (2.6 mmol/L), while no butyrate production was detected in a culture

with P. ruminicola AR29 (Marounek and Dušková, 1999). In vivo comparisons of the

fermentation of feeds high in starch and those that typically contain a high proportion of

pectin (citrus and beet pulps) have shown no difference (Ben-Ghedalia et al., 1989; Leiva

et al., 2000) or an increase in ruminal butyrate concentration (Broderick et al., 2002b;

Voelker and Allen, 2003c) for animals consuming diets containing pulps. Citrus pulp can

contain between 12.5 and 40.2% sugars, and sugar beet pulp between 12.8 and 24.7%

(Hall, 2002). The increase in the proportion of butyrate in these studies may be a result

of the fermentation of sugar rather than of the soluble fiber content. This emphasizes the

need to know the NFC complement of a feedstuff when evaluating the effect of

supplementation on fermentation and animal performance.

Branched chain volatile fatty acids

The branched chain volatile fatty acids (BCVFAs), isobutyric, iso-valeric and 2-

methylbutyric acid result from the deamination of valine, leucine and iso-leucine,

respectively (Van Soest, 1994). Branched chain VFAs serve as carbon skeletons to

ruminal microorganisms for the synthesis of microbial crude protein (MCP) from

ammonia. In fact, the value of amino acids to cellulolytic organisms that have an

obligate need for BCVFAs appears to be mainly as a source of BCVFAs (Stern, 1986).

Sheep fed diets supplemented with sucrose or starch showed decreased proportions

of ruminal BCVFAs compared to those fed a silage control diet (Chamberlain et al.,

1993, Table 1-1), and the proportion of BCVFAs for sheep fed the starch-supplemented

diet (2.8 mol/100 mol) was numerically higher than for those fed the sucrose supplement

(1.8 mol/100 mol; difference was not statistically tested). The ruminal concentrations of

BCVFAs for lactating dairy cows were greater for animals fed a corn control diet

13

compared to those receiving a diet with sucrose (P = 0.02) substituted for corn at 3.2 %

of diet DM (Sannes et al., 2002). Fermentation of corn by mixed ruminal

microorganisms in continuous culture studies gave higher proportions of BCVFAs as

compared to citrus pulp (P = 0.03; [Ariza et al., 2001]) and sugar beet pulp (P > 0.05;

[Mansfield et al., 1994] and P < 0.05; [Bach et al., 1999]). The apparently consistent

thread here is that BCVFA concentrations are less for diets with more sucrose relative to

starch.

Lactic acid

Compared to acetate, butyrate and propionate (average pKa = 4.8), lactate (lactic

acid, pKa = 3.1) is a 10-fold stronger acid (Dawson et al., 1997). An increase in lactate

concentration therefore has a greater potential to decrease ruminal pH. The fermentation

of sugars and starch can yield lactate (Strobel and Russell, 1986), whereas pectin

fermentation is generally not associated with lactate production (Strobel and Russell,

1986; Hatfield and Weimer, 1995). In vitro fermentations of sucrose with mixed ruminal

microorganisms gave a higher lactate concentration compared to fermentations with

starch (P < 0.05; [Strobel and Russell, 1986], Table 1-1). Heldt et al. (1999) also

reported higher ruminal proportions of lactate for steers fed sugar supplements (sucrose,

glucose, fructose) compared to those fed starch. However, in a study with cannulated

steers, animals fed a barley-based diet tended to have higher ruminal concentrations of L-

lactate compared to those receiving a molasses-based diet (P = 0.09; [Moloney et al.,

1994]). This difference in lactate production response may have been a result of a

difference in the source of starch (corn starch vs. barley) and sugar (sucrose, glucose and

fructose vs. molasses) supplemented in the two studies.

14

Although pectin is generally not associated with the production of lactate, pectin

fermentation has been shown to yield small amounts of lactate (Czerkawski and

Breckenridge, 1969). Cultures of B. fibrisolvens 787 and P. ruminicola AR29 both

produced small amounts of lactate (1.5mmol/L and 0.4mmol/L, respectively) from the

fermentation of citrus pectin (Marounek and Dušková, 1999). In vivo studies showed no

effect on lactate production in animals fed diets containing citrus pulp (P = 0.34; [Leiva

et al., 2000]) or sugar beet pulp (P = 0.72; [Voelker and Allen, 2003c]) compared to those

fed corn hominy and high moisture corn supplements, respectively. In general, pectin is

not expected to yield lactate to the extent that sugars and starch may.

Microbial Mass

Microbial composition

The microbial mass that flows from the rumen to the small intestine forms a major

part of the metabolizable nutrient supply to the ruminant animal. The composition of the

microorganisms determines the potential specific nutrient contribution to the small

intestine. Bacteria typically contain 50% protein, 20% RNA, 3% DNA, 9% lipid and

18% carbohydrate, but this composition can change dramatically (Nocek and Russell,

1988). The two main components of microbial mass that contribute to the metabolizable

nutrient supply in the small intestine are MCP and microbial storage carbohydrate (α-

glucan). Bacterial amino nitrogen as a percentage of total nitrogen has been considered

as relatively constant, but it can range from 54.9 to 86.7%, with an average of 66.5%

(Clark et al., 1992). Large changes may also be seen in microbial glycogen content,

especially when cultures are starved for nutrients other than energy (McAllan and Smith,

1974; Nocek and Russell, 1988).

15

There are a variety of factors that can affect microbial growth, of which supply of

carbohydrate and nitrogen (Stern, 1986; Hoover and Stokes, 1991; Clark et al., 1992)

appear to be the most important. Another factor that may affect the efficiency of

microbial growth is pH (Russell et al., 1992). The rest of this discussion will focus on the

synthesis of MCP and glycogen from fermentation of starch, sucrose and pectin. The

effect of nitrogen source (ammonia nitrogen, amino acids) and pH on microbial

fermentation product yield from NFCs will also be considered.

Microbial protein yield

Microbial crude protein has been reported to supply from 34 (Clark et al., 1992);

summary of 31 articles) to 80 (Owens and Bergen, 1983; Stern, 1986) or even 89%

(Clark et al., 1992) of the total amino acid nitrogen entering the small intestine of

ruminants (Owens and Bergen, 1983; Stern, 1986). Microbial crude protein is considered

to have a good amino acid balance relative to the animal’s requirements (Clark et al.,

1992) with a mean true digestibility of 84.7% (Storm et al., 1983). Accordingly, MCP is

an important source of true protein to the animal.

Carbohydrate fermentation in the rumen provides both energy in the form of ATP,

and carbon skeletons for MCP synthesis. The amount of carbohydrate and its rate of

fermentation regulate microbial metabolism and are in turn regulated by the physical and

chemical form of carbohydrates (Stern et al., 1994). Hall and Herejk (2001) compared

the MCP yield from sucrose, starch and pectin in an in vitro fermentation with mixed

ruminal microorganisms. Microbial growth was initiated most rapidly on sucrose,

followed by pectin and starch. Maximal yield was greatest for starch compared to

sucrose and pectin (P < 0.01), which did not differ from each other (P = 0.30). An

explanation offered for the proportional difference between maximal yields of MCP for

16

pectin and starch was the difference between the NFCs in the amount of hexose, and

relative amounts of carbon available to the microorganisms. However, this approach still

overestimated the theoretical maximal yield of MCP from sucrose.

In an in vivo study, Cameron et al. (1991) found no effect on microbial nitrogen

flow to the small intestine and efficiency of microbial crude protein synthesis (MCPeff)

in response to starch and dextrose supplementation to mid-lactation Holstein cows.

Dijkstra et al. (1992) suggested that variation in the results on MCP yield and efficiency

might be due to the lack of correction for microbial α-glucan reaching the small intestine.

For lack of a convenient method to distinguish between dietary starch that escapes

degradation in the rumen and α-glucan (glycogen) stored in ruminal microorganisms,

starch measured as glucose hydrolyzed from α-glucan in the duodenal digesta will

include both fractions. Therefore undigested feed starch content of digesta in the

duodenum will be overestimated, and ruminal digestion of starch will be underestimated,

and that of dextrose, overestimated. Expressing MCP yield as a proportion of the

carbohydrate digested in the rumen, with an underestimation of starch digestion in the

rumen, will lead to overestimation of the MCPeff for starch. The efficiency of microbial

yield from dextrose would be underestimated, because the unfermented dextrose in the

form of glycogen would not be accounted for.

In a continuous culture system, Mansfield et al. (1994) found an increase (P < 0.05)

in non-ammonia nitrogen flow when beet pulp was substituted for corn, but there was no

effect on MCPeff (P > 0.05) and bacterial crude protein production (P > 0.05). If there

was less degradation of crude protein from the feed, this may have resulted in a lower

ammonia nitrogen concentration and less available nitrogen for MCP synthesis, which

17

could explain why no difference in bacterial crude protein production was reported

between beet pulp and corn. In the study by Mansfield et al. (1994) there were indeed

decreased ammonia nitrogen concentrations in fermentations of beet pulp compared to

fermentations of corn (P < 0.05).

Microbial α-glucan

Many species of ruminal bacteria produce polysaccharide, and some can store large

amounts as an intracellular reserve (Thomas, 1960; John, 1984; Lou et al., 1997).

However, studies with P. ruminicola B14 (Russell, 1992) and F. succinogenes S85

(Maglione and Russell, 1997) indicated that ruminal bacteria might have a limited

capacity to store polysaccharides as shown by a decrease of the viable cell number when

the polysaccharide:protein ratio of the cultures exceeded 1.0.

Little information exists on microbial α-glucan production from different

carbohydrate sources. As mentioned earlier, it is difficult to distinguish microbial α-

glucan from dietary starch that passed through the rumen undegraded. McAllan and

Smith (1974) reported a higher microbial α-glucan content for animals on a diet with

more than 70% concentrates (barley and flaked corn). In the same study, time after

feeding also had an effect, with an increased microbial α-glucan content from feeding

through four to six hours after feeding. McAllan and Smith (1974) established a ratio of

individual carbohydrates to nucleic acids in samples of ruminal bacteria to use as an

estimate of the contribution of microbial carbohydrate in the duodenum. This may not be

a very accurate way to quantify microbial α-glucan content. Craig et al. (1987)

confirmed the change in microbial α-glucan content with time after feeding, and added

that particle-associated microbial populations had higher α-glucan content compared to

the liquid-associated populations in the rumen. Therefore, the ratio of individual

18

carbohydrates to nucleic acids will depend on the method of sampling and isolating

ruminal bacteria, as well as time of sampling.

Ruminal microorganisms may incorporate and store carbohydrate as α-glucan

under conditions of excess available carbohydrate (shortly after feeding) and potentially

limiting nitrogen supply. McAllan and Smith (1974) reported diurnal variations in the α-

glucan content of ruminal bacteria. It is possible that when the supply of available

dietary carbohydrate runs out, microorganisms utilize the storage carbohydrate as a

source of energy. This stored carbohydrate can also become available to other

microorganisms upon cell lysis or it can pass to the small intestine and become part of the

glucose supply to the animal.

Other Factors Which Influence Microbial Product Yield

Nitrogen Source

Most ruminal microorganisms can synthesize MCP with a non-protein nitrogen

(NPN) source such as urea, as the sole source of nitrogen (Oltjen, 1969). In a review of

several studies Wallace et al. (1997) concluded that microbial nitrogen derived from

ruminal ammonia nitrogen can range from 40 to 100%. Maximal in vitro microbial

growth has been reported at ammonia nitrogen concentrations of 5 to 8 mg/100 ml (Satter

and Slyter, 1974) in continuous culture studies with purified substrates (starch, cerelose,

wood pulp), a concentrate based diet (cracked corn) or a forage-concentrate combination

(cracked corn, cerelose, lucerne hay, timothy hay). Hume (1970) reported maximum in

vivo MCP synthesis at a ruminal ammonia nitrogen concentration of approximately 9

mg/100ml in sheep fed diets containing urea only or a 50:50 ratio of urea and protein

(casein, gelatin or zein). Ruminal ammonia is a source of nitrogen for MCP synthesis for

both structural and non-structural carbohydrate fermenting microorganisms. In the

19

presence of increased levels of NFCs potential competition between NFC-utilizing

bacteria and cellulolytic bacteria for ammonia could lead to an increase in the theoretical

optimum ammonia concentration required for maximum growth of cellulolytic bacteria.

However, over the longer time of the feeding cycle this apparent negative impact of

increasing NFCs might be alleviated by cross-feeding among different ruminal microbial

populations (McAllister et al., 1994). An example of cross-feeding was shown by Miura

et al. (1980) when Megasphaera elsdenii (lactate utilizer) deaminated protein resulting

from the lysis of Ruminobacter amylophilus (starch utilizer) which provided BCVFA for

the growth of Ruminococcus albus (cellulose and hemicellulose utilizer).

Several studies reported no difference in ruminal ammonia concentration when

comparing different NFC sources (Chamberlain et al., 1993; O'Mara et al., 1997a; Sannes

et al., 2002). When interpreting the response to ruminal ammonia concentration it is

important to keep in mind that this concentration is the net result of protein degradation

in the rumen, ammonia absorption across the ruminal wall, ammonia passage from the

rumen and ammonia incorporation into MCP. Therefore decreased ruminal ammonia

concentration may indicate increased utilization by ruminal microorganisms and potential

increased MCP synthesis. In this case a decrease in ammonia concentration might be

viewed as a positive response. On the other hand a decrease in ammonia concentration

may also indicate a decrease in protein degradation, in which case nitrogen might become

limited and MCP synthesis decreased. If a decrease in ammonia concentration is

accompanied by increased MCP synthesis, it may provide needed nutrients to support

higher milk protein production.

20

Although MCP synthesis can occur with urea as the sole source of nitrogen, the

efficiency of yield may be lower compared to when peptides or amino acids are supplied.

Maeng et al. (1976) reported an optimum ratio of urea nitrogen to amino acid nitrogen of

75:25 for ruminal microbial growth in a series of in vitro fermentations with mixed

ruminal microorganisms in which glucose, cellobiose and starch were the carbohydrate

sources. However, nitrogen used for microbial growth may be derived from sources

other than. Amino acids and peptides, which are breakdown products of dietary protein,

may be directly incorporated into microbial crude protein. Alternatively, amino acids

from dietary origin can be degraded in the rumen to BCVFAs and ammonia, which can

then be used for MCP synthesis.

Both peptides and amino acids have been shown to stimulate MCP synthesis when

substituted for ammonia in vivo (Rooke and Amstrong, 1989) and in vitro (Russell and

Strobel, 1993). The importance of amino acids and peptides from dietary protein

degradation for increasing both MCP production and energetic efficiency has been shown

in several studies with batch culture fermentations (Maeng et al., 1976; Maeng and

Baldwin, 1976a, 1976b). Russell and Sniffen (1984) reported an increase of 18.7% in

ruminal bacteria yield with the addition of amino acids to mixed cultures with

theoretically adequate ammonia concentrations. A yield increase of 28% in vivo (sheep)

has also been reported when true protein was added to urea-containing diets (Hume and

Purser, 1974).

The advantages of peptides and amino acids over NPN for microbial protein

growth may depend on the species of bacteria and energy source (Cruz Soto et al., 1994).

There appears to be a higher requirement for amino acids and peptides by amylolytic

21

organisms (Maeng and Baldwin, 1976a, 1976b) and sugar-utilizing organisms (Hungate,

1966). However, proteins have also been shown to be superior to urea for maintenance

of fiber digestion despite the fact that cellulolytic organisms primarily use ammonia as a

nitrogen source. This may indicate that cellulolytic bacteria may have some requirement

for amino acids or peptides (Hoover, 1986), which may be related to the supply of

BCVFAs. Varga et al. (1988) showed that decreased BCVFAs are responsible for

depressed fiber digestion in continuous culture fermentations of formaldehyde-treated

soybean meal. Gorosito et al. (1985), however, suggested that amino acids or peptides

might increase cell wall digestion more than BCVFAs alone. Pectin-fermenting ruminal

bacteria include species that ferment both structural and non-structural carbohydrates

(e.g. F. succinogenes and P. ruminicola, respectively) and would therefore utilize

ammonia, amino acids or peptides. It appears to be beneficial to supply nitrogen in the

form of amino acids and peptides, whether to be incorporated directly or as source of

BCVFAs, in addition to ammonia to optimize microbial growth.

Fermentation pH

As mentioned previously, NFC supplementation has the potential to decrease

ruminal pH. Low pH in turn may decrease MCPeff (Russell et al., 1992), which may be

related to energy spilling strategies of ruminal microorganisms to cope with excess

available carbohydrate and low pH (Russell and Strobel, 1993). One example of an

energy spilling strategy involves the ability of S. bovis to ferment glucose to lactate

which only yields 2 ATP molecules per glucose molecule as opposed to acetate, formate

and ethanol which yield approximately 3 ATP molecules per glucose molecule (Russell

and Baldwin, 1979). At a low pH, S. bovis decreases its intracellular pH, which favors

lactate production (Russell and Hino, 1985). Low pH has also been shown to increase

22

the maintenance energy cost of ruminal microorganisms and thus decrease microbial cell

yield (Shi and Weimer, 1992).

It appears that cellulolytic bacteria are especially sensitive to low ruminal pH.

However, a moderate decrease in pH from 6.8 to 6.0 does not always affect cellulolytic

numbers (Slyter et al., 1970; Mackie et al., 1978; Leedle et al., 1982; Van der Linden et

al., 1984) and isolated fibrolytic enzyme activity remains high in this range (Stanley and

Kesler, 1959; Smith et al., 1973). On the other hand, a decrease in pH below 6.0 has

been reported to result in loss of fibrolytic activity and decreased numbers of cellulolytic

bacteria in vitro and in vivo (Slyter et al., 1970; Stewart, 1977; Crawford et al., 1980;

Hoover et al., 1984; Mould and Ørskov, 1984; Mould et al., 1984). At a pH between 4.5

and 5.0 there is virtually complete inhibition of fiber digestion (Stewart, 1977; Hoover et

al., 1984; Mould et al., 1984). Russell and Dombrowski (1980) observed washout of

cellulolytic bacteria in continuous culture fermentations at a pH below 6.0. Huhtanen

and Khalili (1992) reported a decrease in cellulolytic and hemicellulolytic enzymes at

decreased ruminal pH, when sucrose was supplemented to cattle on grass-silage based

diets. It is thus not surprising that one of the major results of decreased ruminal pH has

been a decrease in fiber digestion, reported both in vitro and in vivo (Terry et al., 1969;

Mould and Ørskov, 1984; Mould et al., 1984).

NFCs and Ruminal pH, Fiber Digestion and Animal Performance

Ruminal pH and Fiber Digestion

Decreased fiber digestion in vivo is often associated with supplementation of

forage diets with readily fermentable carbohydrate sources. Cameron et al. (1991)

reported decreases in ruminal neutral detergent fiber (NDF) and acid detergent fiber

(ADF) digestion for lactating dairy cows receiving supplements of starch and dextrose

23

(glucose). Heldt et al. (1999) reported a decrease in total tract NDF digestion relative to

control diet-fed animals in steers supplemented at 0.3% BW of DM/d with starch,

sucrose, glucose or fructose with low-quality, tallgrass-prairie hay. The diets in this

study were supplemented with degradable intake protein at 0.031% BW of DM/d, which

may have been below the amount needed to meet ruminal microbe requirements for a

degradable nitrogen source. In a second study, with the same NFC sources, but with

supplemental degradable intake protein of 0.122% BW of DM/d, total tract NDF

digestion increased with NFC supplementation (Heldt et al., 1999). Also, ruminal pH

decreased more in animals consuming the starch diet, and these animals had a lower total

tract NDF digestion compared to animals fed the sugar (sucrose, glucose and fructose)

diets. Some of the decreases noted for fiber digestion may be the result of competition

between NFC and fiber utilizing microorganisms for the nitrogen supply.

A decrease in ruminal fiber digestion is often attributed to a decrease in ruminal pH

(Hoover, 1986), caused by rapid fermentation of NFCs and production of VFAs by

ruminal microorganisms. Several studies have contradicted this concept and reported no

effect on pH and varying effects on fiber digestion as a result of supplementation with

starch or sucrose (Cameron et al., 1991; Aldrich et al., 1993; Casper et al., 1999). In a

two-part study by Khalili and Huhtanen (1991a, b), a decrease in both pH and NDF

digestion was reported in animals fed a grass silage and barley-based diet with

supplementation of sucrose at 1 kg DM/day. However, the negative effect on pH and

NDF digestion was alleviated when sodium bicarbonate was supplemented in

combination with sucrose.

24

The effect of NFC supplementation (especially sucrose and starch) on fiber

digestion might involve more than just the decrease of ruminal pH. It was Mould and

Ørskov (1984) who first coined the term "carbohydrate effect" to describe the initial

impaired fiber digestion at a pH of approximately 6.2. The authors suggested that a

series of events takes place: 1) ruminal microorganisms exhibit a preference for

carbohydrate sources that are more readily available, 2) fermentation of readily available

carbohydrates produce organic acids and ruminal pH decreases and 3) ruminal pH

decreases below 5.5 resulting in a decrease of cellulolytic microorganisms and potentially

completely inhibits fiber digestion. Piwonka and Firkins (1996) also suggested that there

might be a carbohydrate effect related to microbially produced inhibitors, which is

independent from pH.

Substitution of dried, pelleted beet pulp for high moisture corn did not affect

ruminal pH and increased both extent and rate of NDF digestion (Voelker and Allen,

2003b) when fed to Holstein cows on an alfalfa and corn silage diet in early lactation. In

several other studies increased NDF digestion as a result of supplementing sugar beet

pulp (Van Vuuren et al., 1993) or citrus pulp (Zinn and Owens, 1993; Miron et al., 2002)

for barley or corn has been reported.

Animal Performance

There are a multitude of animal studies that compared feedstuffs that differed in

types of NFCs that predominated. The challenge to interpreting these studies is that the

diets were rarely characterized for their carbohydrate fractions and proportional

substitution of carbohydrates was often not equal. Accordingly, one should view these

studies in a general sense to try and understand the impact of various carbohydrates on

ruminal and animal performance, but the results are anything but clear-cut.

25

Dry Matter Intake

Supplementation with NFCs is generally thought to result in decreased dry matter

intake. However, total dry matter intake was not affected by form of NFC

supplementation in several studies (Table 1-2; Charmely et al., 1991; Nombekela and

Murphy, 1995; O'Mara et al., 1997a; O'Mara et al., 1997b; Leiva et al., 2000; McCormick

et al., 2001; Broderick et al., 2002b; Ordway et al., 2002; Sannes et al., 2002; Cherney et

al., 2003; Delahoy et al., 2003). The effect of NFC supplementation on forage intake

alone also gave varied responses (Charmely et al., 1991; O'Mara et al., 1997b; Heldt et

al., 1999; Delahoy et al., 2003). The only report of a decrease (P < 0.05) in forage intake

with lactating dairy cows was in a study by O'Mara et al. (1997b) where perennial

ryegrass was supplemented with molassed beet pulp. Several studies reported no effect

of NFC type on forage intake for a variety of forages and animals, including hay for

steers (Heldt et al., 1999), pasture for lactating dairy cows (Delahoy et al., 2003) and

silage for sheep (Charmely et al., 1991). The varying responses in forage and dry matter

intake among studies investigating the effect of NFC supplementation may be due to

differences in the amount, source and combination of NFC supplemented. However, it

would appear that in general the different NFC sources do not differ from each other in

their effect on dry matter intake, and potentially also forage intake.

Milk Production and Composition

The effect of different NFC sources on milk yield is inconsistent (Table 1-2). In

some studies, substituting sucrose for some portion of corn had no effect on milk yield

(Nombekela and Murphy, 1995; Ordway et al., 2002; Cherney et al., 2003), while Sannes

et al. (2002) reported a decrease (P = 0.02) in milk production. In three studies,

substituting beet pulp for ground corn (Delahoy et al., 2003) and citrus pulp for corn

26

(Solomon et al., 2000) or corn hominy (Leiva et al., 2000) had no effect on milk

production. In other studies, substituting citrus pulp for high moisture ear corn or

cracked shelled corn (P = 0.01 and P = 0.02, respectively; [Broderick et al., 2002b]) or

corn meal (P < 0.01; [Leiva et al., 2000]) decreased milk production.

Milk composition changes in response to different NFC sources also varies (Table

1-2). Sucrose substituted for corn (starch) had no effect on milk fat concentration

(Nombekela and Murphy, 1995; Ordway et al., 2002; Sannes et al., 2002; Cherney et al.,

2003), while it decreased (P = 0.04; [Sannes et al., 2002]), tended to increase (P = 0.07;

[Nombekela and Murphy, 1995] and P = 0.06; [Ordway et al., 2002]), or did not affect

(Cherney et al., 2003) milk fat yield. Cows fed beet pulp substituted for corn had similar

milk fat yields, but increased (P < 0.05) milk fat concentrations (Mansfield et al., 1994).

In contrast cows fed citrus pulp substituted for corn (Solomon et al., 2000), and corn

hominy or corn meal (Leiva et al., 2000) had similar milk fat yields and concentrations.

Varied responses to NFC supplementation in these studies may be due to differences in

carbohydrate composition of feedstuffs such as beet pulp and citrus pulp, and also

variation in composition within a particular feedstuff.

Sucrose supplementation has decreased both milk protein yield (Sannes et al.,

2002) and milk protein percentage (Nombekela and Murphy, 1995) compared to corn.

Beet pulp (Mansfield et al., 1994) and citrus pulp (Solomon et al., 2000; Broderick et al.,

2002b) substituted for corn also decreased milk protein yield and milk protein

concentration. In at least some of the studies (Mansfield et al., 1994; Broderick et al.,

2002b) decreased milk protein yield might have been due to a lower total milk yield.

27

Starch and sucrose appear to be similar in their effects on milk yield, and starch

may increase milk yield compared to pectin. There does not seem to be a consistent

effect on milk composition from different NFC sources.

With the effects of NFC on animal and microbial responses reported in the

literature, and the variation in these responses, it is clear that further knowledge of the

microbial fermentation product yields from different NFC could be a vital resource in

understanding and predicting animal response. It is also necessary to consider factors

that may, in combination with NFC supplementation, affect substrate utilization,

microbial product yield and nutrient supply to the ruminant animal.

In this light, three in vitro fermentation studies with mixed ruminal cultures were

conducted to determine the effect of fermentation pH, nitrogen source and NFC

supplementation on NDF digestion and fermentation product yields. The first two studies

examined the effects of pH and nitrogen source on the fermentation of sucrose and

isolated neutral detergent residue (iNDF). The final study examined the fermentation of

iNDF together with starch, sucrose, pectin and their combinations.

28

Table 1-1. The effects of NFC source on ruminal or fermentation pH and organic acid profile. pH Total VFA1 C2 C3 C4 BCVFA Val Lac Reference and treatment

(% of diet DM or fermentation substrate) mM ---------------------- mol/100 mol ------------------- (Ariza et al., 2001), continuous culture

Citrus pulp (23.6%) --- 104.2 68.9 16.7 11.4 3.0 --- --- Hominy feed (25.3%) --- 101.2 62.6 22.7 11.0 3.7 --- ---

(Bach et al., 1999), continuous culture Control (lush pasture) 6.10 126.4 71.4 17.8 9.6 0.0 --- --- Beet pulp with molasses (44.7%) 6.04 124.3 60.9 20.2 15.6 0.9 --- --- Cracked corn (44.7%) 6.02 141.9 54.3 22.1 18.8 1.2 --- ---

(Ben-Ghedalia et al., 1989), 4 cannulated rams Barley (76.5%) 6.18 82.4 65.0 17.6 14.3 1.9 1.4 --- Dried citrus pulp (84.4%) 6.42 74.4 69.1 14.4 14.2 1.5 1.0 ---

(Broderick et al., 2002b), 6 cannulated lactating cows High moisture ear corn (38.4%) 6.14 102.0 63.3 20.6 11.2 1.6 1.9 --- Cracked shelled corn (38.7%) 6.21 104.1 63.7 19.6 11.7 1.7 1.8 --- High moisture ear corn (19.1%) + Citrus pulp (19.1%) 6.14 107.5 64.0 18.7 12.7 1.4 1.8

---

(Chamberlain et al., 1993), 6 sheep Grass silage control (4 kg/d) 6.43 56.8 62.7 24.5 7.8 3.5 1.5 --- Sucrose (200 g/d) 6.34 57.2 57.3 27.8 11.6 1.8 1.5 --- Starch (200g/d) 6.25 65.4 62.8 24.1 8.9 2.8 1.4 ---

(Charmely et al., 1991), 8 sheep Alfalfa silage 6.73 84.9 70.1 19.2 --- --- --- --- Alfalfa silage + Sucrose (10% of silage DM intake) 6.63 90.6 65.8 21.6 --- --- --- ---

29

Table 1-1 Continued. pH Total VFA C2 C3 C4 BCVFA Val Lac Reference and treatment

(% of diet DM or fermentation substrate) mM ---------------------- mol/100 mol ------------------- (Heldt et al., 1999), 20 cannulated steers

0.031% BW/d RDP supplement Control 6.40 --- 76.1 13.3 9.0 0.9 0.4 0.2 Starch (0.3% BW of DM/d) 6.36 --- 70.3 17.1 10.0 1.9 0.8 0.1 Glucose (0.3% BW of DM/d) 6.28 --- 59.3 15.5 18.6 1.0 1.0 4.5 Fructose (0.3% BW of DM/d) 6.36 --- 58.8 13.5 19.7 1.0 1.0 6.0 Sucrose (0.3% BW of DM/d) 6.23 56.3 15.5 20.9 1.3 1.8 4.3

0.122% BW/d RDP supplement Control 6.56 --- 73.5 14.0 10.6 1.2 0.5 0.3 Starch (0.3% BW of DM/d) 6.13 --- 69.5 16.4 10.3 2.0 1.4 0.5 Glucose (0.3% BW of DM/d) 6.16 --- 61.5 14.1 17.5 1.7 1.7 3.5 Fructose (0.3% BW of DM/d) 6.29 --- 59.8 14.2 18.9 1.6 1.7 3.8 Sucrose (0.3% BW of DM/d) 6.22 --- 59.7 14.4 19.5 1.5 1.8 3.0

(Khalili and Huhtanen, 1991a), 4 cannulated bulls Control (starch) 6.28 105.0 63.6 17.8 14.9 --- 14.8 4.4 Sucrose (1 kg/d) 6.03 104.0 58.9 16.5 19.7 --- 23.5 12.5

(Leiva et al., 2000), 11 lactating cows (3 cannulated) Corn hominy diet (25.3%) 6.24 106.1 67.4 21.4 11.2 --- --- 1.5 Citrus pulp diet (23.6%) 6.19 116.4 67.7 20.8 11.5 --- --- 0.6

(Mansfield et al., 1994), continuous culture Corn (30.2%) --- 112.7 58.7 21.7 15.3 2.3 2.0 0.9 Corn (15.5%) + Beet pulp (15.3%) --- 109.5 61.6 20.5 14.0 1.9 2.0 0.9

(Marounek et al., 1985), in vitro; inoculum from goats Starch (9 g/150 ml, 8 h incubation) --- 76.3 64.5 29.1 6.4 --- --- --- Pectin (2 g/150 ml, 6 h incubation) --- 61.5 81.8 14.8 3.4 --- --- ---

30

Table 1-1. Continued. pH Total VFA C2 C3 C4 BCVFA Val Lac Reference and treatment

(% of diet DM or fermentation substrate) mM ------------------- mol/100 mol ---------------- mg/dl (Moloney et al., 1994), 6 cannulated steers

Barley (61% of DM intake) 6.94 71.2 66.5 15.8 14.0 --- 3.3 42.3 Molasses (61% of DM intake) 6.86 71.7 58.4 16.6 23.0 --- 2.0 28.9

(Piwonka et al., 1994), 6 cannulated heifers -------------------------- mol/100 mol ------------------- Control --- 82.4 70.0 16.7 9.6 --- --- --- Dextrose (5.6%) --- 91.2 68.9 18.1 9.9 --- --- --- Barley (39.7%) --- 90.5 68.7 16.7 11.1 --- --- ---

(Sannes et al., 2002), 16 lactating cows (4 cannulated) ----------------------------- mM --------------------------- Corn (20%) --- 131.0 85.34 26.3 16.2 1.9 --- --- Corn (13.5%) + Sucrose (3.2%) --- 123.0 77.81 27.1 15.5 1.3 --- ---

(Strobel and Russell, 1986) in vitro pH 6.7

Sucrose 6.70 --- 4.7 2.1 1.1 --- --- 3.7 Starch 6.70 --- 5.1 2.9 0.8 --- --- 0.9 Pectin 6.70 --- 10.1 1.3 0.2 --- --- ND2

in vitro pH 6.0 Sucrose 5.50 --- 1.7 1.1 0.7 --- --- 8.3 Starch 5.80 --- 2.7 1.1 0.7 --- --- 4.1 Pectin 5.80 --- 5.0 0.7 0.3 --- --- ND

(Voelker and Allen, 2003c), 8 cannulated lact. cows ------------------------ mol/100 mol ---------------------- Hi moisture corn (35.6%) 5.93 138.0 56.9 27.0 11.3 2.3 --- 0.3 Hi moisture corn (29.5%) + Citrus pulp (6%) 5.97 141.0 59.1 24.9 11.5 2.3 --- 0.3 Hi moisture corn (23.5%) + Citrus pulp (12%) 6.02 142.0 60.2 23.0 12.2 2.3 --- 0.3 Hi moisture corn (11.4%) + Citrus pulp (24%) 5.94 142.0 61.6 22.4 12.3 1.9 --- 0.1

1 VFA = volatile fatty acids; C2 = acetate; C3 = propionate; C4 = butyrate; BCVFA = branched chain VFA; Val = valerate; Lac = lactate 2 ND = not detected

31

Table 1-2. Effects of NFC source on dry matter intake, milk production and milk composition. Reference and treatment DMI1 Milk Fat Protein Fat Protein (% of diet DM) kg/d % (Broderick et al., 2002b), 48 lactating cows

High moisture ear corn (38.4%) 20.9 35.2 1.24 1.04 2.94 3.53 Cracked shelled corn (38.7%) 21.4 35.1 1.19 1.06 3.02 3.38 High moisture ear corn (19.1%) + Citrus pulp (19.1%) 19.7 32.1 1.07 0.88 2.85 3.44

(Charmely et al., 1991), 8 sheep Alfalfa silage 1.25 --- --- --- --- --- Alfalfa silage + Sucrose (10% of silage DMI) 1.22 --- --- --- --- --- Silage intake only Alfalfa silage 1.25 --- --- --- --- --- Alfalfa silage + Sucrose (10% of silage DMI) 1.07 --- --- --- --- ---

(Cherney et al., 2003), 20 lactating cows High moisture corn (35.7%) 21.7 39.8 1.28 1.02 2.58 3.24 High moisture corn (32.1%) + Sucrose (3.6%) 21.4 39.5 1.29 1.02 2.59 3.27 High moisture corn (19.2%) 20.1 37.8 1.30 0.95 2.52 3.44 High moisture corn (17.3%) + Sucrose (1.9%) 20.6 38.9 1.33 0.97 2.49 3.47

(Delahoy et al., 2003), 28 lactating cows Ground corn (70.2%) 20.3 27.6 1.05 0.96 3.23 3.53 Ground corn (34.8%), Beet pulp (18.0%), Wheat middlings (17.4%) 20.2 27.4 1.08 0.95

3.19 3.63

(Fegeros et al., 1995), 26 sheep Maize (28.0%) + Barley (30.0%) --- 0.82 --- --- 5.36 7.04 Maize (20.0%) + (Barley 15.0%) + Dried citrus pulp (30.0%) --- 0.78 --- --- 5.32 7.27

(Friggens et al., 1995), 18 lacating cows Molassed sugar beet pulp (74.5%) --- 14.3 0.60 0.48 3.51 4.19 Molassed sugar beet pulp (37.3%) + Grain (38.2%) --- 14.9 0.57 0.51 3.41 4.05 Grain (Barley and Corn - 76.4%) --- 14.5 0.58 0.50 3.56 3.98

32

Table 1-2. Continued. Reference and treatment DMI Milk Fat Protein Fat Protein (% of diet DM) kg/d % (Leiva et al., 2000) 11 lactating cows (3 cannulated)

Corn hominy diet (25.3%) 21.4 32.8 1.12 0.93 2.83 3.43 Citrus pulp diet (23.6%) 20.9 31.3 1.11 0.85 2.71 3.54

184 lactating cows Corn meal diet (19.5%) 19.5 31.8 1.02 0.96 3.08 3.27 Citrus pulp diet (20.5%) 18.9 27.9 0.97 0.88 3.13 3.45

(Mansfield et al., 1994), 46 lactating cows Corn (30.2%) 21.5 32.2 1.18 0.97 3.64 3.01 Corn (15.5%) + Beet pulp (15.3%) 20.3 31.9 1.21 0.92 3.82 2.90

(McCormick et al., 2001), 32 lactating cows Ground corn (75.0%) 22.8 39.6 1.28 1.14 2.99 3.32 Ground corn (68.9%) + Brown sugar (5.0%) 22.9 38.7 1.30 1.13 2.97 3.39

(Nombekela and Murphy, 1995), 24 lactating cows Ground corn (39.9%) 19.0 28.4 0.96 0.96 3.51 3.40 Ground corn (38.4%) + Sucrose (1.5%) 19.1 29.3 0.97 0.95 3.28 3.30

(O'Mara et al., 1997a), 36 lactating cows Beet pulp (30.0%) 15.2 19.8 0.71 0.62 3.16 3.66 Beet pulp (10.6%) + Ground corn (20.0%) 13.7 21.2 0.78 0.64 3.04 3.64

(O'Mara et al., 1997b), 8 cannulated lactating cows Perennial ryegrass 13.6 --- --- --- --- --- Perennial ryegrass + Molassed beet pulp (3 kg/day) 14.2 --- --- --- --- --- Grass intake only Perennial ryegrass 13.6 --- --- --- --- --- Perennial ryegrass + Molassed beet pulp (3 kg/day) 11.5 --- --- --- --- ---

33

Table 1-2. Continued. Reference and treatment DMI Milk Fat Protein Fat Protein (% of diet DM) kg/d % (Ordway et al., 2002). prepartum diets (carry over effect measured; postpartum diet - 15.1% ground corn)

Ground corn (11.5%) 22.1 45.8 1.66 1.26 2.68 3.54 Ground corn (8.8%) + Sucrose (2.7%) 21.7 45.6 1.72 1.25 2.72 3.76

(Sannes et al., 2002), 16 lactating cows (4 cannulated) Corn (20.0%) 25.7 34.3 1.33 1.07 3.14 3.88 Corn (13.5%) + Sucrose (3.2%) 25.5 33.2 1.27 1.03 3.12 3.83

(Solomon et al., 2000), 20 lactating cows Corn (23.7%) 21.5 36.9 1.22 1.07 2.94 3.33 Dried citrus pulp (23.9%) 20.6 36.4 1.21 1.04 2.88 3.34

(Valk et al., 1990) Study 1 (18 lactating cows)

Beet pulp (82.5%) 19.5 25.8 1.07 0.89 3.43 4.15 Maize meal (47.5%) + Maize bran (50.0%) 20.1 28.4 1.11 0.98 3.48 3.92

Study 2 (27 lactating cows) Beet pulp (78.4%) 21.2 30.9 1.29 1.02 3.32 4.19 Maize meal (87.5%) 20.7 31.6 1.17 1.03 3.27 3.70 Beet pulp (44.0%) + Maize meal (44.0%) 20.7 30.8 1.24 1.02 3.31 4.05

(Voelker and Allen, 2003a), 8 cannulated lactating cows High moisture corn (35.6%) 24.8 36.4 1.34 1.13 3.21 3.72 High moisture corn (29.5%) + Dried citrus pulp (6%) 25.0 36.6 1.40 1.15 3.21 3.84 High moisture corn (23.5%) + Dried citrus pulp (12%) 25.1 35.9 1.39 1.15 3.22 3.90 High moisture corn (11.4%) + Dried citrus pulp (24%) 22.9 35.4 1.33 1.09 3.10 3.81

1 DMI = Dry Matter Intake

34

CHAPTER 2 EFFECT OF PH ON MICROBIAL YIELD AND NEUTRAL DETERGENT FIBER

DIGESTION FROM IN VITRO FERMENTATIONS OF SUCROSE AND ISOLATED NEUTRAL DETERGENT RESIDUE

Introduction

Dairy cattle diets are often supplemented with grains and byproduct feeds that have

a large concentration of non-neutral detergent fiber carbohydrates (NFCs) in order to

increase the energy intake of high producing ruminants. Sucrose, and its constituent

monosaccharides glucose and fructose, are the predominant saccharides of the mono- and

oligosaccharide component of NFCs and are found in byproduct feeds such as molasses

(Kunkle et al., 2000), sugar beet pulp (Hall, 2002) and citrus pulp (Ben-Ghedalia et al.,

1989). Sucrose is readily fermentable in the rumen (Sniffen et al., 1983) and has been

associated with a decrease in ruminal pH (Sutton, 1979; Khalili and Huhtanen, 1991a).

Ruminal pH is considered one of the major modifiers of rumen fermentation (Hoover and

Stokes, 1991). A decrease in ruminal pH below 6.0 has been associated with decreases in

fiber digestion (Khalili and Huhtanen, 1991a, b; Grant and Weidner, 1992), microbial cell

yield (Shi and Weimer, 1992) and efficiency of microbial protein synthesis (MCPeff;

[Russell et al., 1992]), as well as altered volatile fatty acid (VFA) profiles (Sutton, 1979;

Chamberlain et al., 1993; Araba et al., 2002). However, there is evidence that there are

effects of readily fermented carbohydrates that are not pH related (Mould and Ørskov,

1984; Piwonka and Firkins, 1993).

The objectives of this study were to compare the effects of media with neutral or

acidic starting pHs on 1) the yield of microbial fermentation products and on neutral

35

detergent fiber (NDF) digestion, from the in vitro fermentation of sucrose and isolated

NDF, and 2) fermentation pH and NDF digestion of isolated NDF fermented with or

without sucrose supplementation.

Materials and Methods

Substrates

Substrates used were isolated bermudagrass (Cynodon dactylon L.) neutral

detergent residue (iNDF; 92.8% DM, 99.4% OM, 98.6% NDFOM, 5.5% NDFCP) and a

50:50 mixture of iNDF and sucrose (SuNDF). The iNDF was prepared as described by

Hall and Herejk (2001). Sucrose (S5-500, Fisher Scientific, Atlanta, GA; 99.98% DM,

100% OM) and iNDF were analyzed prior to the study for DM and OM (AOAC, 1980),

and iNDF for NDF using heat-stable α-amylase (Termamyl 120L, Novo Nordisk

Biochem, Franklinton, NC; (Van Soest et al., 1991) and CP (AOAC, 1980). A total of

240 mg ± 0.5 mg of substrate, with iNDF and sucrose weighed individually, were

transferred into duplicate 50 ml Nalgene high speed, low density, polyethylene centrifuge

tubes (05-562-13, Fisher Scientific, Atlanta, GA) or Nalgene high speed polypropylene

centrifuge tubes (05-562-10K, Fisher Scientific, Atlanta, GA) depending on the type of

analysis to be performed on the fermentation residues (Table 2-1).

Medium and reducing solution

The pH treatments consisted of one of two iso-nitrogenous media with neutral or

acidic pH, respectively. The Goering and Van Soest (1970) medium served as the neutral

medium (NpH) with initial pH of 6.8, and was modified by adding 4.4 ml of a 1 M citric

acid solution per 100 ml of Goering and Van Soest medium to obtain the acidic medium

(ApH), with initial pH of 5.6 (P. J. Weimer and D. R. Mertens, personal communication).

The media provided 6.85 mg non-protein nitrogen and 3.52 mg amino nitrogen per

36

fermentation tube. Casein acid hydrolysate (A-2427, Sigma Chemical Co., St. Louis,

MO) provided the amino nitrogen source in the medium. The reducing solution was

mixed according to a modification of the Goering and Van Soest (1970) procedure (P. J.

Van Soest, personal communication). For a volume of 100 ml, 0.625 g of L-cysteine

hydrochloride (C-7880, Sigma Chemical Co., St. Louis, MO) and approximately 10

pellets of KOH (P250-3, Fisher Scientific, Atlanta, GA) were dissolved with stirring in

50 ml of distilled water. In a separate 250 ml glass beaker 0.625 g sodium sulfide (S-

4766, Sigma Chemical Co., St. Louis, MO) was dissolved with stirring in 50 ml of

distilled water. The solutions were combined when the contents of both beakers were in

solution, and just before addition of the reducing solution to the fermentation tubes.

Fermentation

Duplicate 24 h in vitro fermentation runs using destructive sampling of mixed

batch cultures were performed according to the method of Goering and Van Soest (1970).

Ruminal inoculum was obtained approximately 3 h post-feeding from a ruminally

cannulated, non-pregnant, non-lactating Holstein cow under protocols approved by the

University of Florida Institutional Animal Care and Use Committee. The donor cow

received a diet of bermudagrass hay (10 kg DM/day), 48% crude protein soybean meal

(900 g/d) and free choice mineral supplement (Ca 17- 20%, P ≥ 9%, NaCl ≤ 25%, Mg ≥

0.25%, Cu ≥ 0.15, Co ≥ 0.01%, I ≥ 0.01%, Mn ≥ 0.2%, Se ≤ 0.004%, Zn ≥ 0.4%, Fl ≤

0.09%). The inoculum was filtered through four layers of cheesecloth and one layer of

glass wool, and maintained under anaerobic conditions at 39°C. Twenty milliliters of the

appropriate medium, 1 ml of reducing solution and 5 ml of filtered rumen fluid were

added to each fermentation tube. After each addition, tube headspace was purged with

CO2. Fermentation tubes were capped with rubber stoppers fitted with gas release valves,

37

incubated (Equatherm Incubator Model C1487, Curtin Matheson Scientific, Inc.,

Houston, TX) under anaerobic conditions at 39°C and destructively sampled at 0, 4, 8,

12, 16, 20 and 24 hours. Tubes were swirled individually every 4 hours.

Sample Handling and Subsequent Analyses

At each sampling hour the fermentation tubes for the specific hour were removed

from the incubator and placed in an ice bath to terminate the fermentation process.

Approximately 5 min after tubes were removed from the incubator pH was recorded on

tubes reserved for NDF analysis. The tubes used for NDF analysis were stored at 10°C

and were analyzed for residual NDFOM (NDF on an ash-free basis) within two days of

completion of the fermentation. For NDF analysis, samples were allowed to equilibrate

to room temperature, the pH was adjusted up or down with minimal amounts of a 0.2 N

NaOH or 1 M citric acid solution, respectively, to obtain a pH of between 6.9 and 7.1.

Samples were quantitatively transferred to 600 ml Berzellius beakers and refluxed with

50 ml of neutral detergent solution and heat-stable α -amylase (Termamyl 120L, Novo

Nordisk Biochem, Franklinton, NC) for 1 h (Van Soest et al., 1991). To ensure

hydrolysis of α-glucan, three doses of 0.2 ml heat-stable α-amylase were used: one with

addition of detergent, one 10 min before removing the sample from the burner and one

added to the Gooch crucible during rinsing with boiling water.

Fermentation tubes reserved for microbial glycogen (GLY), residual sucrose

equivalents (sucrose, and its hydrolysis products: glucose and fructose), organic acids,

ammonia-nitrogen (NH3-N) and total free amino acid analyses were centrifuged at 15,000

x g for 30 min at 5°C. The supernatant was transferred to scintillation vials and stored at

-20°C until analysis for residual sucrose and organic acids by HPLC, and NH3-N and

38

total free amino acid analyses as described by Broderick et al. (2004). The HPLC for

analyzing residual sucrose was equipped with an anion exchange analytical column

(CarboPac™ PA1, Dionex, Sunnyvale, CA), the mobile phase used was 100 mM NaOH,

the flow rate 1.0 ml/min and the injection volume 10 µL. The HPLC for analyzing

organic acids was equipped with an organic acid column (PHX-87H, Bio-Rad

Laboratories, Richmond, CA). The solvent used was 0.015 N H2SO4 / 0.0034 M EDTA,

the flow rate 0.7 ml/min, the column temperature 45°C and the injection volume 50 µL.

The pellets from the high-speed centrifugation were quantitatively transferred to 50

ml glass beakers using no more than 20 ml of a 0.2 N NaOH solution to rinse out the

fermentation tubes and stored at -20°C until analysis for GLY. Beakers were removed

from the freezer and samples were allowed to equilibrate to room temperature.

Microorganisms were lysed with a 0.2 N NaOH solution (brought to a volume of 20 ml in

the 50 ml glass beakers) in a boiling water bath for 15 min. Samples were cooled to

room temperature and then neutralized to pH 7.0 ± 0.1 with 6 N HCl. Samples were

quantitatively transferred from the glass beakers to funnels fitted with glass wool plugs

for filtration into 100 ml volumetric flasks. Beakers, glass wool and funnels were rinsed

with distilled de-ionized water (ddH2O), and then samples were brought to volume with

ddH2O. Four milliliters of a 0.1 M sodium acetate buffer (pH ~ 4.5) and 50 µl of

amyloglucosidase (EC 3.2.1.3, A-3514, Sigma Chemical Co., St. Louis, MO) were added

to 4 ml of sample, incubated at 60ºC for 45 min, and analyzed for α-glucan content as

released glucose corrected for free glucose (Karkalas, 1985).

Microbial crude protein (MCP) was estimated as trichloroacetic acid (TCA)-

precipitated crude protein. Fermentation tubes were individually removed from the ice

39

bath and 5.2 ml of a 120% (w/v) TCA solution were added in two equal increments to

achieve a final concentration of 20.0% TCA. Fermentation tubes were returned to the ice

bath for 45 min after which tubes were centrifuged at 7700 x g for 20 min at 5°C. The

contents of each fermentation tube were then quantitatively transferred into Whatman

541 filter paper (09-851D, Fisher Scientific, Atlanta, GA) in veined funnels set in 125 ml

Erlenmeyer flasks, using approximately 50 ml of chilled 10% TCA to rinse the tubes,

filter and residue. Samples were allowed to filter under gravity. The filtrate for each

tube was filtered through a Whatman GF/A glass fiber filter (09-874-16D, Fisher

Scientific, Atlanta, GA), using 10% TCA to rinse the flask, filter and residue. Both

Whatman 541 and GF/A filters containing the TCA-precipitated material from one

fermentation tube were placed together in a beaker and dried for 24 h at 55°C, before

analysis for crude protein content as Kjeldahl nitrogen content x 6.25 (AOAC, 1980).

Kjeldahl analysis blanks consisted of a Whatman 541 filter and a GF/A filter digested and

distilled together in one Kjeldahl flask. The MCP and GLY contents of each tube were

corrected for fermentation blanks at each hour, and MCP for its content by substrate at

hour 0.

Statistical Analysis

The experimental design was a split-split plot in time with a 2 x 2 factorial

arrangements of treatments (media and substrates). The data were analyzed using the

PROC MIXED procedure of SAS (1999) with fermentation run (R) as a random variable,

and medium (M) and substrate (S) as fixed variables. Fermentation hour (H) was used as

a class variable. Linear or quadratic temporal patterns within media and substrate

treatments were determined using orthogonal contrast statements.

40

The Kenward-Roger method was used to calculate the denominator degrees of

freedom for testing fixed effects. The contrast, NpH vs. ApH, was used for medium

comparisons within substrate (iNDF, SuNDF). All values presented are least squares

means. The model statement used was:

Yijkl = µ + Mi + Sj + MSij + Hk + MHik + SHjk + MSHijk + εijkl

Where:

Yijkl = the dependent variable

µ = overall mean

Mi = medium (i = NpH, ApH)

Sj = substrate (j = iNDF, SuNDF)

Hk = hour (k = 0, 4, 8, 12, 16, 20, 24)

MSij = interaction term for medium and substrate

MHik = interaction term for medium and hour

SHjk = interaction term for substrate and hour

MSHijk = interaction term for medium, substrate and hour

εijkl = residual error

A treatment term (T) consisting of the interaction between substrate and medium

was used in the random statement to obtain appropriate standard errors for the least

squares means. The random statement included the following terms:

Rl + RTlm + RHlk + RTHlmk

Where:

Rl = fermentation run (m = 1, 2)

Tm = treatment (n = 0, 1, 2, 3); number assigned to each M by S combination

41

RTlm = interaction term for fermentation run and treatment

RHlk = interaction term for fermentation run and hour

RTHlmk = interaction term for fermentation run, treatment and hour

The sampling hour of maximum MCP or GLY yield within medium and

fermentation run was defined as the hour with the maximum least squares means for

these measures. Efficiency of MCP yield was expressed as maximum MCP (mg)/organic

matter digested (OMD, mg). Organic matter digested (mg) was calculated as total

sucrose (mg) minus residual sucrose equivalent (mg) plus NDFOM digested (mg), minus

GLY (mg). The sucrose equivalent at any specific sampling hour was calculated as

residual sucrose (mg) + 0.95 x (residual fructose (mg) + residual glucose (mg)). Since

NDF digestion, residual sucrose, MCP and GLY were not measured on the same

fermentation tube, the least squares means for these measurements, within fermentation

run at the hour of maximum MCP, were used to calculate MCP efficiency for individual

treatments. The "hour" term and its interaction terms were omitted from the above

mentioned model to compare minimum values, maximum values, and results at a single

sampling hour.

Results and Discussion

Residual Sucrose

Sucrose disappeared rapidly from the fermentation medium, with only 69 and 40%

of the original 120 mg sucrose recovered at 0 h in NpH and ApH, respectively (Table 2-

2). Even when residual glucose, fructose and the unhydrolyzed sucrose were expressed

as sucrose equivalent, only 83% of the original substrate was accounted for NpH and

ApH at 0 h. Residual sucrose amount, averaged over the 24 h fermentation, did not differ

42

for ApH and NpH, whereas glucose and sucrose equivalent contents were greater and

fructose content tended to be greater for ApH compared to NpH.

Amount of residual sucrose, and sucrose equivalent did not differ between media at

0 h. By 4 h, and through 24 h, no residual sucrose could be detected in either medium.

The ApH fermentation tended to contain more glucose and fructose at 0 h compared to

NpH. At 4 h fructose amount and residual monosaccharide sucrose equivalent (glucose

and fructose) were greater for ApH compared to NpH, whereas glucose was only

numerically higher for ApH. Glucose and fructose were not detected in NpH at 4 h, or in

ApH at 8 h, and in subsequent hours for both media. It appears that ruminal

microorganisms, depending on the pH of the fermentation, do not utilize glucose and

fructose similarly. For NpH, glucose and fructose disappeared from the fermentation by

the same sampling time (4 h). However, for ApH, fructose remained in the fermentation

for a longer period of time compared to glucose (8 and 4 h, respectively).

Early disappearance of sucrose from the fermentations is consistent with reports of

sugar degradation rates of up to 300%/h (Sniffen et al., 1983). In a study by Henning et

al. (1991) glucose was detected through 8 and 12 hours of fermentation when added at

12.5 g /L and 25 g/L of culture medium, respectively. In the current study the initial

concentration of sucrose (4.62 g/L) was only one-sixth to one-third of the amounts used

by Henning et al. (1991), which could explain why no residual sugars were detected as

early as 4 h in NpH. The present study and that of Henning et al. (1991) would indicate

that sucrose or glucose disappearance is rapid, but not instantaneous in fermentations

with ruminal microorganisms. Residual sugar concentrations in the current study

43

confirmed that sucrose is readily utilized by ruminal microorganisms at near neutral pH

(6.8) but may be more slowly utilized at a more acidic pH (5.6).

Microbial Glycogen

Although it is considered part of the microbial cell mass, glycogen represents

available substrate that has been stored, but not yet metabolized by the cells. Maximum

GLY yields for both media were recorded at 4 h, with 6.0 mg and 3.5 mg for NpH and

ApH, respectively (Figure 2-1). In the ApH fermentation, sucrose-utilizing

microorganisms converted less sucrose to GLY (P = 0.04 at 4 h). Though the temporal

pattern for GLY amount tended to differ between media (P = 0.06 for medium by hour

interaction), GLY appeared to decrease through 8 h for both ApH and NpH, and then

remained relatively constant through 24 h.

Ruminal microorganisms can store microbial glycogen under conditions of excess

carbohydrate supply (Thomas, 1960; John, 1984; Lou et al., 1997). Decreased pH has

been shown to increase the maintenance energy required by ruminal microorganisms (Shi

and Weimer, 1992). If more energy were diverted towards maintenance requirements in

the ApH fermentation, there might not have been a relative excess of available

carbohydrates, hence less microbial glycogen storage occurred at the lower pH.

However, the ratio of GLY to MCP (at the hour of maximum GLY yield) was 0.67 and

1.68 for NpH and ApH, respectively. Studies with Prevotella ruminicola B14 (Russell,

1992) and Fibrobacter succinogenes S85 (Maglione and Russell, 1997) indicated a

decreased viable cell number when the polysaccharide:protein ratio of the cultures

exceeded 1.0, which might offer partial explanation for the lower microbial protein yield

(Figure 2-12) and decreased NDF digestion (Figure 2-11) for ApH compared to NpH.

44

Fermentation pH

Temporal patterns for fermentation pH were somewhat similar in both media for

the two substrates (Figure 2-2). The pH of iNDF fermentations increased over the 24 h

fermentation (linear; P = 0.03 and P < 0.01 for NpH and ApH, respectively), whereas that

for SuNDF decreased in the early hours and then increased through 24 h (quadratic

pattern; P = 0.02 and P < 0.01 for NpH and ApH, respectively). However, substrates did

not follow parallel paths for the two media, as indicated by a medium by hour interaction

within substrate (P < 0.01 for both iNDF and SuNDF).

Minimum fermentation pH for ApH, with SuNDF as the substrate, was recorded at

8 h and was lower (P < 0.01) than that for NpH (5.25 and 6.71, respectively), which was

recorded at 4 h. Also, minimum fermentation pH for both media with SuNDF as the

substrate was achieved at the same sampling hour that lactate concentration peaked

(Figure 2-7). Thereafter the pH for these fermentations increased through 24 h.

Minimum fermentation pH for iNDF in both media was recorded at 0 h, and this was

lower (P < 0.01) for ApH compared to NpH (6.07 and 6.99, respectively). The

magnitude of pH change appeared to differ for the two media as well as for the substrates

within each medium. At their minima, the difference in pH between iNDF and SuNDF

was numerically greater for ApH, a difference of 1.16 pH units at 8 h, compared to the

difference of 0.31 pH units at 4 h for NpH. Strobel and Russell (1986) also reported a

decrease in pH after 10 h in vitro fermentation of sucrose when the initial fermentation

pH was 6.0, but not when the initial pH was 6.7.

One explanation for the larger numerical decrease in fermentation pH for SuNDF in

the current study could be the increased lactic (Figure 2-7) and acetic acid (Figure 2-3)

production at 8 h for ApH compared to NpH. Another explanation could be a decreased

45

buffering capacity due to the addition of citric acid to obtain a pH of 5.6 for the ApH

medium. However, Grant and Mertens (1992) reported that a 1 M citric acid solution

used to adjust a phosphate-bicarbonate buffer to a pH of 5.8 was effective in maintaining

the fermentation pH through 24 h for in vitro fermentations of alfalfa silage and corn

grain. It is possible that the alfalfa silage and 1:1 alfalfa silage:corn grain substrates used

in that study did not provide as much rapidly fermenting carbohydrate as did substrates in

the present study, and therefore did not yield as much VFA to decrease pH.

Organic Acids

Temporal patterns for all organic acid concentrations differed for fermentations

containing no substrate (blank fermentations; only inoculum) compared to those

containing SuNDF for ApH and NpH (P < 0.01 for medium by substrate by hour

interactions; Figures 2-3 to 2-7). Organic acid concentrations were not corrected at each

sampling hour for concentration in the blank fermentations (tubes with only medium and

inoculum, but no substrate) since higher acetate (Figure 2-3) and BCVFA (Figure 2-8)

concentrations in blank fermentations for ApH would have resulted in negative net

concentrations of these analytes from 8 h through the end of the fermentations. These

results raise questions about the appropriate use of fermentation blanks to adjust for

treatment values, especially when the fermentation pH is more acidic. It is not clear why

the ApH fermentation for iNDF and fermentation blanks yielded higher acetate

concentrations compared to the ApH fermentation with SuNDF. A partial explanation

could be that the fermentation of plant organic acids such as citric acid primarily yields

acetate (Russell and Van Soest, 1984).

At the end of the 24 h fermentation, with SuNDF as the substrate, there was no

difference between ApH and NpH (P = 0.36) for butyrate concentrations (Figure 2-5),

46

whereas acetate (Figure 2-3), propionate (Figure 2-4) and total VFA concentrations

(Figure 2-6) were greater for ApH (P < 0.01, P = 0.04 and P < 0.01, respectively). In

contrast, Strobel and Russell (1986) recorded decreased acetate and butyrate

concentrations at a more acidic pH and no difference in propionate concentration in

sucrose fermentations. In a continuous culture fermentation of alfalfa hay and corn grain,

Calsamiglia et al. (2002) reported no effect on butyrate and an increase in propionate

proportion at a more acidic pH (5.7). However, in contrast to the current study, the

authors reported decreased total VFA concentrations, and a decrease in the acetate

proportion at the more acidic pH. The differences among studies in VFA production may

be in part due to different fermentation methods (continuous vs. batch culture), and

different sources of inocula.

Lactate was not detected in fermentations with only inoculum. Maximum lactate

concentration did not differ (P = 0.64) between ApH and NpH with SuNDF as the

substrate (28.9 and 27.9 mM, respectively), and this occurred at 4 h for NpH and at 8 h

for ApH (Figure 2-7). It would appear that the lower pH resulted in delayed lactate

production. In contrast, Strobel and Russell (1986) recorded increased lactate

concentrations for sucrose fermented by mixed ruminal microorganisms at pH 6.0

compared to 6.7. In the study by Strobel and Russell (1986) the increased lactate

concentration was recorded after a 10 h fermentation, which would correspond with a

point in the current study where the lactate concentration appears to be greater for the

ApH than for the NpH fermentation.

For NpH, with SuNDF as the substrate, lactate concentration decreased from 4 h

through 12 h and remained at zero till the end of the fermentation. However, for the ApH

47

fermentation lactate was detectable in ApH until 16 h, and it was only at 20 and 24 h that

lactate could no longer be detected. It would appear that lactate utilization might be

delayed at a more acidic pH. Both D(+) and L(-) isomers of lactic acid are produced in

the rumen. However, D-lactate becomes the predominant isomer under more acidic pH

(pH < 6) conditions and is considered to be less degradable compared to L-lactate. Also,

Megasphaera elsdenii, one of the major lactate utilizers in the rumen, is inhibited when

the pH decreases below 5.5 (Dawson et al., 1997).

Protein Degradation Products

Temporal patterns for BCVFA concentrations differed for fermentations containing

no substrate (blank fermentations) compared to those containing SuNDF for ApH and

NpH (P < 0.01 for medium by substrate by hour interaction; Figure 2-8). Higher BCVFA

concentrations in the blank fermentations for ApH from 12 through 24 h could possibly

be due to increased cell lysis and de-amination of microbial protein, leading to higher (at

least numerically) NH3-N (Figure 2-9) and BCVFA concentrations. At the end of the 24

h fermentation of SuNDF, BCVFA concentration tended (P = 0.11) to be lower for ApH

compared to NpH. In contrast to the current study, Calsamiglia et al. (2002) reported

decreased BCVFA concentrations at a more acidic pH (5.7) in a continuous culture

fermentation of alfalfa hay and corn grain.

Total free amino acid and NH3-N concentrations are the net result of protein

breakdown and nitrogen utilization by ruminal microorganisms in vitro. Ammonia

nitrogen concentration, averaged across the 24 h fermentation, was higher (P < 0.01) for

NpH compared to ApH with SuNDF as the substrate (Figure 2-9). In the continuous

culture study by Calsamiglia et al. (2002) NH3-N concentration also decreased at a more

acidic pH (5.7). In another continuous culture fermentation, this time with soybean meal,

48

barley and corn silage as substrates, Erfle et al. (1982) reported decreased ammonia

concentrations at a pH below 6 and attributed this to decreased microbial deaminase

activity. In the same study, protease activity decreased at a more acidic pH, and as a

result free amino acid concentrations decreased. In the current study, total free amino

acid concentration was greater (P < 0.01) for ApH compared to NpH (Figure 2-10). The

lower total free amino acid concentration for NpH in this study may indicate greater

utilization of amino acids for MCP synthesis, which coincided with a higher MCP yield

(P < 0.01) compared to ApH (Figure 2-12).

Neutral Detergent Fiber Digestion

Temporal patterns for NDF digestion differed for substrates within ApH and NpH

(P < 0.01 for both), as well as for ApH and NpH within each substrate (P < 0.01 for

iNDF and SuNDF; Figure 2-11). At 24 h, NDF digestibility for NpH was greater for

SuNDF at 42.4% than for iNDF at 26.4% (P < 0.01). The reverse was true for ApH,

where 24 h NDF digestibility for iNDF (7.8%) tended to be greater (P = 0.15) compared

to SuNDF (2.2%). This would suggest that sucrose addition is more detrimental to fiber

digestion at a more acidic pH, but may actually have a positive effect under more neutral

conditions.

Fermentation pH for ApH decreased to 5.25 at 8 h. Decreased fiber digestion has

been associated with a more acidic pH (Khalili and Huhtanen, 1991b; Kennelly et al.,

1999). Several studies (Stewart, 1977; Hoover et al., 1984; Mould et al., 1984) have

reported almost complete inhibition of fiber digestion at pH 5.0. A decrease in pH or

supplementation of readily fermentable carbohydrates may decrease extent and increase

lag of fiber digestion (Grant, 1994). The current study did not include enough data points

in the early hours of the fermentation to adequately describe the lag phase. Nonetheless,

49

evaluation of the graphed data suggests that sucrose addition in the NpH fermentation

appeared to increase lag over the first 4 h of fermentation compared to fermentation of

iNDF as sole substrate (Figure 2-11).

Decreased fiber digestion can be attributed to a decrease in pH as a result of

increased VFA production, or a "carbohydrate effect" (Mould and Ørskov, 1984). The

carbohydrate effect refers to a preference by ruminal microorganisms for more readily

available carbohydrates. An increased competition for nutrients such as nitrogen among

microbial populations fermenting NFC or NDF might also contribute to reduced fiber

digestion by fibrolytic microorganisms. Mould and Ørskov (1984) suggested that the

carbohydrate effect might be a factor in reducing fiber digestion at a pH as high as 6.2.

However, the addition of sucrose to the NpH fermentation in the current study resulted in

increased fiber digestion. Heldt et al. (1999) also reported an increase in NDF digestion

when supplementing starch, sucrose, glucose or fructose to steers on a tallgrass-prairie

hay diet. In the study by Heldt et al. (1999) pH was only slightly decreased to 6.2, and in

the current study the pH for the neutral fermentation never decreased below 6.7.

Microbial Crude Protein Yield and Efficiency

Microbial crude protein yield was greater (P < 0.01) for NpH compared to ApH

over the 24 h fermentation (Figure 2-12). Maximum MCP yield was recorded at 12 h for

NpH (19.4 mg) and this was almost double the maximum amount achieved by ApH (11.1

mg at 20 h). Synthesis of MCP tended (P = 0.10) to be more efficient for NpH compared

to ApH (0.14 mg and 0.09 mg MCP/mg OMD, respectively).

Other studies have also shown a decrease in bacterial growth at a lower pH (Strobel

and Russell, 1986; Shi and Weimer, 1992; De Veth and Kolver, 2001). Decreased pH

has been associated with a decrease in the efficiency of microbial growth (Russell et al.,

50

1992; Russell and Wilson, 1996), which may be related to a potential increase in ruminal

microorganisms' energy requirements for maintenance at a lower pH (Shi and Weimer,

1992). If more energy is expended on maintenance it will lead to lower efficiency of

microbial protein synthesis.

Conclusions

The fermentation pH alters the yields of products and fermentation of NDF when

sucrose is included in the fermentation. Though sucrose appears to be readily fermented

regardless of pH, the utilization of the monosaccharide constituents (glucose and

fructose) changes depending on pH. Fructose utilization may be delayed at an acidic pH,

whereas glucose utilization does not appear to be affected. At a more neutral pH (6.7)

sucrose supplementation may increase fiber digestion, whereas at an acidic pH (5.6)

sucrose supplementation may decrease fiber digestion. To avoid the potential negative

effect of sucrose supplementation on ruminal pH and fiber digestion adequate effective

fiber should be provided in rations. Microbial protein yield and composition of microbial

mass (microbial protein:glycogen) changed with pH. The potential effects of sucrose

supplementation on ruminal fermentation as they change with pH could be important to

take into consideration when supplementing sucrose to ruminant diets that predispose the

animals to more acidic or more neutral ruminal pH.

Results from this study suggest that consideration be given to the interaction of

sucrose supplementation and ruminal pH for predicting fiber digestion and yield of

potentially metabolizable nutrients to the animal. Animal studies further evaluating the

effects of ruminal pH on substrate utilization and nutrient supply are warranted.

51

Table 2-1. Type and number of fermentation tubes per medium for each sampling hour, indicating the substrate and analysis for which tubes were reserved in an 24 h in vitro fermentation of sucrose and iNDF.

Analysis

Substrate1 pH and residual NDF

Microbial crude protein

Microbial glycogen, residual sucrose, organic acids, NH3-N2

and amino acid nitrogen No substrate - 2 x HSPP3 2 x LDPE4 iNDF 2 x HSPP - - SuNDF 2 x HSPP 2 x HSPP 2 x LDPE

1 iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose + iNDF 2 NH3-N = ammonia nitrogen

3 HSPP = Nalgene high speed, polypropylene 4 LDPE = Nalgene high speed, low density, polyethylene Table 2-2. Residual glucose, fructose, unhydrolyzed sucrose, monosaccharide sucrose

equivalent (glucose+fructose) and sucrose equivalent at 0, 4 and 8 h, and averaged for 24 h in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue with initial medium pH of 6.8 or 5.6.

Time Medium pH Glucose Fructose Unhydrolyzed

Sucrose Glucose+Fructose1

Sucrose eq.2

mg

Hour 0 6.8 8.45 9.31 83.4 16.9 100 5.6 27.0 27.4 48.5 51.6 100

SE3 3.09 3.68 25.0 6.43 18.7P-value (medium) 0.11 0.15 0.36 0.13 1.00

Hour 4 6.8 -0.37 -0.02 -0.29 -0.37 -0.66 5.6 6.11 41.1 -0.24 44.8 44.6

SE 5.47 4.40 0.09 1.08 1.06P-value (medium) 0.54 0.02 0.75 < 0.01 < 0.01

Hour 8 6.8 0.08 -0.05 -0.16 0.03 -0.13 5.6 -0.15 0.13 -0.17 -0.02 -0.19

SE 0.16 0.09 0.05 0.23 0.26P-value (medium) 0.41 0.27 0.82 0.89 0.89

Avg. for 24 h 6.8 1.20 1.31 11.8 2.39 14.2 5.6 5.07 9.81 6.90 14.1 21.0

SE 0.90 1.14 3.60 0.94 2.95P-value (medium) 0.01 0.11 0.37 < 0.01 < 0.01P-value (medium x hour) 0.03 < 0.01 0.15 < 0.01 0.221 Calculated as residual (glucose + fructose) x 0.95 to give residual monosaccharide sucrose equivalent 2 Sucrose equivalent = residual (glucose + fructose) x 0.95 + unhydrolyzed sucrose 3 SE = standard error of least squares means

52

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0 4 8 12 16 20 24Fermentation hour

Mic

robi

al g

lyco

gen

(mg)

Figure 2-1. Microbial glycogen yield (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with initial medium pH of 6.8 (■) or 5.6 (▲). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

5.0

5.5

6.0

6.5

7.0

7.5

8.0


Ferm

enta

tion

pH

Figure 2-2. Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (∆, □) and SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or □) or 5.6 (▲ or ∆). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF.

53

0

10

20

30

40

50

60

70

80

90

100


Ace

tate

(mM

)

Figure 2-3. Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

0

5

10

15

20

25

30

35

40


Prop

iona

te (m

M)

Figure 2-4. Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

54

0

2

4

6

8

10

12

14


But

yrat

e (m

M)

Figure 2-5. Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

0

20

40

60

80

100

120

140


Tota

l VFA

(mM

)

Figure 2-6. Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

55

-5

0

5

10

15

20

25

30

35

0 4 8 12 16 20 24

Fermentation hour

Lact

ate

(mM

)

Figure 2-7. Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0


BC

VFA

(mM

)

Figure 2-8. Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

56

5

10

15

20

25

0 4 8 12 16 20 24

Fermentation hour

Am

mon

ia N

itrog

en (m

M)

Figure 2-9. Ammonia nitrogen concentration (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

0

2

4

6

8

10

12


Tota

l Fre

e A

min

o A

cids

(mM

)

Figure 2-10. Total free amino acid concentration (LSmeans ± standard error) for 24 h in vitro fermentations containing no substrate (○, ×) or SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or ○) or 5.6 (▲ or ×). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

57

50

60

70

80

90

100


Res

idua

l ND

F (%

)

Figure 2-11. Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of (iNDF; ∆, □) and SuNDF (▲, ■) with an initial medium pH of 6.8 (■ or □) or 5.6 (▲ or ∆). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose + iNDF.

0

5

10

15

20

25


Mic

robi

al c

rude

pro

tein

(mg)

Figure 2-12. Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with an initial medium pH of 6.8 (■) or 5.6 (▲). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

58

CHAPTER 3 EFFECT OF NITROGEN SOURCE ON MICROBIAL YIELD AND NEUTRAL DETERGENT FIBER DIGESTION FROM IN VITRO FERMENTATIONS OF

SUCROSE AND ISOLATED NEUTRAL DETERGENT RESIDUE

Introduction

Many ruminal microorganisms can synthesize microbial protein with a non-protein

nitrogen (NPN) source such as urea, as the sole source of nitrogen (Oltjen, 1969). The

efficiency of microbial protein synthesis (MCPeff) with urea as the sole source of

nitrogen may be lower compared to when peptides or amino acids are supplied. The

importance of amino acids and peptides from dietary protein degradation for increasing

microbial protein (MCP) production and energetic efficiency has been shown in several

studies with batch culture fermentations (Maeng et al., 1976; Maeng and Baldwin, 1976a,

1976b). Russell and Sniffen (1984) reported an increase of 18.7% in ruminal bacteria

yield with the addition of amino acids to mixed cultures with theoretically adequate

ammonia concentrations. The advantages of peptides and amino acids may depend on

the species of bacteria and energy source (Cruz Soto et al., 1994). There appears to be a

higher requirement for amino acids or peptides by amylolytic organisms (Maeng and

Baldwin, 1976a, 1976b) and sugar-utilizing organisms (Hungate, 1966), relative to fiber

utilizers. Although cellulolytic organisms primarily use ammonia as nitrogen source,

amino acids and peptides have been shown to increase in situ fiber digestion compared to

ammonia nitrogen (NH3-N) (Yang, 2002). When a rapidly fermented carbohydrate

source, such as sucrose, is available, there could be competition between sugar-utilizing

bacteria and other microbial populations for available nitrogen. This has potential to

59

affect the microbial growth of the different populations and alter neutral detergent fiber

(NDF) digestion. The objective of the present study was to evaluate the effects of

different nitrogen sources on microbial fermentation products and NDF digestion from

the in vitro fermentation of isolated NDF and sucrose. A secondary objective was to

determine the effect of sucrose supplementation in combination with different nitrogen

sources on fermentation pH and NDF digestion compared to fermentation of isolated

NDF only.


Substrates


detergent residue (iNDF; 92.8% dry matter: DM, 99.4% organic matter: OM, 98.6%

neutral detergent fiber OM: NDFOM, 5.4% neutral detergent fiber crude protein:

NDFCP) and a 50:50 mixture of iNDF and sucrose (SuNDF). The iNDF was prepared as

described by Hall and Herejk (2001). Sucrose (S5-500, Fisher Scientific, Atlanta, GA;

99.98% DM, 100% OM) and iNDF were analyzed prior to the onset of the study for DM

and OM (AOAC, 1980), and iNDF for NDF using heat-stable α-amylase (Termamyl

120L, Novo Nordisk Biochem, Franklinton, NC (Van Soest et al., 1991) and crude

protein (AOAC, 1980). A total of 240 mg ± 0.5 mg of substrate, with iNDF and sucrose

weighed individually, were transferred into duplicate 50 ml Nalgene high speed low

density polyethylene (LDPE) centrifuge tubes (05-562-13, Fisher Scientific, Atlanta, GA)

or Nalgene high speed polypropylene (HSPP) centrifuge tubes (05-562-10K, Fisher

Scientific, Atlanta, GA) depending on the type of analysis to be performed on

fermentation residues (Table 3-1).

60

Medium

Treatments consisted of one of three iso-nitrogenous media, containing different

nitrogen sources. The Goering and Van Soest (1970) medium contained NPN (3.52 mg

N) and true protein (6.85 mg N)(B), and was modified to contain only NPN (U) by

substituting urea (0.73 g urea/L of medium) for casein acid hydrolysate, or to contain

only true protein (C) by substituting casein acid hydrolysate (3.79 g casein acid

hydrolysate/L of medium) + sodium bicarbonate (4.25 g sodium bicarbonate/L of

medium) for ammonium bicarbonate.

Fermentation

Duplicate 16 h in vitro fermentation runs using destructive sampling of batch

cultures were performed according to the method of Goering and Van Soest (1970).

Casein acid hydrolysate (A-2427, Sigma Chemical Co., St. Louis, MO) was used as the

amino nitrogen source in B and C media. The reducing solution was mixed according to

a modification of the Goering and Van Soest (1970) procedure (P. J. Van Soest, personal

communication).The reducing solution was modified as described by Van Soest and

Robertson (1985). For a volume of 100 ml, 0.625 g of L-cysteine hydrochloride (C-7880,

Sigma Chemical Co., St. Louis, MO) and approximately 10 pellets of KOH (P250-2,

Fisher Scientific, Atlanta, GA) were dissolved with stirring in 50 ml of distilled water. In

a separate glass beaker 0.625 g sodium sulfide (S-4766, Sigma Chemical Co., St. Louis,

MO) was dissolved with stirring in 50 ml of distilled water. The solutions were

combined when the contents of both beakers were in solution, and just before addition of

the reducing solution to the fermentation tubes.

Rumen inoculum was obtained approximately 3 h post feeding from a ruminally

cannulated, non-pregnant, non-lactating Holstein cow under approved protocols of the

61

University of Florida Institutional Animal Care and Use Committee. The donor cow

received a diet of bermudagrass hay (10 kg DM/day), 48% crude protein soybean meal

(900 g/d) and free choice mineral supplement (Ca 17- 20%, P ≥ 9%, NaCl ≤ 25%, Mg ≥

0.25%, Cu ≥ 0.15, Co ≥ 0.01 %, I ≥ 0.01%, Mn ≥ 0.2%, Se ≤ 0.004%, Zn ≥ 0.4%, Fl ≤

0.09%). The inoculum was filtered through four layers of cheesecloth and one layer of

glass wool and maintained under anaerobic conditions at 39°C. Twenty milliliters of the

appropriate medium, 1 ml of reducing solution and 5 ml of inoculum were added to each

fermentation tube. After each addition, tube headspace was purged with CO2.

Fermentation tubes were capped with rubber stoppers fitted with gas release valves,


Houston, TX) under anaerobic conditions at 39°C and destructively sampled at 0, 4, 8, 12

and 16 hours. Tubes were swirled individually to mix every 4 hours.





tubes reserved for NDF analysis. The tubes used for NDF analysis were stored at 10°C

and were analyzed for residual NDF within two days of completion of the fermentation.

For NDF analysis, samples were allowed to equilibrate to room temperature, were

quantitatively transferred to 600 ml Berzellius beakers and refluxed with 50 ml of neutral

detergent solution and heat-stable α-amylase (Termamyl 120L, Novo Nordisk Biochem,

Franklinton, NC) for 1 h (Van Soest et al., 1991). To ensure removal of α-glucan, three

doses of 0.2 ml heat-stable α-amylase were used: one with addition of detergent, one 10

62

min before removing the sample from the burner and one added to the Gooch crucible

during rinsing with boiling water.


equivalents (sucrose, and its hydrolysis products: glucose and fructose), organic acids,

NH3-N and amino acid analyses were centrifuged at 15,000 x g for 30 min at 5°C. The

supernatant was transferred to scintillation vials and stored at -20°C until analysis for

residual sucrose and organic acids by HPLC, and NH3-N and amino acids by flow-

injection analysis (Broderick et al., 2004). The HPLC for analyzing residual sucrose was

equipped with an anion exchange analytical column (CarboPac™ PA1, Dionex,

Sunnyvale, CA), the mobile phase used was 100 mM NaOH, the flow rate 1.0 ml/min and

the injection volume 10 µL. The HPLC for analyzing organic acids was equipped with

an organic acid column (PHX-87H, Bio-Rad Laboratories, Richmond, CA). The solvent

used was 0.015 N H2SO4 / 0.0034 M EDTA, the flow rate 0.7 ml/min, the column

temperature 45°C and the injection volume 50 µL.



fermentation tubes. Glass beakers were stored at -20°C until further analysis for GLY.

Beakers were removed from the freezer and samples were allowed to equilibrate to room

temperature. Microorganisms were lysed with a 0.2 N NaOH solution (brought to a

volume of 20 ml in the 50 ml glass beakers) in a boiling water bath for 15 min. Samples

were cooled to room temperature and then neutralized to pH 7.0 ± 0.1 with 6 N HCl.

Samples were quantitatively transferred from the glass beakers to funnels fitted with glass

wool plugs for filtration into 100 ml volumetric flasks. Beakers, glass wool and funnels

63

were rinsed with distilled de-ionized water (ddH2O), and then samples were brought to

volume with ddH2O. Four milliliters of a 0.1 M sodium acetate buffer (pH ~ 4.5) and 50

µl of amyloglucosidase (EC 3.2.1.3, A-3514, Sigma Chemical Co., St. Louis, MO) were

added to 4 ml of sample, incubated at 60 ºC for 45 min, and analyzed for α-glucan

content as released glucose corrected for free glucose (Karkalas, 1985).

Microbial crude protein was estimated as trichloroacetic acid (TCA)-precipitated

crude protein. Samples were individually removed from the ice bath and a total of 5.2 ml

of a 120% (w/v) TCA solution were added in two equal increments to achieve a final

concentration of 20.0% TCA. Samples were returned to the ice bath for 45 min after

which tubes were centrifuged at 7700 x g for 20 min at 5°C. Each fermentation tube’s

contents were then quantitatively transferred into Whatman 541 filter paper (09-851D,

Fisher Scientific, Atlanta, GA) in veined funnels set in 125 ml Erlenmeyer flasks, using

approximately 50 ml of chilled 10% TCA to rinse the tubes, filter and residue. Samples

were allowed to filter under gravity. The filtrate was filtered through a Whatman GF/A

glass fiber filter (09-874-16D, Fisher Scientific, Atlanta, GA), using 10% TCA to rinse

the flask, filter and residue. Both Whatman 541 and GF/A filters containing the TCA-

precipitated material from one fermentation tube were placed together in a beaker and

dried for 24 h at 55°C, before analysis for crude protein content as Kjeldahl nitrogen

content x 6.25 (AOAC, 1980). Kjeldahl analysis blanks consisted of a Whatman 541

filter and a GF/A filter digested and distilled together in one Kjeldahl flask. The MCP

and GLY contents of each tube were corrected for fermentation blanks at each hour, and

MCP for its content by substrate at hour 0.

64


The experimental design was a split-split plot in time with a 2 x 2 factorial

arrangements of treatments (media and substrates). The data were analyzed using the

PROC MIXED procedure of SAS (1999) with fermentation run (R) as a random variable,

and medium (M) and substrate (S) as fixed variables. Fermentation hour (H) was used as

a class variable. The Kenward-Roger method was used to calculate the denominator

degrees of freedom for testing fixed effects. Orthogonal contrasts, B and C vs. U, and B

vs. C, were used for medium comparisons across substrates (iNDF, SuNDF) as well as

within substrate. All values presented are least squares means. The model statement

used was:

Yijkl = µ + Mi + Sj + MSij + Hk + MHik + SHjk + MSHijk + εijkl

Where:


µ = overall mean

Mi = medium (i = B, C, U)

Sj = substrate (j = iNDF, SuNDF)

Hk = hour (k = 0, 4, 8, 12, 16)

MSij = interaction term for medium and substrate

MHik = interaction term for medium and hour

SHjk = interaction term for substrate and hour

MSHijk = interaction term for medium, substrate and hour


65

A treatment term (T) consisting of the interaction between substrate (S) and

medium (M) were used in the random statement to obtain appropriate standard errors for

the least squares means. The random statement included the following terms:

Rl + RTlm + RHlk + RTHlmk

Where:

Rl = fermentation run (m = 1, 2)

Tm = treatment (n = 0, 1, 2, 3, 4, 5); number assigned to M by S combinations

RTlm = interaction term for fermentation run and treatment

RHlk = interaction term for fermentation run and hour

RTHklm = interaction term for fermentation run, treatment and hour

The sampling hour of maximum MCP or GLY yield within substrate, medium and

fermentation run were defined as the hour with the maximum least squares means for

these measures. The "hour" term and its interaction terms were omitted from the above

model to compare maximum MCP yield and MCP efficiency, GLY at 4 h, and residual

sucrose, fructose, glucose and sucrose equivalent at 0, 4 and 8 h. The MCP efficiency

was expressed as MCP (mg)/organic matter digested (OMD, mg). Organic matter

digested (mg) was calculated as the total sucrose (mg) minus residual sucrose equivalent

(mg) plus iNDFOM digested (mg), minus GLY (mg). Since NDF digestion, residual

sucrose, MCP and GLY were not measured on the same fermentation tube, the least

squares means for these measurements, within fermentation run at the hour of maximum

MCP, were used to calculate MCPeff for individual treatments. Sucrose equivalent was

calculated as residual sucrose (mg) + 0.95 x (residual fructose (mg) + residual glucose

(mg)). Total volatile fatty acids (VFA) is defined as the sum of acetate, propionate,

66

butyrate and valerate. Organic acid concentrations, but not protein degradation products

(branched chain VFA (BCVFA), total free amino acids and NH3-N), were corrected for

blank fermentations (no substrate, only inoculum). Orthogonal contrasts were used to

make comparisons among media across substrates (iNDF + SuNDF) as well as within

substrate.


Residual Substrate

Residual substrate, defined as the amount (mg) of glucose, fructose and sucrose

that could be detected in the supernatant, did not differ among media over the 16 h

fermentation (P = 0.60, 0.37 and 0.24, respectively). However, there were some

differences among media in the initial sampling hours (Table 3-2). At 0 h, U had more

residual fructose and tended to have more residual glucose than the other treatments.

There was no difference among the media for sucrose content at 0 h, though U was

numerically much smaller than B and C. The great variation and accordingly large

standard errors may have prevented detection of differences. Sucrose equivalents did not

differ among media at 0 h. The percentage of sucrose equivalent remaining at 0 h, as a

proportion of the initial 120 mg sucrose, was 74.6%, 77.9% and 50.9% for B, C and U,

respectively.

Glucose was not detected in either B or C at 4 h, and in U at 8 h, and at subsequent

hours for all media. No fructose was detected for B at 4 h and for C and U at 8 h, and at

subsequent hours for all media. Sucrose was readily degraded regardless of the source of

nitrogen, whereas ruminal microorganisms more readily utilized the monosaccharide

constituents (glucose and fructose) when provided with true protein compared to NH3-N

only. Fructose appeared to be utilized more slowly than glucose.

67

Microbial Glycogen

Maximum GLY accumulation for all treatments was achieved at 4 h, with a similar

steady decline thereafter (P = 0.46 for medium by hour interaction from 4 to 24 h; Figure

3-1). There was no detectable difference (P = 0.54) in maximal glycogen accumulation

among nitrogen sources (7.31, 7.17 and 6.84 mg for B, C and U, respectively). However,

over the 16 h fermentation, U tended to have a lower (P = 0.11) yield of GLY compared

to B and C (3.09, 3.48 and 3.54 mg, respectively), whereas B and C did not differ (P =

0.75).

Microbial glycogen accumulation may have been reduced in U as compared to B

and C as microorganisms had less preformed amino acids to incorporate into MCP and

thus had to expend more energy for MCP synthesis. Alternatively, a lack of sufficient

BCVFAs rather than nitrogen may have reduced the efficiency of substrate use by the

microorganisms provided only with NPN, and the limited amount of amino nitrogen

supplied by the inoculum (Russell and Sniffen, 1984)

Fermentation pH

Fermentation pH was lower (P < 0.01) with SuNDF as compared to iNDF (Figure

3-2). Among the media, mean fermentation pH for U was higher compared to B and C

for both SuNDF (P < 0.01) and iNDF (P < 0.01), while B and C did not differ (SuNDF, P

= 0.67; iNDF, P = 0.24). Aldrich et al. (1993) reported a decrease in ruminal pH for

cows fed a diet containing 65.7% compared to those fed a diet containing 52.4% rumen

available protein in combination with a rapidly degradable carbohydrates source (starch).

However, the comparison of different rumen degradable nitrogen sources (NPN and

amino acids or peptides) on fermentation pH needs further investigation.

68

The higher fermentation pH for U may be a result of the hydrolysis of the added

urea in the NPN fermentation to release ammonia (Figure 3-8), which is alkaline, and the

lower yield of organic acids for this medium. The lower fermentation pH with SuNDF

compared to iNDF as the substrate is likely due to increased organic acid production in

SuNDF fermentations which contained more readily fermented carbohydrate, however,

organic acid concentrations were not measured on iNDF fermentations.

Organic Acids

In general, organic acid production increased in the presence of amino acid

nitrogen compared to NH3-N only (Table 3-3). For fermentations containing SuNDF as

the substrate, concentrations of organic acids at 16 h, with the exception of lactate, were

greater or tended to be greater (acetate) for B and C as compared to U. The organic acid

concentrations of B and C media did not differ at 16 h. Organic acid concentrations did

not follow similar temporal patterns (Figures 3-3 to 3-7) over the 16 h fermentation as

indicated by medium by hour interactions (P < 0.01 for total VFA, propionate, butyrate,

valerate and lactate; P = 0.02 for acetate).

Maximum lactate concentration did not differ (P = 0.58) among media (27.6, 27.1

and 30.2 mM for B, C and U, respectively). However, maximum lactate concentration

was detected at 4 h for B and at 8 h for C and U (Figure 3-7). At the end of the

fermentation, lactate concentration was greater for U compared to B and C, and no lactate

was detected in B and C at 16 h (Table 3-3). Growth rates for Megasphaera elsdenii, a

major ruminal lactate-utilizer, may be stimulated in the presence of peptides and amino

acids (Cruz Soto et al., 1994). The relative decrease in lactate disappearance in

fermentations containing only NPN can be explained by impaired growth of lactate-

69

utilizers due to the lack of amino acids or BCVFAs (needed together with ammonia for

microbial protein synthesis).

Protein Degradation Products

Total free amino acid and NH3-N concentrations in fermentations are the net result

of amount supplied in the medium and inoculum, as well as protein breakdown and

nitrogen utilization by ruminal microorganisms in vitro. At various sampling hours

during the 16 h fermentation NH3-N (Figure 3-8) and total amino acid (Figure 3-9)

concentrations appeared to be higher for blank fermentations (no substrate, only

inoculum) compared to fermentations containing SuNDF. This may have resulted from

increased protein breakdown and decreased utilization in blank fermentations lacking in

fermentable carbohydrate substrates, or increased utilization of protein breakdown

products in fermentations with SuNDF as the substrate.

For the entire fermentation, and at the 16 h endpoint, the NH3-N concentration for

fermentations with SuNDF as the substrate was greater for U (P < 0.01) compared to B

and C, and B was greater than C (P < 0.01). Total free amino acid concentration for the

16 h fermentation, with SuNDF as the substrate, was greater (P < 0.01) for B and C

compared to U, and C was greater than B (P < 0.01). At 16 h, total free amino acid

concentration for B and C was greater (P = 0.02) than for U, and C tended to be greater

than B (P = 0.06). For the most part, relative differences in total amino acid and NH3-N

concentrations among the three fermentations reflected the differences in type of nitrogen

source supplied at the onset: the greater the amount of NPN in the medium, the more

NH3-N, and the more true protein, the more free amino acids.

Branched chain VFAs are also products of protein degradation. The concentration

of BCVFAs remained relatively low and similar among media through the initial 12 h of

70

fermentation (Figure 3-10). It was only at 16 h, with SuNDF as the substrate, that

BCVFA concentration was greater (P < 0.01) for B and C (2.09 and 3.07 mM,

respectively) compared to U (0.28 mM), and C was greater than B (P < 0.01). Increased

microbial lysis and degradation of microbial protein may occur when substrates

supporting growth and maintenance becomes limiting, which could explain the relative

increase in protein degradation products (BCVFA and ammonia) towards the end of the

fermentation.

Most research have been focused on comparing the effect of rumen degradable and

rumen undegradable nitrogen sources on animal performance and on protein breakdown

product concentrations in the rumen. Information on the effect of different rumen

degradable nitrogen sources (NPN vs. amino acids and peptides) on measurements such

as total free amino acid and ammonia nitrogen concentrations both in vitro and in the

rumen needs further investigation.


Fermentation pH did not decline below 6.56 for any treatment, and so it is not

likely that pH had a negative effect on NDF digestion. At 16 h of fermentation, NDF

digestion (100 – residual NDF) did not differ (P = 0.68) among media with iNDF as

substrate (18.5, 16.0 and 16.6% for B, C and U, respectively; Figure 3-11). At 16 h,

digestion of NDF for SuNDF was greater (P = 0.01) for B and C (21.0 and 19.5%,

respectively) compared to U (14.4%), and B and C did not differ (P = 0.45). Digestion of

NDF at 16 h, averaged across media, did not differ (P = 0.28) between iNDF and SuNDF

fermentations.

Proteins may be superior to urea for maintenance of fiber digestion despite the fact

that cellulolytic organisms primarily use ammonia as nitrogen source. This may indicate

71

that cellulolytic bacteria have some requirement for supplementation with amino acids or

peptides (Hoover, 1986), which may be related to the supply of BCVFAs. Gorosito et al.

(1985), however, suggested that amino acids or peptides might increase cell wall

digestion over BCVFAs alone.

There are not enough data points in the early hours to clearly define it, however, the

patterns of the lines suggest that the addition of sucrose increased the lag time of NDF

digestion in the early hours; iNDF declined below 95% residual NDF by 4 h, whereas,

SuNDF did not reach that point until after 8 h. The apparent lag noted for NDF digestion

may be the result of competition between NFC and fiber-utilizing microorganisms for the

nitrogen supply.

The addition of sucrose and true protein increased NDF digestion in the later hours

of the fermentation. Proteolytic activity by bacteria that ferment readily available

carbohydrates (e.g. sucrose; [Wallace et al., 1999]) could increase available ammonia and

BCVFAs, which are growth requirements for cellulolytic bacteria (Hoover, 1986).

Provision of limiting nutrients required by the microorganisms could enhance fiber

digestion.

Microbial Crude Protein Yield and Efficiency

The yield of MCP over the 16 h fermentation was lower for U compared to B and C

with SuNDF (P = 0.01) as the substrate, and tended to be lower when iNDF (P = 0.11)

was fermented alone (Figure 3-12). There was a greater MCP yield with SuNDF

compared to iNDF (P < 0.01) across all media. Maximum MCP yield was greater for B

and C compared to U, and did not differ between B and C, for SuNDF fermentations

(Table 3-4). For iNDF fermentations, however, there was no difference among media for

72

maximum MCP yield. Therefore the benefit of amino acids or peptides for maximum

MCP yield might only be apparent when sucrose is present in the fermentation.

Microbes receiving NPN alone (U) were only 64% as efficient in their yield of

MCP, with SuNDF as the substrate, as those receiving true protein (B and C), whereas B

and C did not differ from each other (Table 3-4). The greater MCP and MCPeff with the

addition of true protein is likely due to direct incorporation of amino acid or peptides into

microbial protein (Cotta and Russell, 1982), or increased availability of carbon skeletons

in the form of BCVFAs to support amino acid synthesis (Russell and Sniffen, 1984). The

importance of amino acids and peptides from dietary protein degradation for increasing

both microbial protein production and energetic efficiency has been shown in several

other studies with batch culture fermentations (Maeng et al., 1976; Maeng and Baldwin,

1976a, 1976b). Russell and Sniffen (1984) reported an increase of 18.7% in ruminal

bacteria yield in mixed culture fermentations with a mixture of carbohydrates (equal parts

of glucose, maltose, sucrose, cellobiose and soluble starch) and the addition of amino

acids with theoretically adequate ammonia concentrations.

Conclusions

Addition of sucrose and source of nitrogen affected in vitro yield of fermentation

products and NDF fermentation. Addition of true protein increased MCP yield and

efficiency of yield from ruminal microorganisms when sucrose was present and had a

positive effect on MCP yield from NDF alone. True protein addition increased NDF

digestion when sucrose was present, and increased total yield of organic acids.

Maximum accumulation of GLY was not affected by nitrogen source when sucrose and

NDF were fermented together. These results imply that the sources of ruminally

73

degradable nitrogen and the inclusion of sucrose may be important to consider in the

prediction of fiber digestion and metabolizable nutrient supply in ruminant diets. Several

animal studies have considered the effect of supplying ruminally degradable nitrogen

compared to ruminally undegradable nitrogen on animal performance. Animal studies

investigating the interaction of ruminally degradable nitrogen source and NFC source on

ruminal measures and animal performance are warranted.

Table 3-1. Type and number of fermentation tubes per medium for one sampling hour, indicating the substrate and analysis for which tubes were reserved in a 16 h in vitro fermentation of sucrose and isolated neutral detergent fiber.

Analysis

Substrate1 pH and residual NDF

Microbial crude protein

Microbial glycogen, residual sucrose, organic acids, NH3-N2

and amino acid nitrogen No substrate (fermentation blank) - 2 x HSPP3 2 x LDPE4

iNDF 2 x HSPP 2 x HSPP - SuNDF 2 x HSPP 2 x HSPP 2 x LDPE

1 iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose + iNDF 2 NH3-N = ammonia nitrogen

3 HSPP = Nalgene high speed, polypropylene 4 LDPE = Nalgene high speed, low density, polyethylene

74

Table 3-2. Residual glucose, fructose, sucrose, monosaccharide sucrose equivalent (glucose + fructose) and sucrose equivalent at 0, 4 and 8 h, and averaged for 16 h in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue with different sources of nitrogen in media.

Treatment1 Glucose Fructose Sucrose Glucose+Fructose2 Sucrose eq.3 Hour 0 Least squares means (mg) B 0.31 1.21 88.0 1.44 89.5 C 0.71 1.75 91.1 2.34 93.4 U 2.69 2.34 56.3 4.78 61.1

SE4 2.10 2.51 26.3 4.34 30.2 P-value Treatment 0.20 < 0.01 0.36 0.08 0.41 Contrasts B and C vs. U 0.11 < 0.01 0.20 0.05 0.24 B vs. C 0.70 0.06 0.89 0.35 0.87 Treatment1 Glucose Fructose Sucrose Glucose+Fructose2 Sucrose eq.3 Hour 4 Least squares means (mg) B -0.47 0.02 -0.34 -0.43 -0.77 C -0.72 5.92 -0.48 4.94 4.46 U 3.14 13.9 0.08 16.2 16.3

SE 1.88 8.62 0.22 9.94 10.0 P-value Treatment 0.32 0.51 0.07 0.48 0.46 Contrasts B and C vs. U 0.18 0.33 0.03 0.29 0.28 B vs. C 0.92 0.61 0.52 0.69 0.69 Hour 8 Least squares means (mg) B 0.13 0.06 -0.07 0.18 0.11 C 0.61 -0.03 -0.14 0.55 0.41 U 0.05 0.03 0.00 0.07 0.07

SE 0.11 0.05 0.04 0.06 0.05 P-value Treatment 0.06 0.55 0.21 0.02 0.02 Contrasts B and C vs. U 0.09 0.89 0.13 0.03 0.04 B vs. C 0.05 0.33 0.32 0.02 0.02 Average for 16 h Least squares means (mg) B 0.35 0.32 17.5 0.63 18.1 C 0.77 1.57 18.1 2.23 20.3 U 1.18 3.26 11.3 4.21 15.5

SE 0.73 2.14 5.28 2.68 7.95 P-value Treatment 0.60 0.37 0.24 0.48 0.79 Contrasts B and C vs. U 0.42 0.21 0.10 0.32 0.58 B vs. C 0.61 0.54 0.88 0.58 0.76 1 B = True protein + Non protein nitrogen; C = True protein only; U = Non-protein nitrogen only 2 Calculated as residual (glucose + fructose) x 0.95 to give residual monosaccharide sucrose equivalent 3 Sucrose equivalent = residual (glucose + fructose) x 0.95 + unhydrolyzed sucrose 4 SE = standard error of least squares means

75

Table 3-3. Organic acid concentrations (least squares means) at 16 h (corrected for blank fermentations) for in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue with different sources of nitrogen in media

Treatment1 Total VFA2 Valerate C2 C3 C4 Lac mM

B 46.0 2.53 20.0 17.8 5.61 0.00 C 43.9 2.54 17.5 18.4 5.44 -0.03 U 26.1 0.14 15.4 8.3 2.39 20.7 SE3 2.17 0.31 1.15 1.08 0.19 1.15

P-value Treatment 0.01 0.03 0.19 0.01 < 0.01 < 0.01 Contrasts B and C vs. U < 0.01 0.01 0.13 < 0.01 < 0.01 < 0.01 B vs. C 0.55 0.97 0.26 0.73 0.40 0.99 1 B = True protein + Non protein nitrogen; C = All true protein; U = Non protein nitrogen 2 VFA = volatile fatty acid; Lac = lactate; C2 = acetate; C3 = propionate; C4 = butyrate; BCVFA = branched chain VFA 3 SE = standard error of least squares means

Table 3-4. Maximum microbial crude protein (MCP) yield (hour of maximum) and efficiency of MCP yield at the point of maximum MCP yield for in vitro fermentations of iNDF and of SuNDF with different source of nitrogen in media. Values are least squares means.

Microbial crude protein Substrate1 Treatment2 Maximum, mg (h) Efficiency, mg/mg OMD4

SuNDF B 14.7 (12) 0.11 C 15.7 (16) 0.11 U 8.85 (12) 0.07 (SE3 = 1.30) (SE = 0.01) P-value Treatment effect < 0.01 0.06 Contrast B and C vs. U < 0.01 0.03 B vs. C 0.15 0.63

iNDF B 3.61 (16) 0.09 C 4.48 (12) 0.17 U 1.77 (16) 0.04 (SE = 1.30) (SE = 0.04) P-value Treatment effect 0.33 0.25 Contrast B and U vs. C 0.27 0.14 B vs. U 0.31 0.52

1 iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose + iNDF 2 B = Non-protein nitrogen + true protein, C = true protein only, U = non-protein nitrogen only 3 Standard error of least squares means 4 OMD = organic matter digested = {[Sucrose OM + NDFOM (mg) digested] – GLY (mg)}; for iNDF as substrate OM digested includes only NDF OM digested

76

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0 4 8 12 16Fermentation hour

Mic

robi

al g

lyco

gen

(mg)

Figure 3-1. Microbial glycogen yield (least squares means ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

6.0

6.5

7.0

7.5

8.0


Ferm

enta

tion

pH

Figure 3-2. Fermentation pH (least squares means ± standard error) for 16 h in vitro fermentations of iNDF (□, ∆, ○) and SuNDF (■, ▲, ●) with media containing nitrogen in the form of non-protein nitrogen + true protein (■ or □), true protein only (▲ or ∆) or non-protein nitrogen only (● or ○). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF.

77

-10

0

10

20

30

40

50

0 4 8 12 16

Fermentation hour

Tota

l VFA

(mM

)

Figure 3-3. Total volatile fatty acid concentrations (LSmeans ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

-5

0

5

10

15

20

25

0 4 8 12 16

Fermentation hour

Ace

tate

(mM

)

Figure 3-4. Acetate concentrations (LSmeans ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

78

-5

0

5

10

15

20

0 4 8 12 16

Fermentation hour

Prop

iona

te (m

M)

Figure 3-5. Propionate concentrations (LSmeans ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0 4 8 12 16

Fermentation hour

But

yrat

e (m

M)

Figure 3-6. Butyrate concentrations (LSmeans ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

79

-5

0

5

10

15

20

25

30

35

0 4 8 12 16

Fermentation hour

Lact

ate

(mM

)

Figure 3-7. Lactate concentrations (LSmeans ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

-5

0

5

10

15

20

25

0 4 8 12 16

Fermentation hour

Am

mon

ia N

itrog

en (m

M)

Figure 3-8. Ammonia nitrogen concentration (LSmeans ± standard error) for 16 h in vitro fermentations with no substrate (□, ∆, ○) or SuNDF as the substrate (■,▲,●) and media containing nitrogen in the form of non-protein nitrogen + true protein (□, ■), true protein only (∆, ▲) or non-protein nitrogen only (○, ●). SuNDF = sucrose+isolated bermudagrass neutral detergent residue.

80

-2

0

2

4

6

8

10

12

14

0 4 8 12 16

Fermentation hour

Tota

l Am

ino

Aci

ds (m

M)

Figure 3-9. Total free amino acid concentration (LSmeans ± standard error) for 16 h in vitro fermentations with no substrate (□, ∆, ○) or SuNDF as the substrate (■,▲,●) and media containing nitrogen in the form of non-protein nitrogen + true protein (□, ■), true protein only (∆, ▲) or non-protein nitrogen only (○, ●). SuNDF = sucrose+isolated bermudagrass neutral detergent residue.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5


BC

VFA

(mM

)

Figure 3-10. Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 16 h in vitro fermentation with no substrate (□, ∆, ○) or SuNDF as the substrate (■,▲,●) and media containing nitrogen in the form of non-protein nitrogen + true protein (□, ■), true protein only (∆, ▲) or non-protein nitrogen only (○, ●). SuNDF = sucrose+isolated bermudagrass neutral detergent residue.

81

A

75

80

85

90

95

100


Res

idua

l ND

F (%

)

B

75

80

85

90

95

100


Res

idua

l ND

F (%

)

Figure 3-11. Residual NDFOM for 16 h in vitro fermentations of iNDF (A; □, ∆, ○) and SuNDF (B; ■, ▲, ●) with media containing nitrogen in the form of non-protein nitrogen + true protein (□, ■), true protein only (∆, ▲) or non-protein nitrogen only (○, ●). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF.

82

-5

0

5

10

15

20


Mic

robi

al c

rude

pro

tein

(mg)

Figure 3-12. Microbial crude protein yield for 16 h in vitro fermentations of iNDF (□, ∆, ○) and SuNDF (■, ▲, ●) with media containing nitrogen in the form of non-protein nitrogen + true protein (■ or □), true protein only (▲ or ∆) or non-protein nitrogen only (● or ○). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF.

83

CHAPTER 4 MICROBIAL PRODUCT YIELD AND NEUTRAL DETERGENT FIBER DIGESTION FROM IN VITRO FERMENTATIONS WITH SUCROSE, STARCH AND PECTIN IN COMBINATION WITH ISOLATED BERMUDAGRASS NEUTRAL DETERGENT

RESIDUE

Introduction

Carbohydrates comprise between 70 and 80% of ruminant diets and are the major

source of energy for both the ruminal microorganisms and the ruminant animal. Non-

neutral detergent fiber carbohydrates (NFCs) can make up 30 to 40% of diet dry matter in

rations for high producing dairy cattle. The NFC fraction in feedstuffs includes sugars,

organic acids, starch, β-glucans, pectic substances and fructans and other carbohydrates

soluble in neutral detergent (Hall et al., 1999). These have often been treated as a

homogenous group regarding fermentation characteristics. However, the fermentation of

different NFCs produces different volatile fatty acid (VFA) profiles (Strobel and Russell,

1986; Mansfield et al., 1994; Ariza et al., 2001) and has varied effects on ruminal pH

(Strobel and Russell, 1986; Khalili and Huhtanen, 1991a), microbial product yield (Hall

and Herejk, 2001; Sannes et al., 2002) and fiber digestion (Heldt et al., 1999; Miron et

al., 2002). The NFCs also vary in their effects on milk yield (O'Mara et al., 1997b; Leiva

et al., 2000; Broderick et al., 2002b) and milk composition (Nombekela and Murphy,

1995; Broderick et al., 2002a). However, differences in animal performance among NFC

sources have not been consistent. Understanding of the fermentation characteristics of

individual NFCs and combinations of NFCs, could help to improve predictions of animal

performance in response to NFC supplementation.

84

The objective of this study was to evaluate the effects of different amounts and

ratios of sucrose, starch and pectin in combination with isolated bermudagrass neutral

detergent fiber (NDF) on microbial product yield and NDF digestion.


Substrates and Treatments


detergent residue (iNDF; 96.4% DM, 99.5% OM, 99.0% NDFOM, 4.6% NDFCP),

sucrose (S5-500, Fisher Scientific, Atlanta, GA; 99.98% DM, 100% OM), corn starch (S-

4126, Sigma Chemical Co., St. Louis, MO; 88.5% DM, 100% OM) and citrus pectin (P-

9135, Sigma Chemical Co., St. Louis, MO; 95.2% DM, 96.0% OM). The iNDF was

prepared from bermudagrass hay ground through the 1 mm screen of a Wiley mill

(Arthur H. Thomas Co., Philadelphia, PA.). Fifty grams of hay were transferred to a 3 L

glass beaker and refluxed with 2 L of neutral detergent solution and 10 ml of heat stable

α-amylase (Termamyl 120L, Novo Nordisk Biochem, Franklinton, NC) for 1 h (Van

Soest et al., 1991). The contents of the glass beaker was filtered through a nylon cloth

(37 µm pore size) and rinsed with boiling distilled water until no foam was visible. The

residue on the nylon cloth was transferred to a 2 L glass beaker and soaked overnight in 1

M ammonium sulfate solution (approximately 5 g of residue DM/200 ml) to remove

residual detergent. The residue was then filtered under vacuum through nylon cloth and

repeatedly rinsed with boiling distilled water until no more foaming from detergent was

visible. The residue was then twice rinsed with acetone and filtered under vacuum until

dry. After drying overnight at 55°C in a forced-air oven, the residue was allowed to

equilibrate with ambient humidity. The iNDF was included in all fermentation tubes

containing substrate at 120 mg ± 0.5 mg (air dry).

85

In each of the fermentations four amounts of two of the three NFC sources were

examined individually and mixtures were also examined (Table 4-1). The weights of

NFCs are expressed on a hexose equivalent weight basis. Fermentation tubes with only

iNDF were used to represent the treatments of 0 mg hexose equivalent NFCs. Hexose

equivalent conversion coefficients used for sucrose, starch and pectin were 0.95, 0.90 and

1.14, respectively. These coefficients are used as multipliers to calculate the amount of

substrate that yields one unit of simple sugars upon complete hydrolysis of the oligo- or

polysaccharides. The pectin value was based upon the manufacturer's analysis

(multiplier to come up with needed mass of pectin = 1/(% galacturaonic acid monomers

[87.6%; as per specification sheet from Sigma Chemical Co. for Lot 108H0913 of P-

9135]). The sucrose multiplier is based upon the molecular weight of sucrose divided by

the weight of its constituent monomers: (342 MW sucrose / 360 MW glucose+fructose) =

0.95. For starch, the multiplier used in starch analyses to convert from released glucose

to starch was used: 0.90. This value reflects the weight of a glucose molecule minus the

weight of a water of hydrolysis by the molecular weight of glucose: (180-18)/180 = 0.90).

In this polysaccharide, the weight of one water molecule is removed per glucose to form

the glycosidic bond between glucose molecules. For use as a classification variable, the

four equally spaced hexose equivalent treatments are referred to as 0, 40, 80, and 120 mg

for all NFC. Fermentation tubes with only iNDF were used to represent the treatments of

0 mg hexose equivalent NFCs. Substrates, with the exception of pectin, were weighed

individually and transferred into duplicate 50 ml centrifuge tubes.

Pectin did not readily go into solution or suspension upon addition of medium, but

formed gel-like clumps. To avoid this problem, three aqueous suspensions of pectin were

86

formulated to deliver the desired amounts of pectin per fermentation tube. On the day

before the start of the fermentation, three separate pectin suspensions were prepared in

individual 1 L glass beakers by adding the desired amount of pectin to half of the amount

of water to be used for solubilizing the casein hydrolysate. These were stirred

continuously with moderate heating for approximately 1 h and no more than 1.5 h, at

which point all pectin was in suspension. Pectin suspensions were then covered with

aluminum foil and kept at 4°C overnight. The next morning, after dissolving the casein

hydrolysate in the remainder of the water, the two solutions were combined and media

preparation completed according to Goering and Van Soest (1970).

Tubes destined for pH + NDF analyses or trichloroacetic acid (TCA) precipitation

were made of Nalgene high-speed, polypropylene (05-562-10K, Fisher Scientific,

Atlanta, GA), and those for residual sucrose + organic acids + microbial glycogen

analyses were made of Nalgene high-speed low-density polyethylene (05-562-13, Fisher

Scientific, Atlanta, GA). Duplicate fermentation tubes were used for each of the three

sets of analyses. This gave six fermentation tubes per treatment per sampling hour.

Fermentation

A series of six 24 h batch culture in vitro fermentation runs were performed using

mixed ruminal microbes as the inoculum according to the method of Goering and Van

Soest (1970). The macro-mineral solution was modified to include 2.22 g NaCl/L (Van

Soest and Robertson, 1985). The reducing solution was mixed according to a

modification of the Goering and Van Soest (1970) procedure (P. J. Van Soest, personal

communication). For every 100 ml of reducing solution, 0.625 g of cysteine hydrochloric

acid (C-7880, Sigma Chemical Co., St. Louis, MO) and 7 pellets of KOH (P250-2, Fisher

Scientific, Atlanta, GA) were dissolved with stirring in 50 ml distilled water. In a

87

separate beaker, 0.625 g of sodium sulfide (S-4766, Sigma Chemical Co., St. Louis, MO)

was dissolved with stirring in 50 ml distilled water. The solutions were combined when

the contents of both beakers were in solution, and just before addition of the reducing

solution to the fermentation tubes. Tryptone (T-9410, Sigma Chemical Co., St. Louis,

MO) was used as the source of amino nitrogen in the medium.

Rumen inoculum was obtained approximately 3 h post-feeding from two ruminally

cannulated, non-pregnant, non-lactating Holstein cows under protocols approved by the

University of Florida Institutional Animal Care and Use Committee. Both donor cows

received bermudagrass hay (10 kg DM/day) and free choice mineral supplement (Ca 17-

20%, P ≥ 9%, NaCl ≤ 25%, Mg ≥ 0.25%, Cu ≥ 0.15, Co ≥ 0.01 %, I ≥ 0.01%, Mn ≥

0.2%, Se ≤ 0.004%, Zn ≥ 0.4%, Fl ≤ 0.09%). In addition, 50 g of trace mineralized salt

and vitamin mix (Ca 2.9%, P 0.18%, Mg 0.02%, K 0.2%, S 2.4%, Co 8250 ppm, Cu

19052 ppm, I 4762 ppm, Fe 7791, Mn 60041 ppm, Se 750 ppm, Zn 45063 ppm, vit. A 26

736 IU/kg, vit. D 8226 IU/kg) and 4 g of vit. E (50 000 UI/kg) was daily mixed in with

the mineral supplement. One donor cow received 48% crude protein soybean meal (900

g/d), while the other donor cow received a supplement containing 48% crude protein

soybean meal (500 g/d), ground corn meal (500 g/d) and dried citrus pulp (500 g/d).

Inoculum was collected into warmed, 3 L glass vessels via the rumen cannula using

a 2.5 cm i.d. hose attached to a vacuum pump. Equal amounts of inocula from the two

donor cows were individually strained through two layers of cheesecloth into a CO2-

purged blender container (BlendMaster Model 50220, Type B15, Hamilton

Beach/Proctor-Silex, Inc., Washington, NC). Inoculum was blended for 15 sec on low

and 45 sec on high while continuously gassed with CO2. It was then strained through

88

four layers of cheesecloth into an Erlenmeyer flask set in a 39°C water bath, with the

headspace continuously gassed with CO2. The inocula from both cows was combined in

the Erlenmeyer flask.

For inoculation of fermentation tubes, enough inoculum was poured out into a 600

ml glass beaker to fill tubes for one sampling hour. The beaker was continuously gassed

with CO2 to maintain anaerobic conditions, and set on a magnetic stir plate for

continuous stirring during inoculation of fermentation tubes. This was to ensure that the

inoculum was consistent for all tubes of the same hour. Twenty milliliters of medium, 1

ml of reducing solution and 5 ml of inoculum were added to each fermentation tube,

including duplicate fermentation blanks (without any substrate) for each sampling hour.

Fermentation tubes were capped with rubber stoppers fitted with gas release valves,


Houston, TX) under anaerobic conditions at 39°C and destructively sampled at 0, 4, 8,

12, 16, 20 and 24 hours. Tubes were individually swirled to mix contents every 4 hours.





tubes reserved for NDF analysis. The tubes reserved for NDF analysis were stored at

10°C and were analyzed for residual NDF within one week of the completion of the

fermentation. For NDF analysis, samples were allowed to equilibrate to room

temperature. Then they were quantitatively transferred to 600 ml Berzellius beakers and

refluxed with 50 ml of neutral detergent solution and heat-stable α-amylase (Termamyl

89

120L, Novo Nordisk Biochem, Franklinton, NC) for 1 h (Van Soest et al., 1991). To

ensure removal of α-glucan, three doses of 0.2 ml heat-stable α-amylase were used: one

with addition of detergent, one 10 min before removing the sample from the burner and

one added to the Gooch crucible during rinsing with boiling water.


equivalents (as sucrose, and its hydrolysis products: glucose and fructose) and organic

acids, were centrifuged at 15,000 x g for 30 min at 5°C. The supernatant was transferred

to serum vials and stored at -20°C until analysis for residual sucrose and organic acids by

HPLC. The HPLC for analyzing residual sucrose was equipped with an anion exchange

analytical column (CarboPac™ PA1, Dionex, Sunnyvale, CA), the mobile phase used

was 100 mM NaOH, the flow rate 1.0 ml/min and the injection volume 10 µL. The

HPLC for analyzing organic acids was equipped with an organic acid column (PHX-87H,

Bio-Rad Laboratories, Richmond, CA). The solvent used was 0.015 N H2SO4 / 0.0034 M

EDTA, the flow rate 0.7 ml/min, the column temperature 45°C and the injection volume

50 µL.



fermentation tubes. These samples were stored at -20°C until further analysis for GLY.

For GLY analysis, beakers were removed from the freezer and samples were allowed to

equilibrate to room temperature. Microorganisms were lysed with a 0.2 N NaOH

solution (brought to a volume of 20 ml in the 50 ml glass beakers) in a boiling water bath

for 15 min. Samples were cooled to room temperature and then neutralized to pH 7.0 ±

0.1 with 6 N HCl. Samples were quantitatively transferred from the glass beakers to

90

funnels fitted with glass wool plugs for filtration into 100 ml volumetric flasks. Beakers,

glass wool and funnels were rinsed with distilled de-ionized water (ddH2O), and then

samples were brought to volume with ddH2O. Two hundred micro-liters of sample and

800 µl of ddH2O were dispensed into borosilicate glass tubes using a semi-automatic

Hamilton Microlab 1000 dilutor (Hamilton Company, Reno, NV). One milliliter of a 0.1

M sodium acetate buffer (pH ~ 4.5) and 50 µl of amyloglucosidase (EC 3.2.1.3, A-1602,

Sigma Chemical Co., St. Louis, MO) were added to the borosilicate glass tubes, which

were then incubated at 60ºC for 45 min, and analyzed for α-glucan content as released

glucose corrected for free glucose (Karkalas, 1985).

Microbial crude protein (MCP) was estimated as TCA-precipitated crude protein.

Fermentation tubes were individually removed from the ice bath and 5.2 ml of a 120%

(w/v) TCA solution were added to achieve a final concentration of 20% TCA. Samples

were returned to the ice bath for 45 min after which tubes were centrifuged at 7700 x g

for 20 min at 5°C. Each fermentation tube's contents were then quantitatively transferred

into Whatman 541 filter paper (09-851D, Fisher Scientific, Atlanta, GA) in veined

funnels set in 125 ml Erlenmeyer flasks. Approximately 50 ml of 10% TCA was used to

rinse the tubes, filter and residue. Samples were allowed to filter under gravity. The

filtrate was filtered through a Whatman GF/A glass fiber filter (09-874-16D, Fisher

Scientific, Atlanta, GA), using 10% TCA to rinse the flask, filter and residue. Both

Whatman 541 and GF/A filters containing TCA-precipitated material from one

fermentation tube were placed together in a beaker and dried for at least 24 h at 55°C,

before analysis for crude protein content as Kjeldahl nitrogen content x 6.25 (AOAC,

1980). Kjeldahl analysis blanks consisted of a Whatman 541 filter and a GF/A filter

91

digested and distilled together in one Kjeldahl flask. The MCP and GLY contents of

each tube were corrected for fermentation blanks at each hour, and MCP for its content

by substrate at hour 0.


Treatment mean comparisons and temporal pattern descriptions

The experimental design was a split-split plot in time. Data were analyzed using

the PROC MIXED procedure of SAS (1999) with fermentation run (R) as a random

variable, and treatment (T) as a fixed variable. Fermentation hour (H) was used as a class

variable. Equally spaced nominal amounts of hexose equivalents (0, 40, 80 and 120 mg)

were used to designate treatments as class variables and polynomial contrasts were used

for evaluating temporal patterns over the 24 h. All values presented are least squares

means. The model statement used was:

Yijkl = µ + Ti + Hj + THij + εijkl

Where:


µ = overall mean

Ti = treatment (i = see Table 4-1)

Hj = hour (j = 0, 4, 8, 12, 16, 20, 24)

THij = interaction term for treatment and hour


The random statement included the following terms:

Rl + RTli + RHlj + RTHlij

Where:

92

Rl = fermentation run (l = 1, 2, 3, 4, 5, 6)

Ti = treatment (i = see Table 4-1)

RTli = interaction term for fermentation run and treatment

RHlj = interaction term for fermentation run and hour

RTHlij = interaction term for fermentation run, treatment and hour

The above model with the hour term omitted and using orthogonal contrasts was

used to determine the linear or quadratic pattern of dependent variable change with

increasing nominal hexose equivalent inclusion of NFCs. The sampling hour of

maximum MCP and GLY yield within treatment was defined as the hour with the

maximum least squares means. The sucrose equivalent at any specific sampling hour was

calculated as residual sucrose (mg) + 0.95 x (residual fructose (mg) + residual glucose

(mg)).

Comparisons of maxima, minima and 24 h data

Heterogeneity of regression is used to examine differences between or among

treatments over time, doses, or other continuous variable. It is used to evaluate whether

slopes of lines do or do not differ. Therefore, heterogeneity of regression analysis was

used for comparisons of increasing hexose equivalent amounts (mg DM) from different

NFCs, on maxima, minima and 24 h values. The data were evaluated for heterogeneity

of regression using type one sums of squares and with the model statement:

Yijkl = µ + Ti + Hj + HHjj + Ck + THij + THHijj + TCik + εijkl

Where:


µ = overall mean

93

Ti = treatment (i = sucrose, starch, pectin)

Hj = linear continuous term for hexose equivalents based on actual hexose

equivalents (mg DM) weighed into fermentation tubes

HHjj = quadratic continuous term for hexose equivalents based on actual hexose

equivalents (mg DM) weighed into fermentation tubes

Ck = hexose equivalent as a class variable (j = 0, 40, 80, 120)

THij = interaction term for treatment and linear continuous hexose equivalents

THHijj = interaction term for treatment and quadratic continuous hexose equivalents

TCik = interaction term for treatment and linear continuous hexose equivalents


The random statement included the following terms:

Rl + RTli

Where:


RTli = interaction term for fermentation run and treatment

The linear form of this model omitted quadratic terms for hexose equivalents

(continuous). Significance of the linear or quadratic term for hexose equivalent defined

the order of the curve. The interaction term of treatment and linear or quadratic hexose

equivalents (continuous) indicates whether slopes differ among treatments. Significance

of the treatment term in this model indicates whether the intercept values differ among

treatments. Accordingly, if the slopes do not differ, but the intercepts do, the analysis did

not detect differences in response per hexose equivalent, however the mean values of the

treatments could be said to differ. The interaction term of treatment and hexose

94

equivalents as a class variable describes the lack of fit of the curves to the data points. If

this term is significant, there is greater lack of fit.

Orthogonal contrasts were performed by recoding the treatments as new variable

names to reflect the comparisons to be made (e.g. for contrast 1 = sucrose+starch vs.

pectin, the new treatment codes were sucrose = 1, starch = 1, pectin = 2, and for contrast

2 = sucrose vs. starch, sucrose = 1 and starch = 2). The same models as above were used

with contrast 1 or 2 substituting for treatment. The interaction of the new term for

treatment and continuous hexose equivalents (linear or quadratic) indicates whether the

slopes of the lines in the contrast differ.

Comparisons of NFC mixtures

The question that needed to be addressed was whether the fermentation of mixtures

of NFCs would give the value that would be expected if the products of the NFC were

additive, or if there is some manner of interaction that makes the yield of products from

the mixture more or less than what would be expected of the individual contributions of

NFC. Nominal hexose equivalents were used to describe a new continuous variable that

converted the hexose equivalents to proportions of the maximum value of hexose

equivalent for each NFC source. For instance 0 mg nominal hexose equivalent = 0, 40

mg = 0.33, 80 mg = 0.67, and 120 mg = 1.0. The model statement used was:

Yijkl = µ + sui + stj + pek + su*stij + su*peik + st*pejk + εijkl

Where:


µ = overall mean

sui = sucrose (i = hexose equivalents from sucrose)

95

stj = starch (j = hexose equivalents from starch)

pek = pectin (k = hexose equivalents from pectin)

su*stij = interaction term for sucrose and starch

su*peik = interaction term for sucrose and pectin

st*pejk = interaction term for starch and pectin


The random statement included the following term:


The intercept and coefficients for each variable can be used to calculate estimates

for the dependent variable using 0, 0.33, 0.67, and 1.00 as the values inserted for su, st, or

pe as a descriptor of the proportional amount of the maximum amount of hexose

equivalent included for each NFC. In the tests of fixed effects, if the interaction terms are

significant, that means that the yields of a product from that combination differs from the

sum of what the individual NFC are predicted to contribute.


Residual Sugars (Sucrose, Glucose, Fructose)

Despite detection of low quantities of glucose, fructose and unhydrolyzed sucrose

in starch and pectin fermentations, it is the residual sugar contents in the fermentations

containing sucrose, which would primarily be of interest as descriptors of remaining

substrate (Table 4-2). At 0 h, varying amounts of sucrose were accounted for when

amount of residual sucrose equivalent was expressed as a percentage of the original

sucrose added to the fermentation (94.0, 96.6, 97.0, 86.0, 88.2, 93.1 and 85.9% for Su40,

Su80, Su120, Su40St80, Su80St40, Su40Pe80 and Su80Pe40, respectively). No residual

sucrose, glucose or fructose could be detected in any of the fermentations at 4 h (Table 4-

96

2), or in subsequent hours. This agrees with degradation data from other in vitro studies

with sucrose as substrate (Chapters 2 and 3). Also, simple sugars are documented in the

Cornell Net Carbohydrate and Protein System model as having a fast degradation rate,

and starch and pectin an intermediate rate (Sniffen et al., 1992). Residual starch and

pectin were not analyzed for in the current study and therefore no inference about the

degradation rates for these two substrates can be made. At 0 h, fermentation of

increasing amounts of sucrose gave a linear increase (P < 0.01) in residual glucose and

fructose content, and a quadratic increase (P < 0.01) in unhydrolyzed sucrose and sucrose

equivalent content.

Microbial Glycogen Yield

For iNDF, sucrose and pectin fermentations, α-glucan content in the fermentation

residue reflects estimates of polysaccharide storage in rumen microorganisms, and can be

referred to as GLY. However, for starch fermentations, α-glucan content also includes

any residual starch that has not been fermented. The method used to analyze for GLY

only resulted in a 77.6% recovery of corn starch (data not reported). Therefore estimates

of GLY will be inflated for starch fermentations and NFC combinations that contain

starch, but these estimates will not account for all the starch that has not been fermented.

Accordingly, comparisons among NFC fermentations for GLY content will be limited to

sucrose and pectin fermentations, and combinations thereof, for lack of a way to

distinguish between GLY and residual starch from fermentations containing starch.

Maximum GLY content differed among treatments (P < 0.01), and it occurred at

different sampling hours for sucrose and pectin fermentations (Table 4-3). Maximum

GLY content increased linearly with increasing amounts of NFC addition for sucrose and

pectin fermentations. Though GLY content per hexose equivalent with increasing NFC

97

inclusion did not differ for sucrose and pectin fermentations, there was a tendency (P =

0.07) for their intercepts to differ, with sucrose having a value more than twice that of

pectin (Table 4-4). There was no interaction between sucrose and pectin in fermentations

that contained mixtures of these NFCs, which suggests that the effect of sucrose and

pectin on GLY accumulation is additive.

In general, GLY content for all treatments decreased from the hour of maximum

yield to 24 h, at which point it could no longer be detected. However, temporal patterns

differed among treatments (P < 0.01 for treatment by hour interaction). Fermentation of

iNDF alone yielded very small amounts of GLY and there was no detectable temporal

pattern in GLY content (Table 4-4). Both pectin and sucrose fermentations showed linear

decreases in GLY content from the hour of maximum yield to 24 h for all amounts added.

As documented in the Cornell Net Carbohydrate and Protein System model, simple

sugars (such as sucrose) are considered to have a fast degradation rate, and pectin an

intermediate rate (Sniffen et al., 1992). Ruminal microorganisms may incorporate and

store carbohydrate as α-glucan under conditions of excess available carbohydrate

(Thomas, 1960; John, 1984; Lou et al., 1997). It is also likely that when the supply of

available dietary carbohydrate runs out, microorganisms utilize the storage carbohydrate

as a source of energy (McAllan and Smith, 1974). This could explain the greater

accumulation of GLY for sucrose compared to pectin fermentations, and the decreased

GLY content in both NFC fermentations from the point of maximum GLY yield through

24 h.

Fermentation pH

Fermentation pH never decreased below 6.60 for any of the treatments during the

24 h fermentations. However, temporal patterns (P < 0.01) for the 24 h fermentation

98

differed among the carbohydrate sources (Table 4-5). Fermentation of iNDF resulted in a

linear increase in pH over the 24 h fermentation, whereas temporal patterns in

fermentation pH for NFCs included linear, quadratic, cubic and quartic patterns.

Addition of individual NFCs (P <0.01) or combinations of NFCs (P < 0.01) to the

fermentation decreased both mean and minimum pH compared to iNDF.

Minimum pH differed among treatments (P < 0.01) and was recorded at different

sampling hours (Table 4-5). The lowest fermentation pH achieved was for Su120 at 4 h.

Fermentation of increased amounts of NFCs gave quadratic decreases in minimum

fermentation pH (Table 4-6). The decrease in minimum fermentation pH per hexose

equivalent with increasing NFC inclusion tended (P = 0.11) to be greater for sucrose and

starch fermentations compared to that of pectin fermentations, and the decrease in

minimum fermentation pH per hexose equivalent for sucrose and starch fermentations

was similar. The intercepts, however, did differ (P < 0.01) among the NFCs.

Fermentation of combinations of NFCs gave a lower minimum pH (P < 0.01) compared

to iNDF fermentation. The sampling hour for which minimum pH of the combinations of

NFCs was recorded varied depending on the component NFCs and their proportions

(Table 4-5). The effect on minimum fermentation pH from the fermentation of NFC

mixtures was not additive, which suggests that the complement and not just the mass of

NFCs in a ration may alter the ruminal pH.

A decrease in ruminal pH is often associated with the rapid fermentation of NFCs.

Several studies have contradicted this concept and reported no effect on pH as a result of

supplementation with starch or sucrose (Cameron et al., 1991; Aldrich et al., 1993;

Casper et al., 1999). In a two-part study by Khalili and Huhtanen (1991a, b), a decrease

99

in both pH and NDF digestion was reported in animals fed a grass silage and barley-

based diet with supplementation of sucrose at 1 kg DM/day. However, the negative

effect on pH and NDF digestion was alleviated when sodium bicarbonate was

supplemented in combination with sucrose. Therefore, NFC supplementation may not

have a negative effect on ruminal pH when the buffering capacity is adequate due to

supplemental buffers or including adequate effective fiber in the ration.

Organic Acids

Maximum VFA concentrations were recorded at 24 h for iNDF, all individual

NFCs and NFC combinations. However, temporal patterns for individual VFA

concentrations varied among treatments (Appendix D, Table D-1). Organic acid

concentrations at 24 h differed among treatments (P < 0.01) and were greater for

individual NFCs (P < 0.01) and NFC combinations (P < 0.01) compared to iNDF (Table

4-7). Fermentation of increased amounts of NFCs gave a quadratic increase in total VFA

concentrations at 24 h, with a similar millimolar VFA increase per additional mg of NFC

hexose equivalent added and similar intercept values for sucrose, starch and pectin

fermentations (Table4-8).

Organic acid concentrations are generally considered to relate to the rate and extent

of carbohydrate fermentation. Similar total VFA production among different NFC

sources have been reported both in vitro (Mansfield et al., 1994; Ariza et al., 2001) and in

vivo (Ben-Ghedalia et al., 1989; Khalili and Huhtanen, 1991a; Sannes et al., 2002;

Voelker and Allen, 2003c), with some exceptions (Bach et al., 1999; Broderick et al.,

2002b). Bach et al. (1999) reported an increase (P > 0.05) in total VFA concentration for

cracked corn compared with beet pulp and molasses in a continuous culture study. In

contrast, total VFA concentration increased (P = 0.01) or tended to increase (P = 0.07)

100

for lactating dairy cows fed a total mixed ration (TMR) containing dried citrus pulp and

high moisture ear corn in a 50:50 ratio compared to cows receiving a TMR containing

high moisture ear corn or cracked shelled corn, respectively (Broderick et al., 2002b).

The varying results in these studies may be due to the fact that the combinations of NFCs

that were compared differed, which may imply that the effect on VFA production from

supplementation with individual NFC sources of other nutrients may not be additive

when supplemented in combinations. In the current study there was no interaction effect

on total VFA concentration at 24 h between sucrose and starch or sucrose and pectin in

fermentations with these combinations of NFC (Table 4-8). However, there was an

interaction effect between starch and pectin in fermentations of starch-pectin

combinations. This would suggest that the effect of starch and pectin on total VFA

concentrations are additive when supplemented together, whereas the effect on total VFA

concentration of sucrose in combination with either starch or pectin is not additive.

Fermentation of increased amounts of NFCs gave a linear increase in acetate

concentrations at 24 h (Table 4-8). The increase in acetate concentration per hexose

equivalent with increasing NFC inclusion was greater for pectin fermentations than that

of starch and sucrose fermentation, whereas that for starch and sucrose fermentations did

not differ. Similar to the findings of this study, Strobel and Russell (1986) reported

greater acetate production for pectin compared to starch and sucrose, and no difference

between sucrose and starch. Several in vivo studies have also associated starch and

sucrose with relative decreases in ruminal acetate concentration (Sutton, 1979; Khalili

and Huhtanen, 1991a; Chamberlain et al., 1993; Moloney et al., 1994; Heldt et al., 1999)

and pectin with increased acetate in the rumen (Broderick et al., 2002b; Voelker and

101

Allen, 2003c). There was no NFC interaction effect on acetate concentrations when

combinations of NFCs were fermented suggesting that their effects are additive.

Fermentation of increased amounts of NFCs gave quadratic increases in propionate

concentration at 24 h (Table 4-8). The increase in propionate concentration per hexose

equivalent with increasing NFC inclusion tended to be greater for sucrose and starch

fermentations than for pectin fermentations, whereas sucrose and starch did not differ.

This is in agreement with similar (P > 0.05) propionate concentrations for starch and

sucrose fermentations with mixed ruminal bacteria, and less propionate yielded from

pectin fermentations (P < 0.05; Strobel and Russell, 1986). The effect of sucrose

compared to starch on ruminal propionate proportion varies among in vivo studies. In

some in vivo studies ruminal molar proportions of propionate did not differ between

sugars and starch (Moloney et al., 1994; Piwonka et al., 1994), whereas others reported

either an increase in the ruminal propionate proportion for sucrose compared to starch

(Chamberlain et al., 1993) or an increase with starch supplementation compared to

supplementation of sugars (sucrose, glucose and fructose) when a higher amount (0.122%

BW/d) of RDP was supplemented (Heldt et al., 1999). Other components of the diet such

as protein may have altered the yield of propionate from NFCs. The effect of individual

NFCs, when fermented in combinations, on propionate concentrations may be additive

since no interaction between any two individual NFCs were recorded.

Fermentation of increased amounts of NFCs gave quadratic increases in butyrate

concentrations at 24 h (Table 4-8). The fermentation of sucrose and starch gave a greater

increase in butyrate concentrations per hexose equivalent with increasing NFC inclusion

compared to pectin fermentations, and that for sucrose and starch fermentations did not

102

differ. This agrees with differences in butyrate production from fermentations of starch,

sucrose and pectin noted by Strobel and Russell (1986). Several in vivo studies also

reported increased butyrate production when sucrose was fermented instead of starch

(Khalili and Huhtanen, 1991a; Moloney et al., 1994), and feeds that typically contain a

substantial proportion of sugar and pectin (citrus and beet pulps) compared to those high

in starch (Broderick et al., 2002b; Voelker and Allen, 2003c). Citrus pulp can contain

between 12.5 and 40.2% sugars, and sugar beet pulp between 12.8 and 24.7% (Hall,

2002). The increase in the proportion of butyrate in these studies may be a result of the

fermentation of sugar rather than of the soluble fiber content. The effect on butyrate

concentrations was additive for sucrose fermented in combination with either starch or

pectin. However, the effect on butyrate concentrations for fermentation of starch in

combination with pectin was not additive.

Fermentation of increased amounts of NFCs gave quadratic increases in BCVFA

concentrations at 24 h (Table 4-8), and individual NFCs did not differ in their effect on

BCVFA concentrations per hexose equivalent with increasing NFC inclusion.

Fermentation of sucrose and starch combinations, and starch and pectin combinations had

an additive response in BCVFA concentrations, whereas combinations of sucrose and

pectin further decreased BCVFA concentrations relative to fermentation of individual

NFC. In continuous culture studies fermentation of corn increased the proportion of

BCVFAs compared to citrus pulp (Ariza et al., 2001) and sugar beet pulp (Mansfield et

al., 1994). Also in an in vivo study the corn control diet increased BCVFA

concentrations in lactating dairy cows compared to those receiving a diet with sucrose

substituted for corn at 3.2% of the diet DM (Sannes et al., 2002). It is not clear why

103

responses in BCVFA concentrations differed among these studies. However, it should be

noted that it is a challenge to compare the effect of NFCs on BCVFA concentrations

among different studies. Branched chain VFAs are products of protein degradation and is

thus dependent on the source and amount of degradable protein in the ration or

fermentation.

Maximum lactate concentrations were greater for individual NFCs (P < 0.01) and

NFC combinations (P < 0.01) compared to iNDF (Table 4-7). The hour at which

maximum lactate concentrations were recorded did not consistently coincide with the

hour of minimum fermentation pH. In general, the hour of minimum fermentation pH for

sucrose fermentation often corresponded with the hour of maximum lactate

concentration, whereas the hour of minimum fermentation pH for starch and pectin

fermentations appeared later in the fermentation than the hour of maximum lactate

concentration.

Maximum lactate concentrations increased quadratically for increasing amounts of

NFCs fermented (Table 4-8). Fermentations of sucrose, starch and pectin had similar

increases in maximum lactate concentrations per hexose equivalent with increasing NFC

inclusion, whereas that of sucrose fermentations was greater than for starch

fermentations. Greater lactate concentrations and proportions have been reported both in

vitro (Strobel and Russell, 1986) and in vivo (Heldt et al., 1999) for fermentation of

sugars compared to fermentation of starch. Compared to acetate, butyrate and propionate

(average pKa = 4.8), lactate (lactic acid, pKa = 3.1) is a 10-fold stronger acid (Dawson et

al., 1997). An increase in lactate concentration therefore has a greater potential to

104

decrease ruminal pH. In the current study, sucrose fermentation gave greater lactate

concentrations and a lower fermentation pH compared to starch and pectin fermentations.

Fermentations of sucrose in combination with starch gave reduced maximum

lactate yields compared to sucrose or starch fermented separately, whereas the

fermentation of sucrose in combination with pectin, and starch in combination with

pectin, had additive responses in maximum lactate concentrations. In general, the

fermentation of combinations of NFCs did not consistently give additive responses in

individual VFA concentrations, which suggests that the complement of NFCs in a

feedstuff or ration may alter the profile of organic acids produced.


Digestion of NDF differed among NFC sources in temporal pattern (P < 0.01) over

the 24 h fermentation (Table 4-9). The majority of the treatments showed a cubic pattern

in NDF digestion over the 24 h fermentation. Exceptions include a quartic pattern for

Su40, a quadratic pattern for St40Pe80, and a linear increase for Su40St80 in NDF

digestion.

At 24 h, NDF digestion was lower (P < 0.01) for fermentation of NFC

combinations compared to that of iNDF fermented alone. Fermentation of increased

amounts of NFCs linearly increased residual NDF at 24 h (Table 4-10). As the amount of

NFC fermented increased, NDF digestion decreased in fermentations supplemented with

starch and pectin, and increased in the sucrose-supplemented fermentation. The

proportion of residual NDF per hexose equivalent with increasing NFC inclusion was

greater for pectin fermentations compared to sucrose and starch fermentations, whereas

those for sucrose and starch fermentations did not differ. Fermentations of sucrose in

combination with starch or pectin had an additive response for NDF digestion, whereas

105

fermenting starch and pectin in combination tended to decrease NDF digestion (increase

proportion remaining) compared to NFC fermented individually.

In contrast to the current study, Cameron et al. (1991) reported decreases in ruminal

NDF and ADF digestion for lactating dairy cows receiving supplements of starch and

dextrose (glucose). Also in contrast, several studies reported increased NDF digestion for

animals fed diets with beet pulp (Van Vuuren et al., 1993; Voelker and Allen, 2003b) or

citrus pulp (Zinn and Owens, 1993; Miron et al., 2002) substituted for high starch (corn

or barley) diets.

A decrease in ruminal fiber digestion is often attributed to a decrease in ruminal pH

(Hoover, 1986), caused by rapid fermentation of NFCs and subsequent production of

VFAs by rumen microorganisms. However, in the current study sucrose fermentations

gave a lower minimum fermentation pH compared to starch and pectin fermentations, yet

sucrose addition increased NDF digestion compared to NDF digestion for pectin

fermentations. The results of the sucrose fermentation agree with the results of Heldt et

al. (1999) who reported an increase in NDF digestion relative to the control diet-fed

animals in steers supplemented at 0.3% BW of DM/d with sucrose, glucose or fructose

with low-quality tallgrass-prairie hay and an apparently adequate supply of ruminally

degradable protein (0.122% BW of DM/d). The results of the pectin fermentations may

be explained in part by a "carbohydrate effect" that describes impaired fiber digestion at a

pH of approximately 6.2 (Mould and Ørskov, 1984). The authors suggested that it is

related to a preference exhibited by ruminal microorganisms for carbohydrate sources

that are more readily available and competition for other nutrients (e.g. nitrogen) among

ruminal microbial populations. Some of the decreases noted for fiber digestion may be

106

the result of competition between NFC and fiber utilizing microorganisms for a nitrogen

supply. There might be a greater similarity in the microbial populations fermenting

pectin and NDF, which are both structural carbohydrates, than for microbial populations

fermenting sucrose and NDF. This may result in a greater competition for nutrients when

pectin and NDF are fermented together, and a potential negative effect on NDF digestion

in such fermentations.

Microbial Crude Protein Yield

The yield of MCP was increased with fermentation of individual NFCs compared

to iNDF fermentation (P < 0.01). Carbohydrates sources also had different (P < 0.01)

temporal patterns of MCP yield over the 24 h fermentation (Table 4-11). Combinations

of NFCs increased MCP yield compared to iNDF (P < 0.01). Fermentation of increased

amounts of NFCs gave linear increases in maximum MCP yields (Table 4-12). The

increase per hexose equivalent with increasing NFC inclusion was greater for pectin

fermentations compared to sucrose and starch fermentations, which did not differ from

each other. In an in vitro fermentation with mixed ruminal microorganisms microbial

protein yield was greater for starch fermentations compared to sucrose and pectin

fermentations, with no difference between the latter two (Hall and Herejk, 2001). In the

current study pectin was added to the fermentations in a suspension, which might have

made it more readily available for fermentation by ruminal microorganisms compare to

pectin in a powder form used in the study by Hall and Herejk (2001).

In general, fermentation of NFC combinations increased maximum MCP yield

compared to iNDF fermentation (P < 0.01). Fermentation of combinations of NFCs

showed no interaction effect on maximum MCP yields (Table 4-12). This would suggest

107

that the effect of individual NFCs on maximum MCP yield is additive when fermented in

combinations.

Conclusions

The various types and combinations of NFCs altered the yield of microbial

products and extent of fiber digestion. Fermentation of sucrose increased GLY content

compared to pectin fermentations, whereas pectin fermentations increased MCP

compared to sucrose and starch. This would suggest that sucrose supplementation has

potential to increase the α-glucan supply and pectin supplementation may increase the

protein supply to the small intestine of ruminants. However the form of the sucrose and

starch subtrates that were fermented differed from that for the pectin. Therefore more

work is required to confirm that pectin supplementation is more effective at increasing

microbial protein yield than sucrose or starch supplementation.

Total VFA production was similar among the NFC sources. However, there was an

effect of NFC source on organic acid profile. Pectin fermentations increased acetate

production, which may suggest an increase in precursors for fatty acid synthesis and

ultimately for milk fat synthesis in the mammary gland. Sucrose and starch

fermentations increased butyrate production, which is a precursor for energy supply

mainly to the heart and skeletal muscle in the form of β-hydroxybutyrate (a ketone body).

Sucrose and starch also increased propionate production, which is a precursor for the

glucogenic energy supply of the ruminant animal. Sucrose fermentations increased

lactate production and decreased pH more than pectin and starch fermentations. This

may imply that sucrose supplementation holds a greater risk for causing ruminal acidosis

and also decreased fiber digestion in ruminants. However, NDF digestion was increased

108

for sucrose fermentations, which supports the contention that factors other than pH may

play a role in the potential effect of NFCs on fiber digestion.

The different effects of fermenting the individual NFCs or mixtures of the NFCs on

microbial fermentation product yield imply that the complement of NFCs in a particular

feedstuff is important when predicting animal response. The treatment of all NFCs as a

uniform entity in ruminant nutrition is not warranted. Further in vitro and in vivo studies

are needed to increase our ability to explain and predict animal response when

supplementing diets with NFC sources.

Table 4-1. Layout of treatments and substrate amounts for a series of three 24 h in vitro fermentations (performed in duplicate) of a mixed batch culture.

Substrate Amount (mg)1 Fermentation A Fermentation B Fermentation C Treatment2 iNDF Su St iNDF St Pe iNDF Su Pe

Blank 0 0 0 0 0 0 0 0 0 iNDF 120 0 0 120 0 0 120 0 0 Su40 120 40 0 - - - 120 40 0 Su80 120 80 0 - - - 120 80 0 Su120 120 120 0 - - - 120 120 0

St40 120 0 35 120 35 0 - - - St80 120 0 71 120 71 0 - - - St120 120 0 106 120 106 0 - - -

Pe40 - - - 120 0 38 120 0 38 Pe80 - - - 120 0 76 120 0 76 Pe120 - - - 120 0 114 120 0 114

Su40St80 120 40 71 - - - - - - Su80St40 120 80 35 - - - - - - St40Pe80 - - - 120 35 76 - - - St80Pe40 - - - 120 71 38 - - - Su40Pe80 - - - - - - 120 40 76 Su80Pe40 - - - - - - 120 80 38 1 iNDF amount on air dry basis; sucrose, starch and pectin amounts expressed as mg nominal hexose equivalent 2 iNDF = isolated bermudagrass neutral detergent residue; Su = sucrose; St = starch; Pe = pectin; numbers reflect nominal amount hexose equivalent of different NFCs

109

Table 4-2. Residual glucose, fructose, unhydrolyzed sucrose and sucrose equivalent (mg) at 0 and 4 h for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means.

Item1 Amount2 Glucose Fructose Sucrose Sucrose. Equivalent4

0 h 4 h 0 h 4 h 0 h 4 h 0 h 4 h iNDF 0.05 -0.01 -0.01 -0.01 0.03 0.03 0.07 0.01 (SE4 = 0.32) (SE = 0.22) (SE = 0.59) (SE = 0.56) Individual NFCs Sucrose 40 7.67 0.19 5.09 -0.01 23.8 -0.09 35.9 0.12

80 8.17 0.18 6.38 -0.02 59.9 0.00 73.7 0.20 120 9.03 0.33 6.81 0.05 95.7 0.01 110.8 0.41

Starch 40 -0.82 -0.01 -0.37 0.00 1.11 -0.06 -0.04 -0.05 80 -0.91 0.05 -0.38 0.04 1.05 -0.01 -0.20 0.09 120 -0.92 -0.01 -0.39 0.04 0.96 -0.07 -0.30 -0.04

Pectin 40 0.73 0.23 0.34 0.11 -0.97 -0.03 0.05 0.26 80 0.89 0.24 0.57 0.03 -0.70 -0.10 0.70 0.12 120 1.54 0.15 0.87 0.05 -0.73 -0.05 1.57 0.11 (SE = 0.35) (SE = 0.25) (SE = 0.69) (SE = 0.68)

NFC combinations Su40St80 10.5 0.01 8.13 0.00 22.8 -0.07 40.4 0.01 Su80St40 10.6 -0.12 8.19 0.00 49.6 -0.05 67.4 -0.09 St40Pe80 0.19 0.16 0.28 0.06 0.15 -0.02 0.57 0.12 St80Pe40 -0.12 0.11 0.02 0.08 0.10 -0.02 -0.02 0.09 Su40Pe80 7.73 0.15 5.27 0.17 23.1 -0.21 35.5 0.09 Su80Pe40 8.79 -0.02 6.37 0.08 50.7 0.09 65.1 0.14

(SE = 0.45) (SE = 0.31) (SE = 0.91) (SE = 0.95) 1 iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 2 Amount of NFC substrate in mg nominal hexose equivalent 3 Sucrose equivalent = residual (glucose + fructose) x 0.95 + unhydrolyzed sucrose 4 SE = standard error of least squares means

110

Table 4-3. Maximum microbial glycogen (GLY) yield (mg), hour of maximum yield and temporal patterns for 24 h in vitro fermentations of iNDF, NFC sources (sucrose and pectin), and combinations of NFCs. Values are least squares means.

Item1 Amount2 Max. GLY, mg (h) Temporal pattern3

iNDF 0.51 (12) No detectable pattern (SE4 = 0.23) Individual NFCs

Sucrose 40 3.08 (4) L (P < 0.01) 80 3.59 (4) L (P < 0.01) 120 4.27 (4) L (P < 0.01) Pectin 40 1.34 (4) L (P < 0.01) 80 1.72 (8) L (P < 0.01) 120 2.43 (8) L (P < 0.01) (SE = 0.26)

NFC combinations Su40Pe80 3.45 (4) L (P < 0.01) Su80Pe40 3.63 (4) L (P < 0.01)

(SE = 0.33) 1 iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; Pe = pectin 2 Amount of NFC substrate in mg nominal hexose equivalent 3 Pattern from hour of maximum GLY yield to 24 h: L = linear 4 SE = standard error of least squares means

111

Table 4-4. Main effects and regression coefficients for maximum microbial glycogen (GLY) yield (mg) for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations.

Main effects1 P-values NFC2 0.07 He3 <0.01 NFC x He 0.47 NFC x HeCL4 0.77

Regression Coefficients Coefficients (P-values) Individual NFCs Sucrose Pectin Intercept 2.59 (0.01) 0.99 (0.25) He 0.01 (<0.01) 0.10 (<0.01)

NFC combination analysis5 Coefficients (P-values) Intercept 1.14 (0.01) Sucrose 3.41 (<0.01) Pectin 1.14 (<0.01) Sucrose x Pectin 0.69 (0.61)

1 Main effects (P-values) and regression coefficients (estimates and P-values) for individual NFCs were obtained with a proc mixed heterogeneity of regression analysis 2 NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 3 He = hexose equivalent (continuous variable) 4 HeCL = hexose equivalent (class variable); NFC x HeCL = lack of fit term: if significant then regression line fits data poorly 5 Regression coefficients (estimates and P-values) for NFC combinations were obtained with a standard proc mixed regression analysis

112

Table 4-5. Fermentation pH (mean, minimum, hour of minimum and temporal pattern) for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means.

pH Item1 Amount2 Mean Minimum (h) Temporal pattern3 iNDF 7.22 7.13 (4) L (P < 0.01) (SE4 = 0.01) (SE = 0.02) Individual NFCs

Sucrose 40 7.13 6.99 (4) Qt (P = 0.04) 80 7.05 6.81 (4) Qt (P < 0.01) 120 6.93 6.68 (4) Qt (P < 0.01)

Starch 40 7.16 7.12 (12) L (P = 0.04) 80 7.09 7.02 (12) Qt (P = 0.03) 120 7.03 6.94 (16) Qd (P = 0.01)

Pectin 40 7.15 7.01 (4) C (P < 0.01) 80 7.07 6.90 (8) C (P < 0.01) 120 6.99 6.76 (8) Qt (P = 0.02) (SE = 0.01) (SE = 0.03)

NFC combinations Su40St80 7.02 6.95 (8) Qd (P = 0.02) Su80St40 7.00 6.85 (4) C (P < 0.01) St40Pe80 7.01 6.86 (8) C (P = 0.05) St80Pe40 7.01 6.92 (12) Qt (P = 0.04) Su40Pe80 7.00 6.81 (8) Qt (P < 0.01) Su80Pe40 7.03 6.82 (4) Qt (P < 0.01)

(SE = 0.02) (SE = 0.03) 1 iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 2 Amount of NFC substrate in mg nominal hexose equivalent 3 Pattern over 24 h fermentation: L = linear; Qd = quadratic; C = cubic; Qt = quartic 4 SE = standard error of least squares means

113

Table 4-6. Main effects and regression coefficients for minimum fermentation pH for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations.

Main effects1 P-values NFC2 <0.01 He3 <0.01 He x He <0.01 NFC x He 0.10 NFC x He x He 0.14 NFC x HeCL4 0.99

Contrasts Intercept Slope Su and St vs. Pe 0.28 0.11 Su vs. St 0.06 0.43

Regression Coefficients5 Coefficients (P-values) Individual NFCs Sucrose Starch Pectin

Intercept 4.80E-08 (<0.01) 6.70E-08 (<0.01) 9.60E-08 (<0.01)He 1.49E-06 (<0.01) 4.65E-07 (<0.01) -3.17E-07 (<0.01)He x He -2.98E-10 (<0.01) 1.33E-09 (<0.01) 8.46E-09 (<0.01)

NFC combination analysis6 Coefficients (P-values) Intercept 6.80E-06 (<0.01) Sucrose 1.47E-07 (<0.01) Starch 6.00E-08 (<0.01) Pectin 9.50E-08 (<0.01) Sucrose x Starch -1.60E-04 (<0.01) Sucrose x Pectin -1.40E-04 (<0.01) Starch x Pectin -7.00E-05 (<0.01)

1 Main effects (P-values) and regression coefficients (estimates and P-values) for individual NFCs were obtained with a proc mixed heterogeneity of regression analysis 2 NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 3 He = hexose equivalent (continuous variable) 4 HeCL = hexose equivalent (class variable); NFC x HeCL = lack of fit term: if significant then regression line fits data poorly 5 Coefficients are [H+] 6 Regression coefficients (estimates and P-values) for NFC combinations were obtained with a standard proc mixed regression analysis

114

Table 4-7. Volatile fatty acid concentrations at 24 h and maximum lactate concentrations (hour of maximum indicated) for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means.

Item1 Amount2 Total VFA3 C2 C3 C4 Val Max. Lac

(h) BCVFA

mM Blank --- --- --- --- --- --- 3.68 iNDF 15.8 9.49 4.26 1.56 0.51 0.00 (4) 4.14 SE4 1.19 0.93 0.36 0.26 0.12 0.34 0.14 Individual NFCs Sucrose 40 28.4 15.0 6.98 4.91 1.55 8.02 (4) 3.39 80 *38.5 17.3 9.94 7.83 *3.44 19.5 (4) *2.90 120 53.5 23.8 18.9 7.37 3.59 31.0 (4) 3.19 Starch 40 **28.7 *16.7 7.82 *3.05 *0.78 0.62 (8) *3.92 80 38.9 21.6 11.8 4.47 1.06 0.72 (8) 3.93 120 47.0 24.9 15.0 5.92 1.19 0.77 (8) 3.80 Pectin 40 28.0 19.8 6.00 1.75 0.33 0.11 (0) 3.83 80 39.8 29.3 8.24 1.77 0.38 0.20 (0) 3.00 120 54.1 40.0 11.3 2.30 0.47 0.19 (0) *3.15

SE

1.35 *1.39

**1.46

1.03 *1.06 0.43 0.30

*0.31 0.14 0.37 0.16 *0.17

NFC combinations Su40St80 49.6 25.8 15.9 6.49 1.58 7.88 (4) 2.92 Su80St40 49.2 22.6 14.5 8.98 3.30 18.4 (4) 3.27 St40Pe80 47.8 32.9 11.5 2.93 0.40 0.14 (4) 2.62 St80Pe40 46.3 29.0 13.2 3.13 0.84 0.33 (8) 3.26 Su40Pe80 50.6 32.3 12.6 4.00 1.60 9.63 (4) 2.78 Su80Pe40 ***61.3 **33.4 15.3 **8.17 **4.34 21.4 (4) **2.98

SE 1.73 ***1.88

1.29 **1.40

0.58 *0.62

0.39 **0.42

0.17 **0.19 0.46 0.21

**0.23 1 Blank = no substrate; iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 2 Amount of NFC substrate in mg nominal hexose equivalent 3 VFA = volatile fatty acid; C2 = acetate; C3 = propionate; C4 = butyrate; Val = valerate; Lac = lactate; BCVFA = branched chain VFA 4 SE = standard error of least squares means *, **, *** Symbols indicate which SE corresponds with the least squares means in any given column

115

Table 4-8. Main effects and regression coefficients for maximum organic acid concentrations for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations.

Total VFA1 C2 C3 C4 Val Max. Lac (h) BCVFA

Main effects2 P-values

NFC3 0.49 <0.01 0.01 <0.01 <0.01 <0.01 0.09 He4 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.05 He x He 0.03 ----- <0.01 <0.01 ----- <0.01 ----- NFC x He <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.17 NFC x He x He 0.25 ----- 0.07 <0.01 ----- 0.06 ----- NFC x HeCL5 0.65 0.08 0.99 0.30 <0.01 <0.01 0.21

Contrasts Intercept; Slope Intercept; Slope Intercept; Slope Intercept; Slope Intercept; Slope Intercept; Slope Intercept; Slope Su and St vs. Pe 0.32 ; 0.83 <0.01 ; <0.01 0.01 ; 0.11 <0.01 ; <0.01 <0.01 ; <0.01 <0.01 ; 0.83 0.28 ; 0.17 Su vs. St 0.96 ; 0.14 0.25 ; 0.68 0.62 ; 0.27 0.11 ; 0.83 0.05 ; 0.96 0.05 ; 0.06 0.18 ; 0.16

Regression coefficients Coefficients (P-values)

Individual NFCs Sucrose: Intercept 25.8 (<0.01) 9.89 (<0.01) 10.1 (<0.01) -1.45 (0.20) 0.80 (0.05) -3.37 (0.05) 3.46 (<0.01)

He -0.05 (0.68) 0.11 (<0.01) -0.15 (<0.01) 0.20 (<0.01) 0.03 (<0.01) 0.28 (<0.01) 0.00 (0.49) He x He 0.00 (<0.01) ----- 0.00 (<0.01) 0.00 (<0.01) ----- 0.00 (0.84) -----

Starch: Intercept 15.2 (0.03) 12.9 (<0.01) 3.10 (0.15) 1.62 (0.17) 0.61 (0.12) 0.06 (0.96) 3.94 (<0.01) He 0.38 (<0.01) 0.12 (<0.01) 0.14 (0.01) 0.04 (0.19) 0.01 (0.05) 0.00 (0.97) 0.00 (0.74) He x He 0.00 (0.33) ----- 0.00 (0.45) 0.00 (0.98) ----- 0.00 (0.97) -----

Pectin: Intercept 17.6 (0.01) 9.37 (<0.01) 4.52 (0.06) 2.21 (0.08) 0.22 (0.50) -0.12 (0.93) 3.97 (<0.01) He 0.22 (0.07) 0.27 (<0.01) 0.03 (0.6) -0.02 (0.49) 0.00 (0.49) 0.00 (0.91) 0.00 (0.02) He x He 0.00 (0.28) ----- 0.00 (0.38) 0.00 (0.34) ----- 0.00 (0.97) -----

116

Table 4-8. Continued

Total VFA1 C2 C3 C4 Val Max. Lac (h) BCVFA

NFC combination analysis6 Coefficients (P-values)

Intercept 15.5 (<0.01) 10.1 (<0.01) 3.48 (<0.01) 1.97 (<0.01) 0.53 (0.06) -0.52 (0.36) 3.98 (<0.01) Sucrose 33.6 (<0.01) 13.3 (<0.01) 13.4 (<0.01) 6.72 (<0.01) 3.53 (<0.01) 30.9 (<0.01) -0.95 (<0.01) Starch 31.6 (<0.01) 16.0 (<0.01) 11.9 (<0.01) 3.79 (<0.01) 0.67 (<0.01) 1.45 (<0.01) -0.19 (0.41) Pectin 36.6 (<0.01) 29.0 (<0.01) 7.51 (<0.01) 0.04 (0.91) -0.23 (0.23) 0.06 (0.90) -1.10 (<0.01) Sucrose x Starch -2.09 (0.80) -1.00 (0.86) -3.71 (0.22) 2.45 (0.19) -0.75 (0.42) -10.9 (<0.01) -0.98 (0.38) Sucrose x Pectin 7.01 (0.41) 4.82 (0.43) -0.12 (0.97) 1.90 (0.33) 3.04 (<0.01) 3.11 (0.17) -0.21 (0.86) Starch x Pectin -17.6 (0.03) -8.96 (0.12) -4.61 (0.13) -3.44 (0.06) -0.67 (0.47) -0.84 (0.71) -2.36 (0.04)

1 VFA = volatile fatty acid; C2 = acetate; C3 = propionate; C4 = butyrate; Val = valerate; Lac = lactate; BCVFA = branched chain VFA 2 Main effects (P-values) and regression coefficients (estimates and P-values) for individual NFCs were obtained with a proc mixed heterogeneity of regression analysis 3 NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 4 He = hexose equivalent (continuous variable) 5 HeCL = hexose equivalent (class variable); NFC x HeCL = lack of fit term: if significant then regression line fits data poorly 6 Regression coefficients (estimates and P-values) for NFC combinations were obtained with a standard proc mixed regression analysis

117

Table 4-9. Residual NDFOM at 24 h and temporal patterns for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means.

Item1 Amount2 Residual NDFOM3 (%) Temporal pattern4

iNDF 61.2 C (P < 0.01) (SE5 = 0.98) Individual NFCs

Sucrose 40 60.8 Qt (P = 0.04) 80 60.7 C (P < 0.01) 120 60.1 C (P < 0.01) Starch 40 60.8 C (P < 0.01) 80 60.9 C (P < 0.01) 120 61.1 C (P < 0.01) Pectin 40 63.8 C (P < 0.01) 80 65.8 C (P < 0.01) 120 66.3 C (P < 0.01) (SE = 1.02)

NFC combinations Su40St80 60.6 L (P < 0.01) Su80St40 61.6 C (P = 0.02) St40Pe80 65.7 Qd (P = 0.01) St80Pe40 64.0 C (P = 0.05) Su40Pe80 65.7 C (P = 0.04) Su80Pe40 63.1 C (P < 0.01)

(SE = 1.12) 1 iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 2 Amount of NFC substrate in mg nominal hexose equivalent 3 NDFOM = neutral detergent fiber organic matter 4 Pattern over 24 h fermentation: L = linear; Qd = quadratic; C = cubic; Qt = quartic 5 SE = standard error of least squares means

118

Table 4-10. Main effects and regression coefficients for residual NDFOM at 24 h for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations.

Main effects1 P-values NFC2 <0.01 He3 0.07 NFC x He <0.01 NFC x HeCL4 0.73

Contrasts Intercept Slope Su and St vs. Pe <0.01 <0.01 Su vs. St 0.98 0.28

Regression coefficients Coefficients (P-values) Individual NFCs Sucrose Starch Pectin

Intercept 61.3 (<0.01) 60.6 (<0.01) 62.9 (<0.01) He 0.00 (0.23) 0.00 (0.61) 0.03 (<0.01)

NFC combination analysis5 Coefficients (P-values) Intercept 61.3 (<0.01) Sucrose -1.14 (0.04) Starch -0.48 (0.38) Pectin 5.58 (<0.01) Sucrose x Starch 2.77 (0.29) Sucrose x Pectin 3.50 (0.18) Starch x Pectin 4.17 (0.11)


119

Table 4-11. Microbial crude protein (MCP) yield (mean, maximum, hour of maximum and temporal pattern) for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means.

Microbial protein yield Item1 Amount2 Mean, mg Max., mg (h) Temporal pattern3

iNDF 2.68 5.12 (16) C (P < 0.01) (SE4 = 0.81) (SE = 1.16) Individual NFCs Sucrose 40 7.15 10.8 (16) Qd (P < 0.01) 80 10.6 15.8 (12) Qd (P < 0.01) 120 12.9 17.9 (12) C (P = 0.04) Starch 40 5.97 12.0 (12) Qt (P < 0.01) 80 9.39 16.7 (16) Qt (P = 0.02) 120 12.0 21.0 (16) Qt (P = 0.02) Pectin 40 7.60 12.0 (12) Qt (P < 0.01) 80 11.3 18.7 (16) Qt (P < 0.01) 120 15.9 25.0 (8) Qt (P < 0.01) (SE = 0.96) (SE = 1.42) NFC combinations

Su40St80 11.0 19.6 (12) Qd (P < 0.01) Su80St40 14.2 21.9 (16) Qd (P < 0.01) St40Pe80 15.4 23.8 (16) Qt (P = 0.01) St80Pe40 15.5 23.7 (16) Qt (P = 0.03) Su40Pe80 14.3 25.3 (16) Qt (P = 0.03) Su80Pe40 14.2 20.3 (16) Qd (P < 0.01) (SE = 1.29) (SE = 2.00) 1 iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 2 Amount of substrate in mg nominal hexose equivalent 3 Pattern over 24 h fermentation: Qd = quadratic; C = cubic; Qt = quartic 4 SE = standard error of least squares means

120

Table 4-12. Main effects and regression coefficients for maximum microbial crude protein (MCP) yield for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations.

Main effects1 P-values NFC2 0.07 He3 <0.01 NFC x He <0.01 NFC x HeCL4 0.46

Contrasts Intercept Slope Su and St vs. Pe 0.12 <0.01 Su vs. St 0.47 0.07

Regression coefficients Coefficients (P-values) Individual NFCs Sucrose Starch Pectin

Intercept 7.33 (<0.01) 9.19 (<0.01) 5.81 (0.02) He 0.09 (<0.01) 0.11 (<0.01) 0.17 (<0.01)

NFC combinations analysis5 Coefficients (P-values) Intercept 6.00 (<0.01) Sucrose 12.4 (<0.01) Starch 15.6 (<0.01) Pectin 19.3 (<0.01) Sucrose x Starch 1.67 (0.67) Sucrose x Pectin 2.87 (0.47) Starch x Pectin 5.10 (0.20)


121

CHAPTER 5 CONCLUSIONS

Results from a series of in vitro fermentations indicate that pH, nitrogen source,

addition of non-neutral detergent fiber carbohydrates (NFCs) and NFC complement may

all be important factors in determining product yield and effect on neutral detergent fiber

(NDF) digestion by ruminal microorganisms. Since ruminal microorganisms and their

fermentation products form a major part of the nutrient supply to the ruminant animal

these factors need to be considered when formulating diets or predicting animal response.

When feeding diets containing sucrose, the current ruminal pH might affect the

relative change in ruminal environment (e.g. pH, organic acid and ammonia nitrogen

concentrations), the degradation of substrates and the amounts of nutrients supplied to the

animal. Sucrose appears to be readily fermented regardless of pH. However, the

utilization of the monosaccharide constituents (glucose and fructose) differs depending

on pH. At a more neutral pH sucrose supplementation may increase fiber digestion,

potentially increasing nutrient supply to the animal, but at acidic pH sucrose

supplementation may decrease fiber digestion. Microbial protein synthesis is higher at a

more neutral pH, also adding to increased nutrient supply. Under acidic conditions,

however, ruminal microorganisms might store more glycogen as a proportion of cell

mass as compared to neutral fermentation conditions, which could decrease microbial cell

viability due to a large carbohydrate to protein ratio. The potential effects of a more

acidic pH and sucrose on ruminal fermentation would be important to take into

consideration when supplementing sucrose to a ruminant diet that already contains

122

substantial amounts of readily fermentable carbohydrate. Therefore it is recommended

that when supplementing sucrose to diets for ruminant animals, diets should include

enough effective fiber or another form of buffering agent, such as sodium bicarbonate, to

ensure that the ruminal pH remains near neutral. Also, animal studies further evaluating

the effects of ruminal pH on substrate utilization and nutrient supply are warranted.

Carbohydrate substrate and nitrogen source may also affect in vitro yield of

fermentation products and NDF fermentation. Addition of true protein increased

microbial crude protein (MCP) yield and efficiency of yield (MCPeff) from ruminal

microorganisms when sucrose was present and had a positive effect on MCP yield from

NDF alone. True protein addition increased NDF digestion when sucrose was present,

and increased total yield of organic acids. Maximum accumulation of microbial

glycogen (GLY) was not affected by nitrogen source when sucrose and NDF were

fermented together. These results imply that it would be advantageous to supply rumen

degradable nitrogen in the form of amino acids or peptides in ruminant diets when a

readily fermentable carbohydrate source such as sucrose is supplemented. Also, the

sources of ruminal degradable nitrogen and the inclusion of sucrose may be important to

consider in the prediction of fiber digestion and metabolizable nutrient supply in

ruminant diets. Animal studies investigating the interaction of ruminally degradable

nitrogen source and NFC source on ruminal measures and animal performance are

warranted.

Various types and combinations of NFCs altered the yield of microbial products

and extent of fiber digestion. Supplementation of sucrose to ruminant diets may increase

the supply of α-glucan to the small intestine, which when hydrolyzed by enzymes in the

123

small intestine and absorbed, becomes part of the metabolizable glucose supply to the

ruminant animal. Supplementation of pectin on the other hand may increase the supply

of metabolizable nitrogen to the small intestine of the ruminant animal in the form of

increased microbial protein synthesis. Among the three NFC sources evaluated sucrose

had the greatest negative effect on pH and therefore may have more potential to cause

ruminal acidosis. This may imply that sucrose also has the greatest potential to decrease

fiber digestion in the rumen. However, sucrose fermentations increased NDF digestion in

the first in vitro study when the fermentation pH was near neutral and in the third in vitro

study. This increase in NDF digestion in the third in vitro study may also be attributed to

the fact that fermentation pH in sucrose fermentations never decreased below 6, a pH

considered critical for maintaining fiber-utilizing microbial populations.

Differences among NFC components regarding microbial fermentation may imply

that the complement of NFCs in a particular feedstuff is important when predicting

animal response. The treatment of all NFCs as a uniform entity in ruminant nutrition is

not warranted. Further in vitro and in vivo studies are needed to increase our ability to

explain and predict animal response when supplementing diets with NFC sources.

124

APPENDIX A ADDITIONAL FIGURES FOR CHAPTER 2

-5

0

5

10

15

20

25

30

0 4 8 12 16 20 24

Fermentation hour

Glu

cose

(mg)

Figure A-1. Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with initial medium pH, before addition of reducing solution or inoculum, of 6.8 (■) or 5.6 (▲). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

125

-5

0

5

10

15

20

25

30

35

40

45

0 4 8 12 16 20 24

Fermentation hour

Fruc

tose

(mg)

Figure A-2. Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with initial medium pH, before addition of reducing solution or inoculum, of 6.8 (■) or 5.6 (▲). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

-20

0

20

40

60

80

100

0 4 8 12 16 20 24

Fermentation hour

Sucr

ose

(mg)

Figure A-3. Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with initial medium pH, before addition of reducing solution or inoculum, of 6.8 (■) or 5.6 (▲). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

126

APPENDIX B ADDITIONAL FIGURES FOR CHAPTER 3

-5

0

5

10

15

20

0 4 8 12 16

Fermentation hour

Glu

cose

(mg)

Figure B-1. Residual glucose content (LSmeans ± standard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

127

-5

0

5

10

15

20

0 4 8 12 16

Fermentation hour

Fruc

tose

(mg)

Figure B-2. Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

-20

0

20

40

60

80

100

120

0 4 8 12 16

Fermentation hour

Sucr

ose

(mg)

Figure B-3. Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein (■), true protein only (▲) or non-protein nitrogen only (●). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

128

APPENDIX C FIGURES FOR CHAPTER 4

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0 4 8 12 16 20 24

Fermentation hour

Glu

cose

(mg)

Figure C-1. Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

129

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0 4 8 12 16 20 24

Fermentation hour

Glu

cose

(mg)

Figure C-2. Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0 4 8 12 16 20 24

Fermentation hour

Glu

cose

(mg)

Figure C-3. Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

130

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0 4 8 12 16 20 24

Fermentation hour

Glu

cose

(mg)

Figure C-4. Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0 4 8 12 16 20 24

Fermentation hour

Glu

cose

(mg)

Figure C-5. Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

131

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0 4 8 12 16 20 24

Fermentation hour

Glu

cose

(mg)

Figure C-6. Residual glucose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

0 4 8 12 16 20 24

Fermentation hour

Fruc

tose

(mg)

Figure C-7. Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

132

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

0 4 8 12 16 20 24

Fermentation hour

Fruc

tose

(mg)

Figure C-8. Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

0 4 8 12 16 20 24

Fermentation hour

Fruc

tose

(mg)

Figure C-9. Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

133

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

0 4 8 12 16 20 24

Fermentation hour

Fruc

tose

(mg)

Figure C-10. Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

0 4 8 12 16 20 24

Fermentation hour

Fruc

tose

(mg)

Figure C-11. Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

134

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

0 4 8 12 16 20 24

Fermentation hour

Fruc

tose

(mg)

Figure C-12. Residual fructose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

-20

0

20

40

60

80

100

0 4 8 12 16 20 24

Fermentation hour

Sucr

ose

(mg)

Figure C-13. Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

135

-20

0

20

40

60

80

100

0 4 8 12 16 20 24

Fermentation hour

Sucr

ose

(mg)

Figure C-14. Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-20

0

20

40

60

80

100

0 4 8 12 16 20 24

Fermentation hour

Sucr

ose

(mg)

Figure C-15. Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

136

-20

0

20

40

60

80

100

0 4 8 12 16 20 24

Fermentation hour

Sucr

ose

(mg)

Figure C-16. Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

-20

0

20

40

60

80

100

0 4 8 12 16 20 24

Fermentation hour

Sucr

ose

(mg)

Figure C-17. Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

137

-20

0

20

40

60

80

100

0 4 8 12 16 20 24

Fermentation hour

Sucr

ose

(mg)

Figure C-18. Residual sucrose content (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

0 4 8 12 16 20 24

Fermentation hour

Mic

robi

al g

lyco

gen

(mg)

Figure C-19. Microbial glycogen yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

138

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

0 4 8 12 16 20 24

Fermentation hour

Mic

robi

al g

lyco

gen

(mg)

Figure C-20. Microbial glycogen yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

0 4 8 12 16 20 24

Fermentation hour

Mic

robi

al g

lyco

gen

(mg)

Figure C-21. Microbial glycogen yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

139

6.6

6.7

6.8

6.9

7.0

7.1

7.2

7.3

7.4


Ferm

enta

tion

pH

Figure C-22. Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

6.6

6.7

6.8

6.9

7.0

7.1

7.2

7.3

7.4


Ferm

enta

tion

pH

Figure C-23. Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

140

6.6

6.7

6.8

6.9

7.0

7.1

7.2

7.3

7.4


Ferm

enta

tion

pH

Figure C-24. Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

6.6

6.7

6.8

6.9

7.0

7.1

7.2

7.3

7.4


Ferm

enta

tion

pH

Figure C-25. Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

141

6.6

6.7

6.8

6.9

7.0

7.1

7.2

7.3

7.4


Ferm

enta

tion

pH

Figure C-26. Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

6.6

6.7

6.8

6.9

7.0

7.1

7.2

7.3

7.4


Ferm

enta

tion

pH

Figure C-27. Fermentation pH (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

142

0

5

10

15

20

25

30

35

40

45


Ace

tate

(mM

)

Figure C-28. Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

0

5

10

15

20

25

30

35

40

45


Ace

tate

(mM

)

Figure C-29. Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

143

0

5

10

15

20

25

30

35

40

45


Ace

tate

(mM

)

Figure C-30. Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

0

5

10

15

20

25

30

35

40

45


Ace

tate

(mM

)

Figure C-31. Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

144

0

5

10

15

20

25

30

35

40

45


Ace

tate

(mM

)

Figure C-32. Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

0

5

10

15

20

25

30

35

40

45


Ace

tate

(mM

)

Figure C-33. Acetate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

145

-5

0

5

10

15

20

25

0 4 8 12 16 20 24

Fermentation hour

Prop

iona

te (m

M)

Figure C-34. Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-5

0

5

10

15

20

25

0 4 8 12 16 20 24

Fermentation hour

Prop

iona

te (m

M)

Figure C-35. Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

146

-5

0

5

10

15

20

25

0 4 8 12 16 20 24

Fermentation hour

Prop

iona

te (m

M)

Figure C-36. Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-5

0

5

10

15

20

25

0 4 8 12 16 20 24

Fermentation hour

Prop

iona

te (m

M)

Figure C-37. Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

147

-5

0

5

10

15

20

25

0 4 8 12 16 20 24

Fermentation hour

Prop

iona

te (m

M)

Figure C-38. Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

-5

0

5

10

15

20

25

0 4 8 12 16 20 24

Fermentation hour

Prop

iona

te (m

M)

Figure C-39. Propionate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

148

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

0 4 8 12 16 20 24

Fermentation hour

But

yrat

e (m

M)

Figure C-40. Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

0 4 8 12 16 20 24

Fermentation hour

But

yrat

e (m

M)

Figure C-41. Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

149

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

0 4 8 12 16 20 24

Fermentation hour

But

yrat

e (m

M)

Figure C-42. Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

0 4 8 12 16 20 24

Fermentation hour

But

yrat

e (m

M)

Figure C-43. Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

150

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

0 4 8 12 16 20 24

Fermentation hour

But

yrat

e (m

M)

Figure C-44. Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

-2.0

0.0

2.0

4.0

6.0

8.0

10.0

0 4 8 12 16 20 24

Fermentation hour

But

yrat

e (m

M)

Figure C-45. Butyrate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

151

-10

0

10

20

30

40

50

60

70

0 4 8 12 16 20 24

Fermentation hour

Tota

l VFA

(mM

)

Figure C-46. Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-10

0

10

20

30

40

50

60

70

0 4 8 12 16 20 24

Fermentation hour

Tota

l VFA

(mM

)

Figure C-47. Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

152

-10

0

10

20

30

40

50

60

70

0 4 8 12 16 20 24

Fermentation hour

Tota

l VFA

(mM

)

Figure C-48. Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-10

0

10

20

30

40

50

60

70

0 4 8 12 16 20 24

Fermentation hour

Tota

l VFA

(mM

)

Figure C-49. Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

153

-10

0

10

20

30

40

50

60

70

0 4 8 12 16 20 24

Fermentation hour

Tota

l VFA

(mM

)

Figure C-50. Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

-10

0

10

20

30

40

50

60

70

0 4 8 12 16 20 24

Fermentation hour

Tota

l VFA

(mM

)

Figure C-51. Total volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

154

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 4 8 12 16 20 24

Fermentation hour

BC

VFA

(mM

)

Figure C-52. Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 4 8 12 16 20 24

Fermentation hour

BC

VFA

(mM

)

Figure C-53. Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

155

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 4 8 12 16 20 24

Fermentation hour

BC

VFA

(mM

)

Figure C-54. Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 4 8 12 16 20 24

Fermentation hour

BC

VFA

(mM

)

Figure C-55. Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

156

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 4 8 12 16 20 24

Fermentation hour

BC

VFA

(mM

)

Figure C-56. Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 4 8 12 16 20 24

Fermentation hour

BC

VFA

(mM

)

Figure C-57. Branched chain volatile fatty acid concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated

157

bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

-5

0

5

10

15

20

25

30

35

0 4 8 12 16 20 24

Fermentation hour

Lact

ate

(mM

)

Figure C-58. Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-5

0

5

10

15

20

25

30

35

0 4 8 12 16 20 24

Fermentation hour

Lact

ate

(mM

)

Figure C-59. Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

158

-5

0

5

10

15

20

25

30

35

0 4 8 12 16 20 24

Fermentation hour

Lact

ate

(mM

)

Figure C-60. Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-5

0

5

10

15

20

25

30

35

0 4 8 12 16 20 24

Fermentation hour

Lact

ate

(mM

)

Figure C-61. Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

159

-5

0

5

10

15

20

25

30

35

0 4 8 12 16 20 24

Fermentation hour

Lact

ate

(mM

)

Figure C-62. Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

-5

0

5

10

15

20

25

30

35

0 4 8 12 16 20 24

Fermentation hour

Lact

ate

(mM

)

Figure C-63. Lactate concentrations (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

160

50

60

70

80

90

100


Res

idua

l ND

F (%

)

Figure C-64. Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

50

60

70

80

90

100


Res

idua

l ND

F (%

)

Figure C-65. Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

161

50

60

70

80

90

100


Res

idua

l ND

F (%

)

Figure C-66. Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

50

60

70

80

90

100


Res

idua

l ND

F (%

)

Figure C-67. Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

162

50

60

70

80

90

100


Res

idua

l ND

F (%

)

Figure C-68. Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

50

60

70

80

90

100


Res

idua

l ND

F (%

)

Figure C-69. Residual NDF OM (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

163

-5

0

5

10

15

20

25

30

0 4 8 12 16 20 24

Fermentation hour

Mic

robi

al p

rote

in (m

g)

Figure C-70. Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + sucrose at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-5

0

5

10

15

20

25

30

0 4 8 12 16 20 24

Fermentation hour

Mic

robi

al p

rote

in (m

g)

Figure C-71. Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + starch at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

164

-5

0

5

10

15

20

25

30

0 4 8 12 16 20 24

Fermentation hour

Mic

robi

al p

rote

in (m

g)

Figure C-72. Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ×), and iNDF + pectin at 40 (ο), 80 (∆) or 120 mg (□) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

-5

0

5

10

15

20

25

30

0 4 8 12 16 20 24

Fermentation hour

Mic

robi

al p

rote

in (m

g)

Figure C-73. Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

165

-5

0

5

10

15

20

25

30

0 4 8 12 16 20 24

Fermentation hour

Mic

robi

al p

rote

in (m

g)

Figure C-74. Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin (□) or starch (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

-5

0

5

10

15

20

25

30

0 4 8 12 16 20 24

Fermentation hour

Mic

robi

al p

rote

in (m

g)

Figure C-75. Microbial crude protein yield (LSmeans ± standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose (□) or pectin (∆) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (▲) and 80:40 (■) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate.

APPENDIX D ADDITIONAL TABLE FOR CHAPTER 4

167

Table D-1. Temporal patterns of organic acid concentrations for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs.

Item1 Amount2 Total VFA3 C2 C3 C4 Val Lac BCVFA Temporal pattern4 (P-value) iNDF (120 mg air dry) C (0.01) C (0.01) C (< 0.01) Qd (0.03) L (< 0.01) NDP C (< 0.01) NFC individual Sucrose 40 L (< 0.01) L (0.01) Qd (< 0.01) Qt (0.05) L (< 0.01) Qt (< 0.01) Qt (0.04)

80 L (< 0.01) L (< 0.01) Qd (< 0.01) C (< 0.01) C (< 0.01) Qt (< 0.01) C (< 0.01) 120 Qt (< 0.01) Qt (< 0.01) Qt (< 0.01) L (< 0.01) C (< 0.01) Qt (< 0.01) C (< 0.01)

Starch 40 C (0.02) C (0.05) C (< 0.01) Qd (0.01) L (< 0.01) Qt (< 0.01) Qt (0.03) 80 Qt (0.05) C (0.03) Qt (< 0.01) L (< 0.01) L (< 0.01) C (< 0.01) Qt (< 0.01)

120 Qt (0.02) Qt (< 0.01) Qt (0.02) L (< 0.01) L (< 0.01) C (0.04) C (< 0.01) Pectin 40 Qt (0.02) Qt (0.04) Qd (0.03) Qd (0.03) L (0.01) C (< 0.01) C (< 0.01)

80 Qt (0.03) Qt (0.01) Qt (< 0.01) C (0.01) Qd (< 0.01) C (0.05) C (< 0.01) 120 Qt (< 0.01) Qt (0.04) Qt (< 0.01) L (< 0.01) Qd (< 0.01) Qt (< 0.01) C (0.02)

NFC combinations Su40St80 Qd (0.05) L (< 0.01) Qd (0.01) L (< 0.01) C (0.02) Qt (< 0.01) C (< 0.01) Su80St40 Qd (< 0.01) L (< 0.01) Qt (0.03) L (< 0.01) C (0.03) Qt (< 0.01) C (0.02) St40Pe80 Qt (< 0.01) Qt (< 0.01) Qt (< 0.01) C (< 0.01) L (< 0.01) L (< 0.01) L (< 0.01) St80Pe40 Qt (0.02) Qd (< 0.01) Qt (< 0.01) C (0.02) L (< 0.01) C (< 0.01) C (0.04) Su40Pe80 Qt (< 0.01) Qt (< 0.01) Qt (< 0.01) C (< 0.01) C (0.05) Qt (< 0.01) C (0.04) Su80Pe40 L (< 0.01) L (< 0.01) L (< 0.01) L (< 0.01) L (< 0.01) Qt (< 0.01) L (< 0.01)

1 iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 2 Amount of NFC substrate in mg hexose equivalent 3 VFA = volatile fatty acid; C2 = acetate; C3 = propionate; C4 = butyrate; Val = valerate; Lac = lactate; BCVFA = branched chain VFA 4 L = linear; Qd = quadratic; C = cubic; Qt = quartic; NDP = no detectable pattern

168

APPENDIX E CHAPTER 2 RAW DATA

Table E-1. Data used for statistical analysis in evaluating the effect of pH on microbial yield and neutral detergen fiber digestion from in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue.

Ferm1 Tube ID Med Sub Hr iNDF

(g DM) Sucrose (g DM)

rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA

(mM)

NH3-N (mM)

1 3 NpH SuNDF 0 0.1114 0.1201 — — 0.1228 — — — — — — — — — — — — —

1 4 NpH SuNDF 0 0.1115 0.1199 — — -0.1227 — — — — — — — — — — — — —

1 7 ApH SuNDF 0 0.1110 0.1200 — — 0.3774 — — — — — — — — — — — — —

1 8 ApH SuNDF 0 0.1118 0.1200 — — -0.3786 — — — — — — — — — — — — —

1 9 NpH BL 0 — — — — — — — — — 0.0000 14.0438 4.1158 2.3390 0.2309 0.4266 0.5722 8.2307 10.6721

1 10 NpH BL 0 — — — — — — — — — 0.0000 13.9550 4.2600 1.9250 0.2500 0.3350 0.4050 8.4490 10.4990

1 11 NpH SuNDF 0 0.1117 0.1200 — — — 0.3707 67.6840 9.2148 9.9029 0.4350 14.3300 4.5450 1.9700 0.2250 0.3100 0.3350 8.1001 11.0689

1 12 NpH SuNDF 0 0.1111 0.1201 — — — -0.0165 76.2848 9.7920 10.1317 0.4500 14.2400 4.6200 1.9350 0.2300 0.2900 0.4000 8.9296 10.4949

1 13 ApH BL 0 — — — — — — — — — 0.0000 14.1397 4.1394 1.9570 0.2613 0.3023 0.4150 7.2441 10.6176

1 14 ApH BL 0 — — — — — — — — — 0.0000 14.1113 4.2247 2.0232 0.0153 0.3160 0.2905 8.6018 12.3786

1 15 ApH SuNDF 0 — — — — — -0.4119 17.1647 31.1368 32.8596 0.3609 14.1887 4.3412 1.9643 0.2011 0.2784 0.4022 8.0978 11.5255

1 16 ApH SuNDF 0 — — — — — -0.4449 12.8350 31.2756 32.2076 0.3752 14.5113 4.6520 1.9842 0.2210 0.2724 0.2981 8.1144 11.9552

1 17 NpH iNDF 0 0.2231 0.0000 0.2104 7.07 — — — — — — — — — — — — — —

1 18 NpH iNDF 0 0.2230 0.0000 0.2138 7.03 — — — — — — — — — — — — — —

1 19 NpH SuNDF 0 0.1116 0.1202 0.1071 7.05 — — — — — — — — — — — — — —

1 20 NpH SuNDF 0 0.1114 0.1199 0.1070 7.02 — — — — — — — — — — — — — —

1 21 ApH iNDF 0 0.2230 0.0000 0.2142 6.13 — — — — — — — — — — — — — —

1 22 ApH iNDF 0 0.2230 0.0000 0.2150 6.13 — — — — — — — — — — — — — —

169



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA

(mM)

NH3-N (mM)

1 23 ApH SuNDF 0 0.1112 0.1203 0.1079 6.13 — — — — — — — — — — — — — —

1 24 ApH SuNDF 0 0.1116 0.1199 0.1086 6.12 — — — — — — — — — — — — — —

1 27 NpH SuNDF 4 0.1118 0.1199 — — 5.8304 — — — — — — — — — — — — —

1 28 NpH SuNDF 4 0.1114 . — — 7.2047 — — — — — — — — — — — — —

1 31 ApH SuNDF 4 0.1116 0.1199 — — 2.1743 — — — — — — — — — — — — —

1 32 ApH SuNDF 4 0.1114 0.1200 — — 1.1084 — — — — — — — — — — — — —

1 33 NpH BL 4 — — — — — — — — — 0.0000 17.4700 5.5950 2.3150 0.3050 0.3850 0.5000 7.4663 14.5975

1 34 NpH BL 4 — — — — — — — — — 0.0000 17.7200 5.6150 2.3050 0.3050 0.4050 0.5400 9.2608 15.8433

1 35 NpH SuNDF 4 0.1114 0.1202 — — — 5.4948 -0.3377 -0.6881 -0.1638 31.0773 22.7308 9.5896 3.7345 0.3813 0.3395 0.4962 5.4841 9.6161

1 36 NpH SuNDF 4 0.1112 0.1198 — — — 7.1012 -0.3693 -0.6764 -0.2370 30.5183 22.5883 8.8958 3.8309 0.3219 0.3970 0.3917 7.1125 12.6730

1 37 ApH BL 4 . . — — — — — — — 0.0157 20.6179 5.1884 2.4192 0.2456 0.4076 0.4807 8.5287 13.7264

1 38 ApH BL 4 . . — — — — — — — 0.0000 18.8501 4.7426 1.9743 0.2664 0.2298 0.4387 7.3615 11.6482

1 39 ApH SuNDF 4 0.1115 0.1202 — — — 2.3190 -0.1010 -1.6201 48.9465 7.3136 21.5669 6.9717 2.7513 0.2992 0.3633 0.4755 9.2936 13.6376

1 40 ApH SuNDF 4 0.1115 0.1199 — — — 3.1510 -0.1490 -1.6201 45.6225 7.9548 22.7940 7.0779 2.8186 0.2676 0.3758 0.4669 8.5886 12.6489

1 41 NpH iNDF 4 0.2230 0.0000 0.2097 7.11 — — — — — — — — — — — — — —

1 42 NpH iNDF 4 0.2228 0.0000 0.2105 7.09 — — — — — — — — — — — — — —

1 43 NpH SuNDF 4 0.1115 0.1202 0.1069 6.74 — — — — — — — — — — — — — —

1 44 NpH SuNDF 4 0.1116 0.1200 0.1070 6.73 — — — — — — — — — — — — — —

1 45 ApH iNDF 4 0.2231 0.0000 0.2125 6.29 — — — — — — — — — — — — — —

1 46 ApH iNDF 4 0.2231 0.0000 0.2142 6.22 — — — — — — — — — — — — — —

1 47 ApH SuNDF 4 0.1118 0.1201 0.1087 6.01 — — — — — — — — — — — — — —

1 48 ApH SuNDF 4 0.1117 0.1200 0.1084 6.01 — — — — — — — — — — — — — —

1 51 NpH SuNDF 8 0.1118 0.1201 — — 20.2040 — — — — — — — — — — — — —

1 52 NpH SuNDF 8 0.1112 0.1203 — — 19.6321 — — — — — — — — — — — — —

1 55 ApH SuNDF 8 0.1114 0.1200 — — 7.9675 — — — — — — — — — — — — —

1 56 ApH SuNDF 8 0.1113 0.1200 — — 6.9017 — — — — — — — — — — — — —

1 57 NpH BL 8 — — — — — — — — — 0.0000 19.5700 6.1000 4.7700 0.4050 0.4550 0.6500 6.7059 14.6006

1 58 NpH BL 8 — — — — — — — — — 0.0000 20.0150 5.8800 3.1250 0.1800 0.4100 0.5900 6.8028 16.2393

170



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA

(mM)

NH3-N (mM)

1 59 NpH SuNDF 8 0.1111 0.1202 — — — 1.6970 -0.1976 0.1583 -0.0040 14.7600 35.7800 20.1250 5.7300 0.7250 0.3900 0.7700 4.7853 12.9973

1 60 NpH SuNDF 8 0.1117 0.1201 — — — 1.4828 -0.2154 0.1519 -0.0214 15.4622 34.2565 19.3290 5.5070 0.3737 0.5554 0.6592 4.7905 12.6039

1 61 ApH BL 8 . . — — — — — — — 0.0000 52.2933 6.2754 2.6938 0.3500 0.4109 0.5377 8.3546 14.5210

1 62 ApH BL 8 . . — — — — — — — 0.0716 55.0462 6.1543 2.4535 0.2402 0.3885 0.4549 7.6446 13.7469

1 63 ApH SuNDF 8 0.1112 0.1201 — — — -0.2307 -0.1750 -0.4103 0.0057 28.4625 35.0037 13.4757 3.5863 0.2174 0.2950 0.5434 6.8616 10.9209

1 64 ApH SuNDF 8 0.1115 0.1201 — — — 1.4252 -0.2529 -0.3208 0.0279 26.2400 34.1200 13.2200 3.1550 0.1050 0.1550 0.4200 6.8102 10.8064

1 65 NpH iNDF 8 0.2226 0.0000 0.2087 7.17 — — — — — — — — — — — — — —

1 66 NpH iNDF 8 0.2230 0.0000 0.2094 7.18 — — — — — — — — — — — — — —

1 67 NpH SuNDF 8 0.1115 0.1197 0.1035 6.80 — — — — — — — — — — — — — —

1 68 NpH SuNDF 8 0.1116 0.1200 0.1032 6.85 — — — — — — — — — — — — — —

1 69 ApH iNDF 8 0.2229 0.0000 0.2109 6.49 — — — — — — — — — — — — — —

1 70 ApH iNDF 8 0.2231 0.0000 0.2120 6.48 — — — — — — — — — — — — — —

1 71 ApH SuNDF 8 0.1115 0.1198 0.1080 5.37 — — — — — — — — — — — — — —

1 72 ApH SuNDF 8 0.1115 0.1199 0.1077 5.38 — — — — — — — — — — — — — —

1 75 NpH SuNDF 12 0.1116 0.1200 — — 21.7672 — — — — — — — — — — — — —

1 76 NpH SuNDF 12 0.1117 0.1203 — — 21.0245 — — — — — — — — — — — — —

1 79 ApH SuNDF 12 0.1114 0.1202 — — 10.6327 — — — — — — — — — — — — —

1 80 ApH SuNDF 12 0.1114 0.1203 — — 11.0411 — — — — — — — — — — — — —

1 81 NpH BL 12 — — — — — — — — — 0.0000 22.7822 5.9063 3.7628 0.6734 0.6066 0.9356 5.5906 17.4949

1 82 NpH BL 12 — — — — — — — — — 0.0000 22.3450 5.9350 3.3600 0.7500 0.5550 0.9750 7.5302 17.8815

1 83 NpH SuNDF 12 0.1112 0.1201 — — — 3.5369 -0.2444 0.5016 -0.0487 0.0000 39.2200 21.1350 9.2900 3.4050 1.0550 1.5200 2.7160 17.3216

1 84 NpH SuNDF 12 0.1115 0.1204 — — — 2.1444 -0.1420 0.1729 -0.0519 0.0000 40.1377 22.6802 9.4351 3.3822 1.0827 1.3096 2.8851 17.3773

1 85 ApH BL 12 — — — — — — — — — 0.0300 78.9650 7.1250 2.9350 0.3200 0.3950 0.7300 8.8617 17.0561

1 86 ApH BL 12 — — — — — — — — — 0.0000 79.3750 6.8000 2.7550 0.3100 0.4450 0.7500 8.1236 16.3443

1 87 ApH SuNDF 12 0.1114 0.1203 — — — 0.8593 -0.0709 -0.1541 0.0197 19.8386 50.0609 21.5207 3.6253 0.2260 0.2862 0.4268 10.0593 15.0383

1 88 ApH SuNDF 12 0.1113 0.1202 — — — 0.4215 -0.1040 0.0689 0.0241 19.0800 53.2900 22.5550 3.6200 0.1850 0.2900 0.3950 7.0308 11.2936

1 89 NpH iNDF 12 0.2226 0.0000 0.2074 7.22 — — — — — — — — — — — — — —

1 90 NpH iNDF 12 0.2228 0.0000 0.2032 7.22 — — — — — — — — — — — — — —

171



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA

(mM)

NH3-N (mM)

1 91 NpH SuNDF 12 0.1112 0.1200 0.0960 6.88 — — — — — — — — — — — — — —

1 92 NpH SuNDF 12 0.1112 0.1202 0.0930 6.86 — — — — — — — — — — — — — —

1 93 ApH iNDF 12 0.2226 0.0000 0.2095 6.62 — — — — — — — — — — — — — —

1 94 ApH iNDF 12 0.2228 0.0000 0.2086 6.60 — — — — — — — — — — — — — —

1 95 ApH SuNDF 12 0.1113 0.1202 0.1098 5.43 — — — — — — — — — — — — — —

1 96 ApH SuNDF 12 0.1118 0.1200 0.1072 5.46 — — — — — — — — — — — — — —

1 99 NpH SuNDF 16 0.1114 0.1202 — — 18.4402 — — — — — — — — — — — — —

1 100 NpH SuNDF 16 0.1112 0.1202 — — 18.5240 — — — — — — — — — — — — —

1 103 ApH SuNDF 16 0.1114 0.1202 — — 12.3577 — — — — — — — — — — — — —

1 104 ApH SuNDF 16 0.1113 0.1201 — — 12.1978 — — — — — — — — — — — — —

1 105 NpH BL 16 — — — — — — — — — 0.0000 22.4910 5.4559 3.3622 0.9781 0.8405 1.5996 4.3989 21.2751

1 106 NpH BL 16 — — — — — — — — — 0.0000 22.3700 5.5900 3.3400 1.0650 0.9350 1.6250 3.5960 19.6991

1 107 NpH SuNDF 16 0.1113 0.1199 — — — 0.5649 -0.0510 0.2483 0.0068 0.0000 45.1700 23.2800 9.3350 4.3500 1.1250 1.5350 — —

1 108 NpH SuNDF 16 0.1115 0.1200 — — — 1.1714 -0.1293 0.2968 -0.0203 0.0000 45.4150 23.3700 9.3550 4.3250 1.0800 1.5400 1.4056 18.4075

1 109 ApH BL 16 — — — — — — — — — 0.0000 81.4600 7.5750 3.3800 0.3950 0.5750 1.0900 7.7167 17.7083

1 110 ApH BL 16 — — — — — — — — — 0.0000 80.6163 7.5995 3.2316 0.2892 0.5022 1.0653 6.7104 16.7367

1 111 ApH SuNDF 16 0.1118 0.1200 — — — 1.7184 0.0451 0.3686 0.1381 11.3850 67.4100 28.8450 3.9450 0.0900 0.3100 0.5150 8.2726 13.2092

1 112 ApH SuNDF 16 0.1116 0.1201 — — — 1.8481 0.0184 0.5717 0.0625 10.7150 71.2200 30.0850 4.0200 0.1900 0.3350 0.4800 9.0806 14.1377

1 113 NpH iNDF 16 0.2226 0.0000 0.1974 7.24 — — — — — — — — — — — — — —

1 114 NpH iNDF 16 0.2228 0.0000 0.1937 7.24 — — — — — — — — — — — — — —

1 115 NpH SuNDF 16 0.1113 0.1201 0.0840 6.87 — — — — — — — — — — — — — —

1 116 NpH SuNDF 16 0.1114 0.1204 0.0809 6.83 — — — — — — — — — — — — — —

1 117 ApH iNDF 16 0.2226 0.0000 0.2075 6.53 — — — — — — — — — — — — — —

1 118 ApH iNDF 16 0.2229 0.0000 0.2052 6.70 — — — — — — — — — — — — — —

1 119 ApH SuNDF 16 0.1117 0.1200 0.1094 5.55 — — — — — — — — — — — — — —

1 120 ApH SuNDF 16 0.1113 0.1199 — 5.58 — — — — — — — — — — — — — —

1 123 NpH SuNDF 20 0.1116 0.1200 — — 17.7011 — — — — — — — — — — — — —

1 124 NpH SuNDF 20 0.1112 0.1202 — — 18.1954 — — — — — — — — — — — — —

172



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA

(mM)

NH3-N (mM)

1 127 ApH SuNDF 20 0.1116 0.1204 — — 10.7883 — — — — — — — — — — — — —

1 128 ApH SuNDF 20 0.1116 0.1200 — — 10.3044 — — — — — — — — — — — — —

1 129 NpH BL 20 — — — — — — — — — 0.0000 21.9066 4.8030 2.9677 1.4915 1.3549 1.9970 1.8837 18.9895

1 130 NpH BL 20 — — — — — — — — — 0.0000 23.0120 4.9836 3.1028 1.6193 1.3427 1.9864 2.0661 22.2596

1 131 NpH SuNDF 20 0.1114 0.1200 — — — 0.4940 -0.1027 -0.0066 -0.0251 0.0000 49.2700 23.6150 10.5050 5.0300 1.2100 1.2200 1.0588 19.5832

1 132 NpH SuNDF 20 0.1114 0.1203 — — — 0.8980 -0.1027 -0.0080 -0.0251 0.0000 50.3900 23.7600 10.6350 5.0550 1.2900 1.4300 1.0409 19.2715

1 133 ApH BL 20 — — — — — — — — — 0.0000 81.4600 7.7800 4.5750 0.4650 1.3100 1.9100 4.8662 20.1194

1 134 ApH BL 20 — — — — — — — — — 0.0000 81.0550 7.9500 4.6300 0.4650 1.3250 1.9850 4.7384 18.4607

1 135 ApH SuNDF 20 0.1117 0.1204 — — — 0.5306 0.1954 0.7880 0.0622 1.6880 89.2863 38.0757 5.1347 0.3032 0.3841 0.4700 6.3289 11.2347

1 136 ApH SuNDF 20 0.1114 0.1201 — — — -0.8196 -0.1438 -0.0518 -0.1380 1.3221 89.0495 38.1287 4.5365 0.2826 0.3230 0.6207 6.0477 10.9487

1 137 NpH iNDF 20 0.2227 0.0000 0.1804 7.32 — — — — — — — — — — — — — —

1 138 NpH iNDF 20 0.2228 0.0000 0.1787 7.32 — — — — — — — — — — — — — —

1 139 NpH SuNDF 20 0.1118 0.1202 0.0728 6.91 — — — — — — — — — — — — — —

1 140 NpH SuNDF 20 0.1116 0.1202 0.0706 6.93 — — — — — — — — — — — — — —

1 141 ApH iNDF 20 0.2229 0.0000 0.2031 6.79 — — — — — — — — — — — — — —

1 142 ApH iNDF 20 0.2232 0.0000 0.2046 6.80 — — — — — — — — — — — — — —

1 143 ApH SuNDF 20 0.1115 0.1201 0.1066 5.78 — — — — — — — — — — — — — —

1 144 ApH SuNDF 20 0.1116 0.1199 0.1073 5.76 — — — — — — — — — — — — — —

1 147 NpH SuNDF 24 0.1115 0.1200 — — 18.8930 — — — — — — — — — — — — —

1 148 NpH SuNDF 24 0.1116 0.1203 — — 17.7395 — — — — — — — — — — — — —

1 151 ApH SuNDF 24 0.1114 0.1201 — — 11.5385 — — — — — — — — — — — — —

1 152 ApH SuNDF 24 0.1111 0.1204 — — 10.2237 — — — — — — — — — — — — —

1 153 NpH BL 24 — — — — — — — — — 0.0000 21.6900 4.4750 2.3800 1.5900 1.3550 1.9400 1.3661 21.2830

1 154 NpH BL 24 — — — — — — — — — 0.0000 21.4800 4.2050 2.6950 1.5450 1.3500 1.8500 1.7411 24.1988

1 155 NpH SuNDF 24 0.1112 0.1201 — — — 0.3705 -0.0562 0.0034 -0.0097 0.0000 51.3050 24.4400 9.9100 5.1150 1.2150 1.7100 0.9474 19.1206

1 156 NpH SuNDF 24 0.1115 0.1201 — — — 0.4696 -0.0562 -0.0016 -0.0097 0.0000 51.4450 24.3450 9.8750 5.2200 1.1500 1.6400 0.9513 19.7636

1 157 ApH BL 24 — — — — — — — — — 0.0000 80.7953 8.1372 5.0507 0.5241 1.3977 2.0012 3.3667 16.8181

1 158 ApH BL 24 — — — — — — — — — 0.0000 79.4688 8.0652 5.0063 0.7851 1.4071 1.8047 3.6369 19.4424

173



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA

(mM)

NH3-N (mM)

1 159 ApH SuNDF 24 0.1119 0.1200 — — — 1.3091 -0.1059 0.8374 -0.0643 0.0502 86.3098 36.8781 6.4347 1.1836 0.4263 0.9579 6.0291 12.1153

1 160 ApH SuNDF 24 0.1116 0.1200 — — — 1.1205 0.1313 0.8242 0.0960 0.0300 86.8100 37.3850 5.0600 0.5550 0.3350 1.0250 6.2091 11.7841

1 161 NpH iNDF 24 0.2231 0.0000 0.1845 7.26 — — — — — — — — — — — — — —

1 162 NpH iNDF 24 0.2228 0.0000 0.1583 6.96 — — — — — — — — — — — — — —

1 163 NpH SuNDF 24 0.1116 0.1201 0.0649 6.90 — — — — — — — — — — — — — —

1 165 ApH iNDF 24 0.2230 0.0000 — 6.78 — — — — — — — — — — — — — —

1 166 ApH iNDF 24 0.2231 0.0000 0.2034 6.86 — — — — — — — — — — — — — —

1 167 ApH SuNDF 24 0.1113 0.1203 0.1062 5.79 — — — — — — — — — — — — — —

1 168 ApH SuNDF 24 0.1113 0.1201 0.1073 5.78 — — — — — — — — — — — — — —

1 171 NpH SuNDF 24 0.1113 0.1202 0.0633 6.95 — — — — — — — — — — — — — —

2 1 NpH iNDF 0 0.2228 0.0000 0.2168 6.95 — — — — — — — — — — — — — —

2 2 NpH iNDF 0 0.2226 0.0000 0.2148 6.91 — — — — — — — — — — — — — —

2 3 NpH SuNDF 0 0.1115 0.1199 0.1115 6.87 — — — — — — — — — — — — — —

2 4 NpH SuNDF 0 0.1113 0.1201 0.1101 6.84 — — — — — — — — — — — — — —

2 5 NpH BL 0 — — — — — — — — — 0.0000 15.8650 4.8250 2.1300 0.2300 0.1400 0.3050 7.9292 10.3062

2 6 NpH BL 0 — — — — — — — — — 0.0000 15.6250 4.8150 2.1450 0.2200 0.1800 0.2600 7.6134 10.9732

2 7 NpH SuNDF 0 0.1118 0.1200 — — — 0.3831 94.3636 7.6349 8.3624 0.3600 16.1100 5.0250 2.2700 0.2050 0.2350 0.2600 8.1881 11.2483

2 8 NpH SuNDF 0 0.1113 0.1200 — — — 0.0783 95.0595 7.1625 8.8500 0.3011 16.7253 3.7054 1.3985 0.1480 0.1633 0.2348 8.0268 11.7330

2 11 NpH SuNDF 0 0.1113 0.1200 — — 0.0821 — — — — — — — — — — — — —

2 12 NpH SuNDF 0 0.1113 0.1200 — — -0.0821 — — — — — — — — — — — — —

2 13 ApH iNDF 0 0.2228 0.0000 0.2182 6.00 — — — — — — — — — — — — — —

2 14 ApH iNDF 0 0.2227 0.0000 0.2180 6.00 — — — — — — — — — — — — — —

2 15 ApH SuNDF 0 0.1116 0.1201 0.1107 6.06 — — — — — — — — — — — — — —

2 16 ApH SuNDF 0 0.1114 0.1201 0.1103 6.09 — — — — — — — — — — — — — —

2 17 ApH BL 0 — — — — — — — — — 0.0000 17.3412 2.9310 1.4186 0.0209 0.1982 0.0209 9.2548 12.9968

2 18 ApH BL 0 — — — — — — — — — 0.0000 17.2250 5.3500 2.3150 0.2300 0.2400 0.2800 8.3045 11.8049

2 19 ApH SuNDF 0 0.1116 0.1201 — — — -0.1153 85.5059 21.7672 21.1033 0.4150 17.9550 5.5350 2.4000 0.2100 0.2350 0.2900 8.0497 11.9899

2 20 ApH SuNDF 0 0.1116 0.1202 — — — -0.1977 78.4755 23.6652 23.3185 0.5150 18.2450 5.6500 2.4350 0.2600 0.2400 0.2950 7.8465 11.6758

174



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA

(mM)

NH3-N (mM)

2 23 ApH SuNDF 0 0.1115 0.1200 — — 0.2499 — — — — — — — — — — — — —

2 24 ApH SuNDF 0 0.1118 0.1201 — — -0.2503 — — — — — — — — — — — — —

2 25 NpH iNDF 4 0.2229 0.0000 0.2117 6.90 — — — — — — — — — — — — — —

2 26 NpH iNDF 4 0.2229 0.0000 0.2123 6.95 — — — — — — — — — — — — — —

2 27 NpH SuNDF 4 0.1113 0.1204 0.1103 6.65 — — — — — — — — — — — — — —

2 28 NpH SuNDF 4 0.1116 0.1201 0.1095 6.71 — — — — — — — — — — — — — —

2 29 NpH BL 4 — — — — — — — — — 0.0000 19.2648 6.1093 2.5485 0.4130 0.2972 0.4483 5.5130 9.6867

2 30 NpH BL 4 — — — — — — — — — 0.0000 18.8400 5.8600 2.8950 0.4100 0.3200 0.5300 6.0710 10.3297

2 31 NpH SuNDF 4 0.1116 0.1202 — — — 7.0433 -0.2189 -0.6062 0.0582 24.8501 26.4564 10.8086 4.2261 0.4561 0.2686 0.3851 7.4138 13.0591

2 32 NpH SuNDF 4 0.1114 0.1200 — — — 4.3002 -0.2317 0.4910 0.2517 25.2000 25.9350 10.7100 4.2200 0.4650 0.2700 0.4550 5.9460 10.9710

2 35 NpH SuNDF 4 0.1117 0.1203 — — 10.1294 — — — — — — — — — — — — —

2 36 NpH SuNDF 4 0.1115 0.1202 — — 9.5610 — — — — — — — — — — — — —

2 37 ApH iNDF 4 0.2227 0.0000 — — — — — — — — — — — — — — — —

2 38 ApH iNDF 4 0.2230 0.0000 0.2179 6.14 — — — — — — — — — — — — — —

2 39 ApH SuNDF 4 0.1115 0.1202 0.1118 5.65 — — — — — — — — — — — — — —

2 40 ApH SuNDF 4 0.1118 0.1202 0.1120 5.52 — — — — — — — — — — — — — —

2 41 ApH BL 4 — — — — — — — — — 0.0000 26.6700 5.9800 2.6450 0.3150 0.2350 0.4500 9.0443 15.6055

2 42 ApH BL 4 — — — — — — — — — 0.0465 27.5058 6.2065 2.6540 0.2995 0.2892 0.4079 6.2693 12.0313

2 43 ApH SuNDF 4 0.1117 0.1202 — — — 4.5721 -0.1983 14.0837 35.7750 19.3912 26.8816 8.6739 3.3128 0.3591 0.2681 0.5058 6.8313 11.1753

2 44 ApH SuNDF 4 0.1113 0.1201 — — — 3.7895 -0.5178 13.6129 33.9265 19.2000 26.7400 8.7000 3.1800 0.2850 0.2100 0.4500 10.7944 16.7407

2 47 ApH SuNDF 4 0.1114 0.1202 — — 2.7532 — — — — — — — — — — — — —

2 48 ApH SuNDF 4 0.1117 0.1201 — — 2.2569 — — — — — — — — — — — — —

2 49 NpH iNDF 8 0.2230 0.0000 0.2075 7.00 — — — — — — — — — — — — — —

2 50 NpH iNDF 8 0.2227 0.0000 0.2071 7.01 — — — — — — — — — — — — — —

2 51 NpH SuNDF 8 0.1117 0.1202 0.1031 6.66 — — — — — — — — — — — — — —

2 52 NpH SuNDF 8 0.1118 0.1200 0.1020 6.62 — — — — — — — — — — — — — —

2 53 NpH BL 8 — — — — — — — — — 0.0000 22.1315 6.2768 3.0316 0.6155 0.3561 0.6358 6.8637 16.3386

2 54 NpH BL 8 — — — — — — — — — 0.0000 22.0150 6.1050 2.9450 0.6650 0.3550 0.6250 6.9025 18.8614

175



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA

(mM)

NH3-N (mM)

2 55 NpH SuNDF 8 0.1114 0.1201 — — — 5.2929 -0.1405 0.0102 -0.0389 6.5700 38.6350 19.2750 6.9800 1.2400 0.3000 0.6800 4.4838 15.2901

2 56 NpH SuNDF 8 0.1114 0.1199 — — — 4.3291 -0.0946 0.0136 -0.1394 6.6900 38.2050 18.9050 7.0250 1.4450 0.2950 0.6800 4.3393 14.4568

2 59 NpH SuNDF 8 0.1115 0.1202 — — 9.6842 — — — — — — — — — — — — —

2 60 NpH SuNDF 8 0.1116 0.1200 — — 10.0153 — — — — — — — — — — — — —

2 61 ApH iNDF 8 0.2227 0.0000 0.2158 6.33 — — — — — — — — — — — — — —

2 62 ApH iNDF 8 0.2227 0.0000 0.2161 6.33 — — — — — — — — — — — — — —

2 63 ApH SuNDF 8 0.1118 0.1201 0.1127 5.13 — — — — — — — — — — — — — —

2 64 ApH SuNDF 8 0.1116 0.1202 0.1121 5.12 — — — — — — — — — — — — — —

2 65 ApH BL 8 — — — — — — — — — 0.0550 56.0400 5.6500 3.1550 0.4000 0.3200 0.6400 6.1963 12.6230

2 66 ApH BL 8 — — — — — — — — — 0.0207 57.8298 6.1249 3.1866 0.3725 0.2793 0.5380 6.4818 12.8710

2 67 ApH SuNDF 8 0.1112 0.1202 — — — 3.1964 -0.1102 0.0686 0.2453 31.1264 40.0997 13.8842 3.4248 0.2880 0.2108 0.4268 6.4481 10.7564

2 68 ApH SuNDF 8 0.1116 0.1203 — — — 2.8174 -0.1308 0.0540 0.2458 29.9500 39.2500 13.5100 3.3500 0.2950 0.2100 0.4550 5.9917 9.8918

2 71 ApH SuNDF 8 0.1116 0.1202 — — 6.9391 — — — — — — — — — — — — —

2 72 ApH SuNDF 8 0.1118 0.1202 — — 6.3605 — — — — — — — — — — — — —

2 73 NpH iNDF 12 0.2226 0.0000 0.1998 6.91 — — — — — — — — — — — — — —

2 74 NpH iNDF 12 0.2229 0.0000 0.1971 7.09 — — — — — — — — — — — — — —

2 75 NpH SuNDF 12 0.1116 0.1201 0.0912 6.69 — — — — — — — — — — — — — —

2 76 NpH SuNDF 12 0.1113 0.1201 0.0907 6.72 — — — — — — — — — — — — — —

2 77 NpH BL 12 — — — — — — — — — 0.0000 23.7500 6.1850 3.5800 1.0500 0.5650 1.1750 4.0947 18.9294

2 78 NpH BL 12 — — — — — — — — — 0.0000 24.3390 6.2666 3.4971 1.0687 0.5619 1.1991 5.0477 20.5911

2 79 NpH SuNDF 12 0.1116 0.1202 — — — 3.1304 -0.1124 0.1143 0.0060 0.0000 43.1600 20.4100 9.6400 3.4900 0.8750 1.3800 1.8108 16.9746

2 80 NpH SuNDF 12 0.1114 0.1201 — — — 2.5044 -0.1168 -0.1015 0.0082 0.0000 42.2989 20.0809 10.3025 3.2107 0.9123 1.4516 1.9397 16.8393

2 83 NpH SuNDF 12 0.1116 0.1202 — — 17.3626 — — — — — — — — — — — — —

2 84 NpH SuNDF 12 0.1118 0.1202 — — 17.5226 — — — — — — — — — — — — —

2 85 ApH iNDF 12 0.2228 0.0000 0.2168 6.59 — — — — — — — — — — — — — —

2 86 ApH iNDF 12 0.2228 0.0000 0.2159 6.61 — — — — — — — — — — — — — —

2 87 ApH SuNDF 12 0.1116 0.1201 0.1117 5.24 — — — — — — — — — — — — — —

2 88 ApH SuNDF 12 0.1115 0.1202 0.1119 5.22 — — — — — — — — — — — — — —

176



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA

(mM)

NH3-N (mM)

2 89 ApH BL 12 — — — — — — — — — 0.0000 75.2350 6.5650 3.5400 0.4350 0.3500 0.7750 5.3788 14.0159

2 90 ApH BL 12 — — — — — — — — — 0.0000 77.0987 6.6758 3.6595 0.4032 0.3675 0.7809 5.8431 15.6357

2 91 ApH SuNDF 12 0.1115 0.1204 — — — 2.6485 -0.0167 0.2642 -0.1373 25.2006 52.2913 19.4543 3.6378 0.3099 0.2337 0.5081 4.5547 8.3301

2 92 ApH SuNDF 12 0.1116 0.1202 — — — 2.2284 -0.2442 0.2919 -0.1356 23.5850 52.1550 20.8950 3.6100 0.3050 0.2100 0.4750 6.6872 11.0473

2 95 ApH SuNDF 12 0.1114 0.1201 — — 6.6981 — — — — — — — — — — — — —

2 96 ApH SuNDF 12 0.1115 0.1201 — — 6.9428 — — — — — — — — — — — — —

2 97 NpH iNDF 16 0.2226 0.0000 0.1836 7.11 — — — — — — — — — — — — — —

2 98 NpH iNDF 16 0.2228 0.0000 0.1792 7.17 — — — — — — — — — — — — — —

2 99 NpH SuNDF 16 0.1115 0.1200 0.0800 6.73 — — — — — — — — — — — — — —

2 100 NpH SuNDF 16 0.1114 0.1201 0.0811 6.71 — — — — — — — — — — — — — —

2 103 NpH SuNDF 16 0.1113 0.1202 — — — — — — — — — — — — — — — —

2 104 NpH SuNDF 16 0.1114 0.1202 — — — — -0.0637 -0.0177 -0.0229 — — — — — — — — —

2 107 NpH SuNDF 16 0.1111 0.1203 — — 18.7264 — — — — — — — — — — — — —

2 108 NpH SuNDF 16 0.1114 0.1204 — — 18.7998 — — — — — — — — — — — — —

2 109 ApH iNDF 16 0.2229 0.0000 0.2121 6.77 — — — — — — — — — — — — — —

2 110 ApH iNDF 16 0.2228 0.0000 0.2136 6.75 — — — — — — — — — — — — — —

2 111 ApH SuNDF 16 0.1117 0.1202 0.1123 5.42 — — — — — — — — — — — — — —

2 112 ApH SuNDF 16 0.1116 0.1202 0.1123 5.39 — — — — — — — — — — — — — —

2 115 ApH SuNDF 16 0.1115 0.1202 — — — — — — — — — — — — — — — —

2 116 ApH SuNDF 16 0.1113 0.1201 — — — — 0.2804 1.2927 0.1411 — — — — — — — — —

2 119 ApH SuNDF 16 0.1115 0.1200 — — 7.3143 — — — — — — — — — — — — —

2 120 ApH SuNDF 16 0.1116 0.1202 — — 7.6373 — — — — — — — — — — — — —

2 121 NpH iNDF 20 0.2227 0.0000 0.1651 7.11 — — — — — — — — — — — — — —

2 122 NpH iNDF 20 0.2227 0.0000 0.1681 7.14 — — — — — — — — — — — — — —

2 123 NpH SuNDF 20 0.1114 0.1202 0.0707 6.79 — — — — — — — — — — — — — —

2 124 NpH SuNDF 20 0.1117 0.1204 0.0720 6.85 — — — — — — — — — — — — — —

2 125 NpH BL 20 — — — — — — — — — 0.0000 27.6431 5.6559 3.8925 2.2894 1.2274 2.0189 1.6588 22.1759

2 126 NpH BL 20 — — — — — — — — — 0.0000 24.7350 4.8150 2.9200 1.9450 1.1850 1.8950 0.9026 15.4703

177



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA

(mM)

NH3-N (mM)

2 127 NpH SuNDF 20 0.1118 0.1202 — — — 1.9689 -0.1334 -0.0238 -0.0120 0.0000 52.5850 22.4800 11.5300 5.0600 1.0000 1.6300 0.7863 17.0543

2 128 NpH SuNDF 20 0.1116 0.1201 — — — 1.7629 -0.1334 -0.0049 -0.0123 0.0000 53.6596 22.1894 11.2509 4.8823 1.0278 1.5871 1.4379 24.1084

2 131 NpH SuNDF 20 0.1118 0.1201 — — 19.7839 — — — — — — — — — — — — —

2 132 NpH SuNDF 20 0.1115 0.1203 — — 19.7036 — — — — — — — — — — — — —

2 133 ApH iNDF 20 0.2231 0.0000 0.2082 6.84 — — — — — — — — — — — — — —

2 134 ApH iNDF 20 0.2228 0.0000 0.2118 6.85 — — — — — — — — — — — — — —

2 135 ApH SuNDF 20 0.1113 0.1200 0.1114 5.66 — — — — — — — — — — — — — —

2 136 ApH SuNDF 20 0.1116 0.1202 0.1133 5.64 — — — — — — — — — — — — — —

2 137 ApH BL 20 — — — — — — — — — 0.0000 87.0264 7.5576 5.5663 0.6991 1.1281 1.8431 1.7051 13.1603

2 138 ApH BL 20 — — — — — — — — — 0.0000 86.8100 7.5100 5.5200 0.6700 1.1300 1.8850 3.5556 20.8428

2 139 ApH SuNDF 20 0.1116 0.1202 — — — 4.1314 0.0825 1.0300 -0.0197 0.0000 73.8350 30.5809 8.9743 2.3724 0.2857 0.8214 5.7512 11.7849

2 140 ApH SuNDF 20 0.1116 0.1199 — — — 3.5547 0.1178 0.9878 -0.0368 0.0000 74.2350 31.5350 8.6700 2.2350 0.2950 0.8050 7.2162 13.7544

2 143 ApH SuNDF 20 0.1117 0.1203 — — 11.8641 — — — — — — — — — — — — —

2 144 ApH SuNDF 20 0.1117 0.1202 — — 11.4553 — — — — — — — — — — — — —

2 145 NpH iNDF 24 0.2227 0.0000 0.1545 7.43 — — — — — — — — — — — — — —

2 146 NpH iNDF 24 0.2230 0.0000 0.1592 7.43 — — — — — — — — — — — — — —

2 147 NpH SuNDF 24 0.1115 0.1204 0.0658 7.19 — — — — — — — — — — — — — —

2 148 NpH SuNDF 24 0.1115 0.1204 0.0629 7.16 — — — — — — — — — — — — — —

2 149 NpH BL 24 — — — — — — — — — 0.0000 21.7700 5.0250 2.1350 1.8900 1.0750 1.8150 0.8949 20.7350

2 150 NpH BL 24 — — — — — — — — — 0.0000 24.2190 5.2247 2.4749 1.9860 1.2171 2.0267 0.8958 20.8142

2 151 NpH SuNDF 24 0.1114 0.1203 — — — 1.2316 -0.0616 -0.0995 -0.0090 0.0000 52.1178 22.1037 10.6412 5.0838 1.0178 1.6526 0.8881 19.3491

2 152 NpH SuNDF 24 0.1115 0.1202 — — — 1.0174 -0.0575 -0.0995 -0.0090 0.0000 55.3150 23.7350 11.1100 4.9650 1.0650 1.7000 0.9239 19.9850

2 155 NpH SuNDF 24 0.1115 0.1200 — — 13.9604 — — — — — — — — — — — — —

2 156 NpH SuNDF 24 0.1116 0.1201 — — 13.7916 — — — — — — — — — — — — —

2 157 ApH iNDF 24 0.2229 0.0000 0.2066 7.15 — — — — — — — — — — — — — —

2 158 ApH iNDF 24 0.2231 0.0000 0.2090 7.18 — — — — — — — — — — — — — —

2 159 ApH SuNDF 24 0.1113 0.1199 0.1112 6.16 — — — — — — — — — — — — — —

2 160 ApH SuNDF 24 0.1115 0.1201 0.1108 6.15 — — — — — — — — — — — — — —

178



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA

(mM)

NH3-N (mM)

2 161 ApH BL 24 — — — — — — — — — 0.0000 88.8063 7.8058 6.1241 0.7699 1.2106 1.9856 2.3308 18.8831

2 162 ApH BL 24 — — — — — — — — — 0.0000 84.7041 7.4894 5.6510 0.6831 1.1352 1.8987 2.8673 20.6115

2 163 ApH SuNDF 24 0.1116 0.1202 — — — 3.6742 0.1027 0.8761 0.1782 0.0000 81.1750 25.8550 11.6350 3.9000 0.7950 1.2850 4.2137 14.8633

2 164 ApH SuNDF 24 0.1117 0.1203 — — — 3.4764 0.0609 1.0014 0.2185 0.0000 79.6400 32.6050 10.1400 3.1900 0.8250 1.4200 4.4632 14.1153

2 167 ApH SuNDF 24 0.1114 0.1203 — — 9.0765 — — — — — — — — — — — — —

2 168 ApH SuNDF 24 0.1112 0.1202 — — 8.9177 — — — — — — — — — — — — — 1 Ferm = fermentation; Med = medium: ApH = acidic medium, NpH = neutral medium; Sub = substrate: BL = no substrate; iNDF = isolated bermudagrass neutral detergent residue, SuNDF = sucrose + iNDF; rNDF = residual NDF; MCP = microbial crude protein; GLY = microbial glycogen; rSuc = residual sucrose; rGlc = residual glucose; rFruc = residual fructose; Lac = lactate; C2 = acetate; C3 = propionate; C4 = butyrate; Val = valerate; Isobut = isobutyrate; Isoval+2MB = isovalerate + 2-Methylbutyrate; AA = free amino acids; NH3-N = ammonia nitrogen

179

APPENDIX F CHAPTER 3 RAW DATA

Table F-1. Data used for statistical analysis in evaluating the effect of nitrogen source on microbial yield and neutral detergen fiber digestion from in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue.



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

1 1 B BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1658 0.2764 7.6161 10.3615

1 2 B BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1600 0.2450 7.4296 10.5887

1 3 B SuNDF 0 0.1114 0.1203 — — — 1.0889 96.1097 2.2895 4.0700 0.2000 0.5246 0.6162 -0.0219 -0.0553 0.1900 0.2300 8.0956 10.8926

1 4 B SuNDF 0 0.1115 0.1201 — — — 0.4244 103.8990 2.3326 3.6539 0.2234 0.5265 0.6298 -0.0510 -0.0187 0.1675 0.2132 4.9905 7.2073

1 7 B SuNDF 0 0.1116 0.1201 — — 0.0801 — — — — — — — — — — — — —

1 8 B SuNDF 0 0.1114 0.1201 — — -0.0800 — — — — — — — — — — — — —

1 9 B iNDF 0 0.2227 0.0000 — — 0.0864 — — — — — — — — — — — — —

1 10 B iNDF 0 0.2229 0.0000 — — -0.0865 — — — — — — — — — — — — —

1 11 B SuNDF 0 0.1116 0.1200 0.1095 6.98 — — — — — — — — — — — — — —

1 12 B SuNDF 0 0.1116 0.1203 0.1103 7.02 — — — — — — — — — — — — — —

1 13 B iNDF 0 0.2226 0.0000 0.2127 7.03 — — — — — — — — — — — — — —

1 14 B iNDF 0 0.2228 0.0000 0.2154 7.07 — — — — — — — — — — — — — —

1 15 C BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.2000 0.3800 10.0151 1.3816

1 16 C BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1800 0.2500 6.8941 0.9627

1 17 C SuNDF 0 0.1114 0.1201 — — — 0.9328 108.9988 1.9854 4.0989 0.2000 0.4325 0.0625 -0.0700 0.0525 0.1600 0.2400 11.0758 1.8644

1 18 C SuNDF 0 0.1114 0.1201 — — — 0.5805 110.2956 2.2641 4.4591 0.1727 -0.2971 -0.2161 -0.2398 -0.0213 0.0914 0.1270 9.0062 1.4813

1 21 C SuNDF 0 0.1116 0.1202 — — -0.1677 — — — — — — — — — — — — —

1 22 C SuNDF 0 0.1114 0.1200 — — 0.1674 — — — — — — — — — — — — —

180



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

1 23 C iNDF 0 0.2226 0.0000 — — -0.1532 — — — — — — — — — — — — —

1 24 C iNDF 0 0.2231 0.0000 — — 0.1535 — — — — — — — — — — — — —

1 25 C SuNDF 0 0.1116 0.1203 0.1085 7.01 — — — — — — — — — — — — — —

1 26 C SuNDF 0 0.1116 0.1203 0.1095 7.02 — — — — — — — — — — — — — —

1 27 C iNDF 0 0.2229 0.0000 0.2131 7.03 — — — — — — — — — — — — — —

1 28 C iNDF 0 0.2228 0.0000 0.2139 7.04 — — — — — — — — — — — — — —

1 29 U BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.0632 0.1947 0.2515 7.0074

1 30 U BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.2048 0.3431 0.2061 5.6065

1 31 U SuNDF 0 0.1117 0.1205 — — — 0.8527 100.9528 4.9920 4.1184 0.1550 0.3092 0.2428 0.0567 0.0261 0.1450 0.2200 0.2541 7.1400

1 32 U SuNDF 0 0.1114 0.1200 — — — 0.6846 91.2548 5.7720 5.2260 0.1600 2.1292 0.1578 0.0717 -0.0389 0.1350 0.2500 0.3000 8.1732

1 35 U SuNDF 0 0.1113 0.1201 — — -1.3532 — — — — — — — — — — — — —

1 36 U SuNDF 0 0.1115 0.1201 — — 1.3542 — — — — — — — — — — — — —

1 37 U iNDF 0 0.2226 0.0000 — — -1.2637 — — — — — — — — — — — — —

1 38 U iNDF 0 0.2230 0.0000 — — 1.2658 — — — — — — — — — — — — —

1 39 U SuNDF 0 0.1115 0.1201 0.1055 7.01 — — — — — — — — — — — — — —

1 40 U SuNDF 0 0.1118 0.1203 0.1094 7.07 — — — — — — — — — — — — — —

1 41 U iNDF 0 0.2231 0.0000 0.2156 7.04 — — — — — — — — — — — — — —

1 42 U iNDF 0 0.2228 0.0000 0.2138 7.07 — — — — — — — — — — — — — —

1 43 B BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.2214 0.4831 5.4986 9.9438

1 44 B BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.2900 0.4900 4.6405 8.4942

1 45 B SuNDF 4 0.1118 0.1201 — — — 7.8060 -0.1630 0.0246 -0.0719 28.2150 0.1300 3.3998 1.2613 -0.0059 0.2450 0.3800 3.7854 4.8287

1 46 B SuNDF 4 0.1114 0.1200 — — — 6.8693 -0.1945 -0.1537 -0.0795 27.8985 0.2760 3.4750 1.2725 0.0467 0.2823 0.3528 5.1141 6.7393

1 49 B SuNDF 4 0.1113 0.1203 — — 11.2125 — — — — — — — — — — — — —

1 50 B SuNDF 4 0.1113 0.1201 — — 11.4634 — — — — — — — — — — — — —

1 51 B iNDF 4 0.2227 0.0000 — — 1.1132 — — — — — — — — — — — — —

1 52 B iNDF 4 0.2231 0.0000 — — 2.4104 — — — — — — — — — — — — —

1 53 B SuNDF 4 0.1116 0.1202 0.1098 6.52 — — — — — — — — — — — — — —

1 54 B SuNDF 4 0.1115 0.1204 0.1080 6.50 — — — — — — — — — — — — — —

181



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

1 55 B iNDF 4 0.2229 0.0000 0.2110 7.13 — — — — — — — — — — — — — —

1 56 B iNDF 4 0.2230 0.0000 0.2098 7.18 — — — — — — — — — — — — — —

1 57 C BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.2650 0.4350 . .

1 58 C BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.2392 0.3715 11.1907 3.4697

1 59 C SuNDF 4 0.1117 0.1203 — — — 7.9746 -0.1569 -0.0215 11.7511 25.1750 4.2899 3.6440 1.6385 -0.0175 0.1900 0.3650 6.5981 0.6904

1 60 C SuNDF 4 0.1116 0.1201 — — — 6.3573 -0.1842 0.0412 10.7450 25.0687 4.3398 3.7385 1.1551 -0.0076 0.2038 0.3617 5.5557 0.6044

1 63 C SuNDF 4 0.1114 0.1200 — — 8.8748 — — — — — — — — — — — — —

1 64 C SuNDF 4 0.1116 0.1200 — — 7.9680 — — — — — — — — — — — — —

1 65 C iNDF 4 0.2228 0.0000 — — -4.5156 — — — — — — — — — — — — —

1 66 C iNDF 4 0.2228 0.0000 — — -3.6942 — — — — — — — — — — — — —

1 67 C SuNDF 4 0.1117 0.1204 0.1078 6.58 — — — — — — — — — — — — — —

1 68 C SuNDF 4 0.1115 0.1200 0.1081 6.45 — — — — — — — — — — — — — —

1 69 C iNDF 4 0.2230 0.0000 0.2103 6.99 — — — — — — — — — — — — — —

1 70 C iNDF 4 0.2227 0.0000 0.2095 6.88 — — — — — — — — — — — — — —

1 71 U BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.1800 0.3150 0.3712 10.9493

1 72 U BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.1950 0.3100 0.4138 11.8845

1 73 U SuNDF 4 0.1118 0.1202 — — — 7.2300 0.3044 6.3809 27.4587 16.5323 3.0809 1.5081 0.4947 -0.0269 0.1709 0.1859 0.3419 8.6223

1 74 U SuNDF 4 0.1119 0.1204 — — — 5.9489 0.0000 6.1776 28.3049 16.9300 3.4025 1.7150 0.8875 -0.0075 0.3400 0.1950 0.5250 13.3896

1 77 U SuNDF 4 0.1115 0.1202 — — 4.0632 — — — — — — — — — — — — —

1 78 U SuNDF 4 0.1117 0.1202 — — 5.5384 — — — — — — — — — — — — —

1 79 U iNDF 4 0.2228 0.0000 — — 0.2064 — — — — — — — — — — — — —

1 80 U iNDF 4 0.2229 0.0000 — — 1.8451 — — — — — — — — — — — — —

1 81 U SuNDF 4 0.1116 0.1203 0.1091 6.62 — — — — — — — — — — — — — —

1 82 U SuNDF 4 0.1116 0.1200 0.1092 6.51 — — — — — — — — — — — — — —

1 83 U iNDF 4 0.2229 0.0000 0.2121 6.81 — — — — — — — — — — — — — —

1 84 U iNDF 4 0.2231 0.0000 0.2107 6.98 — — — — — — — — — — — — — —

1 85 B BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.1700 0.4000 5.2491 11.8492

1 86 B BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.2850 0.5300 — —

182



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

1 87 B SuNDF 8 0.1114 0.1204 — — — 5.4005 -0.1163 0.1987 0.0593 24.0374 8.5604 5.7802 1.5188 -0.0513 0.2651 0.4078 6.3469 9.9714

1 88 B SuNDF 8 0.1116 0.1202 — — — 6.0570 -0.1418 0.1263 0.0440 23.7450 9.9125 6.2550 1.5650 -0.0600 0.1950 0.4650 4.3481 6.8676

1 91 B SuNDF 8 0.1114 0.1203 — — 13.2230 — — — — — — — — — — — — —

1 92 B SuNDF 8 0.1115 0.1203 — — 12.3994 — — — — — — — — — — — — —

1 93 B iNDF 8 0.2231 0.0000 — — 1.8764 — — — — — — — — — — — — —

1 94 B iNDF 8 0.2231 0.0000 — — 2.6936 — — — — — — — — — — — — —

1 95 B SuNDF 8 0.1117 0.1205 0.1077 6.77 — — — — — — — — — — — — — —

1 96 B SuNDF 8 0.1118 0.1203 0.1061 6.75 — — — — — — — — — — — — — —

1 97 B iNDF 8 0.2231 0.0000 0.2054 7.19 — — — — — — — — — — — — — —

1 98 B iNDF 8 0.2230 0.0000 0.2067 7.22 — — — — — — — — — — — — — —

1 99 C BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.2280 0.4713 12.1128 6.4069

1 100 C BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.3150 0.5550 9.2628 4.6730

1 101 C SuNDF 8 0.1115 0.1201 — — — 5.9930 -0.1134 0.4340 0.0584 24.6459 8.2537 7.1776 1.7406 0.0435 0.2241 0.4840 10.9952 2.1030

1 102 C SuNDF 8 0.1113 0.1201 — — — 5.3284 -0.0866 0.4264 0.0624 26.1350 7.2988 4.9814 1.2769 -0.0402 0.2300 0.5000 7.6298 1.4557

1 105 C SuNDF 8 0.1118 0.1201 — — 16.2599 — — — — — — — — — — — — —

1 106 C SuNDF 8 0.1117 0.1204 — — 14.5313 — — — — — — — — — — — — —

1 107 C iNDF 8 0.2231 0.0000 — — 1.0528 — — — — — — — — — — — — —

1 108 C iNDF 8 0.2227 0.0000 — — 1.8139 — — — — — — — — — — — — —

1 109 C SuNDF 8 0.1118 0.1202 0.1076 6.76 — — — — — — — — — — — — — —

1 110 C SuNDF 8 0.1114 0.1204 0.1041 6.78 — — — — — — — — — — — — — —

1 111 C iNDF 8 0.2230 0.0000 0.2052 7.14 — — — — — — — — — — — — — —

1 112 C iNDF 8 0.2228 0.0000 0.2067 7.18 — — — — — — — — — — — — — —

1 113 U BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.1650 0.2450 1.0830 21.8470

1 114 U BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.1844 0.2715 0.7072 16.3340

1 115 U SuNDF 8 0.1115 0.1202 — — — 4.6759 0.0000 0.1543 0.1040 26.9550 6.7430 3.3123 1.3538 0.1532 0.1650 0.2600 0.9145 17.5515

1 116 U SuNDF 8 0.1115 0.1200 — — — 4.3556 0.0000 0.0000 0.0000 27.5300 6.2880 3.4923 1.2788 0.0082 0.1500 0.1950 1.0946 19.2755

1 119 U SuNDF 8 0.1116 0.1201 — — 10.8814 — — — — — — — — — — — — —

1 120 U SuNDF 8 0.1115 0.1200 — — 9.5706 — — — — — — — — — — — — —

183



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

1 121 U iNDF 8 0.2231 0.0000 — — 0.4402 — — — — — — — — — — — — —

1 122 U iNDF 8 0.2231 0.0000 — — — — — — — — — — — — — — — —

1 123 U SuNDF 8 0.1116 0.1205 0.1059 6.92 — — — — — — — — — — — — — —

1 124 U SuNDF 8 0.1114 0.1204 0.1060 6.88 — — — — — — — — — — — — — —

1 125 U iNDF 8 0.2229 0.0000 0.2060 7.45 — — — — — — — — — — — — — —

1 126 U iNDF 8 0.2228 0.0000 0.2068 7.46 — — — — — — — — — — — — — —

1 127 B BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.0000 0.0000 7.5122 4.7885

1 128 B BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.0000 0.0000 11.8436 9.7923

1 129 B SuNDF 12 0.1118 0.1200 — — — — — — — — — — — — 0.0000 0.0000 12.0368 8.0988

1 130 B SuNDF 12 0.1116 0.1201 — — — — — — — — — — — — 0.0000 0.0000 11.0489 9.7332

1 133 B SuNDF 12 0.1118 0.1200 — — — — — — — — — — — — — — — —

1 134 B SuNDF 12 0.1117 0.1201 — — — — — — — — — — — — — — — —

1 135 B iNDF 12 0.2229 0.0000 — — — — — — — — — — — — — — — —

1 136 B iNDF 12 0.2228 0.0000 — — — — — — — — — — — — — — — —

1 137 B SuNDF 12 0.1114 0.1200 0.1062 — — — — — — — — — — — — — — —

1 138 B SuNDF 12 0.1116 0.1203 0.1068 — — — — — — — — — — — — — — —

1 139 B iNDF 12 0.2229 0.0000 0.2119 — — — — — — — — — — — — — — —

1 140 B iNDF 12 0.2231 0.0000 0.2108 — — — — — — — — — — — — — — —

1 141 C BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.3050 0.6850 11.2495 10.1219

1 142 C BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.3641 0.7751 10.8827 10.3974

1 143 C SuNDF 12 0.1114 0.1201 — — — 4.0313 0.1150 1.5471 0.1356 11.0900 15.1458 9.4453 2.7344 0.1353 0.2550 0.6900 8.4321 3.7682

1 144 C SuNDF 12 0.1118 0.1201 — — — 3.7751 0.0017 1.4982 0.1507 10.2700 16.0308 15.4703 2.7694 0.1303 0.2500 0.6950 8.9749 4.4018

1 147 C SuNDF 12 0.1117 0.1200 — — — — — — — — — — — — — — — —

1 148 C SuNDF 12 0.1118 0.1203 — — 14.1618 — — — — — — — — — — — — —

1 149 C iNDF 12 0.2229 0.0000 — — 3.8999 — — — — — — — — — — — — —

1 150 C iNDF 12 0.2229 0.0000 — — 2.2570 — — — — — — — — — — — — —

1 151 C SuNDF 12 0.1118 0.1204 0.0989 6.68 — — — — — — — — — — — — — —

1 152 C SuNDF 12 0.1117 0.1200 0.0946 6.67 — — — — — — — — — — — — — —

184



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

1 153 C iNDF 12 0.2230 0.0000 0.1874 7.14 — — — — — — — — — — — — — —

1 154 C iNDF 12 0.2229 0.0000 0.1974 7.19 — — — — — — — — — — — — — —

1 155 U BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.1871 0.4046 1.2769 22.8742

1 156 U BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.2150 0.3300 1.0929 21.0903

1 157 U SuNDF 12 0.1116 0.1204 — — — 2.8944 0.0000 -0.0023 -0.0027 24.1550 9.4944 5.3598 1.6400 -0.0509 0.1550 0.2150 1.2279 20.1872

1 158 U SuNDF 12 0.1116 0.1203 — — — 2.4300 0.0000 -0.0023 -0.0027 24.8923 8.7713 5.2680 1.5314 -0.0519 0.1582 0.2245 1.2128 20.0421

1 161 U SuNDF 12 0.1116 0.1204 — — 11.0402 — — — — — — — — — — — — —

1 162 U SuNDF 12 0.1117 0.1205 — — 9.8866 — — — — — — — — — — — — —

1 163 U iNDF 12 0.2229 0.0000 — — 0.9415 — — — — — — — — — — — — —

1 164 U iNDF 12 0.2231 0.0000 — — -0.8741 — — — — — — — — — — — — —

1 165 U SuNDF 12 0.1118 0.1203 0.1003 6.92 — — — — — — — — — — — — — —

1 166 U SuNDF 12 0.1113 0.1202 0.1038 6.84 — — — — — — — — — — — — — —

1 167 U iNDF 12 0.2227 0.0000 0.1963 7.40 — — — — — — — — — — — — — —

1 168 U iNDF 12 0.2230 0.0000 0.1997 7.41 — — — — — — — — — — — — — —

1 169 B BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.5300 1.1900 4.8012 19.8484

1 170 B BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.6050 1.2600 3.8819 18.9232

1 171 B SuNDF 16 0.1118 0.1203 — — — 1.3011 0.0019 0.6060 0.0958 0.0000 19.5190 17.7878 5.5122 2.0769 0.7703 1.2335 2.1426 12.6875

1 172 B SuNDF 16 0.1115 0.1202 — — — 1.9256 0.0135 0.6011 0.1361 0.0000 20.0625 16.5150 5.8675 2.1350 0.7550 1.1750 2.3582 14.2038

1 175 B SuNDF 16 0.1115 0.1201 — — 16.4290 — — — — — — — — — — — — —

1 176 B SuNDF 16 0.1114 0.1204 — — 15.6029 — — — — — — — — — — — — —

1 177 B iNDF 16 0.2231 0.0000 — — 5.1579 — — — — — — — — — — — — —

1 178 B iNDF 16 0.2230 0.0000 — — 3.5236 — — — — — — — — — — — — —

1 179 B SuNDF 16 0.1116 0.1204 0.0844 6.78 — — — — — — — — — — — — — —

1 180 B SuNDF 16 0.1118 0.1201 0.0829 6.82 — — — — — — — — — — — — — —

1 181 B iNDF 16 0.2231 0.0000 0.1754 7.11 — — — — — — — — — — — — — —

1 182 B iNDF 16 0.2231 0.0000 0.1795 7.11 — — — — — — — — — — — — — —

1 183 C BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.6852 1.6678 — —

1 184 C BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.8405 1.6558 6.1693 13.3502

185



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

1 185 C SuNDF 16 0.1117 0.1200 — — — 2.7022 -0.0073 0.7594 0.1552 0.0000 19.3760 19.6817 5.4412 2.1682 1.2650 2.1200 3.5002 6.5415

1 186 C SuNDF 16 0.1118 0.1202 — — — 1.4532 -0.0430 0.7459 0.1316 0.0000 19.5860 19.9567 5.7512 2.2682 1.3100 2.0400 4.7521 9.2913

1 189 C SuNDF 16 0.1117 0.1204 — — 16.9135 — — — — — — — — — — — — —

1 190 C SuNDF 16 0.1115 0.1204 — — 16.9167 — — — — — — — — — — — — —

1 191 C iNDF 16 0.2232 0.0000 — — 5.1557 — — — — — — — — — — — — —

1 192 C iNDF 16 0.2231 0.0000 — — 3.9278 — — — — — — — — — — — — —

1 193 C SuNDF 16 0.1114 0.1201 0.0854 6.82 — — — — — — — — — — — — — —

1 194 C SuNDF 16 0.1115 0.1201 0.0829 6.79 — — — — — — — — — — — — — —

1 195 C iNDF 16 0.2231 0.0000 0.1831 7.09 — — — — — — — — — — — — — —

1 196 C iNDF 16 0.2231 0.0000 0.1828 7.05 — — — — — — — — — — — — — —

1 197 U BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.3046 0.2945 1.2034 21.8217

1 198 U BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.1950 0.2500 1.0264 19.9790

1 199 U SuNDF 16 0.1114 0.1201 — — — 1.4612 0.0000 -0.0045 -0.0051 18.7631 16.4570 9.3558 2.7620 0.1220 0.1822 0.1822 1.1479 19.1339

1 200 U SuNDF 16 0.1115 0.1203 — — — 1.7014 0.0000 -0.0045 -0.0051 18.7300 14.8455 9.2419 2.4601 0.1691 0.1500 0.1350 1.2135 19.8234

1 203 U SuNDF 16 0.1119 0.1205 — — 10.0886 — — — — — — — — — — — — —

1 204 U SuNDF 16 0.1114 0.1201 — — 10.1044 — — — — — — — — — — — — —

1 205 U iNDF 16 0.2228 0.0000 — — 3.6154 — — — — — — — — — — — — —

1 206 U iNDF 16 0.2232 0.0000 — — 3.8407 — — — — — — — — — — — — —

1 207 U SuNDF 16 0.1118 0.1202 0.0942 6.93 — — — — — — — — — — — — — —

1 208 U SuNDF 16 0.1116 0.1203 0.0899 6.88 — — — — — — — — — — — — — —

1 209 U iNDF 16 0.2231 0.0000 0.1760 7.30 — — — — — — — — — — — — — —

1 210 U iNDF 16 0.2231 0.0000 0.1868 7.32 — — — — — — — — — — — — — —

2 1 B BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1518 0.1822 8.9943 9.0308

2 2 B BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.0900 0.2050 7.8397 7.9636

2 3 B SuNDF 0 0.1115 0.1201 — — — 0.0841 59.3853 -1.7158 -1.4389 0.2450 1.2438 0.2246 0.1299 0.0242 0.2050 0.1900 7.5064 7.5691

2 4 B SuNDF 0 0.1116 0.1205 — — — 0.1962 92.7013 -1.6710 -1.4389 0.1682 1.0071 0.1857 0.1094 0.0316 0.1122 0.1835 — —

2 7 B SuNDF 0 0.1113 0.1200 — — 2.0956 — — — — — — — — — — — — —

2 8 B SuNDF 0 0.1114 0.1200 — — -2.0965 — — — — — — — — — — — — —

186



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

2 9 B iNDF 0 0.2227 0.0000 — — 1.5665 — — — — — — — — — — — — —

2 10 B iNDF 0 0.2230 0.0000 — — -1.5684 — — — — — — — — — — — — —

2 11 B SuNDF 0 0.1116 0.1202 0.1093 7.06 — — — — — — — — — — — — — —

2 12 B SuNDF 0 0.1116 0.1200 0.1094 7.04 — — — — — — — — — — — — — —

2 13 B iNDF 0 0.2228 0.0000 0.2136 7.04 — — — — — — — — — — — — — —

2 14 B iNDF 0 0.2229 0.0000 0.2139 7.00 — — — — — — — — — — — — — —

2 15 C BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1409 0.2114 11.8680 0.8926

2 16 C BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1800 0.1500 8.9579 0.6736

2 17 C SuNDF 0 0.1117 0.1198 — — — 0.1962 76.3971 -0.4398 -0.6956 0.1850 0.6578 -0.0363 -0.0707 0.0072 0.0750 0.1900 11.9896 0.9974

2 18 C SuNDF 0 0.1115 0.1201 — — — 0.1241 68.7214 -0.9733 -0.8582 0.2079 0.6529 -0.0292 -0.0364 0.0489 0.1673 0.2231 14.6845 1.2873

2 21 C SuNDF 0 0.1115 0.1198 — — 1.3966 — — — — — — — — — — — — —

2 22 C SuNDF 0 0.1112 0.1201 — — -1.3968 — — — — — — — — — — — — —

2 23 C iNDF 0 0.2228 0.0000 — — -0.6139 — — — — — — — — — — — — —

2 24 C iNDF 0 0.2229 0.0000 — — 0.6141 — — — — — — — — — — — — —

2 25 C SuNDF 0 0.1115 0.1202 0.1088 7.06 — — — — — — — — — — — — — —

2 26 C SuNDF 0 0.1116 0.1199 0.1078 6.93 — — — — — — — — — — — — — —

2 27 C iNDF 0 0.2228 0.0000 0.2133 6.99 — — — — — — — — — — — — — —

2 28 C iNDF 0 0.2228 0.0000 0.2129 6.98 — — — — — — — — — — — — — —

2 29 U BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1365 0.1517 0.4707 8.7029

2 30 U BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1150 0.2050 0.3328 5.7084

2 31 U SuNDF 0 0.1117 0.1199 — — — 0.2562 25.1212 0.0000 0.0000 0.1650 0.3230 -0.0156 -0.0048 0.0044 0.1500 0.2050 0.4424 8.2335

2 32 U SuNDF 0 0.1115 0.1200 — — — 0.1681 7.9664 0.0000 0.0000 0.1700 0.3280 0.2244 -0.0898 -0.0506 0.0550 0.0950 0.3313 5.8361

2 35 U SuNDF 0 0.1115 0.1200 — — -0.8892 — — — — — — — — — — — — —

2 36 U SuNDF 0 0.1118 0.1204 — — 0.8918 — — — — — — — — — — — — —

2 37 U iNDF 0 0.2231 0.0000 — — 1.0268 — — — — — — — — — — — — —

2 38 U iNDF 0 0.2231 0.0000 — — -1.0268 — — — — — — — — — — — — —

2 39 U SuNDF 0 0.1116 0.1201 0.1098 7.05 — — — — — — — — — — — — — —

2 40 U SuNDF 0 0.1115 0.1201 0.1084 6.99 — — — — — — — — — — — — — —

187



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

2 41 U iNDF 0 0.2231 0.0000 0.2142 7.01 — — — — — — — — — — — — — —

2 42 U iNDF 0 0.2231 0.0000 0.2128 7.05 — — — — — — — — — — — — — —

2 43 B BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.1300 0.2700 6.9974 8.9578

2 44 B BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.1853 0.2654 3.8949 5.1360

2 45 B SuNDF 4 0.1117 0.1204 — — — 7.6183 -0.7654 -1.2565 0.4098 26.5988 5.1206 2.0789 0.8181 0.0001 0.1333 0.2409 6.7273 6.7012

2 46 B SuNDF 4 0.1115 0.1204 — — — 6.9377 -0.2506 -0.4931 -0.1900 24.6650 3.8989 1.7862 0.9660 -0.0002 0.1550 0.3050 8.4593 8.3316

2 49 B SuNDF 4 0.1116 0.1200 — — 7.8787 — — — — — — — — — — — — —

2 50 B SuNDF 4 0.1116 0.1198 — — 5.7488 — — — — — — — — — — — — —

2 51 B iNDF 4 0.2230 0.0000 — — 0.2798 — — — — — — — — — — — — —

2 52 B iNDF 4 0.2228 0.0000 — — -0.9434 — — — — — — — — — — — — —

2 53 B SuNDF 4 0.1115 0.1197 0.1102 6.73 — — — — — — — — — — — — — —

2 54 B SuNDF 4 0.1117 0.1204 0.1099 6.56 — — — — — — — — — — — — — —

2 55 B iNDF 4 0.2228 0.0000 0.2100 7.17 — — — — — — — — — — — — — —

2 56 B iNDF 4 0.2230 0.0000 0.2095 7.09 — — — — — — — — — — — — — —

2 57 C BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.1750 0.2850 7.8430 1.6254

2 58 C BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.0662 0.3259 13.0860 2.4937

2 59 C SuNDF 4 0.1116 0.1202 — — — 7.2540 -1.2484 -2.1980 -0.7270 22.5850 4.6534 3.3762 0.7530 -0.0068 0.1400 0.2350 . .

2 60 C SuNDF 4 0.1116 0.1200 — — — 7.0939 -0.3451 -0.7110 1.9282 27.4742 14.6051 5.3060 1.9624 0.5430 1.2988 0.3804 9.0842 0.5141

2 63 C SuNDF 4 0.1117 0.1197 — — 7.8431 — — — — — — — — — — — — —

2 64 C SuNDF 4 0.1117 0.1205 — — 8.1535 — — — — — — — — — — — — —

2 65 C iNDF 4 0.2231 0.0000 — — 0.2361 — — — — — — — — — — — — —

2 66 C iNDF 4 0.2231 0.0000 — — 0.6468 — — — — — — — — — — — — —

2 67 C SuNDF 4 0.1116 0.1201 0.1092 6.60 — — — — — — — — — — — — — —

2 68 C SuNDF 4 0.1118 0.1200 0.1091 6.60 — — — — — — — — — — — — — —

2 69 C iNDF 4 0.2229 0.0000 0.2105 7.13 — — — — — — — — — — — — — —

2 70 C iNDF 4 0.2230 0.0000 0.2088 6.96 — — — — — — — — — — — — — —

2 71 U BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.0806 0.2418 0.7180 14.4564

2 72 U BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.7347 0.2503 0.6476 12.7626

188



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

2 73 U SuNDF 4 0.1113 0.1204 — — — 6.8297 0.0000 0.0000 0.0000 18.0891 2.4482 0.7602 0.3818 0.0430 0.1566 0.1768 1.0867 18.6572

2 74 U SuNDF 4 0.1118 0.1200 — — — 7.3421 0.0000 0.0000 0.0000 26.7540 9.5730 2.9343 0.6037 -0.0933 0.0213 0.0414 0.6877 12.6292

2 77 U SuNDF 4 0.1114 0.1201 — — 0.0963 — — — — — — — — — — — — —

2 78 U SuNDF 4 0.1117 0.1197 — — 0.4295 — — — — — — — — — — — — —

2 79 U iNDF 4 0.2228 0.0000 — — -4.2130 — — — — — — — — — — — — —

2 80 U iNDF 4 0.2232 0.0000 — — -4.0778 — — — — — — — — — — — — —

2 81 U SuNDF 4 0.1115 0.1205 0.1081 6.62 — — — — — — — — — — — — — —

2 82 U SuNDF 4 0.1116 0.1200 0.1095 6.80 — — — — — — — — — — — — — —

2 83 U iNDF 4 0.2231 0.0000 0.2118 7.32 — — — — — — — — — — — — — —

2 84 U iNDF 4 0.2231 0.0000 0.2098 7.22 — — — — — — — — — — — — — —

2 85 B BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.1654 0.3558 4.5496 7.1464

2 86 B BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.9418 0.3384 5.8547 9.0172

2 87 B SuNDF 8 0.1117 0.1203 — — — — -0.0172 0.0991 0.1044 26.8058 4.3508 2.8875 0.6461 -0.3111 0.1012 0.1467 5.9177 9.6471

2 88 B SuNDF 8 0.1118 0.1204 — — — — 0.0017 0.0864 0.0385 27.3500 4.2403 2.7308 0.5581 -0.2474 0.1050 0.1850 5.5600 9.2909

2 91 B SuNDF 8 0.1117 0.1201 — — 6.9283 — . . . — — — — — — — — —

2 92 B SuNDF 8 0.1117 0.1202 — — 8.4861 — . . . — — — — — — — — —

2 93 B iNDF 8 0.0000 0.0000 — — 0.4718 — . . . — — — — — — — — —

2 94 B iNDF 8 0.0000 0.0000 — — 0.1432 — . . . — — — — — — — — —

2 95 B SuNDF 8 0.1117 0.1202 0.1069 6.74 — — . . . — — — — — — — — —

2 96 B SuNDF 8 0.1115 0.1200 0.1071 6.76 — — . . . — — — — — — — — —

2 97 B iNDF 8 0.2231 0.0000 0.2089 7.18 — — . . . — — — — — — — — —

2 98 B iNDF 8 0.2228 0.0000 0.2075 7.17 — — . . . — — — — — — — — —

2 99 C BL 8 0.0000 0.0000 — — — — . . . — — — — — 0.1430 0.2605 9.7354 2.9712

2 100 C BL 8 0.0000 0.0000 — — — — . . . — — — — — 0.1500 0.2600 — —

2 101 C SuNDF 8 0.1115 0.1198 — — — 5.3684 -0.1891 0.7839 -0.1310 29.6757 9.6237 5.3019 1.6147 0.2293 0.1930 0.4469 10.7154 1.1109

2 102 C SuNDF 8 0.1116 0.1206 — — — 5.2003 -0.1570 0.7802 -0.1021 — — — — — — — 6.5473 0.7817

2 105 C SuNDF 8 0.1115 0.1199 — — 10.9643 — — — — — — — — — — — — —

2 106 C SuNDF 8 0.1114 0.1201 — — 9.5654 — — — — — — — — — — — — —

189



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

2 107 C iNDF 8 0.2229 0.0000 — — 1.8052 — — — — — — — — — — — — —

2 108 C iNDF 8 0.2228 0.0000 — — 2.3023 — — — — — — — — — — — — —

2 109 C SuNDF 8 0.1118 0.1202 0.1081 6.75 — — — — — — — — — — — — — —

2 110 C SuNDF 8 0.1114 0.1201 0.1077 6.74 — — — — — — — — — — — — — —

2 111 C iNDF 8 0.2229 0.0000 0.2083 7.19 — — — — — — — — — — — — — —

2 112 C iNDF 8 0.2228 0.0000 0.1995 7.23 — — — — — — — — — — — — — —

2 113 U BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.1700 0.2250 1.1214 20.5233

2 114 U BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.9427 0.4204 — —

2 115 U SuNDF 8 0.1114 0.1203 — — — 4.5758 0.0000 0.0168 0.0000 27.9800 5.3323 2.2130 0.7039 -0.3977 0.1000 0.1700 0.7808 13.2187

2 116 U SuNDF 8 0.1116 0.1199 — — — 4.8720 0.0000 0.0194 0.0000 38.3669 16.6379 6.4527 2.4594 0.1783 1.3277 0.6591 0.6853 11.5208

2 119 U SuNDF 8 0.1114 0.1202 — — 5.8836 — — — — — — — — — — — — —

2 120 U SuNDF 8 0.1115 0.1201 — — 5.3089 — — — — — — — — — — — — —

2 121 U iNDF 8 0.2231 0.0000 — — -0.4107 — — — — — — — — — — — — —

2 122 U iNDF 8 0.2229 0.0000 — — -0.8919 — — — — — — — — — — — — —

2 123 U SuNDF 8 0.1117 0.1204 0.1079 6.89 — — — — — — — — — — — — — —

2 124 U SuNDF 8 0.1117 0.1202 0.1070 6.88 — — — — — — — — — — — — — —

2 125 U iNDF 8 0.2230 0.0000 — 7.46 — — — — — — — — — — — — — —

2 126 U iNDF 8 0.2230 0.0000 0.2084 7.40 — — — — — — — — — — — — — —

2 127 B BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.2041 0.3826 — —

2 128 B BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.7605 0.2569 7.8718 13.5572

2 129 B SuNDF 12 0.1116 0.1201 — — — 2.1738 -0.0833 1.0254 0.0933 14.7223 17.7684 13.6165 3.1798 0.5332 1.3204 0.4627 6.1341 8.7851

2 130 B SuNDF 12 0.1114 0.1202 — — — 1.7895 -0.0843 1.0384 0.1131 14.6489 15.7718 12.9972 3.1248 0.4540 1.2944 0.4261 5.9623 8.4822

2 133 B SuNDF 12 0.1116 0.1203 — — 14.3182 — — — — — — — — — — — — —

2 134 B SuNDF 12 0.1116 0.1203 — — 12.5932 — — — — — — — — — — — — —

2 135 B iNDF 12 0.2230 0.0000 — — 2.4567 — — — — — — — — — — — — —

2 136 B iNDF 12 0.2227 0.0000 — — 2.4701 — — — — — — — — — — — — —

2 137 B SuNDF 12 0.1116 0.1198 0.1034 6.79 — — — — — — — — — — — — — —

2 138 B SuNDF 12 0.1118 0.1205 0.1013 6.78 — — — — — — — — — — — — — —

190



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

2 139 B iNDF 12 0.2230 0.0000 0.1987 7.17 — — — — — — — — — — — — — —

2 140 B iNDF 12 0.2228 0.0000 0.2004 7.20 — — — — — — — — — — — — — —

2 141 C BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.2300 0.5000 11.8329 6.1511

2 142 C BL 12 0.0000 0.0000 — — — — — — — — — — — — 1.2291 0.3754 — —

2 143 C SuNDF 12 0.1116 0.1202 — — — 2.0137 0.1108 2.7554 0.0887 17.7450 13.1773 12.4608 1.1735 -0.0495 0.2150 0.4500 9.8320 1.6927

2 144 C SuNDF 12 0.1116 0.1202 — — — 2.2539 0.1129 2.6451 0.0300 18.6508 16.7101 12.3763 2.5894 0.1335 0.0000 0.3948 11.3770 2.3943

2 147 C SuNDF 12 0.1114 0.1203 — — 11.8198 — — — — — — — — — — — — —

2 148 C SuNDF 12 0.1115 0.1204 — — 13.3762 — — — — — — — — — — — — —

2 149 C iNDF 12 0.2229 0.0000 — — 4.8857 — — — — — — — — — — — — —

2 150 C iNDF 12 0.2231 0.0000 — — 3.9695 — — — — — — — — — — — — —

2 151 C SuNDF 12 0.1115 0.1198 0.1016 6.81 — — — — — — — — — — — — — —

2 152 C SuNDF 12 0.1115 0.1202 0.1028 6.75 — — — — — — — — — — — — — —

2 153 C iNDF 12 0.2229 0.0000 0.1976 7.25 — — — — — — — — — — — — — —

2 154 C iNDF 12 0.2229 0.0000 0.2098 7.25 — — — — — — — — — — — — — —

2 155 U BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.1938 0.3621 — —

2 156 U BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.1800 0.3700 0.8864 15.2510

2 157 U SuNDF 12 0.1115 0.1203 — — — 1.9096 0.0000 0.0149 0.0000 26.7600 7.3130 3.3277 1.2936 -0.1206 0.1400 0.2050 1.1826 16.8542

2 158 U SuNDF 12 0.1116 0.1197 — — — 1.3411 0.0000 0.0107 0.0000 30.0198 12.2201 5.9992 2.1148 0.3575 1.1539 0.6028 — —

2 161 U SuNDF 12 0.1114 0.1200 — — 8.2325 — — — — — — — — — — — — —

2 162 U SuNDF 12 0.1114 0.1204 — — 6.2456 — — — — — — — — — — — — —

2 163 U iNDF 12 0.2231 0.0000 — — -3.7376 — — — — — — — — — — — — —

2 164 U iNDF 12 0.2230 0.0000 — — -3.6496 — — — — — — — — — — — — —

2 165 U SuNDF 12 0.1116 0.1201 0.1018 7.00 — — — — — — — — — — — — — —

2 166 U SuNDF 12 0.1114 0.1202 0.1028 7.03 — — — — — — — — — — — — — —

2 167 U iNDF 12 0.2232 0.0000 0.1988 7.53 — — — — — — — — — — — — — —

2 168 U iNDF 12 0.2232 0.0000 0.1991 7.53 — — — — — — — — — — — — — —

2 169 B BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.2450 0.8100 7.6387 16.7876

2 170 B BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.9816 0.3862 5.6819 13.2920

191



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

2 171 B SuNDF 16 0.1115 0.1202 — — — 1.1930 -0.1320 0.9765 0.1480 0.0000 17.2463 18.1684 5.1200 2.6325 0.7350 1.1600 3.6623 11.3550

2 172 B SuNDF 16 0.1117 0.1200 — — — 1.2330 -0.1126 1.0459 0.0103 0.0000 23.2660 18.8919 5.9412 3.2747 1.8643 0.6614 3.3673 11.8837

2 175 B SuNDF 16 0.1117 0.1204 — — 13.0393 — — — — — — — — — — — — —

2 176 B SuNDF 16 0.1118 0.1204 — — 12.0508 — — — — — — — — — — — — —

2 177 B iNDF 16 0.2228 0.0000 — — 2.7531 — — — — — — — — — — — — —

2 178 B iNDF 16 0.2229 0.0000 — — 2.9951 — — — — — — — — — — — — —

2 179 B SuNDF 16 0.1113 0.1201 0.0923 6.89 — — — — — — — — — — — — — —

2 180 B SuNDF 16 0.1117 0.1205 0.0931 6.88 — — — — — — — — — — — — — —

2 181 B iNDF 16 0.2231 0.0000 0.1859 7.28 — — — — — — — — — — — — — —

2 182 B iNDF 16 0.2230 0.0000 0.1865 7.24 — — — — — — — — — — — — — —

2 183 C BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.2050 1.0200 — —

2 184 C BL 16 0.0000 0.0000 — — — — — — — — — — — — 1.1109 0.3125 9.9598 11.0693

2 185 C SuNDF 16 0.1116 0.1200 — — — 0.9328 -0.1478 1.5319 0.1024 -0.0599 15.8866 17.1346 5.3744 2.7667 0.8300 1.8150 5.4314 4.0210

2 186 C SuNDF 16 0.1116 0.1204 — — — 1.3331 -0.1880 1.5069 0.1135 -0.0599 15.2566 16.9346 5.1894 2.9667 1.0200 1.8900 6.7007 5.0380

2 189 C SuNDF 16 0.1116 0.1201 — — 15.2291 — — — — — — — — — — — — —

2 190 C SuNDF 16 0.1116 0.1204 — — 13.7437 — — — — — — — — — — — — —

2 191 C iNDF 16 0.2230 0.0000 — — 1.8832 — — — — — — — — — — — — —

2 192 C iNDF 16 0.2230 0.0000 — — 3.0332 — — — — — — — — — — — — —

2 193 C SuNDF 16 0.1115 0.1200 0.0951 6.85 — — — — — — — — — — — — — —

2 194 C SuNDF 16 0.1116 0.1201 0.0957 6.86 — — — — — — — — — — — — — —

2 195 C iNDF 16 0.2228 0.0000 0.1917 7.22 — — — — — — — — — — — — — —

2 196 C iNDF 16 0.2228 0.0000 0.1912 7.17 — — — — — — — — — — — — — —

2 197 U BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.1400 0.1300 1.3455 19.4901

2 198 U BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.1300 0.2400 1.8095 23.0433

2 199 U SuNDF 16 0.1117 0.1205 — — — 1.1209 0.0000 0.0049 -0.0019 21.9156 12.7881 7.3729 2.3051 0.1545 0.1159 0.1159 1.3812 17.9959

2 200 U SuNDF 16 0.1115 0.1199 — — — 1.2010 0.0000 0.0097 -0.0019 23.5400 17.3025 7.0650 2.0450 0.1275 0.0900 0.1300 — —

2 203 U SuNDF 16 0.1116 0.1204 — — 5.5402 — — — — — — — — — — — — —

2 204 U SuNDF 16 0.1114 0.1203 — — 6.4548 — — — — — — — — — — — — —

192



rNDF

(g OM) pH MCP (mg)

GLY (mg)

rSuc (mg)

rGlc (mg)

rFruc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

Total AA (mM)

NH3-N (mM)

2 205 U iNDF 16 0.2229 0.0000 — — 0.5867 — — — — — — — — — — — — —

2 206 U iNDF 16 0.2230 0.0000 — — -0.9799 — — — — — — — — — — — — —

2 207 U SuNDF 16 0.1114 0.1202 0.0995 7.00 — — — — — — — — — — — — — —

2 208 U SuNDF 16 0.1117 0.1203 0.0986 6.96 — — — — — — — — — — — — — —

2 209 U iNDF 16 0.2228 0.0000 0.1875 7.47 — — — — — — — — — — — — — —

2 210 U iNDF 16 0.2228 0.0000 0.1937 7.50 — — — — — — — — — — — — — — 1 Ferm = fermentation; Med = medium: B = Non-protein + true protein nitrogen, C = True protein nitrogen only, U = Non-protein nitrogen only; Sub = substrate: BL = no substrate; iNDF = isolated bermudagrass neutral detergent residue, SuNDF = sucrose + iNDF; rNDF = residual NDF; MCP = microbial crude protein; GLY = microbial glycogen; rSuc = residual sucrose; rGlc = residual glucose; rFruc = residual fructose; Lac = lactate; C2 = acetate; C3 = propionate; C4 = butyrate; Val = valerate; Isobut = isobutyrate; Isoval+2MB = isovalerate + 2-Methylbutyrate; AA = free amino acids; NH3-N = ammonia nitrogen

193

APPENDIX G CHAPTER 4 RAW DATA

Table G-1. Data used for statistical analysis in evaluating the effect on microbial yield and neutral detergen fiber digestion from in vitro fermentations of different sources (sucrose, starch and pectin), amounts (0, 40, 80 and 120 mg nominal hexose equivalents) and combinations (sucrose+starch, starch+pectin and sucrose+pectin) of NFCs.

Ferm1 Tube ID Trt Hr iNDF

(g DM) Starch (g DM)

Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 1 BL 0 0.0000 0.0000 0.0000 0.0000 7.21 — — — — — — — — — — — — —

1 2 BL 0 0.0000 0.0000 0.0000 0.0000 7.20 — — — — — — — — — — — — —

1 3 NDF 0 0.1156 0.0000 0.0000 0.0000 7.23 97.5345 — — — — — — — — — — — —

1 4 NDF 0 0.1159 0.0000 0.0000 0.0000 7.21 98.6355 — — — — — — — — — — — —

1 5 Su40St0 0 0.1159 0.0000 0.0383 0.0000 7.11 97.7392 — — — — — — — — — — — —

1 6 Su40St0 0 0.1160 0.0000 0.0381 0.0000 7.14 — — — — — — — — — — — — —

1 7 Su80St0 0 0.1156 0.0000 0.0762 0.0000 7.23 98.6127 — — — — — — — — — — — —

1 8 Su80St0 0 0.1159 0.0000 0.0764 0.0000 7.24 98.0977 — — — — — — — — — — — —

1 9 Su120St0 0 0.1157 0.0000 0.1139 0.0000 7.22 97.7226 — — — — — — — — — — — —

1 10 Su120St0 0 0.1159 0.0000 0.1144 0.0000 7.21 98.3666 — — — — — — — — — — — —

1 11 Su0St40 0 0.1157 0.0317 0.0000 0.0000 7.13 99.3386 — — — — — — — — — — — —

1 12 Su0St40 0 0.1160 0.0320 0.0000 0.0000 7.11 99.0908 — — — — — — — — — — — —

1 13 Su0St80 0 0.1161 0.0641 0.0000 0.0000 7.13 99.5454 — — — — — — — — — — — —

1 14 Su0St80 0 0.1162 0.0639 0.0000 0.0000 7.10 98.6582 — — — — — — — — — — — —

1 15 Su0St120 0 0.1159 0.0957 0.0000 0.0000 7.23 98.5459 — — — — — — — — — — — —

1 16 Su0St120 0 0.1161 0.0959 0.0000 0.0000 7.21 98.0243 — — — — — — — — — — — —

1 17 Su40St80 0 0.1157 0.0637 0.0380 0.0000 7.19 102.9297 — — — — — — — — — — — —

194



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 18 Su40St80 0 0.1159 0.0637 0.0385 0.0000 7.12 98.4563 — — — — — — — — — — — —

1 19 Su80St40 0 0.1158 0.0320 0.0762 0.0000 7.11 97.5515 — — — — — — — — — — — —

1 20 Su80St40 0 0.1157 0.0319 0.0759 0.0000 7.13 97.7226 — — — — — — — — — — — —

1 23 NDF 0 0.1161 0.0000 0.0000 0.0000 — — -0.1575 — — — — — — — — — — —

1 24 NDF 0 0.1160 0.0000 0.0000 0.0000 — — 0.1574 — — — — — — — — — — —

1 25 Su40St0 0 0.1159 0.0000 0.0385 0.0000 — — -0.1018 — — — — — — — — — — —

1 26 Su40St0 0 0.1161 0.0000 0.0385 0.0000 — — 0.1019 — — — — — — — — — — —

1 27 Su80St0 0 0.1158 0.0000 0.0762 0.0000 — — 0.1562 — — — — — — — — — — —

1 28 Su80St0 0 0.1157 0.0000 0.0763 0.0000 — — -0.1563 — — — — — — — — — — —

1 29 Su120St0 0 0.1159 0.0000 0.1140 0.0000 — — -0.0520 — — — — — — — — — — —

1 30 Su120St0 0 0.1157 0.0000 0.1142 0.0000 — — 0.0520 — — — — — — — — — — —

1 31 Su0St40 0 0.1161 0.0319 0.0000 0.0000 — — 0.3662 — — — — — — — — — — —

1 32 Su0St40 0 0.1160 0.0320 0.0000 0.0000 — — -0.3664 — — — — — — — — — — —

1 33 Su0St80 0 0.1160 0.0639 0.0000 0.0000 — — 0.7238 — — — — — — — — — — —

1 34 Su0St80 0 0.1159 0.0636 0.0000 0.0000 — — -0.7223 — — — — — — — — — — —

1 35 Su0St120 0 0.1157 0.0957 0.0000 0.0000 — — 0.1077 — — — — — — — — — — —

1 36 Su0St120 0 0.1160 0.0957 0.0000 0.0000 — — -0.1079 — — — — — — — — — — —

1 37 Su40St80 0 0.1159 0.0639 0.0382 0.0000 — — 0.5208 — — — — — — — — — — —

1 38 Su40St80 0 0.1158 0.0639 0.0383 0.0000 — — -0.5208 — — — — — — — — — — —

1 39 Su80St40 0 0.1159 0.0322 0.0763 0.0000 — — -0.7795 — — — — — — — — — — —

1 40 Su80St40 0 0.1160 0.0321 0.0764 0.0000 — — 0.7799 — — — — — — — — — — —

1 41 BL 0 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2154 0.2537

1 42 BL 0 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.1401 0.2007

1 43 NDF 0 0.1161 0.0000 0.0000 0.0000 — — — — 0.0709 0.0112 0.3015 -0.0178 -0.1719 0.1992 -0.0697 0.0185 0.0000 0.2915

1 44 NDF 0 0.1159 0.0000 0.0000 0.0000 — — — — -0.0134 -0.0012 -0.2511 -0.0178 -0.3394 -0.3481 -0.0554 -0.1408 0.1420 0.1882

1 45 Su40St0 0 0.1161 0.0000 0.0384 0.0000 — — — — 10.3684 9.2626 23.2217 0.4119 0.3427 0.1820 0.4238 -0.1076 0.2596 0.1782

1 46 Su40St0 0 0.1157 0.0000 0.0379 0.0000 — — — — 8.0466 8.0042 21.3679 0.3506 -0.4747 -0.3438 -0.1289 0.1942 0.1553 0.2445

1 47 Su80St0 0 0.1158 0.0000 0.0760 0.0000 — — — — 9.1230 7.4985 51.1067 0.4227 0.0378 -0.8901 -0.0610 -0.2119 0.1568 0.0000

195



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 48 Su80St0 0 0.1160 0.0000 0.0765 0.0000 — — — — 12.5706 11.3322 53.5819 0.2187 -0.3988 -0.1942 -0.0356 -0.0150 0.2242 0.2994

1 49 Su120St0 0 0.1157 0.0000 0.1139 0.0000 — — — — 12.6512 8.9090 72.0146 0.3659 -0.2642 -0.1027 -0.1139 -0.0060 0.1718 0.2998

1 50 Su120St0 0 0.1158 0.0000 0.1141 0.0000 — — — — 11.9466 11.1762 90.5539 0.2397 -0.2726 -0.2188 -0.0853 -0.0060 0.2101 0.2548

1 51 Su0St40 0 0.1160 0.0321 0.0000 0.0000 — — — — 0.0036 -0.0062 0.1838 -0.0178 -1.5632 -0.1670 -0.1214 -0.2119 0.0675 0.2435

1 52 Su0St40 0 0.1157 0.0321 0.0000 0.0000 — — — — -0.0108 0.0014 -0.2433 -0.0178 -0.1148 -0.2005 0.0172 0.0634 0.1665 0.3161

1 53 Su0St80 0 0.1161 0.0638 0.0000 0.0000 — — — — 0.0214 0.0119 0.2010 -0.0178 -0.5639 -0.1092 -0.0784 0.0172 0.2203 0.2382

1 54 Su0St80 0 0.1157 0.0639 0.0000 0.0000 — — — — -0.0108 -0.0038 -0.2823 -0.0178 -0.1352 -0.8377 -0.2387 0.0177 0.1918 0.0000

1 55 Su0St120 0 0.1162 0.0955 0.0000 0.0000 — — — — 0.0003 -0.0374 -0.3421 -0.0178 -0.1260 -0.0862 -0.0842 0.0195 0.2128 0.2794

1 56 Su0St120 0 0.1162 0.0955 0.0000 0.0000 — — — — -0.0134 -0.0090 -0.2719 -0.0178 -0.4120 -0.3285 0.0546 -0.2119 0.0000 0.1684

1 57 Su40St80 0 0.1159 0.0640 0.0383 0.0000 — — — — — — — 0.4219 0.3793 0.1699 0.0938 -0.0137 0.1730 0.3827

1 58 Su40St80 0 0.1159 0.0638 0.0384 0.0000 — — — — 9.9186 9.2522 15.5699 0.3498 0.0702 -0.0784 -0.0768 0.0084 0.1800 0.2380

1 59 Su80St40 0 0.1158 0.0320 0.0763 0.0000 — — — — 11.2394 9.6409 50.4697 0.3226 -0.1911 0.2491 -2.1860 -0.0527 0.0000 0.1954

1 60 Su80St40 0 0.1162 0.0322 0.0762 0.0000 — — — — 14.7026 14.0362 43.5979 0.5384 -0.2872 -0.1284 -0.0589 0.0200 0.1951 0.2737

1 61 BL 4 0.0000 0.0000 0.0000 0.0000 7.14 — — — — — — — — — — — — —

1 62 BL 4 0.0000 0.0000 0.0000 0.0000 7.09 — — — — — — — — — — — — —

1 63 NDF 4 0.1159 0.0000 0.0000 0.0000 7.16 95.3193 — — — — — — — — — — — —

1 64 NDF 4 0.1161 0.0000 0.0000 0.0000 7.15 95.7873 — — — — — — — — — — — —

1 65 Su40St0 4 0.1160 0.0000 0.0380 0.0000 6.99 92.3743 — — — — — — — — — — — —

1 66 Su40St0 4 0.1158 0.0000 0.0383 0.0000 6.99 97.8206 — — — — — — — — — — — —

1 67 Su80St0 4 0.1157 0.0000 0.0760 0.0000 6.86 95.3884 — — — — — — — — — — — —

1 68 Su80St0 4 0.1161 0.0000 0.0760 0.0000 6.85 93.0134 — — — — — — — — — — — —

1 69 Su120St0 4 0.1160 0.0000 0.1145 0.0000 6.73 95.7774 — — — — — — — — — — — —

1 70 Su120St0 4 0.1162 0.0000 0.1140 0.0000 6.69 99.0158 — — — — — — — — — — — —

1 71 Su0St40 4 0.1160 0.0320 0.0000 0.0000 7.14 92.5534 — — — — — — — — — — — —

1 72 Su0St40 4 0.1162 0.0318 0.0000 0.0000 7.13 92.8469 — — — — — — — — — — — —

1 73 Su0St80 4 0.1159 0.0640 0.0000 0.0000 7.11 90.8379 — — — — — — — — — — — —

1 74 Su0St80 4 0.1162 0.0637 0.0000 0.0000 7.11 90.8799 — — — — — — — — — — — —

1 75 Su0St120 4 0.1157 0.0957 0.0000 0.0000 7.14 92.5155 — — — — — — — — — — — —

196



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 76 Su0St120 4 0.1158 0.0955 0.0000 0.0000 7.15 91.9002 — — — — — — — — — — — —

1 77 Su40St80 4 0.1161 0.0640 0.0382 0.0000 7.02 90.8659 — — — — — — — — — — — —

1 78 Su40St80 4 0.1160 0.0636 0.0382 0.0000 7.01 92.1952 — — — — — — — — — — — —

1 79 Su80St40 4 0.1157 0.0319 0.0763 0.0000 6.82 91.6177 — — — — — — — — — — — —

1 80 Su80St40 4 0.1161 0.0321 0.0760 0.0000 6.86 92.9240 — — — — — — — — — — — —

1 83 NDF 4 0.1162 0.0000 0.0000 0.0000 — — 3.4324 — — — — — — — — — — —

1 84 NDF 4 0.1163 0.0000 0.0000 0.0000 — — 3.6381 — — — — — — — — — — —

1 85 Su40St0 4 0.1158 0.0000 0.0380 0.0000 — — 6.2121 — — — — — — — — — — —

1 86 Su40St0 4 0.1158 0.0000 0.0385 0.0000 — — 6.7206 — — — — — — — — — — —

1 87 Su80St0 4 0.1157 0.0000 0.0764 0.0000 — — 9.6818 — — — — — — — — — — —

1 88 Su80St0 4 0.1157 0.0000 0.0760 0.0000 — — 10.6259 — — — — — — — — — — —

1 89 Su120St0 4 0.1158 0.0000 0.1143 0.0000 — — 12.4921 — — — — — — — — — — —

1 90 Su120St0 4 0.1159 0.0000 0.1144 0.0000 — — 11.6563 — — — — — — — — — — —

1 91 Su0St40 4 0.1158 0.0323 0.0000 0.0000 — — 4.0550 — — — — — — — — — — —

1 92 Su0St40 4 0.1158 0.0319 0.0000 0.0000 — — 3.0313 — — — — — — — — — — —

1 93 Su0St80 4 0.1161 0.0641 0.0000 0.0000 — — 1.9125 — — — — — — — — — — —

1 94 Su0St80 4 0.1157 0.0640 0.0000 0.0000 — — 4.0092 — — — — — — — — — — —

1 95 Su0St120 4 0.1159 0.0956 0.0000 0.0000 — — 2.2400 — — — — — — — — — — —

1 96 Su0St120 4 0.1161 0.0959 0.0000 0.0000 — — 2.2248 — — — — — — — — — — —

1 97 Su40St80 4 0.1160 0.0638 0.0385 0.0000 — — 5.0382 — — — — — — — — — — —

1 98 Su40St80 4 0.1158 0.0637 0.0383 0.0000 — — 3.7042 — — — — — — — — — — —

1 99 Su80St40 4 0.1162 0.0322 0.0761 0.0000 — — 9.7871 — — — — — — — — — — —

1 100 Su80St40 4 0.1159 0.0321 0.0762 0.0000 — — 9.8985 — — — — — — — — — — —

1 101 BL 4 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2706 0.4626

1 102 BL 4 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2559 0.4521

1 103 NDF 4 0.1163 0.0000 0.0000 0.0000 — — — 0.2851 0.0247 -0.0602 0.0051 0.0434 -1.2708 0.3563 0.1927 0.0449 0.3319 0.6201

1 104 NDF 4 0.1160 0.0000 0.0000 0.0000 — — — -0.1196 -0.0075 0.0758 -0.0446 0.0000 -1.5184 -0.3926 0.1709 0.0663 0.2654 0.3843

1 105 Su40St0 4 0.1161 0.0000 0.0381 0.0000 — — — 2.1689 0.0058 -0.0941 -0.0254 6.6250 1.5650 2.0719 0.2354 0.0284 0.1943 0.7377

197



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 106 Su40St0 4 0.1160 0.0000 0.0382 0.0000 — — — 1.7642 0.0731 0.0394 -0.0394 6.6409 1.3455 2.0920 0.0605 0.1005 0.1518 0.7464

1 107 Su80St0 4 0.1158 0.0000 0.0760 0.0000 — — — 2.9922 0.0204 -0.0974 -0.0296 17.8411 2.2324 3.2346 0.2284 0.0822 0.2847 0.8450

1 108 Su80St0 4 0.1157 0.0000 0.0763 0.0000 — — — 1.3556 0.1225 0.0212 -0.0342 17.0285 2.4493 2.5284 — — 0.0000 0.0000

1 109 Su120St0 4 0.1162 0.0000 0.1143 0.0000 — — — 3.4846 0.0193 -0.0902 0.0182 29.0799 2.8803 4.2755 0.4397 0.0618 0.2665 0.7334

1 110 Su120St0 4 0.1160 0.0000 0.1144 0.0000 — — — 3.1537 0.2187 0.0472 -0.0082 29.1991 2.9650 4.2904 0.4432 0.0976 0.2983 0.5426

1 111 Su0St40 4 0.1159 0.0319 0.0000 0.0000 — — — 17.6205 0.0211 -0.0232 -0.0357 0.0298 -0.9949 — -0.2369 0.1210 0.2799 0.7702

1 112 Su0St40 4 0.1159 0.0323 0.0000 0.0000 — — — 13.1272 -0.0101 0.0654 -0.0472 0.0000 -0.9586 -0.0235 -0.2449 0.0947 0.2540 0.8186

1 113 Su0St80 4 0.1159 0.0636 0.0000 0.0000 — — — 41.0043 0.0253 -0.0213 -0.0403 0.0496 -0.4489 0.3506 -0.1751 0.0852 0.3152 0.6744

1 114 Su0St80 4 0.1158 0.0639 0.0000 0.0000 — — — 40.8488 -0.0101 0.0498 -0.0446 0.0619 -0.4167 0.3913 -0.1735 0.0803 0.3094 0.8054

1 115 Su0St120 4 0.1162 0.0956 0.0000 0.0000 — — — 43.9507 0.0251 -0.0493 -0.0192 0.0000 -0.8668 0.2565 -0.2307 0.0636 0.2522 0.7176

1 116 Su0St120 4 0.1159 0.0957 0.0000 0.0000 — — — 52.5407 -0.0101 0.0858 -0.0339 0.0512 — -0.1259 -0.3831 0.0527 0.2376 1.9426

1 117 Su40St80 4 0.1160 0.0641 0.0379 0.0000 — — — 41.8276 0.1745 -0.0757 -0.0436 6.9572 1.4373 2.4337 0.3484 -0.0022 0.2197 0.6569

1 118 Su40St80 4 0.1159 0.0637 0.0384 0.0000 — — — 25.3155 -0.0101 0.0680 -0.0420 6.7551 1.0645 2.1257 0.1304 -0.0146 0.2738 0.5458

1 119 Su80St40 4 0.1160 0.0321 0.0762 0.0000 — — — 23.4396 0.0105 -0.0769 -0.0213 18.6549 3.0268 3.5701 0.3408 0.1001 0.2725 0.6619

1 120 Su80St40 4 0.1162 0.0323 0.0761 0.0000 — — — 16.6517 0.1771 0.0368 -0.0446 17.3902 2.1067 3.2867 0.3235 -0.0340 0.2615 —

1 121 BL 8 0.0000 0.0000 0.0000 0.0000 7.22 — — — — — — — — — — — — —

1 122 BL 8 0.0000 0.0000 0.0000 0.0000 7.22 — — — — — — — — — — — — —

1 123 NDF 8 0.1161 0.0000 0.0000 0.0000 7.19 86.1236 — — — — — — — — — — — —

1 124 NDF 8 0.1157 0.0000 0.0000 0.0000 7.20 85.6924 — — — — — — — — — — — —

1 125 Su40St0 8 0.1159 0.0000 0.0385 0.0000 7.07 84.2950 — — — — — — — — — — — —

1 126 Su40St0 8 0.1162 0.0000 0.0380 0.0000 7.06 83.9958 — — — — — — — — — — — —

1 127 Su80St0 8 0.1157 0.0000 0.0763 0.0000 6.94 82.4604 — — — — — — — — — — — —

1 128 Su80St0 8 0.1160 0.0000 0.0762 0.0000 6.96 85.4787 — — — — — — — — — — — —

1 129 Su120St0 8 0.1159 0.0000 0.1145 0.0000 6.83 85.8187 — — — — — — — — — — — —

1 130 Su120St0 8 0.1161 0.0000 0.1144 0.0000 6.80 86.1236 — — — — — — — — — — — —

1 131 Su0St40 8 0.1159 0.0319 0.0000 0.0000 7.11 85.0121 — — — — — — — — — — — —

1 132 Su0St40 8 0.1161 0.0319 0.0000 0.0000 7.12 86.5710 — — — — — — — — — — — —

1 133 Su0St80 8 0.1158 0.0642 0.0000 0.0000 7.00 85.9799 — — — — — — — — — — — —

198



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 134 Su0St80 8 0.1161 0.0640 0.0000 0.0000 7.04 85.0498 — — — — — — — — — — — —

1 135 Su0St120 8 0.1162 0.0958 0.0000 0.0000 6.88 86.9461 — — — — — — — — — — — —

1 136 Su0St120 8 0.1160 0.0958 0.0000 0.0000 7.01 86.7325 — — — — — — — — — — — —

1 137 Su40St80 8 0.1160 0.0637 0.0379 0.0000 6.96 84.5832 — — — — — — — — — — — —

1 138 Su40St80 8 0.1158 0.0639 0.0381 0.0000 6.93 83.9167 — — — — — — — — — — — —

1 139 Su80St40 8 0.1157 0.0321 0.0762 0.0000 6.93 84.0764 — — — — — — — — — — — —

1 140 Su80St40 8 0.1157 0.0320 0.0760 0.0000 6.86 84.6151 — — — — — — — — — — — —

1 143 NDF 8 0.1157 0.0000 0.0000 0.0000 — — 3.0803 — — — — — — — — — — —

1 144 NDF 8 0.1162 0.0000 0.0000 0.0000 — — 4.0050 — — — — — — — — — — —

1 145 Su40St0 8 0.1158 0.0000 0.0385 0.0000 — — 9.2717 — — — — — — — — — — —

1 146 Su40St0 8 0.1157 0.0000 0.0385 0.0000 — — 10.5234 — — — — — — — — — — —

1 147 Su80St0 8 0.1160 0.0000 0.0762 0.0000 — — 12.6480 — — — — — — — — — — —

1 148 Su80St0 8 0.1160 0.0000 0.0761 0.0000 — — 12.0250 — — — — — — — — — — —

1 149 Su120St0 8 0.1159 0.0000 0.1143 0.0000 — — 17.0202 — — — — — — — — — — —

1 150 Su120St0 8 0.1158 0.0000 0.1144 0.0000 — — 14.9376 — — — — — — — — — — —

1 151 Su0St40 8 0.1161 0.0319 0.0000 0.0000 — — 4.8435 — — — — — — — — — — —

1 152 Su0St40 8 0.1158 0.0318 0.0000 0.0000 — — 5.0652 — — — — — — — — — — —

1 153 Su0St80 8 0.1162 0.0638 0.0000 0.0000 — — 11.1336 — — — — — — — — — — —

1 154 Su0St80 8 0.1160 0.0639 0.0000 0.0000 — — 7.8043 — — — — — — — — — — —

1 155 Su0St120 8 0.1160 0.0959 0.0000 0.0000 — — 14.5660 — — — — — — — — — — —

1 156 Su0St120 8 0.1159 0.0956 0.0000 0.0000 — — 15.6200 — — — — — — — — — — —

1 157 Su40St80 8 0.1160 0.0636 0.0380 0.0000 — — 11.5743 — — — — — — — — — — —

1 158 Su40St80 8 0.1160 0.0640 0.0383 0.0000 — — 12.1710 — — — — — — — — — — —

1 159 Su80St40 8 0.1158 0.0319 0.0762 0.0000 — — 15.6850 — — — — — — — — — — —

1 160 Su80St40 8 0.1157 0.0319 0.0762 0.0000 — — 16.3148 — — — — — — — — — — —

1 161 BL 8 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3606 1.1007

1 162 BL 8 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3127 1.1325

1 163 NDF 8 0.1157 0.0000 0.0000 0.0000 — — — -0.0797 0.0072 -0.0658 -0.0052 0.0000 2.3201 0.8707 0.6392 0.1641 0.3314 0.8144

199



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 164 NDF 8 0.1157 0.0000 0.0000 0.0000 — — — 1.2300 -0.0065 0.0508 -0.0096 0.0000 2.1723 0.9127 0.3254 0.2023 0.3469 1.1373

1 165 Su40St0 8 0.1161 0.0000 0.0384 0.0000 — — — 1.1502 0.0079 -0.0852 0.0095 3.6633 6.0115 4.5416 1.5659 0.2870 0.4280 0.9382

1 166 Su40St0 8 0.1157 0.0000 0.0380 0.0000 — — — 2.7849 -0.0065 0.0485 0.0086 3.4140 6.2483 4.2409 1.6893 0.3067 0.3425 0.9656

1 167 Su80St0 8 0.1157 0.0000 0.0764 0.0000 — — — 1.9676 0.0023 -0.1083 0.0508 13.4867 8.3573 — 1.7838 0.2106 0.3123 0.9498

1 168 Su80St0 8 0.1162 0.0000 0.0763 0.0000 — — — 2.8706 0.1111 0.0281 -0.0096 12.7279 7.8411 6.2465 1.8173 0.2426 0.3085 0.9616

1 169 Su120St0 8 0.1162 0.0000 0.1144 0.0000 — — — 2.2945 0.0072 -0.0974 0.0098 21.7939 9.9672 9.6332 2.0005 0.1402 0.2888 0.9656

1 170 Su120St0 8 0.1161 0.0000 0.1143 0.0000 — — — 3.2753 0.1392 0.0037 -0.0096 21.2333 10.4404 9.4029 2.2145 0.3093 0.3031 0.6840

1 171 Su0St40 8 0.1162 0.0323 0.0000 0.0000 — — — 5.8091 -0.0057 0.0176 -0.0122 0.0000 5.1501 2.8687 0.6086 0.3160 0.2317 1.1728

1 172 Su0St40 8 0.1160 0.0319 0.0000 0.0000 — — — 8.3468 -0.0065 -0.0116 -0.0096 0.0000 5.6714 2.7883 0.6161 0.3351 0.3301 1.1559

1 173 Su0St80 8 0.1158 0.0640 0.0000 0.0000 — — — 21.4281 -0.0053 0.0205 -0.0098 0.0989 7.1236 4.0171 0.7892 0.2290 0.3216 1.1766

1 174 Su0St80 8 0.1159 0.0637 0.0000 0.0000 — — — 16.9328 -0.0065 -0.0376 -0.0122 0.0397 8.2064 4.3211 1.2388 0.1938 0.3423 0.6092

1 175 Su0St120 8 0.1159 0.0960 0.0000 0.0000 — — — 36.7163 0.0306 0.1294 -0.0100 0.1603 7.1142 1.2390 0.8403 0.2140 0.2739 0.9736

1 176 Su0St120 8 0.1157 0.0959 0.0000 0.0000 — — — 21.5956 -0.0065 -0.0474 -0.0070 0.0000 9.4745 2.9617 0.9064 0.1278 0.0000 1.0353

1 177 Su40St80 8 0.1162 0.0640 0.0379 0.0000 — — — 12.7664 0.0012 -0.0705 0.0117 3.3684 12.3890 9.5252 2.5970 0.3787 0.3072 1.0271

1 178 Su40St80 8 0.1162 0.0636 0.0384 0.0000 — — — 18.1628 -0.0065 -0.1078 0.0008 3.7885 11.1675 8.7575 2.5592 0.4481 0.3272 0.5619

1 179 Su80St40 8 0.1161 0.0322 0.0763 0.0000 — — — 10.2247 -0.0005 -0.0830 0.0275 12.9450 11.0228 8.8042 3.1086 0.4141 0.3083 0.7054

1 180 Su80St40 8 0.1159 0.0321 0.0764 0.0000 — — — 9.0046 -0.0039 -0.0116 -0.0070 12.3310 11.7904 9.1142 2.9403 0.5533 0.3198 1.2016

1 181 BL 12 0.0000 0.0000 0.0000 0.0000 7.22 — — — — — — — — — — — — —

1 182 BL 12 0.0000 0.0000 0.0000 0.0000 7.23 — — — — — — — — — — — — —

1 183 NDF 12 0.1160 0.0000 0.0000 0.0000 7.22 77.0607 — — — — — — — — — — — —

1 184 NDF 12 0.1162 0.0000 0.0000 0.0000 7.21 76.0388 — — — — — — — — — — — —

1 185 Su40St0 12 0.1158 0.0000 0.0380 0.0000 7.10 74.8568 — — — — — — — — — — — —

1 186 Su40St0 12 0.1157 0.0000 0.0380 0.0000 7.12 75.7271 — — — — — — — — — — — —

1 187 Su80St0 12 0.1161 0.0000 0.0765 0.0000 6.99 75.3861 — — — — — — — — — — — —

1 188 Su80St0 12 0.1159 0.0000 0.0761 0.0000 7.01 75.7804 — — — — — — — — — — — —

1 189 Su120St0 12 0.1157 0.0000 0.1139 0.0000 6.87 75.0987 — — — — — — — — — — — —

1 190 Su120St0 12 0.1157 0.0000 0.1142 0.0000 6.84 75.9965 — — — — — — — — — — — —

1 191 Su0St40 12 0.1159 0.0322 0.0000 0.0000 7.13 75.6011 — — — — — — — — — — — —

200



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 192 Su0St40 12 0.1157 0.0318 0.0000 0.0000 7.11 75.1885 — — — — — — — — — — — —

1 193 Su0St80 12 0.1162 0.0640 0.0000 0.0000 7.02 75.5917 — — — — — — — — — — — —

1 194 Su0St80 12 0.1162 0.0639 0.0000 0.0000 6.98 75.4129 — — — — — — — — — — — —

1 195 Su0St120 12 0.1157 0.0957 0.0000 0.0000 6.97 75.2783 — — — — — — — — — — — —

1 196 Su0St120 12 0.1161 0.0957 0.0000 0.0000 6.95 76.2809 — — — — — — — — — — — —

1 197 Su40St80 12 0.1162 0.0636 0.0384 0.0000 6.97 73.6248 — — — — — — — — — — — —

1 198 Su40St80 12 0.1160 0.0637 0.0380 0.0000 6.98 74.8219 — — — — — — — — — — — —

1 199 Su80St40 12 0.1157 0.0323 0.0761 0.0000 6.95 72.8543 — — — — — — — — — — — —

1 200 Su80St40 12 0.1159 0.0322 0.0765 0.0000 6.90 72.5538 — — — — — — — — — — — —

1 203 NDF 12 0.1162 0.0000 0.0000 0.0000 — — 7.2329 — — — — — — — — — — —

1 204 NDF 12 0.1158 0.0000 0.0000 0.0000 — — 5.8882 — — — — — — — — — — —

1 205 Su40St0 12 0.1162 0.0000 0.0382 0.0000 — — 11.5607 — — — — — — — — — — —

1 206 Su40St0 12 0.1162 0.0000 0.0384 0.0000 — — 12.1806 — — — — — — — — — — —

1 207 Su80St0 12 0.1158 0.0000 0.0762 0.0000 — — 16.9204 — — — — — — — — — — —

1 208 Su80St0 12 0.1161 0.0000 0.0762 0.0000 — — 18.6856 — — — — — — — — — — —

1 209 Su120St0 12 0.1157 0.0000 0.1145 0.0000 — — 20.3520 — — — — — — — — — — —

1 210 Su120St0 12 0.1158 0.0000 0.1142 0.0000 — — 21.6045 — — — — — — — — — — —

1 211 Su0St40 12 0.1159 0.0322 0.0000 0.0000 — — 16.2899 — — — — — — — — — — —

1 212 Su0St40 12 0.1161 0.0322 0.0000 0.0000 — — 12.3265 — — — — — — — — — — —

1 213 Su0St80 12 0.1161 0.0640 0.0000 0.0000 — — 14.4625 — — — — — — — — — — —

1 214 Su0St80 12 0.1161 0.0638 0.0000 0.0000 — — 14.2602 — — — — — — — — — — —

1 215 Su0St120 12 0.1159 0.0159 0.0000 0.0000 — — 19.2964 — — — — — — — — — — —

1 216 Su0St120 12 0.1157 0.0957 0.0000 0.0000 — — 19.0585 — — — — — — — — — — —

1 217 Su40St80 12 0.1161 0.0637 0.0379 0.0000 — — 18.2346 — — — — — — — — — — —

1 218 Su40St80 12 0.1160 0.0636 0.0380 0.0000 — — 19.6961 — — — — — — — — — — —

1 219 Su80St40 12 0.1159 0.0322 0.0763 0.0000 — — 20.1496 — — — — — — — — — — —

1 220 Su80St40 12 0.1160 0.0319 0.0762 0.0000 — — 19.9493 — — — — — — — — — — —

1 221 BL 12 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.4192 0.6509

201



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 222 BL 12 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3130 1.5951

1 223 NDF 12 0.1161 0.0000 0.0000 0.0000 — — — 0.0837 0.0000 -0.1018 -0.0037 0.0000 5.0809 2.8546 0.4851 0.3691 0.3885 1.9416

1 224 NDF 12 0.1161 0.0000 0.0000 0.0000 — — — 1.3097 0.0025 -0.0472 0.0001 0.0000 5.1049 2.8607 0.9521 0.3453 0.5757 1.3799

1 225 Su40St0 12 0.1160 0.0000 0.0381 0.0000 — — — 2.2905 0.0003 -0.1008 -0.0005 0.5639 9.2895 6.2842 2.8236 0.6086 0.5106 1.9082

1 226 Su40St0 12 0.1157 0.0000 0.0380 0.0000 — — — 2.1350 0.0077 -0.0502 0.0053 0.5268 8.5322 6.2574 2.8742 0.0359 0.6279 1.9821

1 227 Su80St0 12 0.1160 0.0000 0.0763 0.0000 — — — 2.5377 0.0000 -0.0434 0.0105 4.3168 10.3912 9.0447 4.6128 1.3225 0.3878 1.6012

1 228 Su80St0 12 0.1159 0.0000 0.0764 0.0000 — — — 1.3237 -0.0001 -0.0424 -0.0025 4.0977 10.0681 8.8343 4.9779 1.5765 0.4453 1.6722

1 229 Su120St0 12 0.1157 0.0000 0.1140 0.0000 — — — 2.9524 0.0001 -0.0406 -0.0041 0.0477 16.4213 16.9418 6.6369 2.4931 0.4889 1.8046

1 230 Su120St0 12 0.1157 0.0000 0.1142 0.0000 — — — 2.0493 0.0211 -0.0995 -0.0024 0.0000 14.8775 16.1330 7.6844 2.5056 0.4534 1.5287

1 231 Su0St40 12 0.1160 0.0323 0.0000 0.0000 — — — 4.6668 -0.0001 0.0094 -0.0051 0.0000 8.8385 5.8454 1.2307 0.3593 0.5443 1.9359

1 232 Su0St40 12 0.1161 0.0323 0.0000 0.0000 — — — 4.5851 0.0415 -0.1022 -0.0025 0.0000 — 5.7170 1.3651 0.4038 0.5623 1.9107

1 233 Su0St80 12 0.1162 0.0638 0.0000 0.0000 — — — 7.3620 0.0214 0.0332 -0.0051 0.0000 12.9634 8.2426 1.6159 0.3629 0.0000 1.8746

1 234 Su0St80 12 0.1160 0.0640 0.0000 0.0000 — — — 5.9765 0.0441 -0.0736 -0.0051 0.0000 15.0308 9.9067 1.8641 0.5480 0.6181 1.7873

1 235 Su0St120 12 0.1158 0.0957 0.0000 0.0000 — — — 16.5241 0.0281 0.0477 -0.0051 0.0000 16.0590 11.0437 1.9453 0.4539 0.5094 1.9470

1 236 Su0St120 12 0.1157 0.0957 0.0000 0.0000 — — — 17.0265 0.0103 -0.1022 -0.0025 0.1052 16.0300 12.4017 2.0164 0.5271 0.5980 1.9634

1 237 Su40St80 12 0.1158 0.0640 0.0384 0.0000 — — — 10.9702 0.0147 -0.0028 -0.0033 0.0352 16.2669 12.9217 4.4441 1.0799 0.6104 2.2720

1 238 Su40St80 12 0.1161 0.0637 0.0382 0.0000 — — — 11.7955 0.0233 -0.1022 0.0001 0.0000 15.3237 11.8644 4.5097 1.0910 0.6494 2.2002

1 239 Su80St40 12 0.1159 0.0320 0.0761 0.0000 — — — 7.4517 0.0153 -0.0061 -0.0047 1.1385 15.1422 12.2111 6.8157 2.2330 0.5300 1.9896

1 240 Su80St40 12 0.1159 0.0322 0.0763 0.0000 — — — 8.4405 -0.0001 -0.0969 -0.0025 1.1714 13.8595 11.5596 6.6840 2.1377 0.5499 1.8583

1 241 BL 16 0.0000 0.0000 0.0000 0.0000 7.33 — — — — — — — — — — — — —

1 242 BL 16 0.0000 0.0000 0.0000 0.0000 7.33 — — — — — — — — — — — — —

1 243 NDF 16 0.1159 0.0000 0.0000 0.0000 7.29 69.2824 — — — — — — — — — — — —

1 244 NDF 16 0.1161 0.0000 0.0000 0.0000 7.26 69.3462 — — — — — — — — — — — —

1 245 Su40St0 16 0.1158 0.0000 0.0382 0.0000 7.20 69.4298 — — — — — — — — — — — —

1 246 Su40St0 16 0.1162 0.0000 0.0383 0.0000 7.20 69.5569 — — — — — — — — — — — —

1 247 Su80St0 16 0.1161 0.0000 0.0762 0.0000 7.08 69.4357 — — — — — — — — — — — —

1 248 Su80St0 16 0.1162 0.0000 0.0765 0.0000 7.08 69.1099 — — — — — — — — — — — —

1 249 Su120St0 16 0.1161 0.0000 0.1145 0.0000 6.88 69.1673 — — — — — — — — — — — —

202



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 250 Su120St0 16 0.1157 0.0000 0.1139 0.0000 6.92 68.1410 — — — — — — — — — — — —

1 251 Su0St40 16 0.1158 0.0319 0.0000 0.0000 7.09 67.1872 — — — — — — — — — — — —

1 252 Su0St40 16 0.1158 0.0322 0.0000 0.0000 7.18 68.3533 — — — — — — — — — — — —

1 253 Su0St80 16 0.1157 0.0639 0.0000 0.0000 7.06 69.9365 — — — — — — — — — — — —

1 254 Su0St80 16 0.1157 0.0637 0.0000 0.0000 7.07 68.8592 — — — — — — — — — — — —

1 255 Su0St120 16 0.1157 0.0957 0.0000 0.0000 6.97 69.1285 — — — — — — — — — — — —

1 256 Su0St120 16 0.1160 0.0959 0.0000 0.0000 6.98 69.8517 — — — — — — — — — — — —

1 257 Su40St80 16 0.1160 0.0638 0.0384 0.0000 6.97 68.6875 — — — — — — — — — — — —

1 258 Su40St80 16 0.1161 0.0637 0.0382 0.0000 6.99 67.2882 — — — — — — — — — — — —

1 259 Su80St40 16 0.1160 0.0319 0.0763 0.0000 6.99 67.3442 — — — — — — — — — — — —

1 260 Su80St40 16 0.1161 0.0322 0.0761 0.0000 7.02 67.8251 — — — — — — — — — — — —

1 263 NDF 16 0.1158 0.0000 0.0000 0.0000 — — 6.3057 — — — — — — — — — — —

1 264 NDF 16 0.1160 0.0000 0.0000 0.0000 — — 6.0925 — — — — — — — — — — —

1 265 Su40St0 16 0.1159 0.0000 0.0380 0.0000 — — 9.8022 — — — — — — — — — — —

1 266 Su40St0 16 0.1158 0.0000 0.0384 0.0000 — — 11.2525 — — — — — — — — — — —

1 267 Su80St0 16 0.1159 0.0000 0.0760 0.0000 — — 14.3191 — — — — — — — — — — —

1 268 Su80St0 16 0.1157 0.0000 0.0763 0.0000 — — 14.5254 — — — — — — — — — — —

1 269 Su120St0 16 0.1159 0.0000 0.1141 0.0000 — — 17.8561 — — — — — — — — — — —

1 270 Su120St0 16 0.1160 0.0000 0.1142 0.0000 — — 17.1244 — — — — — — — — — — —

1 271 Su0St40 16 0.1157 0.0323 0.0000 0.0000 — — 9.1084 — — — — — — — — — — —

1 272 Su0St40 16 0.1160 0.0318 0.0000 0.0000 — — 8.7030 — — — — — — — — — — —

1 273 Su0St80 16 0.1159 0.0641 0.0000 0.0000 — — 14.5693 — — — — — — — — — — —

1 274 Su0St80 16 0.1157 0.0639 0.0000 0.0000 — — 14.1643 — — — — — — — — — — —

1 275 Su0St120 16 0.1158 0.0958 0.0000 0.0000 — — 17.9055 — — — — — — — — — — —

1 276 Su0St120 16 0.1162 0.0957 0.0000 0.0000 — — 17.3777 — — — — — — — — — — —

1 277 Su40St80 16 0.1160 0.0640 0.0385 0.0000 — — 14.1412 — — — — — — — — — — —

1 278 Su40St80 16 0.1159 0.0641 0.0384 0.0000 — — 14.3533 — — — — — — — — — — —

1 279 Su80St40 16 0.1157 0.0322 0.0759 0.0000 — — 16.3124 — — — — — — — — — — —

203



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 280 Su80St40 16 0.1162 0.0321 0.0761 0.0000 — — 16.5057 — — — — — — — — — — —

1 281 BL 16 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.6668 1.0817

1 282 BL 16 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3098 2.3044

1 283 NDF 16 0.1159 0.0000 0.0000 0.0000 — — — -0.1110 -0.0378 0.0434 -0.0004 0.0000 7.7127 3.0208 0.7392 0.4274 0.6947 2.5264

1 284 NDF 16 0.1162 0.0000 0.0000 0.0000 — — — -0.5338 0.0436 -0.0290 0.0110 0.0000 7.2881 2.9095 0.6992 0.4412 0.6821 2.4385

1 285 Su40St0 16 0.1157 0.0000 0.0381 0.0000 — — — 0.8579 -0.0448 0.0426 -0.0012 0.0000 10.5100 5.8644 3.0486 0.9929 0.8710 2.7724

1 286 Su40St0 16 0.1158 0.0000 0.0381 0.0000 — — — 1.0488 0.0511 -0.0290 -0.0021 0.0000 10.6479 5.9861 3.0886 0.9192 0.8790 2.6145

1 287 Su80St0 16 0.1159 0.0000 0.0765 0.0000 — — — 1.2686 -0.0466 0.0577 0.0018 0.0000 11.9882 8.9544 6.3548 2.6896 0.8370 2.3877

1 288 Su80St0 16 0.1160 0.0000 0.0761 0.0000 — — — 1.3672 0.0402 0.0022 -0.0021 0.0000 12.5143 9.1795 6.5377 2.6877 0.8104 2.3831

1 289 Su120St0 16 0.1157 0.0000 0.1144 0.0000 — — — 0.3954 -0.0480 -0.0286 0.0008 0.0000 19.1104 17.6872 6.4799 2.8386 0.7921 2.1943

1 290 Su120St0 16 0.1160 0.0000 0.1144 0.0000 — — — 1.2862 -0.0482 -0.0290 0.0031 0.0000 19.0639 17.9059 6.9151 — 0.0000 —

1 291 Su0St40 16 0.1158 0.0318 0.0000 0.0000 — — — 1.6812 -0.0138 -0.0199 0.3353 0.0000 12.2321 6.0215 1.5617 0.4877 0.7767 2.5390

1 292 Su0St40 16 0.1161 0.0322 0.0000 0.0000 — — — 2.0280 0.0013 -0.0264 0.0005 0.0000 12.5210 6.1455 1.3882 0.3994 0.6924 2.3944

1 293 Su0St80 16 0.1162 0.0639 0.0000 0.0000 — — — 10.0260 -0.0197 -0.0211 0.3320 0.0000 15.4864 8.7529 2.1570 0.7375 0.9471 2.7405

1 294 Su0St80 16 0.1161 0.0640 0.0000 0.0000 — — — 5.7984 -0.0066 -0.0290 0.0005 0.0000 16.1144 9.1855 2.2812 0.6493 0.8581 2.7542

1 295 Su0St120 16 0.1160 0.0958 0.0000 0.0000 — — — 7.5760 -0.0368 -0.0290 0.3990 0.0000 21.7728 13.5781 2.6858 0.5786 0.9931 1.5021

1 296 Su0St120 16 0.1158 0.0958 0.0000 0.0000 — — — 7.2227 -0.0092 -0.0264 -0.0021 0.0768 18.4546 11.4882 2.7419 0.7175 0.8998 2.7453

1 297 Su40St80 16 0.1157 0.0639 0.0385 0.0000 — — — 9.9582 -0.0231 -0.0287 0.2198 0.0000 18.0438 13.0212 5.0846 1.3841 0.9456 2.5463

1 298 Su40St80 16 0.1160 0.0638 0.0382 0.0000 — — — 9.2047 -0.0082 -0.0184 0.0006 0.0000 18.0681 12.7086 4.5975 1.2216 0.9420 2.4673

1 299 Su80St40 16 0.1161 0.0320 0.0760 0.0000 — — — 2.7517 -0.0466 -0.0023 0.1756 0.0000 17.6967 12.5707 7.0569 2.6100 0.8731 2.1534

1 300 Su80St40 16 0.1162 0.0322 0.0763 0.0000 — — — 3.0328 -0.0482 -0.0238 0.0005 0.0000 15.9575 12.1591 6.8102 — 0.0000 —

1 301 BL 20 0.0000 0.0000 0.0000 0.0000 7.37 — — — — — — — — — — — — —

1 302 BL 20 0.0000 0.0000 0.0000 0.0000 7.38 — — — — — — — — — — — — —

1 303 NDF 20 0.1160 0.0000 0.0000 0.0000 7.32 64.2993 — — — — — — — — — — — —

1 304 NDF 20 0.1157 0.0000 0.0000 0.0000 7.32 64.9090 — — — — — — — — — — — —

1 305 Su40St0 20 0.1157 0.0000 0.0383 0.0000 7.17 64.7294 — — — — — — — — — — — —

1 306 Su40St0 20 0.1157 0.0000 0.0385 0.0000 7.20 64.6396 — — — — — — — — — — — —

1 307 Su80St0 20 0.1160 0.0000 0.0764 0.0000 7.11 63.1351 — — — — — — — — — — — —

204



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 308 Su80St0 20 0.1158 0.0000 0.0763 0.0000 7.12 63.1506 — — — — — — — — — — — —

1 309 Su120St0 20 0.1161 0.0000 0.1143 0.0000 6.99 63.0827 — — — — — — — — — — — —

1 310 Su120St0 20 0.1157 0.0000 0.1145 0.0000 6.98 63.2930 — — — — — — — — — — — —

1 311 Su0St40 20 0.1162 0.0319 0.0000 0.0000 7.19 64.9079 — — — — — — — — — — — —

1 312 Su0St40 20 0.1162 0.0323 0.0000 0.0000 7.22 64.1926 — — — — — — — — — — — —

1 313 Su0St80 20 0.1162 0.0637 0.0000 0.0000 7.12 64.9973 — — — — — — — — — — — —

1 314 Su0St80 20 0.1159 0.0641 0.0000 0.0000 7.09 64.7113 — — — — — — — — — — — —

1 315 Su0St120 20 0.1158 0.0955 0.0000 0.0000 6.94 65.2137 — — — — — — — — — — — —

1 316 Su0St120 20 0.1161 0.0959 0.0000 0.0000 6.96 66.0355 — — — — — — — — — — — —

1 317 Su40St80 20 0.1159 0.0636 0.0381 0.0000 7.03 65.1595 — — — — — — — — — — — —

1 318 Su40St80 20 0.1160 0.0642 0.0382 0.0000 7.05 62.5978 — — — — — — — — — — — —

1 319 Su80St40 20 0.1158 0.0321 0.0759 0.0000 7.06 64.3167 — — — — — — — — — — — —

1 320 Su80St40 20 0.1160 0.0321 0.0764 0.0000 7.06 63.6725 — — — — — — — — — — — —

1 323 NDF 20 0.1158 0.0000 0.0000 0.0000 — — 8.1799 — — — — — — — — — — —

1 324 NDF 20 0.1161 0.0000 0.0000 0.0000 — — 7.3395 — — — — — — — — — — —

1 325 Su40St0 20 0.1160 0.0000 0.0380 0.0000 — — 11.6742 — — — — — — — — — — —

1 326 Su40St0 20 0.1158 0.0000 0.0380 0.0000 — — 11.1581 — — — — — — — — — — —

1 327 Su80St0 20 0.1157 0.0000 0.0759 0.0000 — — 15.7819 — — — — — — — — — — —

1 328 Su80St0 20 0.1159 0.0000 0.0761 0.0000 — — 14.0050 — — — — — — — — — — —

1 329 Su120St0 20 0.1162 0.0000 0.1142 0.0000 — — 18.4753 — — — — — — — — — — —

1 330 Su120St0 20 0.1158 0.0000 0.1142 0.0000 — — 19.2096 — — — — — — — — — — —

1 331 Su0St40 20 0.1160 0.0321 0.0000 0.0000 — — 8.7931 — — — — — — — — — — —

1 332 Su0St40 20 0.1157 0.0318 0.0000 0.0000 — — 9.1294 — — — — — — — — — — —

1 333 Su0St80 20 0.1157 0.0640 0.0000 0.0000 — — 14.8903 — — — — — — — — — — —

1 334 Su0St80 20 0.1159 0.0641 0.0000 0.0000 — — 14.6734 — — — — — — — — — — —

1 335 Su0St120 20 0.1162 0.0959 0.0000 0.0000 — — 18.2055 — — — — — — — — — — —

1 336 Su0St120 20 0.1162 0.0957 0.0000 0.0000 — — 18.5256 — — — — — — — — — — —

1 337 Su40St80 20 0.1160 0.0639 0.0381 0.0000 — — 14.6821 — — — — — — — — — — —

205



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 338 Su40St80 20 0.1160 0.0636 0.0382 0.0000 — — 13.5446 — — — — — — — — — — —

1 339 Su80St40 20 0.1157 0.0320 0.0759 0.0000 — — 15.6928 — — — — — — — — — — —

1 340 Su80St40 20 0.1160 0.0320 0.0764 0.0000 — — 14.3187 — — — — — — — — — — —

1 341 BL 20 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.4528 3.1574

1 342 BL 20 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.9849 4.0016

1 343 NDF 20 0.1160 0.0000 0.0000 0.0000 — — — -0.4068 -0.0275 0.0051 0.1096 0.0000 11.6201 3.5875 1.2793 0.3066 0.9684 3.0134

1 344 NDF 20 0.1157 0.0000 0.0000 0.0000 — — — 1.0399 0.0191 -0.0049 -0.0452 0.0000 10.5200 — — — — —

1 345 Su40St0 20 0.1162 0.0000 0.0379 0.0000 — — — 1.0664 -0.0223 0.0002 0.1229 0.0000 16.1270 7.2646 3.7863 0.8742 0.9976 2.9302

1 346 Su40St0 20 0.1157 0.0000 0.0381 0.0000 — — — 1.6015 -0.0421 0.0321 -0.0425 0.0000 15.2681 6.8208 3.5462 0.8557 0.8724 2.5819

1 347 Su80St0 20 0.1160 0.0000 0.0765 0.0000 — — — 0.8251 -0.0237 -0.0014 0.1379 0.0000 16.4961 9.4240 6.8103 2.3819 0.8605 2.5309

1 348 Su80St0 20 0.1161 0.0000 0.0761 0.0000 — — — 1.3641 -0.0421 0.0133 -0.0452 0.0000 15.3500 8.4650 5.7277 2.2378 0.0000 2.2802

1 349 Su120St0 20 0.1158 0.0000 0.1141 0.0000 — — — 1.4710 -0.0243 0.1017 -0.0434 0.0000 23.6553 18.6193 6.8047 2.4970 0.6257 2.4647

1 350 Su120St0 20 0.1159 0.0000 0.1142 0.0000 — — — 1.7559 0.0021 -0.0049 -0.0400 0.0000 22.5284 18.8226 7.2400 2.7656 0.8361 2.4283

1 351 Su0St40 20 0.1158 0.0320 0.0000 0.0000 — — — 1.1501 -0.0410 0.1804 -0.0314 0.0000 16.9868 7.0903 2.3568 0.5202 1.0873 2.9327

1 352 Su0St40 20 0.1161 0.0319 0.0000 0.0000 — — — 2.1515 -0.0135 -0.0049 -0.0452 0.0000 16.9933 7.1379 2.4013 0.5120 1.0550 3.0359

1 353 Su0St80 20 0.1156 0.0640 0.0000 0.0000 — — — 2.1349 -0.0383 0.1914 -0.0452 0.0000 20.8936 10.9045 3.4590 — — —

1 354 Su0St80 20 0.1160 0.0641 0.0000 0.0000 — — — 3.1010 -0.0212 -0.0049 -0.0400 0.0000 22.1469 10.8017 3.4687 0.8020 1.0805 2.9506

1 355 Su0St120 20 0.1160 0.0957 0.0000 0.0000 — — — 6.5525 -0.0365 0.2214 -0.0417 0.0000 23.5732 14.1218 4.2968 0.9767 1.1572 2.9681

1 356 Su0St120 20 0.1160 0.0958 0.0000 0.0000 — — — 8.6477 -0.0421 -0.0023 -0.0374 0.0000 24.5438 13.7914 4.0606 0.8042 1.0813 2.8712

1 357 Su40St80 20 0.1161 0.0639 0.0381 0.0000 — — — 3.7715 -0.0388 0.1077 -0.0441 0.0000 24.6762 14.2806 6.1016 1.1889 0.9795 2.6225

1 358 Su40St80 20 0.1162 0.0636 0.0380 0.0000 — — — 4.2918 -0.0212 0.0081 -0.0426 0.0000 24.3999 14.2011 5.9877 1.2041 0.9614 2.6774

1 359 Su80St40 20 0.1160 0.0321 0.0759 0.0000 — — — 2.5475 -0.0398 0.0999 -0.0452 0.0000 — 13.5160 8.1135 2.7694 0.8674 2.6728

1 360 Su80St40 20 0.1161 0.0321 0.0759 0.0000 — — — 2.6186 -0.0421 0.0081 -0.0426 0.0000 23.7106 14.0697 7.9549 2.7050 0.9492 2.6455

1 361 BL 24 0.0000 0.0000 0.0000 0.0000 7.38 — — — — — — — — — — — — —

1 362 BL 24 0.0000 0.0000 0.0000 0.0000 7.45 — — — — — — — — — — — — —

1 363 NDF 24 0.1159 0.0000 0.0000 0.0000 7.31 61.4399 — — — — — — — — — — — —

1 364 NDF 24 0.1158 0.0000 0.0000 0.0000 7.32 60.9529 — — — — — — — — — — — —

1 365 Su40St0 24 0.1157 0.0000 0.0384 0.0000 7.21 58.6695 — — — — — — — — — — — —

206



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 366 Su40St0 24 0.1160 0.0000 0.0381 0.0000 7.22 58.7023 — — — — — — — — — — — —

1 367 Su80St0 24 0.1156 0.0000 0.0760 0.0000 7.14 60.2459 — — — — — — — — — — — —

1 368 Su80St0 24 0.1158 0.0000 0.0764 0.0000 7.09 59.5176 — — — — — — — — — — — —

1 369 Su120St0 24 0.1161 0.0000 0.1144 0.0000 6.99 60.8010 — — — — — — — — — — — —

1 370 Su120St0 24 0.1160 0.0000 0.1139 0.0000 7.00 59.5978 — — — — — — — — — — — —

1 371 Su0St40 24 0.1159 0.0323 0.0000 0.0000 7.18 63.9495 — — — — — — — — — — — —

1 372 Su0St40 24 0.1160 0.0320 0.0000 0.0000 7.21 58.2545 — — — — — — — — — — — —

1 373 Su0St80 24 0.1161 0.0642 0.0000 0.0000 7.06 61.2484 — — — — — — — — — — — —

1 374 Su0St80 24 0.1161 0.0638 0.0000 0.0000 7.07 58.1166 — — — — — — — — — — — —

1 375 Su0St120 24 0.1161 0.0959 0.0000 0.0000 6.95 60.9800 — — — — — — — — — — — —

1 376 Su0St120 24 0.1162 0.0955 0.0000 0.0000 6.92 61.5552 — — — — — — — — — — — —

1 377 Su40St80 24 0.1158 0.0638 0.0380 0.0000 7.01 58.8897 — — — — — — — — — — — —

1 378 Su40St80 24 0.1159 0.0636 0.0382 0.0000 6.97 61.3503 — — — — — — — — — — — —

1 379 Su80St40 24 0.1157 0.0319 0.0765 0.0000 7.03 59.4775 — — — — — — — — — — — —

1 380 Su80St40 24 0.1157 0.0318 0.0763 0.0000 7.02 58.4899 — — — — — — — — — — — —

1 383 NDF 24 0.1158 0.0000 0.0000 0.0000 — — 7.2428 — — — — — — — — — — —

1 384 NDF 24 0.1158 0.0000 0.0000 0.0000 — — 5.9933 — — — — — — — — — — —

1 385 Su40St0 24 0.1160 0.0000 0.0381 0.0000 — — 10.2140 — — — — — — — — — — —

1 386 Su40St0 24 0.1161 0.0000 0.0382 0.0000 — — 9.5846 — — — — — — — — — — —

1 387 Su80St0 24 0.1157 0.0000 0.0761 0.0000 — — 14.0082 — — — — — — — — — — —

1 388 Su80St0 24 0.1158 0.0000 0.0763 0.0000 — — 12.9619 — — — — — — — — — — —

1 389 Su120St0 24 0.1157 0.0000 0.1142 0.0000 — — 16.7120 — — — — — — — — — — —

1 390 Su120St0 24 0.1158 0.0000 0.1139 0.0000 — — 15.4655 — — — — — — — — — — —

1 391 Su0St40 24 0.1161 0.0319 0.0000 0.0000 — — 7.6514 — — — — — — — — — — —

1 392 Su0St40 24 0.1161 0.0323 0.0000 0.0000 — — 8.0539 — — — — — — — — — — —

1 393 Su0St80 24 0.1159 0.0638 0.0000 0.0000 — — 12.0792 — — — — — — — — — — —

1 394 Su0St80 24 0.1162 0.0641 0.0000 0.0000 — — 13.2072 — — — — — — — — — — —

1 395 Su0St120 24 0.1161 0.0958 0.0000 0.0000 — — 17.6899 — — — — — — — — — — —

207



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

1 396 Su0St120 24 0.1157 0.0958 0.0000 0.0000 — — 17.3873 — — — — — — — — — — —

1 397 Su40St80 24 0.1162 0.0637 0.0383 0.0000 — — 12.6960 — — — — — — — — — — —

1 398 Su40St80 24 0.1162 0.0639 0.0379 0.0000 — — 13.5373 — — — — — — — — — — —

1 399 Su80St40 24 0.1158 0.0322 0.0761 0.0000 — — 15.0552 — — — — — — — — — — —

1 400 Su80St40 24 0.1160 0.0318 0.0764 0.0000 — — 14.8470 — — — — — — — — — — —

1 401 BL 24 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.8847 3.2724

1 402 BL 24 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 1.1951 3.4312

1 403 NDF 24 0.1159 0.0000 0.0000 0.0000 — — — 0.3690 -0.0049 0.0485 -0.0006 0.0000 12.5316 4.0314 1.9454 0.2700 1.2099 3.3865

1 404 NDF 24 0.1160 0.0000 0.0000 0.0000 — — — 0.2718 -0.0041 -0.0175 -0.0006 0.0000 11.9886 4.1034 1.9723 -0.2069 1.2220 3.2652

1 405 Su40St0 24 0.1158 0.0000 0.0382 0.0000 — — — 0.6162 -0.0078 0.0492 -0.0006 0.0000 16.0088 3.0446 4.3396 1.6002 1.0883 2.9396

1 406 Su40St0 24 0.1158 0.0000 0.0380 0.0000 — — — 1.0631 -0.0092 -0.0175 -0.0006 0.0000 17.1106 7.5930 4.3053 0.6035 1.0292 2.8651

1 407 Su80St0 24 0.1158 0.0000 0.0759 0.0000 — — — 0.3810 -0.0066 0.0610 0.0024 0.0000 20.0276 12.0903 8.5003 2.1200 1.3339 2.3275

1 408 Su80St0 24 0.1157 0.0000 0.0762 0.0000 — — — 0.8257 -0.0092 -0.0175 -0.0006 0.0000 18.3616 10.9494 7.2208 — — —

1 409 Su120St0 24 0.1158 0.0000 0.1141 0.0000 — — — 0.6999 -0.0068 0.0687 0.0066 0.0000 25.9524 19.2210 7.6991 2.4161 0.9990 2.7593

1 410 Su120St0 24 0.1159 0.0000 0.1143 0.0000 — — — 0.9087 -0.0092 -0.0175 -0.0006 0.0000 25.7634 19.4769 7.9129 2.4929 0.4955 2.8415

1 411 Su0St40 24 0.1158 0.0318 0.0000 0.0000 — — — 0.9451 -0.0082 0.0896 -0.0006 0.0000 16.2604 7.3424 — — — —

1 412 Su0St40 24 0.1157 0.0319 0.0000 0.0000 — — — 1.2233 -0.0092 -0.0175 -0.0006 0.0000 — 7.6667 3.1656 0.1949 1.2085 2.7344

1 413 Su0St80 24 0.1158 0.0637 0.0000 0.0000 — — — 2.3386 -0.0092 0.0072 0.0022 0.0000 22.9572 10.9606 4.2808 0.1470 1.1108 2.9635

1 414 Su0St80 24 0.1158 0.0638 0.0000 0.0000 — — — 2.3349 -0.0092 -0.0175 -0.0006 0.0000 22.5977 11.0057 4.1186 0.2268 1.1338 3.0947

1 415 Su0St120 24 0.1161 0.0958 0.0000 0.0000 — — — 2.0954 -0.0092 0.1451 -0.0006 0.0000 26.4369 14.9978 4.8032 0.3492 1.1845 3.0556

1 416 Su0St120 24 0.1158 0.0957 0.0000 0.0000 — — — 2.8039 -0.0092 -0.0038 -0.0006 0.0000 28.0718 15.1513 4.8255 0.4597 1.2429 3.2008

1 417 Su40St80 24 0.1160 0.0641 0.0384 0.0000 — — — 2.0954 -0.0090 0.0716 0.0101 0.0000 26.9897 15.1338 6.6227 0.8010 1.0783 2.9261

1 418 Su40St80 24 0.1160 0.0637 0.0383 0.0000 — — — 3.5161 -0.0092 -0.0175 -0.0006 0.0000 26.6817 15.3236 6.9383 0.9538 1.0859 1.1448

1 419 Su80St40 24 0.1162 0.0321 0.0760 0.0000 — — — 2.6735 -0.0092 0.0721 0.0006 0.0000 24.6106 14.2321 8.6382 2.2183 0.9747 2.7987

1 420 Su80St40 24 0.1162 0.0320 0.0762 0.0000 — — — — -0.0092 -0.0061 -0.0006 0.0000 25.2280 14.9628 8.3788 2.0313 0.9892 2.6784

2 1 BL 0 0.0000 0.0000 0.0000 0.0000 7.05 — — — — — — — — — — — — —

2 2 BL 0 0.0000 0.0000 0.0000 0.0000 6.99 — — — — — — — — — — — — —

2 3 NDF 0 0.1158 0.0000 0.0000 0.0000 7.05 95.6884 — — — — — — — — — — — —

208



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 4 NDF 0 0.1159 0.0000 0.0000 0.0000 7.06 94.7459 — — — — — — — — — — — —

2 5 Su40St0 0 0.1159 0.0000 0.0384 0.0000 7.02 95.4362 — — — — — — — — — — — —

2 6 Su40St0 0 0.1157 0.0000 0.0384 0.0000 7.04 94.9038 — — — — — — — — — — — —

2 7 Su80St0 0 0.1160 0.0000 0.0763 0.0000 7.06 94.2360 — — — — — — — — — — — —

2 8 Su80St0 0 0.1156 0.0000 0.0759 0.0000 7.04 94.4639 — — — — — — — — — — — —

2 9 Su120St0 0 0.1158 0.0000 0.1142 0.0000 7.06 94.3066 — — — — — — — — — — — —

2 10 Su120St0 0 0.1160 0.0000 0.1141 0.0000 7.04 94.5809 — — — — — — — — — — — —

2 11 Su0St40 0 0.1156 0.0320 0.0000 0.0000 7.07 95.1560 — — — — — — — — — — — —

2 12 Su0St40 0 0.1158 0.0322 0.0000 0.0000 7.03 94.7384 — — — — — — — — — — — —

2 13 Su0St80 0 0.1161 0.0638 0.0000 0.0000 7.08 96.3976 — — — — — — — — — — — —

2 14 Su0St80 0 0.1160 0.0639 0.0000 0.0000 7.07 94.9258 — — — — — — — — — — — —

2 15 Su0St120 0 0.1158 0.0958 0.0000 0.0000 7.07 95.0839 — — — — — — — — — — — —

2 16 Su0St120 0 0.1161 0.0959 0.0000 0.0000 7.05 95.1915 — — — — — — — — — — — —

2 17 Su40St80 0 0.1159 0.0638 0.0381 0.0000 7.06 94.3144 — — — — — — — — — — — —

2 18 Su40St80 0 0.1159 0.0640 0.0383 0.0000 7.07 94.8322 — — — — — — — — — — — —

2 19 Su80St40 0 0.1157 0.0318 0.0764 0.0000 7.08 94.2123 — — — — — — — — — — — —

2 20 Su80St40 0 0.1158 0.0321 0.0763 0.0000 7.13 94.9975 — — — — — — — — — — — —

2 23 NDF 0 0.1157 0.0000 0.0000 0.0000 — — -0.4292 — — — — — — — — — — —

2 24 NDF 0 0.1161 0.0000 0.0000 0.0000 — — 0.4306 — — — — — — — — — — —

2 25 Su40St0 0 0.1160 0.0000 0.0385 0.0000 — — 0.0836 — — — — — — — — — — —

2 26 Su40St0 0 0.1158 0.0000 0.0380 0.0000 — — -0.0832 — — — — — — — — — — —

2 27 Su80St0 0 0.1161 0.0000 0.0759 0.0000 — — 0.1969 — — — — — — — — — — —

2 28 Su80St0 0 0.1157 0.0000 0.0764 0.0000 — — -0.1970 — — — — — — — — — — —

2 29 Su120St0 0 0.1159 0.0000 0.1139 0.0000 — — -0.2883 — — — — — — — — — — —

2 30 Su120St0 0 0.1157 0.0000 0.1145 0.0000 — — 0.2888 — — — — — — — — — — —

2 31 Su0St40 0 0.1160 0.0323 0.0000 0.0000 — — 0.1954 — — — — — — — — — — —

2 32 Su0St40 0 0.1161 0.0322 0.0000 0.0000 — — -0.1954 — — — — — — — — — — —

2 33 Su0St80 0 0.1160 0.0640 0.0000 0.0000 — — -0.1479 — — — — — — — — — — —

209



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 34 Su0St80 0 0.1158 0.0641 0.0000 0.0000 — — 0.1479 — — — — — — — — — — —

2 35 Su0St120 0 0.1161 0.0957 0.0000 0.0000 — — -1.0243 — — — — — — — — — — —

2 36 Su0St120 0 0.1159 0.0959 0.0000 0.0000 — — 1.0247 — — — — — — — — — — —

2 37 Su40St80 0 0.1161 0.0641 0.0385 0.0000 — — -1.2297 — — — — — — — — — — —

2 38 Su40St80 0 0.1159 0.0639 0.0384 0.0000 — — 1.2270 — — — — — — — — — — —

2 39 Su80St40 0 0.1161 0.0319 0.0763 0.0000 — — -0.1448 — — — — — — — — — — —

2 40 Su80St40 0 0.1160 0.0319 0.0765 0.0000 — — 0.1450 — — — — — — — — — — —

2 41 BL 0 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2034 0.5709

2 42 BL 0 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2697 0.5288

2 43 NDF 0 0.1159 0.0000 0.0000 0.0000 — — — 0.5433 -0.4985 -0.0206 -0.3550 0.0000 0.2049 0.0181 0.0070 0.0810 0.2144 0.5087

2 44 NDF 0 0.1158 0.0000 0.0000 0.0000 — — — -0.4595 0.4008 0.0019 -0.2829 0.0000 0.1126 -0.0988 -0.0221 0.0716 0.2232 0.4168

2 45 Su40St0 0 0.1161 0.0000 0.0380 0.0000 — — — 0.7845 11.3991 3.6992 20.5974 0.0953 0.4864 -0.3812 0.0957 -0.1377 0.2386 0.3498

2 46 Su40St0 0 0.1160 0.0000 0.0383 0.0000 — — — 0.2632 15.1782 4.2668 17.9690 0.1421 0.2580 0.1827 0.0162 -0.0295 0.2315 0.4377

2 47 Su80St0 0 0.1161 0.0000 0.0762 0.0000 — — — 1.6510 10.6008 6.7997 60.8350 0.1580 -0.0863 0.2572 -0.0896 0.0520 0.2244 0.5150

2 48 Su80St0 0 0.1162 0.0000 0.0763 0.0000 — — — 0.3430 11.7550 6.0421 53.2300 0.1664 0.4934 — 0.0499 0.0161 0.2304 0.5528

2 49 Su120St0 0 0.1161 0.0000 0.1144 0.0000 — — — 1.1801 12.7134 7.0150 95.0785 0.1593 0.5048 0.1377 -0.6279 0.0199 0.2136 0.3597

2 50 Su120St0 0 0.1160 0.0000 0.1142 0.0000 — — — 0.4268 10.6427 5.8003 97.3364 0.1380 0.9695 0.1331 0.0167 0.0448 0.2184 0.4038

2 51 Su0St40 0 0.1158 0.0323 0.0000 0.0000 — — — 28.0873 -0.5664 -0.0206 -0.3109 0.0000 0.6862 0.6142 0.1935 0.0374 0.1863 0.4446

2 52 Su0St40 0 0.1158 0.0321 0.0000 0.0000 — — — 26.3869 -0.1920 0.0025 -0.2618 0.0000 0.5055 0.0506 0.0241 0.0567 0.1959 0.3905

2 53 Su0St80 0 0.1159 0.0640 0.0000 0.0000 — — — 54.3615 -0.5336 -0.0206 -0.2650 0.0000 0.6786 -0.0097 0.0394 0.0442 0.2392 0.3773

2 54 Su0St80 0 0.1162 0.0641 0.0000 0.0000 — — — 49.8752 -0.5587 0.0075 -0.2720 0.0000 0.3153 -0.1527 0.0153 -0.1377 0.1933 0.5146

2 55 Su0St120 0 0.1159 0.0957 0.0000 0.0000 — — — 83.0925 -0.5318 -0.0206 -0.2703 0.0000 0.7985 0.2875 -0.6750 0.0407 0.2568 0.4077

2 56 Su0St120 0 0.1158 0.0956 0.0000 0.0000 — — — 77.5924 -0.5314 -0.0206 -0.2652 0.0000 0.5046 0.0368 0.0388 -0.1377 0.2245 0.3712

2 57 Su40St80 0 0.1157 0.0636 0.0384 0.0000 — — — 52.2308 8.4941 5.5787 23.0601 0.1559 0.4467 0.0645 -0.0167 -0.1377 0.0000 0.0000

2 58 Su40St80 0 0.1158 0.0639 0.0384 0.0000 — — — 55.6294 9.1267 4.7703 15.4795 0.1632 0.5917 0.2535 0.0014 0.0554 0.2060 0.4711

2 59 Su80St40 0 0.1158 0.0320 0.0761 0.0000 — — — 28.9616 11.3000 6.1695 47.7190 0.1502 0.4901 0.1061 -0.0225 0.0299 0.2305 0.5151

2 60 Su80St40 0 0.1161 0.0321 0.0759 0.0000 — — — 25.9019 10.7150 5.6323 47.6587 0.1673 0.3256 -1.5342 -0.0879 0.0140 0.1649 0.3232

2 61 BL 4 0.0000 0.0000 0.0000 0.0000 7.15 — — — — — — — — — — — — —

210



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 62 BL 4 0.0000 0.0000 0.0000 0.0000 7.14 — — — — — — — — — — — — —

2 63 NDF 4 0.1161 0.0000 0.0000 0.0000 7.14 94.8469 — — — — — — — — — — — —

2 64 NDF 4 0.1160 0.0000 0.0000 0.0000 7.14 94.8396 — — — — — — — — — — — —

2 65 Su40St0 4 0.1157 0.0000 0.0381 0.0000 6.98 94.4716 — — — — — — — — — — — —

2 66 Su40St0 4 0.1160 0.0000 0.0385 0.0000 6.96 93.5463 — — — — — — — — — — — —

2 67 Su80St0 4 0.1160 0.0000 0.0762 0.0000 6.80 93.0135 — — — — — — — — — — — —

2 68 Su80St0 4 0.1157 0.0000 0.0764 0.0000 6.80 93.0023 — — — — — — — — — — — —

2 69 Su120St0 4 0.1161 0.0000 0.1139 0.0000 6.66 91.9180 — — — — — — — — — — — —

2 70 Su120St0 4 0.1159 0.0000 0.1142 0.0000 6.64 93.1064 — — — — — — — — — — — —

2 71 Su0St40 4 0.1158 0.0318 0.0000 0.0000 7.16 93.9612 — — — — — — — — — — — —

2 72 Su0St40 4 0.1159 0.0319 0.0000 0.0000 7.16 93.7104 — — — — — — — — — — — —

2 73 Su0St80 4 0.1158 0.0641 0.0000 0.0000 7.16 94.5657 — — — — — — — — — — — —

2 74 Su0St80 4 0.1160 0.0636 0.0000 0.0000 7.16 93.5463 — — — — — — — — — — — —

2 75 Su0St120 4 0.1159 0.0960 0.0000 0.0000 7.16 94.4007 — — — — — — — — — — — —

2 76 Su0St120 4 0.1162 0.0959 0.0000 0.0000 7.15 93.3910 — — — — — — — — — — — —

2 77 Su40St80 4 0.1161 0.0636 0.0382 0.0000 6.99 93.0379 — — — — — — — — — — — —

2 78 Su40St80 4 0.1159 0.0642 0.0382 0.0000 7.02 92.9338 — — — — — — — — — — — —

2 79 Su80St40 4 0.1161 0.0319 0.0763 0.0000 6.86 93.7270 — — — — — — — — — — — —

2 80 Su80St40 4 0.1158 0.0319 0.0763 0.0000 6.88 93.3566 — — — — — — — — — — — —

2 83 NDF 4 0.1159 0.0000 0.0000 0.0000 — — -0.2922 — — — — — — — — — — —

2 84 NDF 4 0.1161 0.0000 0.0000 0.0000 — — -1.1806 — — — — — — — — — — —

2 85 Su40St0 4 0.1159 0.0000 0.0381 0.0000 — — 2.1058 — — — — — — — — — — —

2 86 Su40St0 4 0.1161 0.0000 0.0384 0.0000 — — 2.2808 — — — — — — — — — — —

2 87 Su80St0 4 0.1160 0.0000 0.0763 0.0000 — — 5.8030 — — — — — — — — — — —

2 88 Su80St0 4 0.1161 0.0000 0.0763 0.0000 — — 7.2649 — — — — — — — — — — —

2 89 Su120St0 4 0.1160 0.0000 0.1140 0.0000 — — 10.0095 — — — — — — — — — — —

2 90 Su120St0 4 0.1162 0.0000 0.1139 0.0000 — — 10.7887 — — — — — — — — — — —

2 91 Su0St40 4 0.1162 0.0321 0.0000 0.0000 — — -2.7834 — — — — — — — — — — —

211



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 92 Su0St40 4 0.1160 0.0322 0.0000 0.0000 — — -2.1940 — — — — — — — — — — —

2 93 Su0St80 4 0.1158 0.0638 0.0000 0.0000 — — 0.5946 — — — — — — — — — — —

2 94 Su0St80 4 0.1160 0.0639 0.0000 0.0000 — — 0.2939 — — — — — — — — — — —

2 95 Su0St120 4 0.1158 0.0960 0.0000 0.0000 — — -0.2934 — — — — — — — — — — —

2 96 Su0St120 4 0.1157 0.0958 0.0000 0.0000 — — -1.1660 — — — — — — — — — — —

2 97 Su40St80 4 0.1158 0.0638 0.0381 0.0000 — — 0.3153 — — — — — — — — — — —

2 98 Su40St80 4 0.1160 0.0642 0.0383 0.0000 — — 2.3414 — — — — — — — — — — —

2 99 Su80St40 4 0.1157 0.0319 0.0764 0.0000 — — 8.2074 — — — — — — — — — — —

2 100 Su80St40 4 0.1160 0.0319 0.0765 0.0000 — — 7.9096 — — — — — — — — — — —

2 101 BL 4 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.1825 0.5306

2 102 BL 4 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.1708 0.0000

2 103 NDF 4 0.1161 0.0000 0.0000 0.0000 — — — -0.0046 -0.0691 0.0372 0.1444 0.0000 0.4166 0.2442 0.4741 0.8014 0.3066 0.5674

2 104 NDF 4 0.1161 0.0000 0.0000 0.0000 — — — 0.3328 0.0509 0.2962 -0.0518 0.0000 0.4521 0.2461 0.3355 0.2582 0.2635 0.3334

2 105 Su40St0 4 0.1160 0.0000 0.0382 0.0000 — — — 3.4056 0.8738 0.0237 -0.0515 6.5976 2.4032 1.8503 0.7569 0.2977 0.2593 0.5173

2 106 Su40St0 4 0.1160 0.0000 0.0379 0.0000 — — — 3.7631 1.0837 0.0231 -0.0430 6.5615 2.3728 1.6500 0.7995 0.1734 0.2578 0.7393

2 107 Su80St0 4 0.1158 0.0000 0.0759 0.0000 — — — 3.8032 -0.0345 -0.0007 0.0394 16.3398 2.9793 2.2068 0.7441 0.2482 0.1207 0.4838

2 108 Su80St0 4 0.1160 0.0000 0.0765 0.0000 — — — 4.6494 1.8076 -0.0007 -0.0518 16.5119 3.3157 2.3860 -0.1856 0.0000 0.0000 0.0000

2 109 Su120St0 4 0.1162 0.0000 0.1144 0.0000 — — — 3.7222 2.4498 0.0114 -0.0518 26.4728 3.5173 2.9295 — 0.2094 0.2536 0.7143

2 110 Su120St0 4 0.1158 0.0000 0.1144 0.0000 — — — 4.5695 -0.0077 0.1283 -0.0471 26.8171 3.9961 3.0278 3.8511 0.0000 0.2908 0.4206

2 111 Su0St40 4 0.1158 0.0319 0.0000 0.0000 — — — 23.7453 0.0866 -0.0007 0.0291 0.0347 0.6213 0.4149 0.5832 0.2690 0.3173 0.8346

2 112 Su0St40 4 0.1160 0.0323 0.0000 0.0000 — — — 24.1346 0.0742 0.0213 0.0115 0.0000 0.7185 0.4598 0.5934 0.7391 0.3038 0.5787

2 113 Su0St80 4 0.1159 0.0637 0.0000 0.0000 — — — 48.4369 -0.1030 0.0056 0.4091 0.0434 1.1063 0.5572 0.6184 0.2641 0.2249 0.6967

2 114 Su0St80 4 0.1160 0.0637 0.0000 0.0000 — — — 47.0560 0.6993 0.0869 -0.0518 0.0000 0.7325 0.1129 0.3648 0.2576 0.2056 0.5406

2 115 Su0St120 4 0.1158 0.0956 0.0000 0.0000 — — — 60.0744 0.0659 -0.0007 -0.0518 0.0328 0.5790 0.5329 0.5215 0.2578 0.3097 0.6387

2 116 Su0St120 4 0.1161 0.0960 0.0000 0.0000 — — — 67.1840 0.0691 -0.0007 -0.0518 0.0702 1.3976 0.7642 0.5802 0.2954 0.2458 0.5629

2 117 Su40St80 4 0.1161 0.0636 0.0385 0.0000 — — — 38.7042 -0.0986 -0.0007 -0.0159 6.7646 2.8258 1.9601 0.9000 0.2819 0.2627 0.7037

2 118 Su40St80 4 0.1157 0.0641 0.0379 0.0000 — — — 44.1809 0.4536 -0.0007 -0.0518 6.4778 2.8485 1.8807 0.9573 0.2816 0.3038 0.5667

2 119 Su80St40 4 0.1160 0.0319 0.0765 0.0000 — — — 27.3890 -0.0631 0.0288 0.0431 16.5966 3.3064 2.4339 0.8517 0.1929 0.2632 0.4044

212



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 120 Su80St40 4 0.1159 0.0322 0.0761 0.0000 — — — 23.8911 -0.1030 0.0071 -0.0518 16.2157 3.2511 2.7413 0.9187 0.3061 0.2543 0.4295

2 121 BL 8 0.0000 0.0000 0.0000 0.0000 7.17 — — — — — — — — — — — — —

2 122 BL 8 0.0000 0.0000 0.0000 0.0000 7.23 — — — — — — — — — — — — —

2 123 NDF 8 0.1161 0.0000 0.0000 0.0000 7.23 90.5396 — — — — — — — — — — — —

2 124 NDF 8 0.1160 0.0000 0.0000 0.0000 7.21 89.5803 — — — — — — — — — — — —

2 125 Su40St0 8 0.1161 0.0000 0.0382 0.0000 7.06 88.8955 — — — — — — — — — — — —

2 126 Su40St0 8 0.1160 0.0000 0.0384 0.0000 7.04 87.5111 — — — — — — — — — — — —

2 127 Su80St0 8 0.1162 0.0000 0.0762 0.0000 6.86 89.1734 — — — — — — — — — — — —

2 128 Su80St0 8 0.1158 0.0000 0.0765 0.0000 6.90 87.8295 — — — — — — — — — — — —

2 129 Su120St0 8 0.1159 0.0000 0.1139 0.0000 6.79 88.1879 — — — — — — — — — — — —

2 130 Su120St0 8 0.1161 0.0000 0.1143 0.0000 6.78 88.3860 — — — — — — — — — — — —

2 131 Su0St40 8 0.1161 0.0318 0.0000 0.0000 7.03 88.9029 — — — — — — — — — — — —

2 132 Su0St40 8 0.1161 0.0322 0.0000 0.0000 7.10 90.5396 — — — — — — — — — — — —

2 133 Su0St80 8 0.1160 0.0637 0.0000 0.0000 7.07 91.5633 — — — — — — — — — — — —

2 134 Su0St80 8 0.1160 0.0639 0.0000 0.0000 7.07 90.7873 — — — — — — — — — — — —

2 135 Su0St120 8 0.1161 0.0957 0.0000 0.0000 7.11 90.8842 — — — — — — — — — — — —

2 136 Su0St120 8 0.1160 0.0956 0.0000 0.0000 7.08 90.3562 — — — — — — — — — — — —

2 137 Su40St80 8 0.1158 0.0641 0.0382 0.0000 6.93 89.5567 — — — — — — — — — — — —

2 138 Su40St80 8 0.1159 0.0638 0.0384 0.0000 6.91 89.3097 — — — — — — — — — — — —

2 139 Su80St40 8 0.1161 0.0319 0.0764 0.0000 6.87 89.3336 — — — — — — — — — — — —

2 140 Su80St40 8 0.1159 0.0319 0.0765 0.0000 6.83 88.5330 — — — — — — — — — — — —

2 143 NDF 8 0.1158 0.0000 0.0000 0.0000 — — 1.3237 — — — — — — — — — — —

2 144 NDF 8 0.1158 0.0000 0.0000 0.0000 — — 1.0308 — — — — — — — — — — —

2 145 Su40St0 8 0.1157 0.0000 0.0383 0.0000 — — 6.4510 — — — — — — — — — — —

2 146 Su40St0 8 0.1160 0.0000 0.0380 0.0000 — — 6.2561 — — — — — — — — — — —

2 147 Su80St0 8 0.1160 0.0000 0.0762 0.0000 — — 9.9560 — — — — — — — — — — —

2 148 Su80St0 8 0.1156 0.0000 0.0759 0.0000 — — 10.9524 — — — — — — — — — — —

2 149 Su120St0 8 0.1159 0.0000 0.1145 0.0000 — — 12.4909 — — — — — — — — — — —

213



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 150 Su120St0 8 0.1161 0.0000 0.1139 0.0000 — — 13.2809 — — — — — — — — — — —

2 151 Su0St40 8 0.1157 0.0322 0.0000 0.0000 — — 2.0637 — — — — — — — — — — —

2 152 Su0St40 8 0.1157 0.0321 0.0000 0.0000 — — 2.2620 — — — — — — — — — — —

2 153 Su0St80 8 0.1160 0.0637 0.0000 0.0000 — — 5.3277 — — — — — — — — — — —

2 154 Su0St80 8 0.1160 0.0642 0.0000 0.0000 — — 3.7532 — — — — — — — — — — —

2 155 Su0St120 8 0.1160 0.0957 0.0000 0.0000 — — 5.4208 — — — — — — — — — — —

2 156 Su0St120 8 0.1160 0.0960 0.0000 0.0000 — — 5.6100 — — — — — — — — — — —

2 157 Su40St80 8 0.1162 0.0639 0.0385 0.0000 — — 10.2957 — — — — — — — — — — —

2 158 Su40St80 8 0.1159 0.0642 0.0383 0.0000 — — 10.3030 — — — — — — — — — — —

2 159 Su80St40 8 0.1157 0.0319 0.0765 0.0000 — — 15.2869 — — — — — — — — — — —

2 160 Su80St40 8 0.1157 0.0319 0.0762 0.0000 — — 15.2918 — — — — — — — — — — —

2 161 BL 8 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.4124 0.5806

2 162 BL 8 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2935 0.7520

2 163 NDF 8 0.1161 0.0000 0.0000 0.0000 — — — 0.2736 -0.0747 0.0146 0.1148 0.0000 0.7421 0.1625 0.1017 0.1979 0.3102 0.6851

2 164 NDF 8 0.1158 0.0000 0.0000 0.0000 — — — 0.2861 -0.0567 0.0000 0.0000 0.0000 0.4711 -0.0574 -0.0186 0.1508 0.2398 0.4714

2 165 Su40St0 8 0.1160 0.0000 0.0383 0.0000 — — — 2.7343 0.0486 0.0908 0.0000 4.6277 1.9902 2.4891 0.4817 0.1688 0.2823 0.7720

2 166 Su40St0 8 0.1158 0.0000 0.0382 0.0000 — — — 2.4503 -0.0500 0.0536 0.0000 4.7763 4.5716 3.2837 0.7886 0.2463 0.1870 0.8229

2 167 Su80St0 8 0.1159 0.0000 0.0760 0.0000 — — — 3.2129 -0.0916 0.0066 0.0998 13.5305 5.0411 4.4608 0.8143 0.2222 0.3257 0.8494

2 168 Su80St0 8 0.1158 0.0000 0.0762 0.0000 — — — 3.0912 0.8002 0.0000 0.0000 14.7060 5.1356 4.1792 0.7104 -0.0802 0.2924 0.7355

2 169 Su120St0 8 0.1158 0.0000 0.1139 0.0000 — — — 3.6877 0.8653 0.0344 0.0000 23.9738 6.7389 5.7650 1.0547 -0.0165 0.3832 0.9227

2 170 Su120St0 8 0.1158 0.0000 0.1141 0.0000 — — — 3.1691 0.9269 0.0931 0.1773 24.0070 6.3245 4.4848 0.8590 0.2140 0.2311 0.7305

2 171 Su0St40 8 0.1160 0.0320 0.0000 0.0000 — — — 14.9257 -0.0835 0.0001 0.1279 0.0000 2.4400 1.3007 0.3267 0.1326 0.3137 0.5200

2 172 Su0St40 8 0.1159 0.0322 0.0000 0.0000 — — — 19.1461 0.0321 0.0000 0.0000 0.0000 2.0077 1.1857 0.1658 -0.1255 0.1419 0.2960

2 173 Su0St80 8 0.1159 0.0642 0.0000 0.0000 — — — 33.3682 -0.0677 0.0458 0.0311 0.0000 3.2487 1.5155 0.2904 0.2094 0.2833 0.8507

2 174 Su0St80 8 0.1162 0.0638 0.0000 0.0000 — — — 34.9633 -0.0374 0.1412 0.0000 0.0349 3.9584 1.9167 0.4287 0.1662 0.3227 0.7978

2 175 Su0St120 8 0.1157 0.0957 0.0000 0.0000 — — — 58.1448 -0.0571 0.0003 0.0000 0.1470 3.1325 1.6798 0.4500 0.0551 0.3219 0.7591

2 176 Su0St120 8 0.1157 0.0959 0.0000 0.0000 — — — 46.1523 0.0211 0.0328 0.0000 0.0806 3.7483 1.8396 0.4240 0.2029 0.3289 0.8189

2 177 Su40St80 8 0.1162 0.0636 0.0380 0.0000 — — — 23.0778 -0.0846 0.0410 0.7407 5.1117 7.1480 5.3454 1.0997 0.2450 0.3179 0.8688

214



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 178 Su40St80 8 0.1157 0.0637 0.0383 0.0000 — — — 14.0327 -0.0916 0.3084 0.0000 5.1395 — 4.5793 0.9653 0.3568 0.3067 0.7355

2 179 Su80St40 8 0.1161 0.0320 0.0765 0.0000 — — — 15.8772 0.7607 0.0652 0.0000 14.4601 7.9305 6.2095 1.1852 0.2352 0.3032 0.7325

2 180 Su80St40 8 0.1162 0.0322 0.0764 0.0000 — — — 5.0859 0.0265 0.0000 0.1384 14.0488 8.0772 6.5859 1.1411 0.2619 0.3042 0.7698

2 181 BL 12 0.0000 0.0000 0.0000 0.0000 7.23 — — — — — — — — — — — — —

2 182 BL 12 0.0000 0.0000 0.0000 0.0000 7.23 — — — — — — — — — — — — —

2 183 NDF 12 0.1161 0.0000 0.0000 0.0000 7.26 81.2789 — — — — — — — — — — — —

2 184 NDF 12 0.1160 0.0000 0.0000 0.0000 7.26 80.3119 — — — — — — — — — — — —

2 185 Su40St0 12 0.1161 0.0000 0.0385 0.0000 7.15 79.5560 — — — — — — — — — — — —

2 186 Su40St0 12 0.1159 0.0000 0.0385 0.0000 7.16 78.6529 — — — — — — — — — — — —

2 187 Su80St0 12 0.1161 0.0000 0.0761 0.0000 7.04 78.8668 — — — — — — — — — — — —

2 188 Su80St0 12 0.1161 0.0000 0.0764 0.0000 7.05 78.3499 — — — — — — — — — — — —

2 189 Su120St0 12 0.1161 0.0000 0.1142 0.0000 6.90 79.2976 — — — — — — — — — — — —

2 190 Su120St0 12 0.1161 0.0000 0.1140 0.0000 6.88 79.2114 — — — — — — — — — — — —

2 191 Su0St40 12 0.1160 0.0322 0.0000 0.0000 7.16 80.3981 — — — — — — — — — — — —

2 192 Su0St40 12 0.1161 0.0322 0.0000 0.0000 7.16 80.4175 — — — — — — — — — — — —

2 193 Su0St80 12 0.1159 0.0637 0.0000 0.0000 7.03 80.5513 — — — — — — — — — — — —

2 194 Su0St80 12 0.1158 0.0642 0.0000 0.0000 6.98 80.3593 — — — — — — — — — — — —

2 195 Su0St120 12 0.1159 0.0960 0.0000 0.0000 6.99 81.7593 — — — — — — — — — — — —

2 196 Su0St120 12 0.1161 0.0960 0.0000 0.0000 6.88 82.6573 — — — — — — — — — — — —

2 197 Su40St80 12 0.1157 0.0640 0.0384 0.0000 6.95 78.6111 — — — — — — — — — — — —

2 198 Su40St80 12 0.1159 0.0639 0.0385 0.0000 6.95 78.6529 — — — — — — — — — — — —

2 199 Su80St40 12 0.1160 0.0321 0.0765 0.0000 6.93 77.9840 — — — — — — — — — — — —

2 200 Su80St40 12 0.1160 0.0319 0.0764 0.0000 6.91 76.7769 — — — — — — — — — — — —

2 203 NDF 12 0.1158 0.0000 0.0000 0.0000 — — 4.5462 — — — — — — — — — — —

2 204 NDF 12 0.1159 0.0000 0.0000 0.0000 — — 4.5414 — — — — — — — — — — —

2 205 Su40St0 12 0.1161 0.0000 0.0382 0.0000 — — 8.3922 — — — — — — — — — — —

2 206 Su40St0 12 0.1160 0.0000 0.0385 0.0000 — — 8.5791 — — — — — — — — — — —

2 207 Su80St0 12 0.1158 0.0000 0.0759 0.0000 — — 14.0716 — — — — — — — — — — —

215



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 208 Su80St0 12 0.1162 0.0000 0.0765 0.0000 — — 14.5313 — — — — — — — — — — —

2 209 Su120St0 12 0.1157 0.0000 0.1142 0.0000 — — 17.2864 — — — — — — — — — — —

2 210 Su120St0 12 0.1160 0.0000 0.1140 0.0000 — — 17.2845 — — — — — — — — — — —

2 211 Su0St40 12 0.1158 0.0319 0.0000 0.0000 — — 10.6627 — — — — — — — — — — —

2 212 Su0St40 12 0.1157 0.0319 0.0000 0.0000 — — 10.9620 — — — — — — — — — — —

2 213 Su0St80 12 0.1160 0.0639 0.0000 0.0000 — — 14.1114 — — — — — — — — — — —

2 214 Su0St80 12 0.1161 0.0639 0.0000 0.0000 — — 16.6476 — — — — — — — — — — —

2 215 Su0St120 12 0.1160 0.0956 0.0000 0.0000 — — 21.4394 — — — — — — — — — — —

2 216 Su0St120 12 0.1160 0.0958 0.0000 0.0000 — — 19.2850 — — — — — — — — — — —

2 217 Su40St80 12 0.1159 0.0639 0.0383 0.0000 — — 18.9050 — — — — — — — — — — —

2 218 Su40St80 12 0.1160 0.0636 0.0383 0.0000 — — 20.2776 — — — — — — — — — — —

2 219 Su80St40 12 0.1161 0.0320 0.0759 0.0000 — — 22.6112 — — — — — — — — — — —

2 220 Su80St40 12 0.1158 0.0322 0.0762 0.0000 — — 20.0693 — — — — — — — — — — —

2 221 BL 12 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2365 1.1549

2 222 BL 12 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.4746 1.3227

2 223 NDF 12 0.1161 0.0000 0.0000 0.0000 — — — 0.5928 -0.1623 -0.0043 0.0201 0.0000 3.5673 1.4733 0.4179 0.1698 0.4189 1.2735

2 224 NDF 12 0.1162 0.0000 0.0000 0.0000 — — — 0.5255 -0.1835 -0.0036 -0.0358 0.0000 4.4002 1.6123 0.5665 0.1680 0.4579 0.7884

2 225 Su40St0 12 0.1159 0.0000 0.0380 0.0000 — — — 2.2584 0.2612 0.0015 -0.0397 1.7557 8.6840 5.1241 2.2978 0.5180 0.4312 1.4498

2 226 Su40St0 12 0.1161 0.0000 0.0379 0.0000 — — — 2.2065 0.1857 -0.0045 -0.0316 1.6069 8.3658 4.9792 2.3144 0.6845 0.4694 1.6386

2 227 Su80St0 12 0.1159 0.0000 0.0765 0.0000 — — — 2.6540 0.2676 -0.0222 -0.0383 7.1313 9.9638 7.4751 3.1985 0.7260 0.5239 0.9273

2 228 Su80St0 12 0.1158 0.0000 0.0762 0.0000 — — — 2.3702 0.1807 -0.0248 -0.0156 7.3026 10.9037 7.5104 3.1804 0.9034 0.4201 1.3262

2 229 Su120St0 12 0.1160 0.0000 0.1141 0.0000 — — — — -0.0218 -0.0292 0.0203 — — — — — — —

2 230 Su120St0 12 0.1161 0.0000 0.1139 0.0000 — — — 2.4481 0.0035 -0.0338 -0.0249 4.6125 17.4724 16.0477 3.8994 1.2275 0.3560 1.2864

2 231 Su0St40 12 0.1160 0.0318 0.0000 0.0000 — — — 4.0744 -0.1835 -0.0354 0.0372 0.0000 8.7703 4.8227 1.1356 0.2542 0.5931 1.4600

2 232 Su0St40 12 0.1157 0.0319 0.0000 0.0000 — — — 3.3246 -0.1835 -0.0374 -0.0280 0.0000 8.8680 4.8931 1.1017 0.2508 0.6352 1.4401

2 233 Su0St80 12 0.1161 0.0636 0.0000 0.0000 — — — 4.1574 -0.1835 -0.0396 -0.0097 0.0000 13.5384 9.0134 1.3364 0.2960 0.5243 1.4643

2 234 Su0St80 12 0.1158 0.0641 0.0000 0.0000 — — — 5.0875 -0.1835 -0.0414 -0.0304 0.0000 13.9667 9.0626 1.4400 0.3672 0.5223 1.5346

2 235 Su0St120 12 0.1159 0.0957 0.0000 0.0000 — — — 13.0236 -0.1835 -0.0426 -0.0418 0.0324 16.5182 10.0698 1.8695 0.3855 0.5249 1.5286

216



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 236 Su0St120 12 0.1159 0.0957 0.0000 0.0000 — — — 14.7571 -0.1835 -0.0426 -0.0418 0.0000 16.1743 10.6442 1.9757 0.4192 0.5406 1.5396

2 237 Su40St80 12 0.1161 0.0638 0.0380 0.0000 — — — 8.3550 -0.1835 -0.0426 -0.0222 0.3379 16.5174 12.3931 3.7858 0.8749 0.5445 1.6524

2 238 Su40St80 12 0.1157 0.0642 0.0381 0.0000 — — — 8.7555 -0.1835 -0.0426 -0.0418 0.0440 16.7056 12.6087 3.8122 0.8778 0.5425 1.7565

2 239 Su80St40 12 0.1160 0.0318 0.0763 0.0000 — — — 5.1919 -0.1835 -0.0426 -0.0418 6.0687 14.8671 11.7172 5.3904 1.5970 0.5145 0.9400

2 240 Su80St40 12 0.1157 0.0318 0.0765 0.0000 — — — 6.7589 -0.1835 -0.0331 -0.0216 7.2785 14.0033 10.7279 4.6877 1.4847 0.4962 1.2789

2 241 BL 16 0.0000 0.0000 0.0000 0.0000 7.34 — — — — — — — — — — — — —

2 242 BL 16 0.0000 0.0000 0.0000 0.0000 7.38 — — — — — — — — — — — — —

2 243 NDF 16 0.1160 0.0000 0.0000 0.0000 7.34 72.3798 — — — — — — — — — — — —

2 244 NDF 16 0.1159 0.0000 0.0000 0.0000 7.32 71.4909 — — — — — — — — — — — —

2 245 Su40St0 16 0.1161 0.0000 0.0384 0.0000 7.23 70.8552 — — — — — — — — — — — —

2 246 Su40St0 16 0.1161 0.0000 0.0384 0.0000 7.25 71.4583 — — — — — — — — — — — —

2 247 Su80St0 16 0.1160 0.0000 0.0760 0.0000 7.15 72.2936 — — — — — — — — — — — —

2 248 Su80St0 16 0.1160 0.0000 0.0762 0.0000 7.02 72.2074 — — — — — — — — — — — —

2 249 Su120St0 16 0.1159 0.0000 0.1140 0.0000 7.04 71.7497 — — — — — — — — — — — —

2 250 Su120St0 16 0.1159 0.0000 0.1140 0.0000 7.02 71.0594 — — — — — — — — — — — —

2 251 Su0St40 16 0.1157 0.0321 0.0000 0.0000 7.22 72.3015 — — — — — — — — — — — —

2 252 Su0St40 16 0.1161 0.0322 0.0000 0.0000 7.28 73.1812 — — — — — — — — — — — —

2 253 Su0St80 16 0.1157 0.0637 0.0000 0.0000 7.16 73.2522 — — — — — — — — — — — —

2 254 Su0St80 16 0.1157 0.0639 0.0000 0.0000 7.14 72.6472 — — — — — — — — — — — —

2 255 Su0St120 16 0.1157 0.0959 0.0000 0.0000 7.07 74.4623 — — — — — — — — — — — —

2 256 Su0St120 16 0.1161 0.0957 0.0000 0.0000 7.07 74.2149 — — — — — — — — — — — —

2 257 Su40St80 16 0.1161 0.0640 0.0385 0.0000 7.08 71.1998 — — — — — — — — — — — —

2 258 Su40St80 16 0.1159 0.0642 0.0380 0.0000 7.07 72.7852 — — — — — — — — — — — —

2 259 Su80St40 16 0.1161 0.0320 0.0761 0.0000 7.05 67.3232 — — — — — — — — — — — —

2 260 Su80St40 16 0.1160 0.0322 0.0765 0.0000 7.08 71.2590 — — — — — — — — — — — —

2 263 NDF 16 0.1161 0.0000 0.0000 0.0000 — — 3.9460 — — — — — — — — — — —

2 264 NDF 16 0.1157 0.0000 0.0000 0.0000 — — 3.4768 — — — — — — — — — — —

2 265 Su40St0 16 0.1157 0.0000 0.0380 0.0000 — — 8.5140 — — — — — — — — — — —

217



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 266 Su40St0 16 0.1161 0.0000 0.0383 0.0000 — — 8.0951 — — — — — — — — — — —

2 267 Su80St0 16 0.1161 0.0000 0.0761 0.0000 — — 13.1786 — — — — — — — — — — —

2 268 Su80St0 16 0.1157 0.0000 0.0760 0.0000 — — 13.5832 — — — — — — — — — — —

2 269 Su120St0 16 0.1157 0.0000 0.1142 0.0000 — — 16.2122 — — — — — — — — — — —

2 270 Su120St0 16 0.1156 0.0000 0.1139 0.0000 — — 18.1738 — — — — — — — — — — —

2 271 Su0St40 16 0.1160 0.0319 0.0000 0.0000 — — 10.0702 — — — — — — — — — — —

2 272 Su0St40 16 0.1160 0.0319 0.0000 0.0000 — — 10.2685 — — — — — — — — — — —

2 273 Su0St80 16 0.1160 0.0641 0.0000 0.0000 — — 18.3055 — — — — — — — — — — —

2 274 Su0St80 16 0.1158 0.0637 0.0000 0.0000 — — 17.4416 — — — — — — — — — — —

2 275 Su0St120 16 0.1159 0.0957 0.0000 0.0000 — — 20.3654 — — — — — — — — — — —

2 276 Su0St120 16 0.1160 0.0957 0.0000 0.0000 — — 21.2401 — — — — — — — — — — —

2 277 Su40St80 16 0.1157 0.0639 0.0381 0.0000 — — 20.2850 — — — — — — — — — — —

2 278 Su40St80 16 0.1160 0.0641 0.0385 0.0000 — — 20.2565 — — — — — — — — — — —

2 279 Su80St40 16 0.1158 0.0321 0.0765 0.0000 — — 23.7765 — — — — — — — — — — —

2 280 Su80St40 16 0.1161 0.0318 0.0764 0.0000 — — 23.8769 — — — — — — — — — — —

2 281 BL 16 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.5874 1.9229

2 282 BL 16 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 1.1033 1.8552

2 283 NDF 16 0.1158 0.0000 0.0000 0.0000 — — — 0.3594 -0.0550 0.0000 -0.0087 0.0000 6.3607 2.5954 0.6102 0.1848 0.5711 2.0101

2 284 NDF 16 0.1157 0.0000 0.0000 0.0000 — — — 0.2071 -0.0662 0.0000 -0.0087 0.0000 5.9348 2.4117 3.1836 0.1170 0.5730 1.5940

2 285 Su40St0 16 0.1160 0.0000 0.0384 0.0000 — — — 1.7837 -0.0009 0.0000 0.0169 0.0000 11.4152 5.6979 3.1313 0.7342 0.7060 1.9145

2 286 Su40St0 16 0.1157 0.0000 0.0383 0.0000 — — — 1.4109 0.0129 0.0042 0.0053 0.0285 11.2017 6.0003 2.8499 0.7638 0.6594 2.1862

2 287 Su80St0 16 0.1159 0.0000 0.0760 0.0000 — — — 2.0249 -0.0093 0.0000 0.0045 0.2224 11.4786 8.1334 5.7685 2.6739 0.5648 1.8261

2 288 Su80St0 16 0.1159 0.0000 0.0764 0.0000 — — — 1.8103 -0.0230 0.0000 -0.0008 1.6423 11.7581 7.8162 5.5898 2.4207 0.6625 1.9916

2 289 Su120St0 16 0.1161 0.0000 0.1139 0.0000 — — — 2.0249 0.0158 0.0000 -0.0087 0.0000 18.7200 16.7983 5.1168 2.7974 0.8670 2.1451

2 290 Su120St0 16 0.1159 0.0000 0.1140 0.0000 — — — 2.0499 -0.0083 0.0000 -0.0087 0.0000 19.5953 17.9146 5.0136 2.5802 0.8291 1.9934

2 291 Su0St40 16 0.1157 0.0319 0.0000 0.0000 — — — 3.2871 -0.0308 0.0000 -0.0065 0.0000 11.2198 5.9500 1.4299 0.4034 0.7450 2.2876

2 292 Su0St40 16 0.1160 0.0318 0.0000 0.0000 — — — 2.7686 -0.0408 0.0000 -0.0066 0.0000 11.0011 5.8734 1.3089 0.3441 0.6519 2.2097

2 293 Su0St80 16 0.1160 0.0642 0.0000 0.0000 — — — 5.1881 -0.0145 0.0000 -0.0087 0.0000 16.0265 9.8803 1.8700 0.5368 0.6756 2.4155

218



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 294 Su0St80 16 0.1158 0.0637 0.0000 0.0000 — — — 5.8074 -0.0084 0.0000 0.0038 0.0000 15.7574 9.4943 1.9576 0.3876 0.7221 2.3309

2 295 Su0St120 16 0.1158 0.0957 0.0000 0.0000 — — — 14.6931 0.0136 0.0000 0.0039 0.0716 18.6191 12.4569 2.6989 0.6344 0.7644 2.1866

2 296 Su0St120 16 0.1158 0.0955 0.0000 0.0000 — — — 7.8041 0.0116 0.0000 0.0611 0.0549 17.8707 12.2594 2.3946 0.6313 0.8098 2.3953

2 297 Su40St80 16 0.1162 0.0641 0.0385 0.0000 — — — 6.2206 -0.0364 0.0000 0.0291 0.0000 18.5922 13.3358 4.4623 1.4319 0.8633 2.1862

2 298 Su40St80 16 0.1157 0.0638 0.0381 0.0000 — — — 6.5262 -0.0054 0.0000 -0.0040 0.0000 19.4611 13.2081 4.4123 1.4454 0.8991 2.1920

2 299 Su80St40 16 0.1161 0.0319 0.0760 0.0000 — — — 2.8220 -0.0553 0.0000 0.0280 0.0000 17.2240 12.4217 6.8617 3.0839 0.8502 2.0150

2 300 Su80St40 16 0.1159 0.0321 0.0765 0.0000 — — — 4.5354 -0.0178 0.0000 -0.0053 0.0000 16.2368 12.1335 6.5498 3.3018 0.8818 2.3210

2 301 BL 20 0.0000 0.0000 0.0000 0.0000 7.33 — — — — — — — — — — — — —

2 302 BL 20 0.0000 0.0000 0.0000 0.0000 7.41 — — — — — — — — — — — — —

2 303 NDF 20 0.1159 0.0000 0.0000 0.0000 7.15 63.5091 — — — — — — — — — — — —

2 304 NDF 20 0.1161 0.0000 0.0000 0.0000 7.31 66.3326 — — — — — — — — — — — —

2 305 Su40St0 20 0.1158 0.0000 0.0383 0.0000 7.23 64.6847 — — — — — — — — — — — —

2 306 Su40St0 20 0.1158 0.0000 0.0381 0.0000 7.26 64.8574 — — — — — — — — — — — —

2 307 Su80St0 20 0.1158 0.0000 0.0765 0.0000 7.16 63.3892 — — — — — — — — — — — —

2 308 Su80St0 20 0.1160 0.0000 0.0759 0.0000 7.17 63.1114 — — — — — — — — — — — —

2 309 Su120St0 20 0.1162 0.0000 0.1145 0.0000 7.07 63.1788 — — — — — — — — — — — —

2 310 Su120St0 20 0.1162 0.0000 0.1139 0.0000 7.09 63.6953 — — — — — — — — — — — —

2 311 Su0St40 20 0.1157 0.0320 0.0000 0.0000 7.27 65.5165 — — — — — — — — — — — —

2 312 Su0St40 20 0.1160 0.0323 0.0000 0.0000 7.12 63.6287 — — — — — — — — — — — —

2 313 Su0St80 20 0.1161 0.0639 0.0000 0.0000 7.09 64.0928 — — — — — — — — — — — —

2 314 Su0St80 20 0.1161 0.0642 0.0000 0.0000 7.17 64.7819 — — — — — — — — — — — —

2 315 Su0St120 20 0.1158 0.0959 0.0000 0.0000 7.05 64.7710 — — — — — — — — — — — —

2 316 Su0St120 20 0.1160 0.0960 0.0000 0.0000 7.08 65.1806 — — — — — — — — — — — —

2 317 Su40St80 20 0.1158 0.0641 0.0384 0.0000 7.10 63.4756 — — — — — — — — — — — —

2 318 Su40St80 20 0.1161 0.0636 0.0381 0.0000 7.08 63.0538 — — — — — — — — — — — —

2 319 Su80St40 20 0.1157 0.0318 0.0764 0.0000 7.13 65.4300 — — — — — — — — — — — —

2 320 Su80St40 20 0.1160 0.0322 0.0761 0.0000 7.13 64.9220 — — — — — — — — — — — —

2 323 NDF 20 0.1158 0.0000 0.0000 0.0000 — — 2.5932 — — — — — — — — — — —

219



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 324 NDF 20 0.1157 0.0000 0.0000 0.0000 — — 2.5003 — — — — — — — — — — —

2 325 Su40St0 20 0.1160 0.0000 0.0385 0.0000 — — 7.9932 — — — — — — — — — — —

2 326 Su40St0 20 0.1157 0.0000 0.0384 0.0000 — — 6.1539 — — — — — — — — — — —

2 327 Su80St0 20 0.1159 0.0000 0.0761 0.0000 — — 12.6959 — — — — — — — — — — —

2 328 Su80St0 20 0.1160 0.0000 0.0761 0.0000 — — 12.6931 — — — — — — — — — — —

2 329 Su120St0 20 0.1159 0.0000 0.1143 0.0000 — — 18.7448 — — — — — — — — — — —

2 330 Su120St0 20 0.1157 0.0000 0.1139 0.0000 — — 18.1717 — — — — — — — — — — —

2 331 Su0St40 20 0.1159 0.0323 0.0000 0.0000 — — 7.3272 — — — — — — — — — — —

2 332 Su0St40 20 0.1158 0.0319 0.0000 0.0000 — — 9.2986 — — — — — — — — — — —

2 333 Su0St80 20 0.1162 0.0638 0.0000 0.0000 — — 14.9874 — — — — — — — — — — —

2 334 Su0St80 20 0.1156 0.0638 0.0000 0.0000 — — 17.2492 — — — — — — — — — — —

2 335 Su0St120 20 0.1160 0.0959 0.0000 0.0000 — — 21.2360 — — — — — — — — — — —

2 336 Su0St120 20 0.1161 0.0959 0.0000 0.0000 — — 21.2338 — — — — — — — — — — —

2 337 Su40St80 20 0.1157 0.0642 0.0381 0.0000 — — 18.7138 — — — — — — — — — — —

2 338 Su40St80 20 0.1161 0.0639 0.0385 0.0000 — — 19.2827 — — — — — — — — — — —

2 339 Su80St40 20 0.1159 0.0323 0.0763 0.0000 — — 21.4317 — — — — — — — — — — —

2 340 Su80St40 20 0.1158 0.0322 0.0759 0.0000 — — 21.9295 — — — — — — — — — — —

2 341 BL 20 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.8941 2.5390

2 342 BL 20 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.8702 2.5797

2 343 NDF 20 0.1160 0.0000 0.0000 0.0000 — — — 0.2415 -0.0799 0.0000 0.0039 0.0000 11.5151 4.5698 1.3453 0.5212 0.9089 2.7344

2 344 NDF 20 0.1161 0.0000 0.0000 0.0000 — — — 0.3214 -0.0979 0.0000 0.0016 0.0000 10.9420 4.1973 1.4126 0.5351 0.9477 2.7512

2 345 Su40St0 20 0.1160 0.0000 0.0385 0.0000 — — — 0.8844 -0.0822 0.0289 0.1329 0.0000 16.2880 7.9851 4.1140 1.3595 1.0653 2.7591

2 346 Su40St0 20 0.1158 0.0000 0.0379 0.0000 — — — 0.9681 -0.1042 0.0010 0.0005 0.0000 15.6213 7.8361 3.9338 1.2690 0.9960 2.7848

2 347 Su80St0 20 0.1158 0.0000 0.0760 0.0000 — — — 1.0441 -0.1817 0.0022 -0.0013 0.0000 17.8713 9.7951 7.2012 2.9847 0.9207 2.4168

2 348 Su80St0 20 0.1158 0.0000 0.0765 0.0000 — — — 1.1279 -0.1817 0.0252 -0.0013 0.0000 18.2500 9.8619 7.7617 3.5303 0.9339 2.5573

2 349 Su120St0 20 0.1157 0.0000 0.1145 0.0000 — — — 0.9681 -0.1817 0.0004 0.0005 0.0000 24.7114 17.7408 7.2502 3.6421 0.8306 2.1888

2 350 Su120St0 20 0.1161 0.0000 0.1145 0.0000 — — — 1.3713 -0.1817 0.0000 -0.0013 0.0000 23.6731 18.5149 6.8512 3.5503 0.7874 2.2382

2 351 Su0St40 20 0.1161 0.0319 0.0000 0.0000 — — — 1.0460 -0.1494 0.0028 -0.0003 0.0000 16.9095 7.8579 2.2672 0.6132 1.0236 2.7728

220



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 352 Su0St40 20 0.1161 0.0320 0.0000 0.0000 — — — 1.4512 -0.1423 0.0057 0.0149 0.0000 15.8925 7.7146 2.0843 0.5313 0.9282 2.6714

2 353 Su0St80 20 0.1158 0.0636 0.0000 0.0000 — — — 2.0025 -0.1725 0.0000 0.0042 0.0000 22.8247 12.3194 2.8647 0.7331 1.0515 2.7358

2 354 Su0St80 20 0.1161 0.0637 0.0000 0.0000 — — — 1.9304 -0.1505 0.0006 -0.0013 0.0000 22.9178 12.8730 2.9002 0.7144 1.1242 2.6368

2 355 Su0St120 20 0.1157 0.0956 0.0000 0.0000 — — — 2.8011 -0.1541 0.0010 -0.0011 0.0000 26.8035 16.5037 3.4805 0.8429 1.0165 2.5402

2 356 Su0St120 20 0.1159 0.0957 0.0000 0.0000 — — — 2.2460 -0.1817 0.0025 0.0074 0.0000 27.1951 16.4961 3.6513 0.8189 1.3077 2.5180

2 357 Su40St80 20 0.1160 0.0641 0.0380 0.0000 — — — 3.1225 -0.1448 0.0000 -0.0013 0.0000 25.7251 15.9157 5.2703 1.4950 0.9106 2.4685

2 358 Su40St80 20 0.1161 0.0641 0.0379 0.0000 — — — 2.8109 -0.1444 0.0000 0.0023 0.0000 26.4709 15.9613 5.4508 1.5556 0.9631 2.4020

2 359 Su80St40 20 0.1157 0.0320 0.0763 0.0000 — — — 2.2538 -0.1544 0.0066 0.0146 0.0751 23.0787 13.8056 7.6827 3.1817 0.9011 2.2769

2 360 Su80St40 20 0.1157 0.0319 0.0764 0.0000 — — — 2.2538 -0.1732 0.0000 -0.0013 0.0000 22.1301 13.0821 7.9450 3.2096 0.8584 2.2203

2 361 BL 24 0.0000 0.0000 0.0000 0.0000 7.58 — — — — — — — — — — — — —

2 362 BL 24 0.0000 0.0000 0.0000 0.0000 7.58 — — — — — — — — — — — — —

2 363 NDF 24 0.1160 0.0000 0.0000 0.0000 7.46 62.8528 — — — — — — — — — — — —

2 364 NDF 24 0.1158 0.0000 0.0000 0.0000 7.42 61.9211 — — — — — — — — — — — —

2 365 Su40St0 24 0.1161 0.0000 0.0385 0.0000 7.34 61.6807 — — — — — — — — — — — —

2 366 Su40St0 24 0.1158 0.0000 0.0380 0.0000 7.33 61.2302 — — — — — — — — — — — —

2 367 Su80St0 24 0.1162 0.0000 0.0763 0.0000 7.15 59.5637 — — — — — — — — — — — —

2 368 Su80St0 24 0.1158 0.0000 0.0761 0.0000 7.25 61.4029 — — — — — — — — — — — —

2 369 Su120St0 24 0.1161 0.0000 0.1140 0.0000 7.12 59.8716 — — — — — — — — — — — —

2 370 Su120St0 24 0.1161 0.0000 0.1142 0.0000 7.13 59.9577 — — — — — — — — — — — —

2 371 Su0St40 24 0.1157 0.0318 0.0000 0.0000 7.32 61.7134 — — — — — — — — — — — —

2 372 Su0St40 24 0.1159 0.0321 0.0000 0.0000 7.31 60.6615 — — — — — — — — — — — —

2 373 Su0St80 24 0.1160 0.0640 0.0000 0.0000 7.05 59.3178 — — — — — — — — — — — —

2 374 Su0St80 24 0.1158 0.0640 0.0000 0.0000 7.25 61.7484 — — — — — — — — — — — —

2 375 Su0St120 24 0.1159 0.0956 0.0000 0.0000 7.13 61.5244 — — — — — — — — — — — —

2 376 Su0St120 24 0.1158 0.0955 0.0000 0.0000 7.03 62.7847 — — — — — — — — — — — —

2 377 Su40St80 24 0.1157 0.0637 0.0385 0.0000 7.17 60.5033 — — — — — — — — — — — —

2 378 Su40St80 24 0.1157 0.0641 0.0379 0.0000 7.15 60.5898 — — — — — — — — — — — —

2 379 Su80St40 24 0.1161 0.0321 0.0764 0.0000 7.18 62.0252 — — — — — — — — — — — —

221



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 380 Su80St40 24 0.1161 0.0322 0.0760 0.0000 7.21 59.9577 — — — — — — — — — — — —

2 383 NDF 24 0.1161 0.0000 0.0000 0.0000 — — -1.2782 — — — — — — — — — — —

2 384 NDF 24 0.1159 0.0000 0.0000 0.0000 — — 0.4890 — — — — — — — — — — —

2 385 Su40St0 24 0.1157 0.0000 0.0384 0.0000 — — 4.9333 — — — — — — — — — — —

2 386 Su40St0 24 0.1157 0.0000 0.0380 0.0000 — — 1.0437 — — — — — — — — — — —

2 387 Su80St0 24 0.1159 0.0000 0.0762 0.0000 — — 7.9570 — — — — — — — — — — —

2 388 Su80St0 24 0.1159 0.0000 0.0761 0.0000 — — 8.1552 — — — — — — — — — — —

2 389 Su120St0 24 0.1160 0.0000 0.1140 0.0000 — — 10.0095 — — — — — — — — — — —

2 390 Su120St0 24 0.1160 0.0000 0.1145 0.0000 — — 15.2718 — — — — — — — — — — —

2 391 Su0St40 24 0.1160 0.0319 0.0000 0.0000 — — 1.0376 — — — — — — — — — — —

2 392 Su0St40 24 0.1162 0.0318 0.0000 0.0000 — — 4.0642 — — — — — — — — — — —

2 393 Su0St80 24 0.1160 0.0636 0.0000 0.0000 — — 13.6792 — — — — — — — — — — —

2 394 Su0St80 24 0.1160 0.0637 0.0000 0.0000 — — 12.7003 — — — — — — — — — — —

2 395 Su0St120 24 0.1161 0.0958 0.0000 0.0000 — — 11.4220 — — — — — — — — — — —

2 396 Su0St120 24 0.1158 0.0956 0.0000 0.0000 — — 13.2900 — — — — — — — — — — —

2 397 Su40St80 24 0.1161 0.0637 0.0379 0.0000 — — 9.1038 — — — — — — — — — — —

2 398 Su40St80 24 0.1161 0.0638 0.0379 0.0000 — — 8.8079 — — — — — — — — — — —

2 399 Su80St40 24 0.1158 0.0319 0.0763 0.0000 — — 16.8984 — — — — — — — — — — —

2 400 Su80St40 24 0.1159 0.0320 0.0764 0.0000 — — 19.1397 — — — — — — — — — — —

2 401 BL 24 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 1.0406 2.8190

2 402 BL 24 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.9987 2.6317

2 403 NDF 24 0.1157 0.0000 0.0000 0.0000 — — — 0.0799 -0.0105 -0.0041 0.0209 0.0000 9.3804 4.2139 1.8233 0.4553 0.9885 2.8245

2 404 NDF 24 0.1159 0.0000 0.0000 0.0000 — — — 0.1656 0.0102 -0.0041 -0.0012 0.0000 8.8129 4.1437 1.4456 0.3928 0.9480 2.8552

2 405 Su40St0 24 0.1161 0.0000 0.0384 0.0000 — — — 0.7188 0.0334 -0.0041 -0.0012 0.0000 17.2047 8.0942 5.0370 1.3614 1.0152 2.7371

2 406 Su40St0 24 0.1161 0.0000 0.0381 0.0000 — — — 0.8026 -0.0169 -0.0018 0.0001 0.0000 15.9961 7.9940 4.9414 1.2307 0.9244 2.5526

2 407 Su80St0 24 0.1160 0.0000 0.0762 0.0000 — — — 1.0383 -0.0087 -0.0041 -0.0012 0.0000 18.1155 9.4403 7.7346 2.8520 0.7748 2.3511

2 408 Su80St0 24 0.1161 0.0000 0.0759 0.0000 — — — 1.0421 0.0196 -0.0027 -0.0012 0.0000 19.4250 10.1080 8.1235 2.9839 0.9006 2.3944

2 409 Su120St0 24 0.1159 0.0000 0.1145 0.0000 — — — 0.8045 0.0255 -0.0035 0.0058 0.0000 25.5566 17.4273 8.2461 3.7559 0.7926 2.2225

222



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

2 410 Su120St0 24 0.1161 0.0000 0.1140 0.0000 — — — 0.7285 -0.0053 0.0049 0.0116 0.0000 25.4512 16.9985 8.2921 3.7073 0.8667 2.2393

2 411 Su0St40 24 0.1158 0.0322 0.0000 0.0000 — — — 0.4909 0.0027 -0.0032 -0.0012 0.0000 17.9434 8.7686 3.5541 0.9894 1.1743 2.9219

2 412 Su0St40 24 0.1161 0.0320 0.0000 0.0000 — — — 0.9662 0.0094 -0.0041 -0.0012 0.0000 17.4540 8.1644 3.4234 0.9256 1.1141 2.9183

2 413 Su0St80 24 0.1159 0.0639 0.0000 0.0000 — — — 0.7344 -0.0042 0.0154 0.0058 0.0000 26.2267 13.3746 4.3213 0.8880 1.1925 2.7058

2 414 Su0St80 24 0.1162 0.0642 0.0000 0.0000 — — — 1.2058 -0.0169 0.0191 -0.0012 0.0000 26.3364 13.5421 4.2800 0.8885 1.1743 2.7130

2 415 Su0St120 24 0.1160 0.0957 0.0000 0.0000 — — — 1.2973 0.0204 -0.0032 -0.0012 0.0000 27.5612 17.0886 8.1142 1.0790 1.6925 1.6827

2 416 Su0St120 24 0.1158 0.0959 0.0000 0.0000 — — — 1.5291 0.0030 -0.0041 -0.0012 0.0000 31.0772 18.1173 4.8674 0.9238 1.1656 2.5473

2 417 Su40St80 24 0.1157 0.0642 0.0384 0.0000 — — — 0.8259 -0.0149 0.0165 -0.0012 0.0000 30.2452 17.8855 6.4567 1.5125 1.1183 2.4269

2 418 Su40St80 24 0.1158 0.0639 0.0380 0.0000 — — — 1.7707 -0.0169 -0.0005 -0.0012 0.0000 26.4753 15.7937 6.1840 1.3912 0.9768 1.7231

2 419 Su80St40 24 0.1161 0.0319 0.0764 0.0000 — — — 0.6584 0.0186 -0.0041 -0.0012 0.0000 21.0815 14.6649 10.2009 4.2377 0.8821 2.2939

2 420 Su80St40 24 0.1159 0.0320 0.0763 0.0000 — — — 1.5330 0.0122 -0.0041 -0.0012 0.0736 26.5215 14.7120 8.9437 3.0542 0.9659 2.3015

3 1 BL 0 0.0000 0.0000 0.0000 0.0000 7.15 — — — — — — — — — — — — —

3 2 BL 0 0.0000 0.0000 0.0000 0.0000 7.13 — — — — — — — — — — — — —

3 3 NDF 0 0.1157 0.0000 0.0000 0.0000 7.16 93.6505 — — — — — — — — — — — —

3 4 NDF 0 0.1157 0.0000 0.0000 0.0000 7.15 93.3912 — — — — — — — — — — — —

3 5 St40Pe0 0 0.1159 0.0318 0.0000 0.0000 7.13 93.4947 — — — — — — — — — — — —

3 6 St40Pe0 0 0.1159 0.0319 0.0000 0.0000 7.18 92.8907 — — — — — — — — — — — —

3 7 St80Pe0 0 0.1160 0.0641 0.0000 0.0000 7.17 91.5202 — — — — — — — — — — — —

3 8 St80Pe0 0 0.1161 0.0641 0.0000 0.0000 7.13 92.4779 — — — — — — — — — — — —

3 9 St120Pe0 0 0.1157 0.0957 0.0000 0.0000 7.15 93.4776 — — — — — — — — — — — —

3 10 St120Pe0 0 0.1157 0.0956 0.0000 0.0000 7.15 92.8726 — — — — — — — — — — — —

3 11 St0Pe40 0 0.1159 0.0000 0.0000 0.0435 7.05 93.4947 — — — — — — — — — — — —

3 12 St0Pe40 0 0.1158 0.0000 0.0000 0.0435 7.07 92.3635 — — — — — — — — — — — —

3 13 St0Pe91 0 0.1161 0.0000 0.0000 0.0869 7.06 93.2532 — — — — — — — — — — — —

3 14 St0Pe91 0 0.1157 0.0000 0.0000 0.0869 7.08 93.9962 — — — — — — — — — — — —

3 15 St0Pe120 0 0.1160 0.0000 0.0000 0.1304 7.13 93.4170 — — — — — — — — — — — —

3 16 St0Pe120 0 0.1160 0.0000 0.0000 0.1304 7.14 93.5032 — — — — — — — — — — — —

3 17 St40Pe91 0 0.1157 0.0321 0.0000 0.0869 7.07 95.2927 — — — — — — — — — — — —

223



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 18 St40Pe91 0 0.1161 0.0322 0.0000 0.0869 7.07 95.0623 — — — — — — — — — — — —

3 19 St80Pe40 0 0.1157 0.0641 0.0000 0.0435 7.08 93.6505 — — — — — — — — — — — —

3 20 St80Pe40 0 0.1162 0.0642 0.0000 0.0435 7.06 94.2948 — — — — — — — — — — — —

3 23 NDF 0 0.1160 0.0000 0.0000 0.0000 — — 0.4865 — — — — — — — — — — —

3 24 NDF 0 0.1159 0.0000 0.0000 0.0000 — — -0.4861 — — — — — — — — — — —

3 25 St40Pe0 0 0.1158 0.0322 0.0000 0.0000 — — 0.5322 — — — — — — — — — — —

3 26 St40Pe0 0 0.1159 0.0318 0.0000 0.0000 — — -0.5310 — — — — — — — — — — —

3 27 St80Pe0 0 0.1157 0.0637 0.0000 0.0000 — — 0.0133 — — — — — — — — — — —

3 28 St80Pe0 0 0.1161 0.0642 0.0000 0.0000 — — -0.0134 — — — — — — — — — — —

3 29 St120Pe0 0 0.1158 0.0960 0.0000 0.0000 — — -0.9761 — — — — — — — — — — —

3 30 St120Pe0 0 0.1161 0.0957 0.0000 0.0000 — — 0.9762 — — — — — — — — — — —

3 31 St0Pe40 0 0.1159 0.0000 0.0000 0.0435 — — -0.2410 — — — — — — — — — — —

3 32 St0Pe40 0 0.1161 0.0000 0.0000 0.0435 — — 0.2413 — — — — — — — — — — —

3 33 St0Pe91 0 0.1161 0.0000 0.0000 0.0869 — — -0.1027 — — — — — — — — — — —

3 34 St0Pe91 0 0.1157 0.0000 0.0000 0.0869 — — 0.1025 — — — — — — — — — — —

3 35 St0Pe120 0 0.1157 0.0000 0.0000 0.1304 — — -0.3846 — — — — — — — — — — —

3 36 St0Pe120 0 0.1162 0.0000 0.0000 0.1304 — — 0.3854 — — — — — — — — — — —

3 37 St40Pe91 0 0.1160 0.0320 0.0000 0.0869 — — 0.0990 — — — — — — — — — — —

3 38 St40Pe91 0 0.1162 0.0319 0.0000 0.0869 — — -0.0990 — — — — — — — — — — —

3 39 St80Pe40 0 0.1159 0.0639 0.0000 0.0435 — — -0.7291 — — — — — — — — — — —

3 40 St80Pe40 0 0.1162 0.0639 0.0000 0.0435 — — 0.7300 — — — — — — — — — — —

3 41 BL 0 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.1918 0.2521

3 42 BL 0 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2687 0.2378

3 43 NDF 0 0.1159 0.0000 0.0000 0.0000 — — — 0.0395 0.0033 0.0821 0.2825 0.0000 0.1916 -0.3264 -0.3328 0.0790 0.1745 0.0446

3 44 NDF 0 0.1162 0.0000 0.0000 0.0000 — — — -0.1913 -0.0002 -0.0432 -0.2302 0.0000 0.2649 -0.0714 -0.3309 0.0507 0.1716 0.3349

3 45 St40Pe0 0 0.1161 0.0318 0.0000 0.0000 — — — 20.1600 0.0169 0.1556 0.7461 0.0000 -0.0268 -0.2375 -0.3477 -0.1713 0.1788 0.2187

3 46 St40Pe0 0 0.1161 0.0318 0.0000 0.0000 — — — 21.0790 -0.0002 -0.0475 -0.2273 0.0000 0.0605 -0.1615 -0.1797 -0.0676 0.2670 0.2958

3 47 St80Pe0 0 0.1156 0.0638 0.0000 0.0000 — — — 37.3665 0.0141 0.1174 0.3096 0.0000 0.0347 -0.1266 -0.2321 -0.3502 0.0000 0.0000

224



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 48 St80Pe0 0 0.1161 0.0637 0.0000 0.0000 — — — 50.3659 -0.0002 -0.0502 -0.2769 0.0000 0.3402 0.0845 -0.2983 -0.3502 0.1556 0.1542

3 49 St120Pe0 0 0.1158 0.0960 0.0000 0.0000 — — — 55.8317 0.0176 0.1245 0.0766 0.0000 1.4305 -1.1731 -0.2614 -0.2529 0.1056 0.0000

3 50 St120Pe0 0 0.1159 0.0960 0.0000 0.0000 — — — 71.7783 -0.0002 -0.0518 -0.2531 0.0000 0.1081 0.3416 -0.2988 -0.0759 0.2115 0.2444

3 51 St0Pe40 0 0.1157 0.0000 0.0000 0.0435 — — — 0.0452 0.0207 0.1111 0.1878 0.0850 0.4738 0.1724 -0.0086 0.0508 0.1214 0.2280

3 52 St0Pe40 0 0.1159 0.0000 0.0000 0.0435 — — — -0.0972 -0.0002 -0.0525 -0.2478 0.1126 0.6104 -1.4049 -0.0220 0.3389 0.1108 0.2823

3 53 St0Pe91 0 0.1162 0.0000 0.0000 0.0869 — — — 0.4380 0.1426 0.0561 0.4964 0.3475 1.7953 0.4377 -0.2137 0.0341 0.0708 0.2515

3 54 St0Pe91 0 0.1161 0.0000 0.0000 0.0869 — — — 0.2963 -0.0002 0.0299 -0.2568 0.2210 0.7228 0.4476 -0.2157 0.0324 0.0889 0.2556

3 55 St0Pe120 0 0.1156 0.0000 0.0000 0.1304 — — — 0.5970 0.3698 0.1718 0.5122 0.2145 0.4619 -0.2017 -0.2636 0.0168 0.1505 0.2405

3 56 St0Pe120 0 0.1158 0.0000 0.0000 0.1304 — — — 11.4767 0.8776 0.2152 -0.2768 0.2317 0.8296 -0.2887 -0.3117 -0.0065 0.0396 0.3052

3 57 St40Pe91 0 0.1161 0.0322 0.0000 0.0869 — — — 15.5293 0.1689 0.1683 0.2834 0.1346 0.4555 0.2329 -0.2152 -0.1069 0.0905 0.2557

3 58 St40Pe91 0 0.1161 0.0323 0.0000 0.0869 — — — 11.0121 -0.0002 0.0115 -0.2842 0.1501 0.3250 0.2393 -0.2640 -0.0620 0.0702 0.2834

3 59 St80Pe40 0 0.1157 0.0642 0.0000 0.0435 — — — 21.5777 -0.0002 -0.0887 0.6106 0.0743 0.0822 -0.0514 -0.2578 -0.0592 0.1123 0.4097

3 60 St80Pe40 0 0.1162 0.0640 0.0000 0.0435 — — — 37.6230 -0.0002 -0.0638 -0.2691 0.0802 0.2725 0.1349 -0.2260 0.0699 0.1262 0.2184

3 61 BL 4 0.0000 0.0000 0.0000 0.0000 7.17 — — — — — — — — — — — — —

3 62 BL 4 0.0000 0.0000 0.0000 0.0000 7.16 — — — — — — — — — — — — —

3 63 NDF 4 0.1156 0.0000 0.0000 0.0000 7.12 92.4310 — — — — — — — — — — — —

3 64 NDF 4 0.1159 0.0000 0.0000 0.0000 7.12 92.2003 — — — — — — — — — — — —

3 65 St40Pe0 4 0.1157 0.0323 0.0000 0.0000 7.14 90.6253 — — — — — — — — — — — —

3 66 St40Pe0 4 0.1162 0.0319 0.0000 0.0000 7.17 92.3151 — — — — — — — — — — — —

3 67 St80Pe0 4 0.1158 0.0638 0.0000 0.0000 7.19 91.7590 — — — — — — — — — — — —

3 68 St80Pe0 4 0.1160 0.0641 0.0000 0.0000 7.17 92.1237 — — — — — — — — — — — —

3 69 St120Pe0 4 0.1161 0.0960 0.0000 0.0000 7.16 90.6689 — — — — — — — — — — — —

3 70 St120Pe0 4 0.1161 0.0960 0.0000 0.0000 7.17 92.3056 — — — — — — — — — — — —

3 71 St0Pe40 4 0.1157 0.0000 0.0000 0.0435 7.05 93.3048 — — — — — — — — — — — —

3 72 St0Pe40 4 0.1162 0.0000 0.0000 0.0435 7.03 93.0037 — — — — — — — — — — — —

3 73 St0Pe91 4 0.1160 0.0000 0.0000 0.0869 7.01 92.0375 — — — — — — — — — — — —

3 74 St0Pe91 4 0.1157 0.0000 0.0000 0.0869 7.04 92.5269 — — — — — — — — — — — —

3 75 St0Pe120 4 0.1159 0.0000 0.0000 0.1304 7.02 94.7027 — — — — — — — — — — — —

225



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 76 St0Pe120 4 0.1161 0.0000 0.0000 0.1304 7.12 92.3056 — — — — — — — — — — — —

3 77 St40Pe91 4 0.1162 0.0321 0.0000 0.0869 7.04 93.3480 — — — — — — — — — — — —

3 78 St40Pe91 4 0.1161 0.0323 0.0000 0.0869 7.02 93.7701 — — — — — — — — — — — —

3 79 St80Pe40 4 0.1159 0.0640 0.0000 0.0435 7.01 93.3221 — — — — — — — — — — — —

3 80 St80Pe40 4 0.1161 0.0642 0.0000 0.0435 7.03 92.5641 — — — — — — — — — — — —

3 83 NDF 4 0.1161 0.0000 0.0000 0.0000 — — -0.2497 — — — — — — — — — — —

3 84 NDF 4 0.1160 0.0000 0.0000 0.0000 — — -0.2459 — — — — — — — — — — —

3 85 St40Pe0 4 0.1159 0.0322 0.0000 0.0000 — — 0.1874 — — — — — — — — — — —

3 86 St40Pe0 4 0.1162 0.0323 0.0000 0.0000 — — 0.1754 — — — — — — — — — — —

3 87 St80Pe0 4 0.1158 0.0637 0.0000 0.0000 — — -1.4057 — — — — — — — — — — —

3 88 St80Pe0 4 0.1158 0.0638 0.0000 0.0000 — — -1.3109 — — — — — — — — — — —

3 89 St120Pe0 4 0.1162 0.0959 0.0000 0.0000 — — -0.2516 — — — — — — — — — — —

3 90 St120Pe0 4 0.1161 0.0960 0.0000 0.0000 — — -0.2514 — — — — — — — — — — —

3 91 St0Pe40 4 0.1161 0.0000 0.0000 0.0435 — — 1.4619 — — — — — — — — — — —

3 92 St0Pe40 4 0.1160 0.0000 0.0000 0.0435 — — 1.2696 — — — — — — — — — — —

3 93 St0Pe91 4 0.1162 0.0000 0.0000 0.0869 — — 1.6037 — — — — — — — — — — —

3 94 St0Pe91 4 0.1161 0.0000 0.0000 0.0869 — — 1.6062 — — — — — — — — — — —

3 95 St0Pe120 4 0.1160 0.0000 0.0000 0.1304 — — 2.5870 — — — — — — — — — — —

3 96 St0Pe120 4 0.1157 0.0000 0.0000 0.1304 — — 2.5937 — — — — — — — — — — —

3 97 St40Pe91 4 0.1157 0.0322 0.0000 0.0869 — — 2.7872 — — — — — — — — — — —

3 98 St40Pe91 4 0.1157 0.0323 0.0000 0.0869 — — 2.6873 — — — — — — — — — — —

3 99 St80Pe40 4 0.1161 0.0640 0.0000 0.0435 — — 3.4155 — — — — — — — — — — —

3 100 St80Pe40 4 0.1161 0.0640 0.0000 0.0435 — — 3.4155 — — — — — — — — — — —

3 101 BL 4 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2205 0.3756

3 102 BL 4 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2007 0.3901

3 103 NDF 4 0.1159 0.0000 0.0000 0.0000 — — — -0.0066 0.0000 -0.0105 0.0669 0.0000 0.1343 -1.1781 0.1153 0.0086 0.2110 0.4775

3 104 NDF 4 0.1159 0.0000 0.0000 0.0000 — — — 0.7971 0.0000 0.0097 -0.0592 0.0000 0.5944 0.3345 0.1821 -0.0327 0.2784 0.3962

3 105 St40Pe0 4 0.1161 0.0319 0.0000 0.0000 — — — 22.8632 0.0000 -0.0105 -0.1177 0.0000 0.6052 0.3278 0.1653 -0.0171 0.2311 0.4320

226



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 106 St40Pe0 4 0.1158 0.0323 0.0000 0.0000 — — — 16.0721 0.0000 0.0094 -0.0904 0.0829 0.8500 0.2737 0.6595 -0.0894 1.5412 0.4485

3 107 St80Pe0 4 0.1159 0.0642 0.0000 0.0000 — — — 40.6195 0.0181 0.1575 -0.1177 0.0915 0.9419 -0.0024 0.1840 0.0430 0.2116 0.4829

3 108 St80Pe0 4 0.1160 0.0642 0.0000 0.0000 — — — 45.5184 0.0000 0.0075 -0.0612 0.0517 0.7518 0.2942 0.1830 -0.1122 0.2167 0.3605

3 109 St120Pe0 4 0.1157 0.0959 0.0000 0.0000 — — — 57.9831 0.0207 0.1779 -0.1177 0.0525 0.5121 0.2559 0.1590 -0.1585 0.2134 0.3747

3 110 St120Pe0 4 0.1157 0.0956 0.0000 0.0000 — — — 70.3209 0.0000 0.0023 -0.1040 0.0000 -0.2368 -1.1182 -0.1776 -0.4765 0.0000 0.0000

3 111 St0Pe40 4 0.1156 0.0000 0.0000 0.0435 — — — 0.6275 0.0180 0.1939 -0.1177 0.0000 3.0499 1.0068 0.0830 0.0105 0.1394 0.3915

3 112 St0Pe40 4 0.1159 0.0000 0.0000 0.0435 — — — 0.2538 0.0000 -0.0003 -0.0937 0.0391 3.1405 1.6165 0.4316 -0.0211 0.2442 0.0800

3 113 St0Pe91 4 0.1159 0.0000 0.0000 0.0869 — — — 0.8651 0.0000 -0.0105 -0.1177 0.0000 3.3092 0.1117 0.1539 -0.4765 0.0000 0.3482

3 114 St0Pe91 4 0.1161 0.0000 0.0000 0.0869 — — — 0.4112 0.0000 -0.0021 -0.1013 0.0735 4.4041 1.6123 0.1659 0.2112 0.0567 0.3944

3 115 St0Pe120 4 0.1162 0.0000 0.0000 0.1304 — — — 0.8613 0.0362 0.1769 -0.1177 0.1754 4.4365 1.5200 0.1991 -0.1189 0.0000 0.3896

3 116 St0Pe120 4 0.1160 0.0000 0.0000 0.1304 — — — 0.0253 0.0000 0.0103 -0.1018 0.3669 4.0512 1.8280 0.1239 -0.0223 0.0344 0.4335

3 117 St40Pe91 4 0.1157 0.0319 0.0000 0.0869 — — — 15.1671 0.0229 0.2326 0.0551 0.2342 3.9045 1.4976 0.1362 0.0105 0.0766 0.3792

3 118 St40Pe91 4 0.1160 0.0322 0.0000 0.0869 — — — 13.8069 0.0000 0.0121 -0.0994 0.0000 1.4059 0.0917 0.2110 0.0240 0.2467 0.6915

3 119 St80Pe40 4 0.1159 0.0642 0.0000 0.0435 — — — 19.4855 0.0226 0.2140 0.0418 0.1397 3.8671 1.5061 0.1715 -0.0856 0.1468 0.4164

3 120 St80Pe40 4 0.1159 0.0639 0.0000 0.0435 — — — 42.8538 0.0000 0.0062 -0.1151 0.1277 4.2332 1.2675 0.2229 -0.1471 0.0921 0.4219

3 121 BL 8 0.0000 0.0000 0.0000 0.0000 7.16 — — — — — — — — — — — — —

3 122 BL 8 0.0000 0.0000 0.0000 0.0000 7.15 — — — — — — — — — — — — —

3 123 NDF 8 0.1159 0.0000 0.0000 0.0000 7.20 88.7056 — — — — — — — — — — — —

3 124 NDF 8 0.1159 0.0000 0.0000 0.0000 7.18 88.7919 — — — — — — — — — — — —

3 125 St40Pe0 8 0.1159 0.0323 0.0000 0.0000 7.10 88.7056 — — — — — — — — — — — —

3 126 St40Pe0 8 0.1162 0.0319 0.0000 0.0000 7.13 88.7430 — — — — — — — — — — — —

3 127 St80Pe0 8 0.1158 0.0637 0.0000 0.0000 7.09 88.8658 — — — — — — — — — — — —

3 128 St80Pe0 8 0.1157 0.0638 0.0000 0.0000 7.10 89.3721 — — — — — — — — — — — —

3 129 St120Pe0 8 0.1157 0.0956 0.0000 0.0000 7.07 88.5077 — — — — — — — — — — — —

3 130 St120Pe0 8 0.1161 0.0960 0.0000 0.0000 7.04 89.8505 — — — — — — — — — — — —

3 131 St0Pe40 8 0.1157 0.0000 0.0000 0.0435 7.02 89.1992 — — — — — — — — — — — —

3 132 St0Pe40 8 0.1157 0.0000 0.0000 0.0435 7.03 89.2856 — — — — — — — — — — — —

3 133 St0Pe91 8 0.1158 0.0000 0.0000 0.0869 6.88 89.5567 — — — — — — — — — — — —

227



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 134 St0Pe91 8 0.1157 0.0000 0.0000 0.0869 6.89 89.1128 — — — — — — — — — — — —

3 135 St0Pe120 8 0.1159 0.0000 0.0000 0.1304 6.67 90.3451 — — — — — — — — — — — —

3 136 St0Pe120 8 0.1162 0.0000 0.0000 0.1304 6.67 90.5506 — — — — — — — — — — — —

3 137 St40Pe91 8 0.1160 0.0322 0.0000 0.0869 6.87 90.3562 — — — — — — — — — — — —

3 138 St40Pe91 8 0.1157 0.0323 0.0000 0.0869 6.84 90.8414 — — — — — — — — — — — —

3 139 St80Pe40 8 0.1157 0.0642 0.0000 0.0435 6.96 90.4093 — — — — — — — — — — — —

3 140 St80Pe40 8 0.1158 0.0637 0.0000 0.0435 6.97 90.3340 — — — — — — — — — — — —

3 143 NDF 8 0.1161 0.0000 0.0000 0.0000 — — 0.8244 — — — — — — — — — — —

3 144 NDF 8 0.1159 0.0000 0.0000 0.0000 — — 0.8322 — — — — — — — — — — —

3 145 St40Pe0 8 0.1159 0.0323 0.0000 0.0000 — — 3.7000 — — — — — — — — — — —

3 146 St40Pe0 8 0.1159 0.0322 0.0000 0.0000 — — 3.7028 — — — — — — — — — — —

3 147 St80Pe0 8 0.1161 0.0640 0.0000 0.0000 — — 4.0448 — — — — — — — — — — —

3 148 St80Pe0 8 0.1157 0.0641 0.0000 0.0000 — — 4.0544 — — — — — — — — — — —

3 149 St120Pe0 8 0.1162 0.0955 0.0000 0.0000 — — 9.1345 — — — — — — — — — — —

3 150 St120Pe0 8 0.1158 0.0956 0.0000 0.0000 — — 6.7011 — — — — — — — — — — —

3 151 St0Pe40 8 0.1162 0.0000 0.0000 0.0435 — — 8.0992 — — — — — — — — — — —

3 152 St0Pe40 8 0.1157 0.0000 0.0000 0.0435 — — 8.6999 — — — — — — — — — — —

3 153 St0Pe91 8 0.1160 0.0000 0.0000 0.0869 — — 15.7679 — — — — — — — — — — —

3 154 St0Pe91 8 0.1157 0.0000 0.0000 0.0869 — — 15.8730 — — — — — — — — — — —

3 155 St0Pe120 8 0.1159 0.0000 0.0000 0.1304 — — 19.7756 — — — — — — — — — — —

3 156 St0Pe120 8 0.1158 0.0000 0.0000 0.1304 — — 20.2661 — — — — — — — — — — —

3 157 St40Pe91 8 0.1159 0.0321 0.0000 0.0869 — — 17.5297 — — — — — — — — — — —

3 158 St40Pe91 8 0.1159 0.0322 0.0000 0.0869 — — 17.0392 — — — — — — — — — — —

3 159 St80Pe40 8 0.1161 0.0642 0.0000 0.0435 — — 12.7865 — — — — — — — — — — —

3 160 St80Pe40 8 0.1161 0.0642 0.0000 0.0435 — — 12.7865 — — — — — — — — — — —

3 161 BL 8 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — — —

3 162 BL 8 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3768 0.5172

3 163 NDF 8 0.1159 0.0000 0.0000 0.0000 — — — -0.6246 0.0012 0.0450 0.0563 0.0000 -0.6548 0.4329 0.0658 -0.0290 0.2819 0.6624

228



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 164 NDF 8 0.1160 0.0000 0.0000 0.0000 — — — 0.6318 -0.0037 -0.0228 0.0013 0.1019 -0.9535 0.2749 0.0964 -0.0006 0.1674 0.3081

3 165 St40Pe0 8 0.1159 0.0319 0.0000 0.0000 — — — 13.0547 0.0120 0.1195 0.0856 0.0000 1.6678 1.3474 0.2941 0.0888 0.2683 0.5586

3 166 St40Pe0 8 0.1161 0.0323 0.0000 0.0000 — — — 17.8803 -0.0037 -0.0251 -0.0080 0.0000 1.4587 1.1799 0.3369 -0.2022 0.2368 0.6535

3 167 St80Pe0 8 0.1157 0.0642 0.0000 0.0000 — — — 34.8957 -0.0012 0.0522 0.0831 0.0732 0.9180 1.2547 0.2273 -0.1583 0.2538 0.5331

3 168 St80Pe0 8 0.1158 0.0637 0.0000 0.0000 — — — 42.2874 -0.0037 -0.0251 -0.0116 0.0972 1.5224 1.5426 0.3337 -0.0469 0.2451 0.6426

3 169 St120Pe0 8 0.1159 0.0957 0.0000 0.0000 — — — 51.1596 0.0102 0.0710 0.0874 0.0949 2.4619 1.5857 0.3267 -0.0532 0.2277 0.5283

3 170 St120Pe0 8 0.1161 0.0957 0.0000 0.0000 — — — 59.8430 -0.0037 -0.0251 -0.0158 0.0552 2.1719 1.6309 0.3143 -0.0313 0.2393 0.6782

3 171 St0Pe40 8 0.1158 0.0000 0.0000 0.0435 — — — 0.0862 0.0194 0.0899 0.1486 0.0000 9.8972 2.4931 0.0511 0.0012 0.0725 0.4036

3 172 St0Pe40 8 0.1157 0.0000 0.0000 0.0435 — — — 1.6588 0.1139 -0.0251 -0.0158 0.0000 10.5496 2.6632 0.1088 0.0028 0.1701 0.4389

3 173 St0Pe91 8 0.1158 0.0000 0.0000 0.0869 — — — 0.4004 0.0066 0.1065 0.1143 0.0322 16.0614 3.6274 0.1673 -0.3504 0.1154 0.6037

3 174 St0Pe91 8 0.1157 0.0000 0.0000 0.0869 — — — 2.3672 0.3042 0.0133 -0.0158 0.0000 17.7373 4.0517 0.2016 0.1195 0.1120 0.4880

3 175 St0Pe120 8 0.1161 0.0000 0.0000 0.1304 — — — 0.4042 0.0059 0.0879 0.3557 0.0882 17.5125 4.3834 0.2866 -0.0821 0.1092 0.3544

3 176 St0Pe120 8 0.1157 0.0000 0.0000 0.1304 — — — 1.8969 0.3113 0.0153 -0.0158 0.0638 18.3867 4.2414 0.3134 -0.0896 0.0856 0.2071

3 177 St40Pe91 8 0.1159 0.0319 0.0000 0.0869 — — — 9.5200 0.0196 0.0007 0.2962 0.0723 19.3893 4.8109 0.3813 -0.1090 0.0000 0.3570

3 178 St40Pe91 8 0.1157 0.0319 0.0000 0.0869 — — — 13.4727 0.9398 0.0127 -0.0158 0.0901 19.2073 4.8823 0.4032 -0.0310 0.1156 0.2197

3 179 St80Pe40 8 0.1161 0.0637 0.0000 0.0435 — — — 24.3699 0.0295 0.0061 0.1904 0.3532 12.5424 4.3499 0.7084 -0.4214 0.2034 0.4675

3 180 St80Pe40 8 0.1162 0.0637 0.0000 0.0435 — — — 30.2451 0.8643 -0.0251 -0.0158 0.4747 11.9719 4.7094 0.8228 -0.0336 0.1110 0.4783

3 181 BL 12 0.0000 0.0000 0.0000 0.0000 7.26 — — — — — — — — — — — — —

3 182 BL 12 0.0000 0.0000 0.0000 0.0000 7.26 — — — — — — — — — — — — —

3 183 NDF 12 0.1159 0.0000 0.0000 0.0000 7.27 82.0182 — — — — — — — — — — — —

3 184 NDF 12 0.1158 0.0000 0.0000 0.0000 7.26 82.5183 — — — — — — — — — — — —

3 185 St40Pe0 12 0.1160 0.0322 0.0000 0.0000 7.11 82.4673 — — — — — — — — — — — —

3 186 St40Pe0 12 0.1159 0.0319 0.0000 0.0000 7.06 81.0690 — — — — — — — — — — — —

3 187 St80Pe0 12 0.1161 0.0642 0.0000 0.0000 7.04 82.4850 — — — — — — — — — — — —

3 188 St80Pe0 12 0.1161 0.0637 0.0000 0.0000 7.01 82.7434 — — — — — — — — — — — —

3 189 St120Pe0 12 0.1161 0.0958 0.0000 0.0000 6.90 83.1741 — — — — — — — — — — — —

3 190 St120Pe0 12 0.1157 0.0956 0.0000 0.0000 6.95 82.5006 — — — — — — — — — — — —

3 191 St0Pe40 12 0.1162 0.0000 0.0000 0.0435 7.12 84.9988 — — — — — — — — — — — —

229



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 192 St0Pe40 12 0.1157 0.0000 0.0000 0.0435 7.11 84.6614 — — — — — — — — — — — —

3 193 St0Pe91 12 0.1162 0.0000 0.0000 0.0869 7.03 86.8063 — — — — — — — — — — — —

3 194 St0Pe91 12 0.1157 0.0000 0.0000 0.0869 7.02 86.6494 — — — — — — — — — — — —

3 195 St0Pe120 12 0.1158 0.0000 0.0000 0.1304 6.89 85.3682 — — — — — — — — — — — —

3 196 St0Pe120 12 0.1161 0.0000 0.0000 0.1304 6.90 88.3429 — — — — — — — — — — — —

3 197 St40Pe91 12 0.1160 0.0320 0.0000 0.0869 6.95 87.9853 — — — — — — — — — — — —

3 198 St40Pe91 12 0.1159 0.0319 0.0000 0.0869 6.95 87.8859 — — — — — — — — — — — —

3 199 St80Pe40 12 0.1161 0.0642 0.0000 0.0435 6.89 84.8971 — — — — — — — — — — — —

3 200 St80Pe40 12 0.1162 0.0642 0.0000 0.0435 6.93 84.9988 — — — — — — — — — — — —

3 203 NDF 12 0.1160 0.0000 0.0000 0.0000 — — 2.1954 — — — — — — — — — — —

3 204 NDF 12 0.1160 0.0000 0.0000 0.0000 — — 2.5860 — — — — — — — — — — —

3 205 St40Pe0 12 0.1158 0.0322 0.0000 0.0000 — — 8.9789 — — — — — — — — — — —

3 206 St40Pe0 12 0.1158 0.0322 0.0000 0.0000 — — 8.4907 — — — — — — — — — — —

3 207 St80Pe0 12 0.1162 0.0638 0.0000 0.0000 — — 9.3205 — — — — — — — — — — —

3 208 St80Pe0 12 0.1160 0.0641 0.0000 0.0000 — — 9.8064 — — — — — — — — — — —

3 209 St120Pe0 12 0.1160 0.0956 0.0000 0.0000 — — 12.9456 — — — — — — — — — — —

3 210 St120Pe0 12 0.1158 0.0959 0.0000 0.0000 — — 12.0625 — — — — — — — — — — —

3 211 St0Pe40 12 0.1158 0.0000 0.0000 0.0435 — — 8.7945 — — — — — — — — — — —

3 212 St0Pe40 12 0.1160 0.0000 0.0000 0.0435 — — 8.7886 — — — — — — — — — — —

3 213 St0Pe91 12 0.1161 0.0000 0.0000 0.0869 — — 13.5195 — — — — — — — — — — —

3 214 St0Pe91 12 0.1160 0.0000 0.0000 0.0869 — — 12.8384 — — — — — — — — — — —

3 215 St0Pe120 12 0.1161 0.0000 0.0000 0.1304 — — 19.2829 — — — — — — — — — — —

3 216 St0Pe120 12 0.1159 0.0000 0.0000 0.1304 — — 19.0921 — — — — — — — — — — —

3 217 St40Pe91 12 0.1159 0.0319 0.0000 0.0869 — — 18.6106 — — — — — — — — — — —

3 218 St40Pe91 12 0.1157 0.0322 0.0000 0.0869 — — 18.8995 — — — — — — — — — — —

3 219 St80Pe40 12 0.1157 0.0637 0.0000 0.0435 — — 19.3453 — — — — — — — — — — —

3 220 St80Pe40 12 0.1160 0.0641 0.0000 0.0435 — — 19.4303 — — — — — — — — — — —

3 221 BL 12 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.0000 0.0000

230



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 222 BL 12 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3666 0.9720

3 223 NDF 12 0.1159 0.0000 0.0000 0.0000 — — — -0.0366 0.0002 -0.0063 -0.0185 0.0000 3.0507 1.4695 0.3492 0.4690 0.3590 0.8310

3 224 NDF 12 0.1157 0.0000 0.0000 0.0000 — — — 0.6730 -0.0053 0.0177 -0.0130 0.0000 2.9028 1.7981 0.1733 -0.0088 0.4579 0.9756

3 225 St40Pe0 12 0.1161 0.0319 0.0000 0.0000 — — — 2.8736 0.0171 -0.0076 0.2517 0.0000 8.5539 5.0496 1.1159 0.3326 0.3730 1.1604

3 226 St40Pe0 12 0.1162 0.0319 0.0000 0.0000 — — — 3.2742 -0.0053 -0.0031 -0.0144 0.0000 8.3506 4.9466 1.0495 0.3545 0.3892 1.1976

3 227 St80Pe0 12 0.1160 0.0637 0.0000 0.0000 — — — 9.7171 0.0045 0.0028 -0.0198 0.0651 11.3981 7.4032 2.3448 0.2809 0.4314 0.5897

3 228 St80Pe0 12 0.1158 0.0638 0.0000 0.0000 — — — 9.4172 -0.0053 -0.0212 -0.0199 0.0000 10.7010 7.7676 1.7434 0.2353 0.3786 1.1326

3 229 St120Pe0 12 0.1158 0.0956 0.0000 0.0000 — — — 20.4039 0.0119 -0.0196 -0.0182 0.2256 13.1401 9.4445 2.3766 0.3802 0.4201 1.0598

3 230 St120Pe0 12 0.1158 0.0959 0.0000 0.0000 — — — 17.6124 -0.0053 -0.0136 -0.0199 0.1699 14.4674 9.2933 2.2926 0.2791 0.2691 1.1243

3 231 St0Pe40 12 0.1159 0.0000 0.0000 0.0435 — — — 0.4366 0.0343 0.0005 0.1201 0.0000 13.6572 3.6237 0.7428 0.3763 0.2171 1.4254

3 232 St0Pe40 12 0.1162 0.0000 0.0000 0.0435 — — — 0.9936 -0.0053 -0.0212 -0.0199 0.0000 13.0290 4.0756 0.6419 0.3739 0.3613 0.6720

3 233 St0Pe91 12 0.1161 0.0000 0.0000 0.0869 — — — 0.6703 0.0599 -0.0021 0.1871 0.0000 28.8467 8.6890 0.8983 0.3155 0.4771 1.4258

3 234 St0Pe91 12 0.1159 0.0000 0.0000 0.0869 — — — 1.2278 -0.0053 -0.0212 -0.0199 0.0387 21.3793 6.0536 0.6877 -0.0026 0.2698 1.2420

3 235 St0Pe120 12 0.1157 0.0000 0.0000 0.1304 — — — 0.5956 0.0708 0.0143 0.2024 0.0515 33.5313 9.7983 1.1276 0.2003 0.2967 1.3247

3 236 St0Pe120 12 0.1158 0.0000 0.0000 0.1304 — — — 1.8613 0.0077 -0.0212 -0.0199 0.0000 33.8283 9.2902 1.0152 0.1021 0.2517 0.6287

3 237 St40Pe91 12 0.1162 0.0323 0.0000 0.0869 — — — 6.0195 0.0546 -0.0024 0.0729 0.0347 30.4612 9.1176 1.6635 0.4489 0.4515 0.9855

3 238 St40Pe91 12 0.1157 0.0320 0.0000 0.0869 — — — 5.1689 -0.0053 -0.0212 -0.0199 0.0309 28.3992 9.3172 1.6947 0.3696 0.3144 1.3633

3 239 St80Pe40 12 0.1162 0.0642 0.0000 0.0435 — — — 14.6639 0.0528 0.0002 0.2241 0.0986 20.2912 9.9730 2.2624 0.2662 0.3444 1.2460

3 240 St80Pe40 12 0.1158 0.0642 0.0000 0.0435 — — — 11.4713 -0.0053 0.0063 0.0038 0.0000 20.4339 9.1064 2.0035 0.3451 0.0000 0.9381

3 241 BL 16 0.0000 0.0000 0.0000 0.0000 7.38 — — — — — — — — — — — — —

3 242 BL 16 0.0000 0.0000 0.0000 0.0000 7.32 — — — — — — — — — — — — —

3 243 NDF 16 0.1161 0.0000 0.0000 0.0000 7.26 72.9658 — — — — — — — — — — — —

3 244 NDF 16 0.1161 0.0000 0.0000 0.0000 7.07 70.3814 — — — — — — — — — — — —

3 245 St40Pe0 16 0.1157 0.0322 0.0000 0.0000 7.11 73.8141 — — — — — — — — — — — —

3 246 St40Pe0 16 0.1159 0.0323 0.0000 0.0000 7.10 72.6558 — — — — — — — — — — — —

3 247 St80Pe0 16 0.1160 0.0642 0.0000 0.0000 6.92 71.3883 — — — — — — — — — — — —

3 248 St80Pe0 16 0.1161 0.0637 0.0000 0.0000 6.95 74.3442 — — — — — — — — — — — —

3 249 St120Pe0 16 0.1161 0.0956 0.0000 0.0000 6.86 74.9472 — — — — — — — — — — — —

231



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 250 St120Pe0 16 0.1157 0.0960 0.0000 0.0000 6.86 74.7648 — — — — — — — — — — — —

3 251 St0Pe40 16 0.1161 0.0000 0.0000 0.0435 7.19 77.4454 — — — — — — — — — — — —

3 252 St0Pe40 16 0.1162 0.0000 0.0000 0.0435 7.19 77.8115 — — — — — — — — — — — —

3 253 St0Pe91 16 0.1159 0.0000 0.0000 0.0869 7.12 79.4726 — — — — — — — — — — — —

3 254 St0Pe91 16 0.1160 0.0000 0.0000 0.0869 7.11 78.6306 — — — — — — — — — — — —

3 255 St0Pe120 16 0.1161 0.0000 0.0000 0.1304 7.00 80.2882 — — — — — — — — — — — —

3 256 St0Pe120 16 0.1161 0.0000 0.0000 0.1304 6.97 82.0973 — — — — — — — — — — — —

3 257 St40Pe91 16 0.1160 0.0320 0.0000 0.0869 7.00 80.3550 — — — — — — — — — — — —

3 258 St40Pe91 16 0.1157 0.0319 0.0000 0.0869 6.99 79.5187 — — — — — — — — — — — —

3 259 St80Pe40 16 0.1157 0.0638 0.0000 0.0435 6.96 76.3206 — — — — — — — — — — — —

3 260 St80Pe40 16 0.1156 0.0639 0.0000 0.0435 6.94 76.4708 — — — — — — — — — — — —

3 263 NDF 16 0.1157 0.0000 0.0000 0.0000 — — 2.4511 — — — — — — — — — — —

3 264 NDF 16 0.1160 0.0000 0.0000 0.0000 — — 3.0254 — — — — — — — — — — —

3 265 St40Pe0 16 0.1162 0.0321 0.0000 0.0000 — — 8.7254 — — — — — — — — — — —

3 266 St40Pe0 16 0.1160 0.0320 0.0000 0.0000 — — 8.7343 — — — — — — — — — — —

3 267 St80Pe0 16 0.1157 0.0637 0.0000 0.0000 — — 13.4890 — — — — — — — — — — —

3 268 St80Pe0 16 0.1157 0.0641 0.0000 0.0000 — — 12.9894 — — — — — — — — — — —

3 269 St120Pe0 16 0.1157 0.0956 0.0000 0.0000 — — 18.0799 — — — — — — — — — — —

3 270 St120Pe0 16 0.1158 0.0956 0.0000 0.0000 — — 17.5891 — — — — — — — — — — —

3 271 St0Pe40 16 0.1159 0.0000 0.0000 0.0435 — — 9.0357 — — — — — — — — — — —

3 272 St0Pe40 16 0.1160 0.0000 0.0000 0.0435 — — 9.2281 — — — — — — — — — — —

3 273 St0Pe91 16 0.1160 0.0000 0.0000 0.0869 — — 15.0355 — — — — — — — — — — —

3 274 St0Pe91 16 0.1161 0.0000 0.0000 0.0869 — — 14.2518 — — — — — — — — — — —

3 275 St0Pe120 16 0.1160 0.0000 0.0000 0.1304 — — 19.7246 — — — — — — — — — — —

3 276 St0Pe120 16 0.1160 0.0000 0.0000 0.1304 — — 20.4081 — — — — — — — — — — —

3 277 St40Pe91 16 0.1158 0.0319 0.0000 0.0869 — — 19.9314 — — — — — — — — — — —

3 278 St40Pe91 16 0.1161 0.0319 0.0000 0.0869 — — 20.6075 — — — — — — — — — — —

3 279 St80Pe40 16 0.1162 0.0638 0.0000 0.0435 — — 19.2852 — — — — — — — — — — —

232



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 280 St80Pe40 16 0.1159 0.0638 0.0000 0.0435 — — 19.6815 — — — — — — — — — — —

3 281 BL 16 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.4598 1.4304

3 282 BL 16 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.5132 1.2932

3 283 NDF 16 0.1156 0.0000 0.0000 0.0000 — — — 0.4317 -0.0086 -0.0004 0.0759 0.0000 3.8692 2.4812 0.0097 0.1211 0.4466 1.3526

3 284 NDF 16 0.1160 0.0000 0.0000 0.0000 — — — 0.9089 0.0021 -0.0135 -0.0757 0.0000 5.0774 2.6912 -0.4861 0.2489 0.5120 1.5709

3 285 St40Pe0 16 0.1158 0.0323 0.0000 0.0000 — — — 3.0277 0.0151 0.0166 0.1513 0.0000 11.2570 6.1185 1.7079 0.4539 0.5993 1.7921

3 286 St40Pe0 16 0.1161 0.0323 0.0000 0.0000 — — — 3.5907 -0.0187 0.0072 -0.0757 0.0000 11.6828 6.0595 1.5960 0.2082 0.5081 1.8524

3 287 St80Pe0 16 0.1161 0.0640 0.0000 0.0000 — — — 4.4455 0.0142 0.0236 0.1552 0.0000 14.5123 9.5037 2.4086 0.4892 0.4936 1.8521

3 288 St80Pe0 16 0.1157 0.0640 0.0000 0.0000 — — — 3.3565 -0.0187 -0.0148 -0.0757 0.0000 16.0057 9.6407 2.4798 0.3407 0.5517 1.8674

3 289 St120Pe0 16 0.1160 0.0956 0.0000 0.0000 — — — 7.1986 0.0166 0.0242 0.1305 0.0000 19.0975 13.2864 3.7952 0.6089 0.5943 1.4856

3 290 St120Pe0 16 0.1158 0.0958 0.0000 0.0000 — — — 6.0364 -0.0187 -0.0148 -0.0757 0.0000 18.9790 12.7019 3.5184 0.4658 0.4582 1.8350

3 291 St0Pe40 16 0.1157 0.0000 0.0000 0.0435 — — — 0.6674 0.0167 0.0117 0.0933 0.0000 13.5559 3.9262 0.8570 -0.2535 0.4762 1.7465

3 292 St0Pe40 16 0.1159 0.0000 0.0000 0.0435 — — — 1.2276 -0.0187 -0.0148 -0.0757 0.0000 12.7151 3.6588 0.8654 -0.6105 0.5810 1.9581

3 293 St0Pe91 16 0.1160 0.0000 0.0000 0.0869 — — — 0.4355 0.0261 0.0001 0.1030 0.0000 23.9988 6.8783 1.0932 -0.4541 0.6166 1.9965

3 294 St0Pe91 16 0.1157 0.0000 0.0000 0.0869 — — — 1.1527 -0.0187 -0.0148 -0.0757 0.0000 25.4458 6.5105 1.2187 0.4616 0.7604 2.0319

3 295 St0Pe120 16 0.1157 0.0000 0.0000 0.1304 — — — 0.8283 0.0399 -0.0137 0.1387 0.0000 36.3108 8.4245 1.3994 0.1750 0.6583 1.0639

3 296 St0Pe120 16 0.1158 0.0000 0.0000 0.1304 — — — 1.3101 -0.0187 -0.0148 -0.0757 0.0000 34.9065 9.7501 1.4102 -0.2582 0.5359 1.7598

3 297 St40Pe91 16 0.1162 0.0321 0.0000 0.0869 — — — 2.5603 0.0212 -0.0078 0.0955 0.0000 30.4942 10.5229 2.0714 0.4802 0.6298 1.8490

3 298 St40Pe91 16 0.1158 0.0321 0.0000 0.0869 — — — 3.5965 -0.0187 -0.0148 -0.0757 0.0000 32.8672 10.1582 2.0321 0.5506 0.6187 1.9286

3 299 St80Pe40 16 0.1159 0.0637 0.0000 0.0435 — — — 3.5814 0.0120 -0.0109 0.1704 0.0000 27.1706 12.0634 2.3944 0.5792 0.6768 1.3487

3 300 St80Pe40 16 0.1161 0.0642 0.0000 0.0435 — — — 2.4159 -0.0187 -0.0148 -0.0757 0.0000 25.2115 11.6780 2.2724 0.4647 0.6198 1.6776

3 301 BL 20 0.0000 0.0000 0.0000 0.0000 7.57 — — — — — — — — — — — — —

3 302 BL 20 0.0000 0.0000 0.0000 0.0000 7.54 — — — — — — — — — — — — —

3 303 NDF 20 0.1157 0.0000 0.0000 0.0000 7.41 69.5356 — — — — — — — — — — — —

3 304 NDF 20 0.1157 0.0000 0.0000 0.0000 7.42 70.6592 — — — — — — — — — — — —

3 305 St40Pe0 20 0.1162 0.0322 0.0000 0.0000 7.29 69.5914 — — — — — — — — — — — —

3 306 St40Pe0 20 0.1157 0.0323 0.0000 0.0000 7.26 68.5848 — — — — — — — — — — — —

3 307 St80Pe0 20 0.1159 0.0642 0.0000 0.0000 7.12 68.7296 — — — — — — — — — — — —

233



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 308 St80Pe0 20 0.1160 0.0639 0.0000 0.0000 7.14 68.8449 — — — — — — — — — — — —

3 309 St120Pe0 20 0.1161 0.0959 0.0000 0.0000 6.94 67.4094 — — — — — — — — — — — —

3 310 St120Pe0 20 0.1162 0.0960 0.0000 0.0000 7.03 69.6775 — — — — — — — — — — — —

3 311 St0Pe40 20 0.1159 0.0000 0.0000 0.0435 7.30 73.2167 — — — — — — — — — — — —

3 312 St0Pe40 20 0.1157 0.0000 0.0000 0.0435 7.33 73.6844 — — — — — — — — — — — —

3 313 St0Pe91 20 0.1162 0.0000 0.0000 0.0869 7.25 75.6166 — — — — — — — — — — — —

3 314 St0Pe91 20 0.1161 0.0000 0.0000 0.0869 7.24 73.9565 — — — — — — — — — — — —

3 315 St0Pe120 20 0.1160 0.0000 0.0000 0.1304 7.11 74.5353 — — — — — — — — — — — —

3 316 St0Pe120 20 0.1159 0.0000 0.0000 0.1304 7.12 75.2876 — — — — — — — — — — — —

3 317 St40Pe91 20 0.1161 0.0320 0.0000 0.0869 7.15 77.3162 — — — — — — — — — — — —

3 318 St40Pe91 20 0.1157 0.0319 0.0000 0.0869 7.14 76.2774 — — — — — — — — — — — —

3 319 St80Pe40 20 0.1162 0.0639 0.0000 0.0435 7.12 74.4116 — — — — — — — — — — — —

3 320 St80Pe40 20 0.1159 0.0637 0.0000 0.0435 7.14 73.3029 — — — — — — — — — — — —

3 323 NDF 20 0.1162 0.0000 0.0000 0.0000 — — 1.0159 — — — — — — — — — — —

3 324 NDF 20 0.1157 0.0000 0.0000 0.0000 — — -0.5272 — — — — — — — — — — —

3 325 St40Pe0 20 0.1161 0.0322 0.0000 0.0000 — — 7.2121 — — — — — — — — — — —

3 326 St40Pe0 20 0.1160 0.0320 0.0000 0.0000 — — 6.7325 — — — — — — — — — — —

3 327 St80Pe0 20 0.1157 0.0640 0.0000 0.0000 — — 11.9669 — — — — — — — — — — —

3 328 St80Pe0 20 0.1157 0.0642 0.0000 0.0000 — — 13.7189 — — — — — — — — — — —

3 329 St120Pe0 20 0.1157 0.0957 0.0000 0.0000 — — 17.1499 — — — — — — — — — — —

3 330 St120Pe0 20 0.1157 0.0955 0.0000 0.0000 — — 15.6898 — — — — — — — — — — —

3 331 St0Pe40 20 0.1162 0.0000 0.0000 0.0435 — — 6.1462 — — — — — — — — — — —

3 332 St0Pe40 20 0.1162 0.0000 0.0000 0.0435 — — 6.8297 — — — — — — — — — — —

3 333 St0Pe91 20 0.1161 0.0000 0.0000 0.0869 — — 11.6641 — — — — — — — — — — —

3 334 St0Pe91 20 0.1161 0.0000 0.0000 0.0869 — — 11.3712 — — — — — — — — — — —

3 335 St0Pe120 20 0.1157 0.0000 0.0000 0.1304 — — 17.0459 — — — — — — — — — — —

3 336 St0Pe120 20 0.1160 0.0000 0.0000 0.1304 — — 17.9180 — — — — — — — — — — —

3 337 St40Pe91 20 0.1157 0.0321 0.0000 0.0869 — — 17.1440 — — — — — — — — — — —

234



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 338 St40Pe91 20 0.1160 0.0319 0.0000 0.0869 — — 16.5552 — — — — — — — — — — —

3 339 St80Pe40 20 0.1160 0.0642 0.0000 0.0435 — — 18.3544 — — — — — — — — — — —

3 340 St80Pe40 20 0.1158 0.0637 0.0000 0.0435 — — 18.9528 — — — — — — — — — — —

3 341 BL 20 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.7203 2.1399

3 342 BL 20 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.6581 2.0864

3 343 NDF 20 0.1159 0.0000 0.0000 0.0000 — — — -1.2136 -0.0841 -0.0026 0.0635 0.0000 8.6571 4.1928 1.7060 0.5773 0.9152 2.4534

3 344 NDF 20 0.1157 0.0000 0.0000 0.0000 — — — 0.9865 -0.0173 0.0061 -0.0482 0.0000 6.5422 3.3288 1.0853 0.3131 0.9315 2.2381

3 345 St40Pe0 20 0.1161 0.0318 0.0000 0.0000 — — — 0.2788 -0.0830 -0.0034 0.1255 0.0000 11.2328 6.2047 2.3395 0.5603 0.9734 2.4799

3 346 St40Pe0 20 0.1157 0.0323 0.0000 0.0000 — — — 1.0671 -0.0193 -0.0168 -0.0482 0.0000 11.0825 6.5759 2.2557 0.5779 0.9178 2.5528

3 347 St80Pe0 20 0.1158 0.0637 0.0000 0.0000 — — — 0.7540 -0.0830 -0.0164 0.1082 0.0000 15.7004 10.1172 4.0420 0.7508 1.2107 2.6007

3 348 St80Pe0 20 0.1159 0.0641 0.0000 0.0000 — — — 0.5968 -0.0464 0.0100 -0.0482 0.0000 17.1870 10.4596 4.3700 1.0992 1.0742 2.8403

3 349 St120Pe0 20 0.1162 0.0955 0.0000 0.0000 — — — 1.4628 -0.0817 -0.0173 0.1072 0.0000 17.1463 12.3189 5.3574 1.0333 1.0049 2.3224

3 350 St120Pe0 20 0.1157 0.0956 0.0000 0.0000 — — — 1.3052 -0.0218 0.0047 -0.0482 0.0000 16.6429 13.8082 5.4364 0.9441 0.8309 2.0293

3 351 St0Pe40 20 0.1162 0.0000 0.0000 0.0435 — — — -0.5852 -0.0650 -0.0155 0.0925 0.0000 12.5781 4.3473 1.2345 0.3293 0.8657 2.0550

3 352 St0Pe40 20 0.1157 0.0000 0.0000 0.0435 — — — -1.2960 -0.0844 0.0026 -0.0482 0.0000 12.3334 4.3641 1.3294 0.3004 0.9570 2.2917

3 353 St0Pe91 20 0.1158 0.0000 0.0000 0.0869 — — — -0.5852 -0.0605 -0.0173 0.1089 0.0000 24.2928 7.4207 2.1488 0.5187 0.7883 2.1381

3 354 St0Pe91 20 0.1157 0.0000 0.0000 0.0869 — — — -0.5051 -0.0844 -0.0173 -0.0482 0.0000 23.5055 6.7738 1.8162 0.1677 0.8249 2.0679

3 355 St0Pe120 20 0.1159 0.0000 0.0000 0.1304 — — — -0.4281 -0.0539 0.0017 0.1721 0.0000 34.0711 10.0770 2.1079 0.2637 0.7258 1.9042

3 356 St0Pe120 20 0.1161 0.0000 0.0000 0.1304 — — — 0.0773 -0.0844 -0.0173 -0.0482 0.0000 36.3763 11.9023 2.8272 0.7089 0.6558 1.9028

3 357 St40Pe91 20 0.1162 0.0323 0.0000 0.0869 — — — 0.8325 -0.0440 -0.0141 0.1132 0.0000 30.4317 12.8069 3.0447 0.4202 0.8540 2.0285

3 358 St40Pe91 20 0.1159 0.0322 0.0000 0.0869 — — — 0.2052 -0.0844 -0.0173 -0.0482 0.0000 30.0541 10.7502 2.8796 0.4617 0.8115 1.9813

3 359 St80Pe40 20 0.1157 0.0639 0.0000 0.0435 — — — 1.6889 -0.0619 -0.0151 0.1044 0.0000 26.1215 13.7700 4.2312 0.8484 0.8995 2.1110

3 360 St80Pe40 20 0.1161 0.0642 0.0000 0.0435 — — — 0.6793 -0.0490 -0.0138 -0.0482 0.0000 27.0469 12.7730 3.7367 0.6464 0.9223 2.1353

3 361 BL 24 0.0000 0.0000 0.0000 0.0000 7.52 — — — — — — — — — — — — —

3 362 BL 24 0.0000 0.0000 0.0000 0.0000 7.56 — — — — — — — — — — — — —

3 363 NDF 24 0.1159 0.0000 0.0000 0.0000 7.15 62.7756 — — — — — — — — — — — —

3 364 NDF 24 0.1162 0.0000 0.0000 0.0000 7.29 63.7383 — — — — — — — — — — — —

3 365 St40Pe0 24 0.1160 0.0321 0.0000 0.0000 7.13 63.7581 — — — — — — — — — — — —

235



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 366 St40Pe0 24 0.1159 0.0320 0.0000 0.0000 7.16 64.3288 — — — — — — — — — — — —

3 367 St80Pe0 24 0.1161 0.0640 0.0000 0.0000 7.06 63.5328 — — — — — — — — — — — —

3 368 St80Pe0 24 0.1159 0.0642 0.0000 0.0000 7.04 63.4659 — — — — — — — — — — — —

3 369 St120Pe0 24 0.1159 0.0960 0.0000 0.0000 6.95 62.3442 — — — — — — — — — — — —

3 370 St120Pe0 24 0.1162 0.0957 0.0000 0.0000 6.97 63.9965 — — — — — — — — — — — —

3 371 St0Pe40 24 0.1160 0.0000 0.0000 0.0435 7.23 66.6895 — — — — — — — — — — — —

3 372 St0Pe40 24 0.1162 0.0000 0.0000 0.0435 7.26 69.0749 — — — — — — — — — — — —

3 373 St0Pe91 24 0.1162 0.0000 0.0000 0.0869 7.15 70.1078 — — — — — — — — — — — —

3 374 St0Pe91 24 0.1157 0.0000 0.0000 0.0869 7.17 68.6713 — — — — — — — — — — — —

3 375 St0Pe120 24 0.1162 0.0000 0.0000 0.1304 7.01 68.9889 — — — — — — — — — — — —

3 376 St0Pe120 24 0.1159 0.0000 0.0000 0.1304 7.04 67.4353 — — — — — — — — — — — —

3 377 St40Pe91 24 0.1159 0.0319 0.0000 0.0869 7.06 69.5925 — — — — — — — — — — — —

3 378 St40Pe91 24 0.1160 0.0322 0.0000 0.0869 7.04 69.4484 — — — — — — — — — — — —

3 379 St80Pe40 24 0.1162 0.0641 0.0000 0.0435 7.03 67.0952 — — — — — — — — — — — —

3 380 St80Pe40 24 0.1159 0.0640 0.0000 0.0435 7.04 66.1409 — — — — — — — — — — — —

3 383 NDF 24 0.1157 0.0000 0.0000 0.0000 — — 0.5469 — — — — — — — — — — —

3 384 NDF 24 0.1158 0.0000 0.0000 0.0000 — — 0.1525 — — — — — — — — — — —

3 385 St40Pe0 24 0.1157 0.0322 0.0000 0.0000 — — 5.8572 — — — — — — — — — — —

3 386 St40Pe0 24 0.1161 0.0319 0.0000 0.0000 — — 5.3679 — — — — — — — — — — —

3 387 St80Pe0 24 0.1162 0.0640 0.0000 0.0000 — — 10.0960 — — — — — — — — — — —

3 388 St80Pe0 24 0.1161 0.0640 0.0000 0.0000 — — 8.9273 — — — — — — — — — — —

3 389 St120Pe0 24 0.1158 0.0959 0.0000 0.0000 — — 15.0896 — — — — — — — — — — —

3 390 St120Pe0 24 0.1161 0.0956 0.0000 0.0000 — — 16.3608 — — — — — — — — — — —

3 391 St0Pe40 24 0.1157 0.0000 0.0000 0.0435 — — 7.3328 — — — — — — — — — — —

3 392 St0Pe40 24 0.1158 0.0000 0.0000 0.0435 — — 7.2321 — — — — — — — — — — —

3 393 St0Pe91 24 0.1161 0.0000 0.0000 0.0869 — — 16.5466 — — — — — — — — — — —

3 394 St0Pe91 24 0.1162 0.0000 0.0000 0.0869 — — 13.6146 — — — — — — — — — — —

3 395 St0Pe120 24 0.1157 0.0000 0.0000 0.1304 — — 18.8036 — — — — — — — — — — —

236



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

3 396 St0Pe120 24 0.1160 0.0000 0.0000 0.1304 — — 18.8945 — — — — — — — — — — —

3 397 St40Pe91 24 0.1160 0.0320 0.0000 0.0869 — — 17.2365 — — — — — — — — — — —

3 398 St40Pe91 24 0.1159 0.0319 0.0000 0.0869 — — 17.3412 — — — — — — — — — — —

3 399 St80Pe40 24 0.1157 0.0642 0.0000 0.0435 — — 18.5554 — — — — — — — — — — —

3 400 St80Pe40 24 0.1159 0.0638 0.0000 0.0435 — — 18.4609 — — — — — — — — — — —

3 401 BL 24 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.9641 2.2651

3 402 BL 24 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.9808 1.9020

3 403 NDF 24 0.1158 0.0000 0.0000 0.0000 — — — 0.3897 0.0014 -0.0005 0.0295 0.0000 7.1141 3.7228 0.5872 0.3452 1.1701 2.2824

3 404 NDF 24 0.1158 0.0000 0.0000 0.0000 — — — -1.0981 0.0050 0.0016 -0.0361 0.0000 6.9392 3.6780 1.2650 0.2031 1.1054 3.1285

3 405 St40Pe0 24 0.1160 0.0320 0.0000 0.0000 — — — 0.4684 0.0211 -0.0016 0.1250 0.0000 12.3609 7.4033 2.1606 0.0221 1.0939 2.4742

3 406 St40Pe0 24 0.1159 0.0323 0.0000 0.0000 — — — 0.6335 0.0625 -0.0021 -0.0361 0.0000 17.8774 8.1441 2.7860 0.9530 1.2382 2.6353

3 407 St80Pe0 24 0.1158 0.0638 0.0000 0.0000 — — — 0.4703 0.0003 0.0003 0.1321 0.0000 18.9410 12.2004 4.6992 1.6565 1.0896 2.6421

3 408 St80Pe0 24 0.1162 0.0639 0.0000 0.0000 — — — 0.4031 0.0735 -0.0024 -0.0266 0.0000 18.2881 11.9152 3.2667 0.8348 1.0548 2.5804

3 409 St120Pe0 24 0.1159 0.0956 0.0000 0.0000 — — — 0.5510 0.0012 -0.0014 0.1346 0.0000 23.8510 14.8981 6.1736 1.3672 1.1804 2.4888

3 410 St120Pe0 24 0.1162 0.0960 0.0000 0.0000 — — — 0.6412 -0.0006 -0.0024 -0.0318 0.0000 22.1751 16.6883 5.9739 1.7613 1.1013 2.7000

3 411 St0Pe40 24 0.1159 0.0000 0.0000 0.0435 — — — -0.0825 0.0013 -0.0024 0.1310 0.0000 14.6001 5.2462 1.2702 0.3925 1.2084 2.0350

3 412 St0Pe40 24 0.1162 0.0000 0.0000 0.0435 — — — -0.7064 0.0634 -0.0020 -0.0361 0.0000 15.5926 5.4573 1.4354 0.4201 0.4041 2.0916

3 413 St0Pe91 24 0.1160 0.0000 0.0000 0.0869 — — — -0.0019 -0.0006 -0.0024 0.1262 0.0000 25.6890 7.7288 1.9419 0.4771 0.8127 1.9426

3 414 St0Pe91 24 0.1157 0.0000 0.0000 0.0869 — — — -0.9368 0.0956 0.0008 -0.0361 0.0000 26.0537 7.9973 1.9036 0.5021 0.8258 1.9835

3 415 St0Pe120 24 0.1156 0.0000 0.0000 0.1304 — — — 0.3148 0.0011 -0.0012 0.1304 0.0000 34.7266 10.5264 2.1364 0.6090 0.6794 1.7208

3 416 St0Pe120 24 0.1159 0.0000 0.0000 0.1304 — — — -0.7007 0.0245 0.0011 -0.0086 0.0000 35.6101 10.7376 2.2463 0.5934 0.7262 1.9263

3 417 St40Pe91 24 0.1160 0.0323 0.0000 0.0869 — — — 0.3974 0.0011 -0.0024 0.1278 0.0000 30.9549 11.8701 2.9752 0.5763 0.7917 1.9213

3 418 St40Pe91 24 0.1158 0.0323 0.0000 0.0869 — — — 1.1134 0.1162 -0.0018 -0.0361 0.0000 30.9373 11.5980 2.9545 0.4447 0.7941 1.8040

3 419 St80Pe40 24 0.1162 0.0641 0.0000 0.0435 — — — 0.9483 -0.0006 -0.0024 0.1170 0.0000 25.1862 13.3158 3.4818 0.8591 0.8654 2.0556

3 420 St80Pe40 24 0.1161 0.0640 0.0000 0.0435 — — — 1.1096 0.0686 -0.0022 -0.0028 0.0000 23.3165 12.7823 3.3399 0.7492 0.7967 1.5480

4 1 BL 0 0.0000 0.0000 0.0000 0.0000 7.24 — — — — — — — — — — — — —

4 2 BL 0 0.0000 0.0000 0.0000 0.0000 7.21 — — — — — — — — — — — — —

4 3 NDF 0 0.1155 0.0000 0.0000 0.0000 7.20 93.2874 — — — — — — — — — — — —

237



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 4 NDF 0 0.1157 0.0000 0.0000 0.0000 7.18 93.1319 — — — — — — — — — — — —

4 5 St40Pe0 0 0.1161 0.0319 0.0000 0.0000 7.22 93.3394 — — — — — — — — — — — —

4 6 St40Pe0 0 0.1159 0.0323 0.0000 0.0000 7.25 93.4947 — — — — — — — — — — — —

4 7 St80Pe0 0 0.1159 0.0637 0.0000 0.0000 7.22 94.1850 — — — — — — — — — — — —

4 8 St80Pe0 0 0.1157 0.0641 0.0000 0.0000 7.20 93.3912 — — — — — — — — — — — —

4 9 St120Pe0 0 0.1160 0.0957 0.0000 0.0000 7.19 92.4686 — — — — — — — — — — — —

4 10 St120Pe0 0 0.1157 0.0956 0.0000 0.0000 7.21 92.6997 — — — — — — — — — — — —

4 11 St0Pe40 0 0.1162 0.0000 0.0000 0.0435 7.22 93.9505 — — — — — — — — — — — —

4 12 St0Pe40 0 0.1158 0.0000 0.0000 0.0435 7.19 93.8316 — — — — — — — — — — — —

4 13 St0Pe91 0 0.1160 0.0000 0.0000 0.0869 7.18 93.1583 — — — — — — — — — — — —

4 14 St0Pe91 0 0.1161 0.0000 0.0000 0.0869 7.18 93.6840 — — — — — — — — — — — —

4 15 St0Pe120 0 0.1161 0.0000 0.0000 0.1304 7.15 94.8039 — — — — — — — — — — — —

4 16 St0Pe120 0 0.1158 0.0000 0.0000 0.1304 7.14 94.2634 — — — — — — — — — — — —

4 17 St40Pe91 0 0.1160 0.0320 0.0000 0.0869 7.20 94.4516 — — — — — — — — — — — —

4 18 St40Pe91 0 0.1157 0.0320 0.0000 0.0869 7.18 93.7369 — — — — — — — — — — — —

4 19 St80Pe40 0 0.1161 0.0641 0.0000 0.0435 7.15 94.2008 — — — — — — — — — — — —

4 20 St80Pe40 0 0.1161 0.0640 0.0000 0.0435 7.16 93.5978 — — — — — — — — — — — —

4 23 NDF 0 0.1160 0.0000 0.0000 0.0000 — — 0.2949 — — — — — — — — — — —

4 24 NDF 0 0.1161 0.0000 0.0000 0.0000 — — -0.2952 — — — — — — — — — — —

4 25 St40Pe0 0 0.1158 0.0319 0.0000 0.0000 — — -0.3417 — — — — — — — — — — —

4 26 St40Pe0 0 0.1159 0.0319 0.0000 0.0000 — — 0.3417 — — — — — — — — — — —

4 27 St80Pe0 0 0.1161 0.0637 0.0000 0.0000 — — -0.5404 — — — — — — — — — — —

4 28 St80Pe0 0 0.1158 0.0637 0.0000 0.0000 — — 0.5395 — — — — — — — — — — —

4 29 St120Pe0 0 0.1159 0.0958 0.0000 0.0000 — — 0.1485 — — — — — — — — — — —

4 30 St120Pe0 0 0.1161 0.0958 0.0000 0.0000 — — -0.1486 — — — — — — — — — — —

4 31 St0Pe40 0 0.1157 0.0000 0.0000 0.0435 — — 0.0012 — — — — — — — — — — —

4 32 St0Pe40 0 0.1158 0.0000 0.0000 0.0435 — — -0.0012 — — — — — — — — — — —

4 33 St0Pe91 0 0.1160 0.0000 0.0000 0.0869 — — -0.2441 — — — — — — — — — — —

238



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 34 St0Pe91 0 0.1160 0.0000 0.0000 0.0869 — — 0.2441 — — — — — — — — — — —

4 35 St0Pe120 0 0.1157 0.0000 0.0000 0.1304 — — 0.0000 — — — — — — — — — — —

4 36 St0Pe120 0 0.1157 0.0000 0.0000 0.1304 — — 0.0000 — — — — — — — — — — —

4 37 St40Pe91 0 0.1161 0.0322 0.0000 0.0869 — — -0.3980 — — — — — — — — — — —

4 38 St40Pe91 0 0.1160 0.0319 0.0000 0.0869 — — 0.3968 — — — — — — — — — — —

4 39 St80Pe40 0 0.1160 0.0638 0.0000 0.0435 — — 0.3431 — — — — — — — — — — —

4 40 St80Pe40 0 0.1158 0.0642 0.0000 0.0435 — — -0.3434 — — — — — — — — — — —

4 41 BL 0 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2201 0.2747

4 42 BL 0 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3367 0.2960

4 43 NDF 0 0.1160 0.0000 0.0000 0.0000 — — — -1.1019 0.6284 -0.2379 -0.1954 0.0000 0.2582 -0.0248 0.2653 0.0371 0.3444 0.3231

4 44 NDF 0 0.1161 0.0000 0.0000 0.0000 — — — -0.2284 0.0048 -0.3067 -0.1640 0.0000 -0.0892 -0.0313 0.0046 -0.0060 0.2220 0.3973

4 45 St40Pe0 0 0.1158 0.0319 0.0000 0.0000 — — — 25.8296 0.4480 -0.2752 -0.1806 0.0000 -0.0588 -0.0705 -0.0087 0.0047 0.2070 0.3098

4 46 St40Pe0 0 0.1157 0.0321 0.0000 0.0000 — — — 26.2385 -0.0188 -0.3067 -0.1543 0.0000 -0.2272 -0.0851 -0.0018 -0.0157 0.2166 0.2760

4 47 St80Pe0 0 0.1160 0.0640 0.0000 0.0000 — — — 53.2218 0.0232 -0.3067 -0.1965 0.0000 -0.0035 -0.0003 0.0306 -0.0041 0.2086 0.2273

4 48 St80Pe0 0 0.1159 0.0639 0.0000 0.0000 — — — 53.5500 0.0079 -0.3067 -0.1958 0.0000 -0.3050 -0.0241 -0.0205 0.1503 0.1998 0.2776

4 49 St120Pe0 0 0.1157 0.0957 0.0000 0.0000 — — — 71.8832 0.0081 -0.3067 -0.1962 0.0000 0.1779 0.0369 -0.0717 0.0261 0.1899 0.2861

4 50 St120Pe0 0 0.1158 0.0958 0.0000 0.0000 — — — 46.3896 -0.0191 -0.3045 -0.1623 0.0000 0.1965 -0.0752 -0.0991 0.0641 0.0381 0.1181

4 51 St0Pe40 0 0.1158 0.0000 0.0000 0.0435 — — — -0.0749 0.0284 -0.2939 -0.1965 0.0596 0.1501 0.2284 -0.0984 0.0195 0.1221 0.3005

4 52 St0Pe40 0 0.1162 0.0000 0.0000 0.0435 — — — 0.0902 0.0256 -0.3067 -0.1962 0.0755 0.2983 0.6952 -0.0051 0.0333 0.1398 0.3154

4 53 St0Pe91 0 0.1159 0.0000 0.0000 0.0869 — — — -0.3110 0.3605 0.5937 0.0900 0.1692 0.8902 0.6579 0.1611 0.0002 0.0965 0.2379

4 54 St0Pe91 0 0.1161 0.0000 0.0000 0.0869 — — — 0.4838 0.6674 0.0848 0.0677 0.1195 0.6452 0.5447 0.0080 -0.0020 0.1066 0.2586

4 55 St0Pe120 0 0.1161 0.0000 0.0000 0.1304 — — — -1.0981 1.3726 0.9315 0.0581 0.0972 0.6692 0.2837 -0.0799 0.0033 0.0487 0.2852

4 56 St0Pe120 0 0.1160 0.0000 0.0000 0.1304 — — — 0.5625 1.4006 0.9473 0.0801 0.1368 0.8415 0.6304 0.0112 -0.0061 0.0677 0.3614

4 57 St40Pe91 0 0.1158 0.0321 0.0000 0.0869 — — — 25.7566 0.4271 0.5477 0.1383 0.1025 0.3095 0.5547 -0.2238 0.0262 0.0911 0.2774

4 58 St40Pe91 0 0.1160 0.0321 0.0000 0.0869 — — — 18.6000 0.6842 0.1071 0.0291 0.2276 0.1988 0.3914 -0.0399 -0.0107 0.0743 0.2669

4 59 St80Pe40 0 0.1162 0.0637 0.0000 0.0435 — — — 48.2728 0.0314 -0.3067 -0.1965 0.0652 0.0026 0.3806 -0.0666 -0.0216 0.1045 0.2680

4 60 St80Pe40 0 0.1160 0.0640 0.0000 0.0435 — — — 40.0142 0.0257 0.2492 -0.1965 0.1065 0.2610 0.5217 -0.0652 0.0256 0.0510 0.3176

4 61 BL 4 0.0000 0.0000 0.0000 0.0000 7.21 — — — — — — — — — — — — —

239



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 62 BL 4 0.0000 0.0000 0.0000 0.0000 7.23 — — — — — — — — — — — — —

4 63 NDF 4 0.1161 0.0000 0.0000 0.0000 7.22 92.2626 — — — — — — — — — — — —

4 64 NDF 4 0.1160 0.0000 0.0000 0.0000 7.19 93.1152 — — — — — — — — — — — —

4 65 St40Pe0 4 0.1162 0.0320 0.0000 0.0000 7.21 92.5303 — — — — — — — — — — — —

4 66 St40Pe0 4 0.1159 0.0319 0.0000 0.0000 7.22 91.8983 — — — — — — — — — — — —

4 67 St80Pe0 4 0.1157 0.0639 0.0000 0.0000 7.23 91.4465 — — — — — — — — — — — —

4 68 St80Pe0 4 0.1160 0.0638 0.0000 0.0000 7.20 92.3393 — — — — — — — — — — — —

4 69 St120Pe0 4 0.1159 0.0959 0.0000 0.0000 7.24 92.0709 — — — — — — — — — — — —

4 70 St120Pe0 4 0.1161 0.0956 0.0000 0.0000 7.17 92.5210 — — — — — — — — — — — —

4 71 St0Pe40 4 0.1158 0.0000 0.0000 0.0435 7.08 92.8385 — — — — — — — — — — — —

4 72 St0Pe40 4 0.1160 0.0000 0.0000 0.0435 7.09 92.5979 — — — — — — — — — — — —

4 73 St0Pe91 4 0.1161 0.0000 0.0000 0.0869 7.05 93.2963 — — — — — — — — — — — —

4 74 St0Pe91 4 0.1157 0.0000 0.0000 0.0869 7.04 91.6193 — — — — — — — — — — — —

4 75 St0Pe120 4 0.1162 0.0000 0.0000 0.1304 7.03 92.4442 — — — — — — — — — — — —

4 76 St0Pe120 4 0.1160 0.0000 0.0000 0.1304 7.03 92.2530 — — — — — — — — — — — —

4 77 St40Pe91 4 0.1163 0.0322 0.0000 0.0869 7.05 92.9696 — — — — — — — — — — — —

4 78 St40Pe91 4 0.1159 0.0321 0.0000 0.0869 7.06 92.1572 — — — — — — — — — — — —

4 79 St80Pe40 4 0.1160 0.0639 0.0000 0.0435 7.08 92.7703 — — — — — — — — — — — —

4 80 St80Pe40 4 0.1160 0.0637 0.0000 0.0435 7.10 93.2876 — — — — — — — — — — — —

4 83 NDF 4 0.1161 0.0000 0.0000 0.0000 — — -0.9787 — — — — — — — — — — —

4 84 NDF 4 0.1161 0.0000 0.0000 0.0000 — — -2.1505 — — — — — — — — — — —

4 85 St40Pe0 4 0.1159 0.0321 0.0000 0.0000 — — 1.2132 — — — — — — — — — — —

4 86 St40Pe0 4 0.1161 0.0320 0.0000 0.0000 — — -0.6450 — — — — — — — — — — —

4 87 St80Pe0 4 0.1157 0.0638 0.0000 0.0000 — — 0.5397 — — — — — — — — — — —

4 88 St80Pe0 4 0.1162 0.0640 0.0000 0.0000 — — 0.7219 — — — — — — — — — — —

4 89 St120Pe0 4 0.1159 0.0956 0.0000 0.0000 — — 1.0330 — — — — — — — — — — —

4 90 St120Pe0 4 0.1158 0.0959 0.0000 0.0000 — — -0.1443 — — — — — — — — — — —

4 91 St0Pe40 4 0.1160 0.0000 0.0000 0.0435 — — 4.5836 — — — — — — — — — — —

240



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 92 St0Pe40 4 0.1162 0.0000 0.0000 0.0435 — — 3.9930 — — — — — — — — — — —

4 93 St0Pe91 4 0.1161 0.0000 0.0000 0.0869 — — 3.6578 — — — — — — — — — — —

4 94 St0Pe91 4 0.1160 0.0000 0.0000 0.0869 — — 4.6384 — — — — — — — — — — —

4 95 St0Pe120 4 0.1158 0.0000 0.0000 0.1304 — — 5.2711 — — — — — — — — — — —

4 96 St0Pe120 4 0.1161 0.0000 0.0000 0.1304 — — 4.2885 — — — — — — — — — — —

4 97 St40Pe91 4 0.1161 0.0319 0.0000 0.0869 — — 3.7113 — — — — — — — — — — —

4 98 St40Pe91 4 0.1160 0.0321 0.0000 0.0869 — — 4.1971 — — — — — — — — — — —

4 99 St80Pe40 4 0.1158 0.0637 0.0000 0.0435 — — 4.9378 — — — — — — — — — — —

4 100 St80Pe40 4 0.1159 0.0640 0.0000 0.0435 — — 5.5171 — — — — — — — — — — —

4 101 BL 4 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2373 0.4850

4 102 BL 4 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.1765 0.4394

4 103 NDF 4 0.1160 0.0000 0.0000 0.0000 — — — -0.7458 -0.0009 -0.1606 -0.0054 0.0000 2.8082 0.7829 -0.0190 -0.0263 0.2427 0.4690

4 104 NDF 4 0.1159 0.0000 0.0000 0.0000 — — — 0.9838 -0.0277 -0.1470 0.1101 0.0000 -0.2129 -0.1461 -0.3435 -0.3381 0.1894 0.4894

4 105 St40Pe0 4 0.1161 0.0321 0.0000 0.0000 — — — 24.0606 -0.0187 -0.1430 0.0017 0.0483 0.2203 -0.0496 0.0826 -0.0708 0.3107 0.4726

4 106 St40Pe0 4 0.1159 0.0321 0.0000 0.0000 — — — 25.8689 0.0017 -0.1640 -0.0058 0.0407 -0.1671 -0.2089 -0.3437 -0.0857 0.2105 0.4485

4 107 St80Pe0 4 0.1157 0.0637 0.0000 0.0000 — — — 49.5676 0.0045 -0.0475 0.0173 0.0000 0.4028 0.2725 -0.1717 -0.0822 0.1978 0.5366

4 108 St80Pe0 4 0.1159 0.0642 0.0000 0.0000 — — — 48.3083 -0.0081 -0.1480 -0.0045 0.0000 -0.3893 -0.0816 -0.3379 -0.2273 0.1571 0.3689

4 109 St120Pe0 4 0.1158 0.0957 0.0000 0.0000 — — — 73.8960 -0.0374 -0.1671 -0.0058 0.0403 0.2497 0.1130 -0.1867 -0.1173 0.2063 0.3933

4 110 St120Pe0 4 0.1160 0.0956 0.0000 0.0000 — — — 64.0537 0.0005 -0.0173 -0.0058 0.0000 0.4073 0.0376 -0.2364 -0.0780 0.1968 0.4190

4 111 St0Pe40 4 0.1162 0.0000 0.0000 0.0435 — — — 0.5961 0.0149 -0.1381 0.2086 0.0467 3.4793 0.9800 -0.3311 -0.0330 0.1225 0.5101

4 112 St0Pe40 4 0.1159 0.0000 0.0000 0.0435 — — — 2.2451 -0.0356 -0.1671 0.0055 0.0000 4.1421 1.1538 -0.2313 -0.1308 0.1266 0.3494

4 113 St0Pe91 4 0.1159 0.0000 0.0000 0.0869 — — — 1.1470 0.1351 -0.1671 -0.0058 0.0567 4.4399 1.4942 -0.1203 -0.0750 0.0000 0.3975

4 114 St0Pe91 4 0.1161 0.0000 0.0000 0.0869 — — — 2.5657 0.1652 -0.0739 0.0192 0.2217 4.2357 1.8440 -0.2033 -0.0525 0.0519 0.5679

4 115 St0Pe120 4 0.1159 0.0000 0.0000 0.1304 — — — 1.5444 0.2003 0.0125 0.0292 0.2406 5.6666 2.7254 -0.1339 -0.0444 0.0000 0.4187

4 116 St0Pe120 4 0.1158 0.0000 0.0000 0.1304 — — — 2.6425 -0.0374 -0.1671 0.0992 0.0000 6.1604 1.3145 -0.1779 -0.0913 0.0000 0.2834

4 117 St40Pe91 4 0.1159 0.0319 0.0000 0.0869 — — — 24.3811 0.1716 -0.1273 -0.0058 0.2041 2.6941 1.1631 -0.2375 -0.0350 0.0782 0.6700

4 118 St40Pe91 4 0.1159 0.0321 0.0000 0.0869 — — — 13.1142 0.1672 -0.1211 0.0090 0.1136 4.8931 1.4455 -0.1157 -0.1442 0.2859 0.3678

4 119 St80Pe40 4 0.1160 0.0637 0.0000 0.0435 — — — 51.6179 0.1791 0.0226 -0.0058 0.1232 4.3239 1.3144 -0.1802 -0.1652 0.1023 0.3239

241



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 120 St80Pe40 4 0.1157 0.0641 0.0000 0.0435 — — — 44.9296 -0.0374 -0.1671 0.0237 0.2695 4.5225 1.2670 -0.2216 -0.0358 0.1022 0.5893

4 121 BL 8 0.0000 0.0000 0.0000 0.0000 7.27 — — — — — — — — — — — — —

4 122 BL 8 0.0000 0.0000 0.0000 0.0000 7.21 — — — — — — — — — — — — —

4 123 NDF 8 0.1161 0.0000 0.0000 0.0000 7.28 87.3092 — — — — — — — — — — — —

4 124 NDF 8 0.1159 0.0000 0.0000 0.0000 7.28 87.1956 — — — — — — — — — — — —

4 125 St40Pe0 8 0.1157 0.0323 0.0000 0.0000 7.24 86.7358 — — — — — — — — — — — —

4 126 St40Pe0 8 0.1159 0.0321 0.0000 0.0000 7.28 86.9367 — — — — — — — — — — — —

4 127 St80Pe0 8 0.1160 0.0642 0.0000 0.0000 7.29 87.2093 — — — — — — — — — — — —

4 128 St80Pe0 8 0.1158 0.0638 0.0000 0.0000 7.23 88.7363 — — — — — — — — — — — —

4 129 St120Pe0 8 0.1160 0.0957 0.0000 0.0000 7.22 87.2093 — — — — — — — — — — — —

4 130 St120Pe0 8 0.1158 0.0960 0.0000 0.0000 7.19 87.9591 — — — — — — — — — — — —

4 131 St0Pe40 8 0.1161 0.0956 0.0000 0.0435 7.12 88.4291 — — — — — — — — — — — —

4 132 St0Pe40 8 0.1161 0.0000 0.0000 0.0435 7.12 87.7399 — — — — — — — — — — — —

4 133 St0Pe91 8 0.1159 0.0000 0.0000 0.0869 7.00 88.3173 — — — — — — — — — — — —

4 134 St0Pe91 8 0.1160 0.0000 0.0000 0.0869 6.99 89.0199 — — — — — — — — — — — —

4 135 St0Pe120 8 0.1159 0.0000 0.0000 0.1304 6.86 88.0585 — — — — — — — — — — — —

4 136 St0Pe120 8 0.1160 0.0000 0.0000 0.1304 6.83 88.9336 — — — — — — — — — — — —

4 137 St40Pe91 8 0.1157 0.0319 0.0000 0.0869 6.97 88.0323 — — — — — — — — — — — —

4 138 St40Pe91 8 0.1157 0.0319 0.0000 0.0869 6.94 87.8595 — — — — — — — — — — — —

4 139 St80Pe40 8 0.1158 0.0640 0.0000 0.0435 7.08 88.3045 — — — — — — — — — — — —

4 140 St80Pe40 8 0.1157 0.0639 0.0000 0.0435 7.05 87.6866 — — — — — — — — — — — —

4 143 NDF 8 0.1162 0.0000 0.0000 0.0000 — — 1.8489 — — — — — — — — — — —

4 144 NDF 8 0.1160 0.0000 0.0000 0.0000 — — 2.8338 — — — — — — — — — — —

4 145 St40Pe0 8 0.1157 0.0322 0.0000 0.0000 — — 6.6845 — — — — — — — — — — —

4 146 St40Pe0 8 0.1159 0.0319 0.0000 0.0000 — — 5.8076 — — — — — — — — — — —

4 147 St80Pe0 8 0.1162 0.0641 0.0000 0.0000 — — 6.6768 — — — — — — — — — — —

4 148 St80Pe0 8 0.1159 0.0638 0.0000 0.0000 — — 6.6878 — — — — — — — — — — —

4 149 St120Pe0 8 0.1158 0.0960 0.0000 0.0000 — — 7.6658 — — — — — — — — — — —

242



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 150 St120Pe0 8 0.1158 0.0957 0.0000 0.0000 — — 8.6480 — — — — — — — — — — —

4 151 St0Pe40 8 0.1160 0.0000 0.0000 0.0435 — — 10.7356 — — — — — — — — — — —

4 152 St0Pe40 8 0.1161 0.0000 0.0000 0.0435 — — 10.5379 — — — — — — — — — — —

4 153 St0Pe91 8 0.1160 0.0000 0.0000 0.0869 — — 16.0634 — — — — — — — — — — —

4 154 St0Pe91 8 0.1157 0.0000 0.0000 0.0869 — — 15.4898 — — — — — — — — — — —

4 155 St0Pe120 8 0.1158 0.0000 0.0000 0.1304 — — 24.1175 — — — — — — — — — — —

4 156 St0Pe120 8 0.1160 0.0000 0.0000 0.1304 — — 23.8205 — — — — — — — — — — —

4 157 St40Pe91 8 0.1160 0.0319 0.0000 0.0869 — — 20.4124 — — — — — — — — — — —

4 158 St40Pe91 8 0.1159 0.0323 0.0000 0.0869 — — 20.9905 — — — — — — — — — — —

4 159 St80Pe40 8 0.1157 0.0638 0.0000 0.0435 — — 19.3901 — — — — — — — — — — —

4 160 St80Pe40 8 0.1160 0.0637 0.0000 0.0435 — — 14.3087 — — — — — — — — — — —

4 161 BL 8 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3053 0.8713

4 162 BL 8 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2637 0.8825

4 163 NDF 8 0.1161 0.0000 0.0000 0.0000 — — — -1.3347 0.0000 -0.0103 -0.0029 0.0000 0.7537 0.3866 0.0804 -0.0350 0.0000 0.7708

4 164 NDF 8 0.1161 0.0000 0.0000 0.0000 — — — 0.3178 0.0000 -0.0040 0.0091 0.0000 -0.6552 0.0270 — -0.1660 0.2339 0.4194

4 165 St40Pe0 8 0.1159 0.0320 0.0000 0.0000 — — — 12.8423 0.0000 -0.0094 -0.0005 0.0000 3.4505 1.6378 0.3675 -0.0193 0.1772 0.7358

4 166 St40Pe0 8 0.1158 0.0322 0.0000 0.0000 — — — 19.8179 0.0000 -0.0119 -0.0463 0.0000 2.9016 1.1624 0.4362 -0.0258 0.2699 0.7326

4 167 St80Pe0 8 0.1162 0.0639 0.0000 0.0000 — — — 38.1958 0.0000 -0.0087 -0.0394 0.3686 1.7270 0.9984 0.3340 -0.2764 0.2472 0.7479

4 168 St80Pe0 8 0.1158 0.0638 0.0000 0.0000 — — — 41.9841 0.0000 -0.0150 -0.0395 0.0397 -3.3724 1.4408 0.3717 -0.0925 0.2498 0.7643

4 169 St120Pe0 8 0.1159 0.0957 0.0000 0.0000 — — — 70.7904 0.0000 -0.0054 -0.0420 0.4639 3.0634 1.4223 0.4474 -0.1626 0.2622 0.7428

4 170 St120Pe0 8 0.1160 0.0957 0.0000 0.0000 — — — 66.0401 0.0000 -0.0172 -0.0298 0.1920 3.4438 1.7296 0.5231 -0.0321 0.2383 0.8121

4 171 St0Pe40 8 0.1159 0.0000 0.0000 0.0435 — — — -0.6244 0.0000 -0.0187 -0.0292 0.0000 11.3252 2.1173 0.2463 -0.1032 0.0000 0.6703

4 172 St0Pe40 8 0.1157 0.0000 0.0000 0.0435 — — — 0.7107 0.0000 -0.0195 -0.0484 0.0000 12.3671 3.4525 0.2748 -0.0807 0.1388 0.6603

4 173 St0Pe91 8 0.1158 0.0000 0.0000 0.0869 — — — 0.0091 0.0774 -0.0220 -0.0577 0.0000 20.2089 4.8700 0.1023 -0.1517 0.0658 0.6067

4 174 St0Pe91 8 0.1160 0.0000 0.0000 0.0869 — — — 2.3610 0.0866 -0.0281 -0.0577 0.0000 21.8238 3.3918 0.0246 -0.2421 0.0000 0.2464

4 175 St0Pe120 8 0.1158 0.0000 0.0000 0.1304 — — — 1.8981 0.0847 -0.0306 -0.0448 0.0000 29.4087 6.6338 0.3993 -0.1565 0.0517 0.4130

4 176 St0Pe120 8 0.1158 0.0000 0.0000 0.1304 — — — 2.8401 0.1069 -0.0349 -0.0219 0.0331 30.9694 5.6607 0.3805 -0.3413 0.1787 0.4647

4 177 St40Pe91 8 0.1162 0.0322 0.0000 0.0869 — — — 16.1576 0.0852 -0.0380 -0.0491 0.1872 21.5737 5.5896 0.5290 -0.1296 0.0954 0.4334

243



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 178 St40Pe91 8 0.1158 0.0319 0.0000 0.0869 — — — 20.1361 0.0794 -0.0405 -0.0525 0.1814 22.6297 6.3328 0.7547 -0.1357 0.0806 0.5664

4 179 St80Pe40 8 0.1160 0.0642 0.0000 0.0435 — — — 35.8346 0.0816 -0.0398 -0.0577 0.4394 14.8341 5.3433 0.8532 -0.0392 0.1535 0.6175

4 180 St80Pe40 8 0.1159 0.0640 0.0000 0.0435 — — — 34.3654 0.0692 -0.0448 -0.0577 0.1728 15.6260 5.3545 0.9058 -0.1635 0.1643 0.7496

4 181 BL 12 0.0000 0.0000 0.0000 0.0000 7.22 — — — — — — — — — — — — —

4 182 BL 12 0.0000 0.0000 0.0000 0.0000 7.22 — — — — — — — — — — — — —

4 183 NDF 12 0.1161 0.0000 0.0000 0.0000 7.23 80.8051 — — — — — — — — — — — —

4 184 NDF 12 0.1161 0.0000 0.0000 0.0000 7.25 80.8913 — — — — — — — — — — — —

4 185 St40Pe0 12 0.1157 0.0321 0.0000 0.0000 7.13 81.0745 — — — — — — — — — — — —

4 186 St40Pe0 12 0.1160 0.0323 0.0000 0.0000 7.15 80.0101 — — — — — — — — — — — —

4 187 St80Pe0 12 0.1162 0.0640 0.0000 0.0000 7.04 81.1684 — — — — — — — — — — — —

4 188 St80Pe0 12 0.1160 0.0640 0.0000 0.0000 7.07 81.2172 — — — — — — — — — — — —

4 189 St120Pe0 12 0.1161 0.0958 0.0000 0.0000 6.95 82.3557 — — — — — — — — — — — —

4 190 St120Pe0 12 0.1160 0.0960 0.0000 0.0000 7.00 82.4242 — — — — — — — — — — — —

4 191 St0Pe40 12 0.1158 0.0000 0.0000 0.0435 7.10 83.8569 — — — — — — — — — — — —

4 192 St0Pe40 12 0.1160 0.0000 0.0000 0.0435 7.11 82.7691 — — — — — — — — — — — —

4 193 St0Pe91 12 0.1160 0.0000 0.0000 0.0869 6.98 84.2348 — — — — — — — — — — — —

4 194 St0Pe91 12 0.1160 0.0000 0.0000 0.0869 7.00 82.7691 — — — — — — — — — — — —

4 195 St0Pe120 12 0.1160 0.0000 0.0000 0.1304 6.87 84.9245 — — — — — — — — — — — —

4 196 St0Pe120 12 0.1161 0.0000 0.0000 0.1304 6.86 83.6479 — — — — — — — — — — — —

4 197 St40Pe91 12 0.1157 0.0323 0.0000 0.0869 6.89 82.4574 — — — — — — — — — — — —

4 198 St40Pe91 12 0.1157 0.0321 0.0000 0.0869 6.89 83.6675 — — — — — — — — — — — —

4 199 St80Pe40 12 0.1160 0.0642 0.0000 0.0435 6.92 82.2518 — — — — — — — — — — — —

4 200 St80Pe40 12 0.1162 0.0638 0.0000 0.0435 6.93 80.7381 — — — — — — — — — — — —

4 203 NDF 12 0.1159 0.0000 0.0000 0.0000 — — 3.3263 — — — — — — — — — — —

4 204 NDF 12 0.1161 0.0000 0.0000 0.0000 — — 3.8061 — — — — — — — — — — —

4 205 St40Pe0 12 0.1159 0.0319 0.0000 0.0000 — — 10.6901 — — — — — — — — — — —

4 206 St40Pe0 12 0.1161 0.0319 0.0000 0.0000 — — 10.3919 — — — — — — — — — — —

4 207 St80Pe0 12 0.1158 0.0638 0.0000 0.0000 — — 15.5759 — — — — — — — — — — —

244



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 208 St80Pe0 12 0.1161 0.0642 0.0000 0.0000 — — 15.5631 — — — — — — — — — — —

4 209 St120Pe0 12 0.1160 0.0960 0.0000 0.0000 — — 19.3797 — — — — — — — — — — —

4 210 St120Pe0 12 0.1159 0.0957 0.0000 0.0000 — — 19.1921 — — — — — — — — — — —

4 211 St0Pe40 12 0.1160 0.0000 0.0000 0.0435 — — 13.3721 — — — — — — — — — — —

4 212 St0Pe40 12 0.1162 0.0000 0.0000 0.0435 — — 13.4651 — — — — — — — — — — —

4 213 St0Pe91 12 0.1161 0.0000 0.0000 0.0869 — — 18.0123 — — — — — — — — — — —

4 214 St0Pe91 12 0.1160 0.0000 0.0000 0.0869 — — 18.5047 — — — — — — — — — — —

4 215 St0Pe120 12 0.1161 0.0000 0.0000 0.1304 — — 24.6973 — — — — — — — — — — —

4 216 St0Pe120 12 0.1157 0.0000 0.0000 0.1304 — — 24.4125 — — — — — — — — — — —

4 217 St40Pe91 12 0.1160 0.0322 0.0000 0.0869 — — 23.1385 — — — — — — — — — — —

4 218 St40Pe91 12 0.1158 0.0319 0.0000 0.0869 — — 23.9363 — — — — — — — — — — —

4 219 St80Pe40 12 0.1157 0.0637 0.0000 0.0435 — — 25.3484 — — — — — — — — — — —

4 220 St80Pe40 12 0.1157 0.0637 0.0000 0.0435 — — 25.4460 — — — — — — — — — — —

4 221 BL 12 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.4078 1.6224

4 222 BL 12 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3679 2.1341

4 223 NDF 12 0.1157 0.0000 0.0000 0.0000 — — — -0.0023 0.0000 -0.0087 0.0151 0.0000 3.4269 1.6428 0.7120 0.1535 0.3743 1.4587

4 224 NDF 12 0.1157 0.0000 0.0000 0.0000 — — — 0.4777 0.0000 -0.0018 0.0000 0.0000 3.0857 1.5971 0.5968 0.1078 0.4059 1.4474

4 225 St40Pe0 12 0.1159 0.0319 0.0000 0.0000 — — — 11.6560 0.0000 -0.0124 0.0198 0.0000 0.2722 1.3025 1.2554 -0.0061 0.3545 1.4006

4 226 St40Pe0 12 0.1157 0.0322 0.0000 0.0000 — — — 12.2728 0.0000 -0.0174 0.0200 0.0000 3.4475 2.3855 1.8725 0.3955 0.5174 2.0688

4 227 St80Pe0 12 0.1158 0.0639 0.0000 0.0000 — — — 15.7507 0.0000 -0.0189 0.0000 0.0000 8.7909 6.0313 2.5437 0.4438 0.3873 1.7006

4 228 St80Pe0 12 0.1159 0.0639 0.0000 0.0000 — — — 18.4061 0.0000 -0.0178 0.0081 0.0000 8.3280 5.6335 2.3467 0.2983 0.4180 1.7301

4 229 St120Pe0 12 0.1160 0.0958 0.0000 0.0000 — — — 28.5878 0.0000 -0.0178 0.0225 0.0000 10.2548 7.1034 3.7141 0.3259 1.2270 1.6231

4 230 St120Pe0 12 0.1160 0.0959 0.0000 0.0000 — — — 32.0087 0.0000 -0.0172 0.0153 0.0000 9.7925 6.5901 3.0859 0.2754 0.2754 1.5500

4 231 St0Pe40 12 0.1158 0.0000 0.0000 0.0435 — — — -0.3920 0.0000 -0.0189 0.0103 0.0000 13.8535 4.4915 0.9303 0.2014 0.3206 2.0108

4 232 St0Pe40 12 0.1160 0.0000 0.0000 0.0435 — — — 0.8706 0.0000 -0.0189 0.0015 0.0000 14.0645 4.3517 0.9320 0.2097 0.4661 2.0096

4 233 St0Pe91 12 0.1157 0.0000 0.0000 0.0869 — — — 0.0821 0.0114 -0.0189 0.0072 0.0000 23.4815 7.0586 1.1326 0.0803 0.4579 2.0183

4 234 St0Pe91 12 0.1159 0.0000 0.0000 0.0869 — — — 1.1063 0.0121 -0.0189 0.0054 0.0000 21.7310 6.5106 0.9290 -0.2907 0.3553 1.9023

4 235 St0Pe120 12 0.1161 0.0000 0.0000 0.1304 — — — 0.1628 0.0087 -0.0189 0.0000 0.0000 34.0469 10.4204 1.3107 0.0981 0.0731 1.7346

245



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 236 St0Pe120 12 0.1161 0.0000 0.0000 0.1304 — — — 1.2673 0.0040 -0.0189 0.0120 0.0000 35.8852 8.7566 1.3385 0.1618 0.6532 1.7779

4 237 St40Pe91 12 0.1157 0.0321 0.0000 0.0869 — — — 5.2001 0.0112 -0.0178 0.0089 0.0000 26.1175 9.0178 2.1848 0.2838 0.4848 1.8599

4 238 St40Pe91 12 0.1160 0.0320 0.0000 0.0869 — — — 5.4380 0.0000 -0.0189 0.0021 0.0000 30.4272 9.6469 2.6404 0.2461 0.6801 1.9470

4 239 St80Pe40 12 0.1161 0.0638 0.0000 0.0435 — — — 10.7173 0.0114 -0.0189 0.0065 0.0000 20.3841 9.7936 3.8081 0.4501 0.6089 2.0145

4 240 St80Pe40 12 0.1161 0.0637 0.0000 0.0435 — — — 8.3475 0.0000 -0.0189 0.0118 0.0000 22.4092 10.3011 3.7071 0.6465 0.2842 2.0909

4 241 BL 16 0.0000 0.0000 0.0000 0.0000 7.40 — — — — — — — — — — — — —

4 242 BL 16 0.0000 0.0000 0.0000 0.0000 7.36 — — — — — — — — — — — — —

4 243 NDF 16 0.1160 0.0000 0.0000 0.0000 7.30 73.1989 — — — — — — — — — — — —

4 244 NDF 16 0.1160 0.0000 0.0000 0.0000 7.28 72.8540 — — — — — — — — — — — —

4 245 St40Pe0 16 0.1159 0.0323 0.0000 0.0000 7.18 74.3816 — — — — — — — — — — — —

4 246 St40Pe0 16 0.1161 0.0321 0.0000 0.0000 7.16 72.7935 — — — — — — — — — — — —

4 247 St80Pe0 16 0.1162 0.0640 0.0000 0.0000 7.05 73.2496 — — — — — — — — — — — —

4 248 St80Pe0 16 0.1161 0.0640 0.0000 0.0000 7.08 74.2580 — — — — — — — — — — — —

4 249 St120Pe0 16 0.1160 0.0956 0.0000 0.0000 6.86 72.9402 — — — — — — — — — — — —

4 250 St120Pe0 16 0.1161 0.0957 0.0000 0.0000 6.88 73.9134 — — — — — — — — — — — —

4 251 St0Pe40 16 0.1159 0.0000 0.0000 0.0435 7.21 74.7267 — — — — — — — — — — — —

4 252 St0Pe40 16 0.1158 0.0000 0.0000 0.0435 7.21 75.0480 — — — — — — — — — — — —

4 253 St0Pe91 16 0.1161 0.0000 0.0000 0.0869 7.08 72.4489 — — — — — — — — — — — —

4 254 St0Pe91 16 0.1159 0.0000 0.0000 0.0869 7.11 75.2445 — — — — — — — — — — — —

4 255 St0Pe120 16 0.1156 0.0000 0.0000 0.1304 7.00 77.0763 — — — — — — — — — — — —

4 256 St0Pe120 16 0.1157 0.0000 0.0000 0.1304 6.98 77.7900 — — — — — — — — — — — —

4 257 St40Pe91 16 0.1161 0.0321 0.0000 0.0869 7.03 75.9809 — — — — — — — — — — — —

4 258 St40Pe91 16 0.1157 0.0321 0.0000 0.0869 7.04 75.8885 — — — — — — — — — — — —

4 259 St80Pe40 16 0.1160 0.0641 0.0000 0.0435 6.94 75.0095 — — — — — — — — — — — —

4 260 St80Pe40 16 0.1157 0.0637 0.0000 0.0435 6.98 75.3699 — — — — — — — — — — — —

4 263 NDF 16 0.1158 0.0000 0.0000 0.0000 — — 5.8694 — — — — — — — — — — —

4 264 NDF 16 0.1158 0.0000 0.0000 0.0000 — — 5.6741 — — — — — — — — — — —

4 265 St40Pe0 16 0.1157 0.0320 0.0000 0.0000 — — 14.3061 — — — — — — — — — — —

246



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 266 St40Pe0 16 0.1158 0.0320 0.0000 0.0000 — — 13.7175 — — — — — — — — — — —

4 267 St80Pe0 16 0.1161 0.0641 0.0000 0.0000 — — 20.3497 — — — — — — — — — — —

4 268 St80Pe0 16 0.1161 0.0639 0.0000 0.0000 — — 21.3297 — — — — — — — — — — —

4 269 St120Pe0 16 0.1161 0.0959 0.0000 0.0000 — — 25.2385 — — — — — — — — — — —

4 270 St120Pe0 16 0.1160 0.0960 0.0000 0.0000 — — 26.0199 — — — — — — — — — — —

4 271 St0Pe40 16 0.1160 0.0000 0.0000 0.0435 — — 13.9580 — — — — — — — — — — —

4 272 St0Pe40 16 0.1157 0.0000 0.0000 0.0435 — — 14.3557 — — — — — — — — — — —

4 273 St0Pe91 16 0.1160 0.0000 0.0000 0.0869 — — 18.7000 — — — — — — — — — — —

4 274 St0Pe91 16 0.1161 0.0000 0.0000 0.0869 — — 18.5982 — — — — — — — — — — —

4 275 St0Pe120 16 0.1158 0.0000 0.0000 0.1304 — — 24.8011 — — — — — — — — — — —

4 276 St0Pe120 16 0.1161 0.0000 0.0000 0.1304 — — 24.8926 — — — — — — — — — — —

4 277 St40Pe91 16 0.1159 0.0322 0.0000 0.0869 — — 27.2428 — — — — — — — — — — —

4 278 St40Pe91 16 0.1160 0.0322 0.0000 0.0869 — — 27.4351 — — — — — — — — — — —

4 279 St80Pe40 16 0.1162 0.0637 0.0000 0.0435 — — 28.1715 — — — — — — — — — — —

4 280 St80Pe40 16 0.1160 0.0639 0.0000 0.0435 — — 27.7812 — — — — — — — — — — —

4 281 BL 16 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.6587 2.4533

4 282 BL 16 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.6553 2.6470

4 283 NDF 16 0.1161 0.0000 0.0000 0.0000 — — — -0.1558 0.0078 0.0067 -0.0121 0.0000 3.4674 1.8448 1.5337 0.2572 0.5570 1.2746

4 284 NDF 16 0.1161 0.0000 0.0000 0.0000 — — — 0.6338 -0.0079 -0.0019 -0.0121 0.0000 3.8391 1.9605 0.5000 0.2328 0.5401 2.1670

4 285 St40Pe0 16 0.1159 0.0321 0.0000 0.0000 — — — 1.5739 0.0016 0.0021 -0.0121 0.0000 9.6877 5.6267 1.8553 0.3979 0.6972 2.5690

4 286 St40Pe0 16 0.1160 0.0320 0.0000 0.0000 — — — 2.2093 -0.0008 0.0084 -0.0121 0.0000 9.4318 5.1099 2.0404 0.6016 0.7279 2.5056

4 287 St80Pe0 16 0.1161 0.0637 0.0000 0.0000 — — — 7.7265 -0.0079 -0.0019 -0.0121 0.0000 12.4167 7.7550 3.3396 0.6625 0.7486 2.5798

4 288 St80Pe0 16 0.1159 0.0641 0.0000 0.0000 — — — 4.4134 -0.0027 -0.0019 -0.0121 0.0000 14.0349 8.9446 3.0059 0.7098 0.7424 2.5728

4 289 St120Pe0 16 0.1158 0.0960 0.0000 0.0000 — — — 8.1930 0.0149 0.0136 -0.0121 0.0000 16.8106 11.5482 4.5541 0.8462 0.6953 2.5211

4 290 St120Pe0 16 0.1160 0.0959 0.0000 0.0000 — — — 10.6886 -0.0079 0.0112 -0.0071 0.0000 14.4035 9.9425 4.7042 0.7556 0.7097 2.4416

4 291 St0Pe40 16 0.1159 0.0000 0.0000 0.0435 — — — -0.0041 -0.0079 -0.0019 -0.0121 0.0000 13.4116 4.5391 0.6871 0.3449 0.5989 2.2356

4 292 St0Pe40 16 0.1159 0.0000 0.0000 0.0435 — — — 1.4196 -0.0079 -0.0019 -0.0121 0.0000 13.7975 4.4951 0.6536 0.3587 0.5877 2.2409

4 293 St0Pe91 16 0.1161 0.0000 0.0000 0.0869 — — — 0.1610 -0.0066 -0.0019 -0.0121 0.0000 24.6910 8.0300 1.1368 0.3920 0.5832 2.2876

247



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 294 St0Pe91 16 0.1159 0.0000 0.0000 0.0869 — — — 1.5020 -0.0079 -0.0019 -0.0121 0.0000 25.4601 8.1444 1.2377 0.2541 0.5505 2.0984

4 295 St0Pe120 16 0.1158 0.0000 0.0000 0.1304 — — — 0.7042 -0.0079 -0.0019 -0.0121 0.0000 36.2458 11.2648 1.2103 0.1962 0.6124 1.9392

4 296 St0Pe120 16 0.1159 0.0000 0.0000 0.1304 — — — 1.5806 0.0043 -0.0019 -0.0081 0.0000 36.5066 9.6701 0.8934 0.0592 0.4392 1.6487

4 297 St40Pe91 16 0.1161 0.0319 0.0000 0.0869 — — — 0.9461 -0.0079 -0.0019 0.0261 0.0000 34.1386 10.4076 2.3151 0.4007 0.5173 0.7884

4 298 St40Pe91 16 0.1161 0.0323 0.0000 0.0869 — — — 2.0521 — — — 0.0000 — — — — — —

4 299 St80Pe40 16 0.1159 0.0640 0.0000 0.0435 — — — 1.1899 -0.0079 -0.0019 -0.0121 0.0000 23.3991 11.9159 3.3067 0.8526 0.6053 2.2712

4 300 St80Pe40 16 0.1160 0.0641 0.0000 0.0435 — — — 2.5275 0.0033 -0.0019 -0.0121 0.0000 24.6206 11.5280 3.6509 0.8391 0.6370 2.2235

4 301 BL 20 0.0000 0.0000 0.0000 0.0000 7.43 — — — — — — — — — — — — —

4 302 BL 20 0.0000 0.0000 0.0000 0.0000 7.42 — — — — — — — — — — — — —

4 303 NDF 20 0.1158 0.0000 0.0000 0.0000 7.30 67.5778 — — — — — — — — — — — —

4 304 NDF 20 0.1161 0.0000 0.0000 0.0000 7.32 65.9449 — — — — — — — — — — — —

4 305 St40Pe0 20 0.1158 0.0322 0.0000 0.0000 7.19 66.0233 — — — — — — — — — — — —

4 306 St40Pe0 20 0.1157 0.0321 0.0000 0.0000 7.19 66.0783 — — — — — — — — — — — —

4 307 St80Pe0 20 0.1161 0.0640 0.0000 0.0000 7.11 66.9787 — — — — — — — — — — — —

4 308 St80Pe0 20 0.1158 0.0637 0.0000 0.0000 6.98 61.7900 — — — — — — — — — — — —

4 309 St120Pe0 20 0.1160 0.0959 0.0000 0.0000 6.90 64.7065 — — — — — — — — — — — —

4 310 St120Pe0 20 0.1160 0.0960 0.0000 0.0000 6.96 67.1205 — — — — — — — — — — — —

4 311 St0Pe40 20 0.1157 0.0000 0.0000 0.0435 7.27 69.2763 — — — — — — — — — — — —

4 312 St0Pe40 20 0.1161 0.0000 0.0000 0.0435 7.27 69.9938 — — — — — — — — — — — —

4 313 St0Pe91 20 0.1161 0.0000 0.0000 0.0869 7.16 69.0462 — — — — — — — — — — — —

4 314 St0Pe91 20 0.1161 0.0000 0.0000 0.0869 7.06 64.9111 — — — — — — — — — — — —

4 315 St0Pe120 20 0.1158 0.0000 0.0000 0.1304 7.04 72.5004 — — — — — — — — — — — —

4 316 St0Pe120 20 0.1157 0.0000 0.0000 0.1304 7.03 71.8693 — — — — — — — — — — — —

4 317 St40Pe91 20 0.1159 0.0322 0.0000 0.0869 7.03 69.5925 — — — — — — — — — — — —

4 318 St40Pe91 20 0.1157 0.0319 0.0000 0.0869 7.05 69.5356 — — — — — — — — — — — —

4 319 St80Pe40 20 0.1157 0.0641 0.0000 0.0435 7.04 69.7949 — — — — — — — — — — — —

4 320 St80Pe40 20 0.1157 0.0637 0.0000 0.0435 7.04 69.9678 — — — — — — — — — — — —

4 323 NDF 20 0.1162 0.0000 0.0000 0.0000 — — 6.0478 — — — — — — — — — — —

248



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 324 NDF 20 0.1161 0.0000 0.0000 0.0000 — — 5.8568 — — — — — — — — — — —

4 325 St40Pe0 20 0.1158 0.0323 0.0000 0.0000 — — 13.5149 — — — — — — — — — — —

4 326 St40Pe0 20 0.1159 0.0323 0.0000 0.0000 — — 12.3405 — — — — — — — — — — —

4 327 St80Pe0 20 0.1161 0.0641 0.0000 0.0000 — — 19.1779 — — — — — — — — — — —

4 328 St80Pe0 20 0.1160 0.0637 0.0000 0.0000 — — 19.3821 — — — — — — — — — — —

4 329 St120Pe0 20 0.1160 0.0957 0.0000 0.0000 — — 24.0745 — — — — — — — — — — —

4 330 St120Pe0 20 0.1161 0.0960 0.0000 0.0000 — — 22.6000 — — — — — — — — — — —

4 331 St0Pe40 20 0.1160 0.0000 0.0000 0.0435 — — 12.4933 — — — — — — — — — — —

4 332 St0Pe40 20 0.1161 0.0000 0.0000 0.0435 — — 13.4674 — — — — — — — — — — —

4 333 St0Pe91 20 0.1157 0.0000 0.0000 0.0869 — — 17.3452 — — — — — — — — — — —

4 334 St0Pe91 20 0.1158 0.0000 0.0000 0.0869 — — 17.8293 — — — — — — — — — — —

4 335 St0Pe120 20 0.1161 0.0000 0.0000 0.1304 — — 24.2091 — — — — — — — — — — —

4 336 St0Pe120 20 0.1162 0.0000 0.0000 0.1304 — — 23.4259 — — — — — — — — — — —

4 337 St40Pe91 20 0.1158 0.0319 0.0000 0.0869 — — 24.3269 — — — — — — — — — — —

4 338 St40Pe91 20 0.1161 0.0319 0.0000 0.0869 — — 24.4131 — — — — — — — — — — —

4 339 St80Pe40 20 0.1161 0.0642 0.0000 0.0435 — — 25.1380 — — — — — — — — — — —

4 340 St80Pe40 20 0.1159 0.0642 0.0000 0.0435 — — 26.1180 — — — — — — — — — — —

4 341 BL 20 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.9521 3.0002

4 342 BL 20 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.9072 2.6230

4 343 NDF 20 0.1157 0.0000 0.0000 0.0000 — — — -0.3178 -0.0064 -0.0006 0.0000 0.0000 10.2100 3.9197 1.6511 0.4804 0.9130 3.1703

4 344 NDF 20 0.1161 0.0000 0.0000 0.0000 — — — 1.1814 -0.0116 -0.0016 0.0000 0.0000 8.7949 3.3064 1.1909 0.3055 0.8196 2.9066

4 345 St40Pe0 20 0.1159 0.0321 0.0000 0.0000 — — — -0.2352 0.0093 0.0025 0.0000 0.0000 11.4097 5.5622 3.5973 0.4608 0.8560 2.8096

4 346 St40Pe0 20 0.1158 0.0320 0.0000 0.0000 — — — 1.2753 0.0013 0.0038 0.0000 0.0000 10.9394 5.2950 3.4916 0.4372 0.8145 2.7487

4 347 St80Pe0 20 0.1161 0.0642 0.0000 0.0000 — — — -0.0740 -0.0064 -0.0016 0.0112 0.0000 13.8195 7.7961 6.0454 0.7285 0.8013 2.5245

4 348 St80Pe0 20 0.1161 0.0642 0.0000 0.0000 — — — 1.3539 -0.0089 -0.0016 0.0000 0.0000 14.3329 8.3491 6.5955 1.1377 1.0169 2.9418

4 349 St120Pe0 20 0.1161 0.0957 0.0000 0.0000 — — — 0.4789 0.0135 0.0051 0.0000 0.0000 14.4393 9.3388 9.8649 1.2634 0.7380 2.7166

4 350 St120Pe0 20 0.1158 0.0955 0.0000 0.0000 — — — 2.2930 0.0141 0.0090 0.0055 0.0000 16.9814 11.2083 8.6523 1.2373 0.7312 2.6160

4 351 St0Pe40 20 0.1157 0.0000 0.0000 0.0435 — — — -0.9397 -0.0116 -0.0016 0.0229 0.0000 15.8811 5.3370 1.6526 -0.0536 0.7385 2.5604

249



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 352 St0Pe40 20 0.1161 0.0000 0.0000 0.0435 — — — 0.9552 -0.0001 -0.0016 0.0000 0.0000 17.3339 5.1823 1.7407 0.0576 0.7834 2.4869

4 353 St0Pe91 20 0.1159 0.0000 0.0000 0.0869 — — — -0.3101 -0.0116 -0.0016 0.0000 0.0000 24.8776 7.7966 1.8064 -0.1837 0.6709 2.1851

4 354 St0Pe91 20 0.1160 0.0000 0.0000 0.0869 — — — 1.1929 -0.0116 -0.0010 0.0115 0.0000 28.8734 8.0987 1.7648 -0.1533 0.6998 2.1689

4 355 St0Pe120 20 0.1160 0.0000 0.0000 0.1304 — — — 0.0835 -0.0066 0.0274 0.0000 0.0000 34.6848 10.6689 1.8390 -0.2434 0.4960 2.0361

4 356 St0Pe120 20 0.1160 0.0000 0.0000 0.1304 — — — 1.9787 -0.0116 -0.0002 0.0279 0.0000 34.5908 11.2308 1.7771 -0.3113 0.5208 1.9212

4 357 St40Pe91 20 0.1159 0.0321 0.0000 0.0869 — — — -0.0740 0.0173 0.0041 0.0000 0.0000 32.7825 11.0186 3.1509 -0.0790 0.3410 1.9659

4 358 St40Pe91 20 0.1161 0.0319 0.0000 0.0869 — — — 2.2930 0.0043 -0.0004 0.0000 0.0000 32.0947 11.1974 3.3269 -0.0128 0.5667 1.9262

4 359 St80Pe40 20 0.1161 0.0641 0.0000 0.0435 — — — 0.0086 -0.0116 -0.0016 0.0000 0.0000 22.8206 10.8848 5.0061 0.3629 0.6018 2.2438

4 360 St80Pe40 20 0.1158 0.0641 0.0000 0.0435 — — — 2.2930 0.0047 -0.0014 0.0000 0.0000 25.0776 11.4683 4.8443 0.3250 0.6154 2.2353

4 361 BL 24 0.0000 0.0000 0.0000 0.0000 7.46 — — — — — — — — — — — — —

4 362 BL 24 0.0000 0.0000 0.0000 0.0000 7.47 — — — — — — — — — — — — —

4 363 NDF 24 0.1162 0.0000 0.0000 0.0000 7.31 59.1333 — — — — — — — — — — — —

4 364 NDF 24 0.1161 0.0000 0.0000 0.0000 7.31 60.5608 — — — — — — — — — — — —

4 365 St40Pe0 24 0.1159 0.0319 0.0000 0.0000 7.21 59.1946 — — — — — — — — — — — —

4 366 St40Pe0 24 0.1158 0.0319 0.0000 0.0000 7.21 58.9848 — — — — — — — — — — — —

4 367 St80Pe0 24 0.1157 0.0640 0.0000 0.0000 7.09 59.7254 — — — — — — — — — — — —

4 368 St80Pe0 24 0.1158 0.0640 0.0000 0.0000 7.11 62.4393 — — — — — — — — — — — —

4 369 St120Pe0 24 0.1159 0.0957 0.0000 0.0000 6.97 59.8849 — — — — — — — — — — — —

4 370 St120Pe0 24 0.1159 0.0960 0.0000 0.0000 7.00 60.1438 — — — — — — — — — — — —

4 371 St0Pe40 24 0.1159 0.0000 0.0000 0.0435 7.24 61.3518 — — — — — — — — — — — —

4 372 St0Pe40 24 0.1160 0.0000 0.0000 0.0435 7.24 62.5079 — — — — — — — — — — — —

4 373 St0Pe91 24 0.1160 0.0000 0.0000 0.0869 7.16 63.3701 — — — — — — — — — — — —

4 374 St0Pe91 24 0.1159 0.0000 0.0000 0.0869 7.16 62.6462 — — — — — — — — — — — —

4 375 St0Pe120 24 0.1160 0.0000 0.0000 0.1304 7.05 64.5771 — — — — — — — — — — — —

4 376 St0Pe120 24 0.1160 0.0000 0.0000 0.1304 7.05 65.6980 — — — — — — — — — — — —

4 377 St40Pe91 24 0.1160 0.0323 0.0000 0.0869 7.06 63.1976 — — — — — — — — — — — —

4 378 St40Pe91 24 0.1158 0.0319 0.0000 0.0869 7.08 63.1302 — — — — — — — — — — — —

4 379 St80Pe40 24 0.1157 0.0639 0.0000 0.0435 7.05 64.1335 — — — — — — — — — — — —

250



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 380 St80Pe40 24 0.1160 0.0637 0.0000 0.0435 7.03 61.4733 — — — — — — — — — — — —

4 383 NDF 24 0.1161 0.0000 0.0000 0.0000 — — 4.5873 — — — — — — — — — — —

4 384 NDF 24 0.1157 0.0000 0.0000 0.0000 — — 4.5065 — — — — — — — — — — —

4 385 St40Pe0 24 0.1158 0.0323 0.0000 0.0000 — — 11.0737 — — — — — — — — — — —

4 386 St40Pe0 24 0.1160 0.0323 0.0000 0.0000 — — 10.1895 — — — — — — — — — — —

4 387 St80Pe0 24 0.1161 0.0637 0.0000 0.0000 — — 17.8178 — — — — — — — — — — —

4 388 St80Pe0 24 0.1157 0.0642 0.0000 0.0000 — — 16.9378 — — — — — — — — — — —

4 389 St120Pe0 24 0.1160 0.0956 0.0000 0.0000 — — 20.8539 — — — — — — — — — — —

4 390 St120Pe0 24 0.1162 0.0957 0.0000 0.0000 — — 21.9201 — — — — — — — — — — —

4 391 St0Pe40 24 0.1157 0.0000 0.0000 0.0435 — — 11.8168 — — — — — — — — — — —

4 392 St0Pe40 24 0.1158 0.0000 0.0000 0.0435 — — 12.0098 — — — — — — — — — — —

4 393 St0Pe91 24 0.1159 0.0000 0.0000 0.0869 — — 17.1417 — — — — — — — — — — —

4 394 St0Pe91 24 0.1162 0.0000 0.0000 0.0869 — — 17.2270 — — — — — — — — — — —

4 395 St0Pe120 24 0.1159 0.0000 0.0000 0.1304 — — 21.8695 — — — — — — — — — — —

4 396 St0Pe120 24 0.1158 0.0000 0.0000 0.1304 — — 21.5786 — — — — — — — — — — —

4 397 St40Pe91 24 0.1158 0.0319 0.0000 0.0869 — — 22.4689 — — — — — — — — — — —

4 398 St40Pe91 24 0.1160 0.0320 0.0000 0.0869 — — 22.4604 — — — — — — — — — — —

4 399 St80Pe40 24 0.1162 0.0640 0.0000 0.0435 — — 23.1865 — — — — — — — — — — —

4 400 St80Pe40 24 0.1159 0.0639 0.0000 0.0435 — — 22.5098 — — — — — — — — — — —

4 401 BL 24 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.6371 2.6339

4 402 BL 24 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.9478 2.6044

4 403 NDF 24 0.1160 0.0000 0.0000 0.0000 — — — 0.1887 0.0262 -0.0016 0.0036 0.0000 10.3274 4.7663 2.0419 1.1791 0.8773 2.9641

4 404 NDF 24 0.1158 0.0000 0.0000 0.0000 — — — 0.4482 0.0202 -0.0016 -0.0027 0.0000 10.7964 4.5435 2.0708 1.2434 0.5880 2.8574

4 405 St40Pe0 24 0.1159 0.0321 0.0000 0.0000 — — — 0.5035 0.0259 0.0005 -0.0058 0.0000 16.1513 7.2639 3.3242 1.3035 0.8253 2.6712

4 406 St40Pe0 24 0.1157 0.0319 0.0000 0.0000 — — — 0.8411 0.0316 0.0044 -0.0019 0.0000 16.3821 7.5335 3.7647 1.2868 0.7902 2.7838

4 407 St80Pe0 24 0.1161 0.0637 0.0000 0.0000 — — — 0.4325 0.0330 0.0081 -0.0002 0.0000 17.1418 10.9389 5.8403 1.8661 0.9648 2.7779

4 408 St80Pe0 24 0.1161 0.0638 0.0000 0.0000 — — — 0.9982 -0.0180 0.0046 0.0049 0.0000 19.3522 9.7720 5.6070 1.4823 1.0116 2.7740

4 409 St120Pe0 24 0.1158 0.0960 0.0000 0.0000 — — — 0.9009 -0.0180 0.0016 -0.0009 0.0000 17.2674 10.3596 6.2203 1.3924 0.8160 2.3742

251



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

4 410 St120Pe0 24 0.1158 0.0960 0.0000 0.0000 — — — 1.0768 -0.0180 0.0017 0.0041 0.0000 21.7833 12.3159 7.0806 1.6834 0.7617 2.7036

4 411 St0Pe40 24 0.1159 0.0000 0.0000 0.0435 — — — 0.2674 -0.0180 0.0149 0.0051 0.0000 21.0745 6.8091 2.1212 1.0398 0.8941 2.5087

4 412 St0Pe40 24 0.1159 0.0000 0.0000 0.0435 — — — 0.6053 -0.0180 0.0162 -0.0047 0.0000 21.4374 6.5968 2.0978 0.9681 0.7405 2.4859

4 413 St0Pe91 24 0.1160 0.0000 0.0000 0.0869 — — — 0.2732 -0.0180 0.0282 0.0023 0.0000 29.1561 8.9771 2.1900 0.9690 0.6884 2.2521

4 414 St0Pe91 24 0.1158 0.0000 0.0000 0.0869 — — — 0.6820 -0.0180 0.0392 0.0028 0.0000 29.0235 9.4486 2.3752 1.2372 0.7135 2.3564

4 415 St0Pe120 24 0.1157 0.0000 0.0000 0.1304 — — — 0.4325 -0.0180 0.0070 -0.0016 0.0000 38.0296 12.0154 2.3130 1.0131 0.6129 1.9915

4 416 St0Pe120 24 0.1159 0.0000 0.0000 0.1304 — — — 0.9196 -0.0180 0.0172 -0.0015 0.0000 36.8890 12.0106 2.1700 0.9380 0.6044 2.1470

4 417 St40Pe91 24 0.1157 0.0321 0.0000 0.0869 — — — 0.4363 -0.0180 0.0443 0.0128 0.0000 28.0271 9.6933 2.7549 0.7701 0.1795 0.9260

4 418 St40Pe91 24 0.1157 0.0321 0.0000 0.0869 — — — 1.0768 -0.0180 0.0289 0.0299 0.0000 33.4927 12.0967 3.4624 0.9774 0.7250 2.0590

4 419 St80Pe40 24 0.1161 0.0640 0.0000 0.0435 — — — 0.5150 -0.0180 0.0184 0.0002 0.0000 30.9281 13.5060 5.2787 1.5539 0.9230 2.4871

4 420 St80Pe40 24 0.1158 0.0640 0.0000 0.0435 — — — 1.2301 0.0159 -0.0016 -0.0042 0.0000 28.5010 12.5860 0.8394 1.3846 0.8082 2.2598

5 1 BL 0 0.0000 0.0000 0.0000 0.0000 7.21 — — — — — — — — — — — — —

5 2 BL 0 0.0000 0.0000 0.0000 0.0000 7.19 — — — — — — — — — — — — —

5 3 NDF 0 0.1160 0.0000 0.0000 0.0000 7.18 94.2360 — — — — — — — — — — — —

5 4 NDF 0 0.1157 0.0000 0.0000 0.0000 7.16 94.5581 — — — — — — — — — — — —

5 5 Su40Pe0 0 0.1157 0.0000 0.0382 0.0000 7.14 93.6073 — — — — — — — — — — — —

5 6 Su40Pe0 0 0.1159 0.0000 0.0382 0.0000 7.11 93.4515 — — — — — — — — — — — —

5 7 Su80Pe0 0 0.1160 0.0000 0.0760 0.0000 7.13 93.3739 — — — — — — — — — — — —

5 8 Su80Pe0 0 0.1158 0.0000 0.0761 0.0000 7.12 94.1339 — — — — — — — — — — — —

5 9 Su120Pe0 0 0.1160 0.0000 0.1144 0.0000 7.11 93.7187 — — — — — — — — — — — —

5 10 Su120Pe0 0 0.1157 0.0000 0.1140 0.0000 7.14 93.2616 — — — — — — — — — — — —

5 11 Su0Pe40 0 0.1158 0.0000 0.0000 0.0435 7.17 93.7021 — — — — — — — — — — — —

5 12 Su0Pe40 0 0.1161 0.0000 0.0000 0.0435 7.14 94.1578 — — — — — — — — — — — —

5 13 Su0Pe91 0 0.1160 0.0000 0.0000 0.0869 7.20 94.2360 — — — — — — — — — — — —

5 14 Su0Pe91 0 0.1161 0.0000 0.0000 0.0869 7.22 94.1578 — — — — — — — — — — — —

5 15 Su0Pe120 0 0.1158 0.0000 0.0000 0.1304 7.17 94.3066 — — — — — — — — — — — —

5 16 Su0Pe120 0 0.1159 0.0000 0.0000 0.1304 7.18 94.4007 — — — — — — — — — — — —

5 17 Su40Pe91 0 0.1159 0.0000 0.0380 0.0869 7.19 94.8322 — — — — — — — — — — — —

252



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 18 Su40Pe91 0 0.1161 0.0000 0.0385 0.0869 7.20 94.5024 — — — — — — — — — — — —

5 19 Su80Pe40 0 0.1157 0.0000 0.0764 0.0435 7.19 93.6937 — — — — — — — — — — — —

5 20 Su80Pe40 0 0.1158 0.0000 0.0760 0.0435 7.19 93.3566 — — — — — — — — — — — —

5 23 NDF 0 0.1156 0.0000 0.0000 0.0000 — — 0.1050 — — — — — — — — — — —

5 24 NDF 0 0.1159 0.0000 0.0000 0.0000 — — -0.1053 — — — — — — — — — — —

5 25 Su40Pe0 0 0.1162 0.0000 0.0383 0.0000 — — 0.5335 — — — — — — — — — — —

5 26 Su40Pe0 0 0.1158 0.0000 0.0384 0.0000 — — -0.5325 — — — — — — — — — — —

5 27 Su80Pe0 0 0.1157 0.0000 0.0764 0.0000 — — -0.7250 — — — — — — — — — — —

5 28 Su80Pe0 0 0.1161 0.0000 0.0765 0.0000 — — 0.7269 — — — — — — — — — — —

5 29 Su120Pe0 0 0.1159 0.0000 0.1140 0.0000 — — 2.9284 — — — — — — — — — — —

5 30 Su120Pe0 0 0.1159 0.0000 0.1139 0.0000 — — -2.9271 — — — — — — — — — — —

5 31 Su0Pe40 0 0.1158 0.0000 0.0000 0.0435 — — 1.0273 — — — — — — — — — — —

5 32 Su0Pe40 0 0.1160 0.0000 0.0000 0.0435 — — -1.0285 — — — — — — — — — — —

5 33 Su0Pe91 0 0.1162 0.0000 0.0000 0.0869 — — 0.0930 — — — — — — — — — — —

5 34 Su0Pe91 0 0.1158 0.0000 0.0000 0.0869 — — -0.0928 — — — — — — — — — — —

5 35 Su0Pe120 0 0.1158 0.0000 0.0000 0.1304 — — 0.4405 — — — — — — — — — — —

5 36 Su0Pe120 0 0.1160 0.0000 0.0000 0.1304 — — -0.4409 — — — — — — — — — — —

5 37 Su40Pe91 0 0.1159 0.0000 0.0384 0.0869 — — 0.0470 — — — — — — — — — — —

5 38 Su40Pe91 0 0.1160 0.0000 0.0380 0.0869 — — -0.0470 — — — — — — — — — — —

5 39 Su80Pe40 0 0.1160 0.0000 0.0761 0.0435 — — -0.8301 — — — — — — — — — — —

5 40 Su80Pe40 0 0.1161 0.0000 0.0760 0.0435 — — 0.8300 — — — — — — — — — — —

5 41 BL 0 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.1296 0.2892

5 42 BL 0 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.1895 0.2166

5 43 NDF 0 0.1161 0.0000 0.0000 0.0000 — — — 0.4721 0.0000 0.0000 -0.0529 -0.0164 0.0336 0.1520 -0.0200 0.0949 0.1817 0.2630

5 44 NDF 0 0.1162 0.0000 0.0000 0.0000 — — — -0.6320 0.0000 0.0000 -0.0529 -0.0164 -0.1774 0.2769 -0.1150 0.1151 0.1468 0.1622

5 45 Su40Pe0 0 0.1161 0.0000 0.0381 0.0000 — — — 1.1939 3.0573 2.7992 26.3618 0.2046 0.1330 0.3537 -0.0615 0.0805 0.1620 0.2657

5 46 Su40Pe0 0 0.1161 0.0000 0.0380 0.0000 — — — 0.0117 1.2184 3.1663 26.2356 0.0327 0.0690 0.2729 0.0677 -0.1320 0.1560 0.3236

5 47 Su80Pe0 0 0.1158 0.0000 0.0765 0.0000 — — — 1.4358 1.2511 3.7430 59.7846 0.1772 0.0786 0.2161 -0.1937 -0.0250 0.2502 0.3406

253



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 48 Su80Pe0 0 0.1157 0.0000 0.0765 0.0000 — — — 0.4916 5.2463 4.1725 60.5036 0.0951 -0.0987 0.0547 -0.1030 -0.2914 0.0000 0.0000

5 49 Su120Pe0 0 0.1162 0.0000 0.1144 0.0000 — — — 1.3597 5.0294 4.1007 97.7092 0.1242 -0.2238 0.1127 0.0415 -0.0179 0.1454 0.2556

5 50 Su120Pe0 0 0.1159 0.0000 0.1143 0.0000 — — — 0.4155 5.4610 4.5219 97.2672 0.0841 -0.1200 0.2972 -0.0409 0.0365 0.1715 0.1714

5 51 Su0Pe40 0 0.1159 0.0000 0.0000 0.0435 — — — 0.9578 0.0000 0.0334 -0.0438 0.0414 0.0472 0.2728 -0.0359 -0.0240 0.0000 0.2703

5 52 Su0Pe40 0 0.1157 0.0000 0.0000 0.0435 — — — -0.8720 0.0000 0.0493 -0.0079 0.1439 0.4058 -1.1132 0.0390 0.1098 0.0494 0.2918

5 53 Su0Pe91 0 0.1159 0.0000 0.0000 0.0869 — — — 0.6398 0.0000 0.0631 0.2536 0.1047 0.5047 0.6408 -0.0115 -0.1568 0.0000 0.2416

5 54 Su0Pe91 0 0.1161 0.0000 0.0000 0.0869 — — — -0.7081 0.1143 0.0626 -0.0095 0.0831 0.8816 0.3568 -0.0801 0.0911 0.0000 0.3371

5 55 Su0Pe120 0 0.1160 0.0000 0.0000 0.1304 — — — 0.1600 0.2713 0.2160 -0.0529 0.1797 3.7888 1.8581 0.3147 0.0758 0.0000 0.3025

5 56 Su0Pe120 0 0.1159 0.0000 0.0000 0.1304 — — — -0.4682 0.4197 0.2265 -0.0117 0.2662 0.1830 -0.1985 -0.2887 0.2257 0.0000 0.2004

5 57 Su40Pe91 0 0.1162 0.0000 0.0385 0.0869 — — — 0.8778 5.8354 4.6602 25.1442 0.0646 0.0004 0.2029 -0.3107 0.0313 0.0000 0.1954

5 58 Su40Pe91 0 0.1161 0.0000 0.0385 0.0869 — — — -0.3043 6.2553 4.7324 24.2453 0.1304 0.2462 1.0253 0.0467 -0.0581 0.0000 0.2846

5 59 Su80Pe40 0 0.1157 0.0000 0.0762 0.0435 — — — 1.3597 7.1750 5.8189 54.9183 0.2874 0.0206 -1.6604 -0.0585 0.0131 0.0664 0.3415

5 60 Su80Pe40 0 0.1157 0.0000 0.0761 0.0435 — — — 0.0995 7.3559 6.0190 56.6734 0.3078 0.5549 0.8120 -0.0840 -0.2914 0.0000 0.0000

5 61 BL 4 0.0000 0.0000 0.0000 0.0000 7.12 — — — — — — — — — — — — —

5 62 BL 4 0.0000 0.0000 0.0000 0.0000 6.94 — — — — — — — — — — — — —

5 63 NDF 4 0.1160 0.0000 0.0000 0.0000 7.23 92.9428 — — — — — — — — — — — —

5 64 NDF 4 0.1159 0.0000 0.0000 0.0000 7.22 93.1927 — — — — — — — — — — — —

5 65 Su40Pe0 4 0.1160 0.0000 0.0382 0.0000 7.03 91.8219 — — — — — — — — — — — —

5 66 Su40Pe0 4 0.1161 0.0000 0.0383 0.0000 7.01 92.1764 — — — — — — — — — — — —

5 67 Su80Pe0 4 0.1157 0.0000 0.0761 0.0000 6.80 92.0515 — — — — — — — — — — — —

5 68 Su80Pe0 4 0.1161 0.0000 0.0761 0.0000 6.76 92.6933 — — — — — — — — — — — —

5 69 Su120Pe0 4 0.1158 0.0000 0.1139 0.0000 6.65 92.0612 — — — — — — — — — — — —

5 70 Su120Pe0 4 0.1160 0.0000 0.1143 0.0000 6.68 92.3393 — — — — — — — — — — — —

5 71 Su0Pe40 4 0.1162 0.0000 0.0000 0.0435 7.01 92.0999 — — — — — — — — — — — —

5 72 Su0Pe40 4 0.1158 0.0000 0.0000 0.0435 7.05 91.4567 — — — — — — — — — — — —

5 73 Su0Pe91 4 0.1162 0.0000 0.0000 0.0869 6.98 92.0138 — — — — — — — — — — — —

5 74 Su0Pe91 4 0.1159 0.0000 0.0000 0.0869 7.01 91.7258 — — — — — — — — — — — —

5 75 Su0Pe120 4 0.1160 0.0000 0.0000 0.1304 6.96 91.7357 — — — — — — — — — — — —

254



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 76 Su0Pe120 4 0.1157 0.0000 0.0000 0.1304 7.02 91.9651 — — — — — — — — — — — —

5 77 Su40Pe91 4 0.1161 0.0000 0.0384 0.0869 6.91 92.6933 — — — — — — — — — — — —

5 78 Su40Pe91 4 0.1158 0.0000 0.0380 0.0869 6.89 92.1476 — — — — — — — — — — — —

5 79 Su80Pe40 4 0.1160 0.0000 0.0764 0.0435 6.76 92.0806 — — — — — — — — — — — —

5 80 Su80Pe40 4 0.1157 0.0000 0.0759 0.0435 6.79 91.7058 — — — — — — — — — — — —

5 83 NDF 4 0.1157 0.0000 0.0000 0.0000 — — -3.3177 — — — — — — — — — — —

5 84 NDF 4 0.1161 0.0000 0.0000 0.0000 — — -4.0213 — — — — — — — — — — —

5 85 Su40Pe0 4 0.1157 0.0000 0.0380 0.0000 — — 1.4346 — — — — — — — — — — —

5 86 Su40Pe0 4 0.1161 0.0000 0.0385 0.0000 — — 0.3353 — — — — — — — — — — —

5 87 Su80Pe0 4 0.1157 0.0000 0.0765 0.0000 — — 3.3736 — — — — — — — — — — —

5 88 Su80Pe0 4 0.1160 0.0000 0.0763 0.0000 — — 2.2971 — — — — — — — — — — —

5 89 Su120Pe0 4 0.1159 0.0000 0.1140 0.0000 — — 4.4908 — — — — — — — — — — —

5 90 Su120Pe0 4 0.1157 0.0000 0.1140 0.0000 — — 5.3764 — — — — — — — — — — —

5 91 Su0Pe40 4 0.1159 0.0000 0.0000 0.0435 — — 1.1224 — — — — — — — — — — —

5 92 Su0Pe40 4 0.1158 0.0000 0.0000 0.0435 — — 1.8085 — — — — — — — — — — —

5 93 Su0Pe91 4 0.1161 0.0000 0.0000 0.0869 — — 0.7789 — — — — — — — — — — —

5 94 Su0Pe91 4 0.1157 0.0000 0.0000 0.0869 — — 0.6908 — — — — — — — — — — —

5 95 Su0Pe120 4 0.1157 0.0000 0.0000 0.1304 — — 3.2736 — — — — — — — — — — —

5 96 Su0Pe120 4 0.1158 0.0000 0.0000 0.1304 — — 4.5418 — — — — — — — — — — —

5 97 Su40Pe91 4 0.1162 0.0000 0.0385 0.0869 — — 4.4365 — — — — — — — — — — —

5 98 Su40Pe91 4 0.1158 0.0000 0.0384 0.0869 — — 5.6142 — — — — — — — — — — —

5 99 Su80Pe40 4 0.1160 0.0000 0.0762 0.0435 — — 7.3711 — — — — — — — — — — —

5 100 Su80Pe40 4 0.1160 0.0000 0.0760 0.0435 — — 8.5458 — — — — — — — — — — —

5 101 BL 4 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.0000 0.4640

5 102 BL 4 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2283 0.5647

5 103 NDF 4 0.1157 0.0000 0.0000 0.0000 — — — 1.1548 0.0000 0.0035 -0.0269 0.0000 0.1674 0.7599 0.0591 0.0607 0.2230 0.3945

5 104 NDF 4 0.1157 0.0000 0.0000 0.0000 — — — 1.4084 0.0000 0.0008 -0.0170 0.0000 0.4149 0.9267 -0.0309 0.0693 0.2134 0.2971

5 105 Su40Pe0 4 0.1159 0.0000 0.0384 0.0000 — — — 4.6818 0.0000 0.0044 0.0098 9.0227 1.8695 2.2052 0.2747 -0.1795 0.1836 0.5062

255



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 106 Su40Pe0 4 0.1161 0.0000 0.0385 0.0000 — — — 4.1453 0.0000 0.0021 0.0205 9.0026 1.7377 2.2198 0.3284 0.1048 0.2387 0.4194

5 107 Su80Pe0 4 0.1157 0.0000 0.0760 0.0000 — — — 4.8457 0.1067 0.0070 0.0380 20.7789 2.4129 2.7643 0.4766 -0.2958 0.0000 0.0000

5 108 Su80Pe0 4 0.1161 0.0000 0.0765 0.0000 — — — 4.8730 0.0787 0.0065 0.0162 21.6829 2.3320 2.5515 0.4512 0.1021 0.1812 0.4764

5 109 Su120Pe0 4 0.1159 0.0000 0.1143 0.0000 — — — 5.8854 0.2893 0.0038 0.0078 33.3120 2.4794 2.6787 0.6297 0.0449 0.1895 0.2099

5 110 Su120Pe0 4 0.1160 0.0000 0.1142 0.0000 — — — 5.5167 0.3438 0.0011 -0.0004 32.8229 2.3747 2.5572 0.4530 0.2502 0.1918 0.3701

5 111 Su0Pe40 4 0.1161 0.0000 0.0000 0.0435 — — — 2.5984 0.7249 0.3725 0.2219 0.0000 3.2150 1.6736 -0.0319 0.1093 0.0769 0.3839

5 112 Su0Pe40 4 0.1160 0.0000 0.0000 0.0435 — — — 2.4501 0.7284 0.1983 0.0781 0.0358 4.6349 -0.0651 0.0859 0.0610 0.1187 0.4060

5 113 Su0Pe91 4 0.1160 0.0000 0.0000 0.0869 — — — 2.3585 0.6266 0.3172 -0.0428 0.1078 5.2798 2.8350 0.2796 0.6953 0.0000 0.4369

5 114 Su0Pe91 4 0.1158 0.0000 0.0000 0.0869 — — — 2.9339 0.5642 0.2431 0.0052 0.0000 3.4106 0.6995 -0.4861 -0.2958 0.0000 0.0000

5 115 Su0Pe120 4 0.1160 0.0000 0.0000 0.1304 — — — 2.7545 0.5231 0.0560 0.0757 0.1692 5.7284 1.9574 0.1609 0.0389 0.2005 0.2821

5 116 Su0Pe120 4 0.1161 0.0000 0.0000 0.1304 — — — 3.9737 0.5685 0.2560 0.2133 0.3065 4.9546 2.4774 0.2018 0.0081 0.0000 0.4106

5 117 Su40Pe91 4 0.1160 0.0000 0.0380 0.0869 — — — 5.9576 0.3854 0.3192 -0.0428 9.8398 4.7265 3.2370 0.3032 0.1201 0.0636 0.4520

5 118 Su40Pe91 4 0.1162 0.0000 0.0384 0.0869 — — — 4.3892 0.3656 0.3209 -0.0428 10.9118 5.5534 2.6787 0.2990 0.0160 0.0437 0.5091

5 119 Su80Pe40 4 0.1161 0.0000 0.0761 0.0435 — — — 4.0381 0.0201 0.0188 0.6123 19.7638 2.5567 2.1167 0.2001 0.0687 0.0000 0.3634

5 120 Su80Pe40 4 0.1158 0.0000 0.0762 0.0435 — — — 4.5531 0.0229 0.0044 -0.0428 23.3963 5.6542 3.2786 0.5733 0.1189 0.1470 0.4033

5 121 BL 8 0.0000 0.0000 0.0000 0.0000 7.21 — — — — — — — — — — — — —

5 122 BL 8 0.0000 0.0000 0.0000 0.0000 6.98 — — — — — — — — — — — — —

5 123 NDF 8 0.1156 0.0000 0.0000 0.0000 7.18 86.9379 — — — — — — — — — — — —

5 124 NDF 8 0.1160 0.0000 0.0000 0.0000 7.19 87.2524 — — — — — — — — — — — —

5 125 Su40Pe0 8 0.1160 0.0000 0.0381 0.0000 7.04 85.7867 — — — — — — — — — — — —

5 126 Su40Pe0 8 0.1160 0.0000 0.0380 0.0000 7.06 86.3040 — — — — — — — — — — — —

5 127 Su80Pe0 8 0.1160 0.0000 0.0761 0.0000 6.94 86.1316 — — — — — — — — — — — —

5 128 Su80Pe0 8 0.1157 0.0000 0.0762 0.0000 6.95 85.8283 — — — — — — — — — — — —

5 129 Su120Pe0 8 0.1157 0.0000 0.1144 0.0000 6.80 87.2112 — — — — — — — — — — — —

5 130 Su120Pe0 8 0.1158 0.0000 0.1144 0.0000 6.80 86.9659 — — — — — — — — — — — —

5 131 Su0Pe40 8 0.1161 0.0000 0.0000 0.0435 7.03 87.2661 — — — — — — — — — — — —

5 132 Su0Pe40 8 0.1157 0.0000 0.0000 0.0435 7.06 87.9027 — — — — — — — — — — — —

5 133 Su0Pe91 8 0.1162 0.0000 0.0000 0.0869 6.92 88.4848 — — — — — — — — — — — —

256



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 134 Su0Pe91 8 0.1161 0.0000 0.0000 0.0869 6.91 87.1799 — — — — — — — — — — — —

5 135 Su0Pe120 8 0.1157 0.0000 0.0000 0.1304 6.76 89.5449 — — — — — — — — — — — —

5 136 Su0Pe120 8 0.1161 0.0000 0.0000 0.1304 6.80 90.0228 — — — — — — — — — — — —

5 137 Su40Pe91 8 0.1158 0.0000 0.0385 0.0869 6.84 88.4340 — — — — — — — — — — — —

5 138 Su40Pe91 8 0.1162 0.0000 0.0384 0.0869 6.81 88.6569 — — — — — — — — — — — —

5 139 Su80Pe40 8 0.1158 0.0000 0.0763 0.0435 6.84 87.3113 — — — — — — — — — — — —

5 140 Su80Pe40 8 0.1158 0.0000 0.0760 0.0435 6.85 87.3113 — — — — — — — — — — — —

5 143 NDF 8 0.1159 0.0000 0.0000 0.0000 — — 1.8965 — — — — — — — — — — —

5 144 NDF 8 0.1161 0.0000 0.0000 0.0000 — — 1.7889 — — — — — — — — — — —

5 145 Su40Pe0 8 0.1159 0.0000 0.0383 0.0000 — — 13.4804 — — — — — — — — — — —

5 146 Su40Pe0 8 0.1160 0.0000 0.0384 0.0000 — — 11.0335 — — — — — — — — — — —

5 147 Su80Pe0 8 0.1157 0.0000 0.0763 0.0000 — — 15.1457 — — — — — — — — — — —

5 148 Su80Pe0 8 0.1156 0.0000 0.0761 0.0000 — — 15.3488 — — — — — — — — — — —

5 149 Su120Pe0 8 0.1162 0.0000 0.1140 0.0000 — — 14.0015 — — — — — — — — — — —

5 150 Su120Pe0 8 0.1159 0.0000 0.1141 0.0000 — — 15.0823 — — — — — — — — — — —

5 151 Su0Pe40 8 0.1157 0.0000 0.0000 0.0435 — — 12.3085 — — — — — — — — — — —

5 152 Su0Pe40 8 0.1157 0.0000 0.0000 0.0435 — — 12.7967 — — — — — — — — — — —

5 153 Su0Pe91 8 0.1159 0.0000 0.0000 0.0869 — — 21.5343 — — — — — — — — — — —

5 154 Su0Pe91 8 0.1157 0.0000 0.0000 0.0869 — — 19.0002 — — — — — — — — — — —

5 155 Su0Pe120 8 0.1159 0.0000 0.0000 0.1304 — — 31.0525 — — — — — — — — — — —

5 156 Su0Pe120 8 0.1160 0.0000 0.0000 0.1304 — — 31.1489 — — — — — — — — — — —

5 157 Su40Pe91 8 0.1156 0.0000 0.0380 0.0869 — — 25.9815 — — — — — — — — — — —

5 158 Su40Pe91 8 0.1160 0.0000 0.0383 0.0869 — — 26.4614 — — — — — — — — — — —

5 159 Su80Pe40 8 0.1157 0.0000 0.0759 0.0435 — — 21.1969 — — — — — — — — — — —

5 160 Su80Pe40 8 0.1156 0.0000 0.0761 0.0435 — — 21.1955 — — — — — — — — — — —

5 161 BL 8 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2256 0.6357

5 162 BL 8 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2312 0.6804

5 163 NDF 8 0.1161 0.0000 0.0000 0.0000 — — — 0.2409 -0.0126 -0.0413 -0.0742 0.0000 0.8838 0.3502 -0.0152 0.0537 0.2198 0.7192

257



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 164 NDF 8 0.1162 0.0000 0.0000 0.0000 — — — 0.6447 -0.0358 -0.0199 -0.1168 0.0000 0.4866 -0.1787 -0.1251 -0.1484 0.2440 0.3951

5 165 Su40Pe0 8 0.1157 0.0000 0.0384 0.0000 — — — 1.7645 -0.1500 0.1186 -0.1168 5.9859 1.1569 2.2951 0.4072 -0.0183 0.2417 0.5972

5 166 Su40Pe0 8 0.1157 0.0000 0.0381 0.0000 — — — 2.8081 -0.3430 -0.1886 -0.0604 6.8523 4.8567 2.3979 1.0118 0.1080 0.1038 0.6655

5 167 Su80Pe0 8 0.1157 0.0000 0.0759 0.0000 — — — 2.8842 -0.1510 0.2298 -0.1168 19.1647 4.7195 4.9243 0.7547 -0.3149 0.2093 0.6606

5 168 Su80Pe0 8 0.1159 0.0000 0.0762 0.0000 — — — 3.4519 -0.1678 0.2446 -0.1168 18.5013 5.5676 4.5890 0.8611 -0.0054 0.2049 0.6394

5 169 Su120Pe0 8 0.1158 0.0000 0.1140 0.0000 — — — 4.0119 -0.3145 -0.1667 0.0334 30.6210 6.3894 6.7937 0.9094 0.0046 0.4014 0.3303

5 170 Su120Pe0 8 0.1159 0.0000 0.1142 0.0000 — — — 3.6118 -0.1716 0.3662 -0.1168 29.0940 5.6523 6.5569 0.8782 0.0106 0.2105 0.6014

5 171 Su0Pe40 8 0.1159 0.0000 0.0000 0.0435 — — — 2.4804 -0.0687 0.1913 -0.1168 0.0000 11.0448 2.6557 0.1017 0.0464 0.1212 0.5624

5 172 Su0Pe40 8 0.1160 0.0000 0.0000 0.0435 — — — 1.3645 -0.3429 -0.1886 0.0845 0.0000 2.8352 1.4806 0.1870 -0.2621 0.0743 0.0836

5 173 Su0Pe91 8 0.1159 0.0000 0.0000 0.0869 — — — 2.9642 0.0000 0.3202 -0.1168 0.0000 20.9011 4.0948 0.1381 -0.0915 0.0924 0.1401

5 174 Su0Pe91 8 0.1157 0.0000 0.0000 0.0869 — — — 2.4082 -0.0139 0.2762 -0.1168 0.0000 19.6341 3.9641 0.1661 -0.0876 0.0774 2.6198

5 175 Su0Pe120 8 0.1159 0.0000 0.0000 0.1304 — — — 3.8479 -0.2817 -0.0110 -0.0848 0.0000 25.6979 4.7410 0.3667 -0.1128 0.1024 0.4644

5 176 Su0Pe120 8 0.1161 0.0000 0.0000 0.1304 — — — 2.9681 -0.3153 -0.1886 0.2370 0.0000 27.6367 5.7255 0.3321 -0.1052 0.0730 0.4986

5 177 Su40Pe91 8 0.1162 0.0000 0.0379 0.0869 — — — 4.4077 -0.0908 0.2846 -0.1168 7.5050 24.0772 7.4412 0.7772 -0.1261 0.0816 0.4630

5 178 Su40Pe91 8 0.1159 0.0000 0.0381 0.0869 — — — 3.8518 -0.1067 0.3024 -0.0704 7.9591 23.8117 7.4490 0.7547 -0.0429 0.0802 0.3468

5 179 Su80Pe40 8 0.1160 0.0000 0.0764 0.0435 — — — 4.7276 -0.3278 -0.1886 0.0248 18.4604 15.3012 7.7199 0.7270 -0.1907 0.0941 0.6094

5 180 Su80Pe40 8 0.1161 0.0000 0.0760 0.0435 — — — 3.6118 -0.2132 0.2706 -0.1168 18.4980 16.0661 7.9540 0.8042 -0.1025 0.1261 0.6826

5 181 BL 12 0.0000 0.0000 0.0000 0.0000 7.27 — — — — — — — — — — — — —

5 182 BL 12 0.0000 0.0000 0.0000 0.0000 7.27 — — — — — — — — — — — — —

5 183 NDF 12 0.1160 0.0000 0.0000 0.0000 7.26 80.3550 — — — — — — — — — — — —

5 184 NDF 12 0.1159 0.0000 0.0000 0.0000 7.28 80.5944 — — — — — — — — — — — —

5 185 Su40Pe0 12 0.1161 0.0000 0.0380 0.0000 7.02 78.3930 — — — — — — — — — — — —

5 186 Su40Pe0 12 0.1162 0.0000 0.0383 0.0000 7.16 79.4469 — — — — — — — — — — — —

5 187 Su80Pe0 12 0.1160 0.0000 0.0763 0.0000 7.01 80.9585 — — — — — — — — — — — —

5 188 Su80Pe0 12 0.1161 0.0000 0.0762 0.0000 7.01 80.9774 — — — — — — — — — — — —

5 189 Su120Pe0 12 0.1157 0.0000 0.1142 0.0000 6.85 81.7659 — — — — — — — — — — — —

5 190 Su120Pe0 12 0.1157 0.0000 0.1142 0.0000 6.84 81.8524 — — — — — — — — — — — —

5 191 Su0Pe40 12 0.1157 0.0000 0.0000 0.0435 7.16 82.3710 — — — — — — — — — — — —

258



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 192 Su0Pe40 12 0.1162 0.0000 0.0000 0.0435 7.16 82.2013 — — — — — — — — — — — —

5 193 Su0Pe91 12 0.1157 0.0000 0.0000 0.0869 7.03 83.9268 — — — — — — — — — — — —

5 194 Su0Pe91 12 0.1160 0.0000 0.0000 0.0869 7.05 83.8899 — — — — — — — — — — — —

5 195 Su0Pe120 12 0.1162 0.0000 0.0000 0.1304 6.95 84.5253 — — — — — — — — — — — —

5 196 Su0Pe120 12 0.1161 0.0000 0.0000 0.1304 6.93 84.4233 — — — — — — — — — — — —

5 197 Su40Pe91 12 0.1162 0.0000 0.0384 0.0869 6.98 83.4064 — — — — — — — — — — — —

5 198 Su40Pe91 12 0.1157 0.0000 0.0383 0.0869 6.97 82.9760 — — — — — — — — — — — —

5 199 Su80Pe40 12 0.1159 0.0000 0.0764 0.0435 6.93 81.4573 — — — — — — — — — — — —

5 200 Su80Pe40 12 0.1161 0.0000 0.0761 0.0435 6.97 83.0449 — — — — — — — — — — — —

5 203 NDF 12 0.1160 0.0000 0.0000 0.0000 — — 1.9892 — — — — — — — — — — —

5 204 NDF 12 0.1161 0.0000 0.0000 0.0000 — — 3.1560 — — — — — — — — — — —

5 205 Su40Pe0 12 0.1161 0.0000 0.0382 0.0000 — — 8.6929 — — — — — — — — — — —

5 206 Su40Pe0 12 0.1156 0.0000 0.0383 0.0000 — — 9.1920 — — — — — — — — — — —

5 207 Su80Pe0 12 0.1160 0.0000 0.0764 0.0000 — — 18.2602 — — — — — — — — — — —

5 208 Su80Pe0 12 0.1157 0.0000 0.0762 0.0000 — — 15.3437 — — — — — — — — — — —

5 209 Su120Pe0 12 0.1157 0.0000 0.1140 0.0000 — — 14.7996 — — — — — — — — — — —

5 210 Su120Pe0 12 0.1157 0.0000 0.1143 0.0000 — — 15.8633 — — — — — — — — — — —

5 211 Su0Pe40 12 0.1158 0.0000 0.0000 0.0435 — — 11.9153 — — — — — — — — — — —

5 212 Su0Pe40 12 0.1161 0.0000 0.0000 0.0435 — — 11.8098 — — — — — — — — — — —

5 213 Su0Pe91 12 0.1156 0.0000 0.0000 0.0869 — — 17.8308 — — — — — — — — — — —

5 214 Su0Pe91 12 0.1159 0.0000 0.0000 0.0869 — — 17.4330 — — — — — — — — — — —

5 215 Su0Pe120 12 0.1161 0.0000 0.0000 0.1304 — — 28.3158 — — — — — — — — — — —

5 216 Su0Pe120 12 0.1159 0.0000 0.0000 0.1304 — — 26.9512 — — — — — — — — — — —

5 217 Su40Pe91 12 0.1160 0.0000 0.0380 0.0869 — — 24.8050 — — — — — — — — — — —

5 218 Su40Pe91 12 0.1162 0.0000 0.0380 0.0869 — — 24.4120 — — — — — — — — — — —

5 219 Su80Pe40 12 0.1157 0.0000 0.0759 0.0435 — — 21.0016 — — — — — — — — — — —

5 220 Su80Pe40 12 0.1157 0.0000 0.0764 0.0435 — — 20.6040 — — — — — — — — — — —

5 221 BL 12 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3424 1.1322

259



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 222 BL 12 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3264 1.1610

5 223 NDF 12 0.1161 0.0000 0.0000 0.0000 — — — 1.1236 0.0170 0.0119 -0.0448 0.0000 3.1734 1.5225 0.4858 0.1711 0.0443 1.1259

5 224 NDF 12 0.1160 0.0000 0.0000 0.0000 — — — 0.1619 0.0583 0.0222 -0.0098 0.0000 3.8038 1.5651 0.4741 0.1455 0.3558 1.2544

5 225 Su40Pe0 12 0.1161 0.0000 0.0384 0.0000 — — — 2.4872 -0.0610 0.1800 0.0308 2.4019 7.8035 4.9123 2.3540 0.6148 0.4103 1.4866

5 226 Su40Pe0 12 0.1158 0.0000 0.0382 0.0000 — — — 1.2836 -0.0622 0.1516 0.0813 2.4605 8.1585 4.9652 2.2833 0.6666 0.3119 1.6441

5 227 Su80Pe0 12 0.1158 0.0000 0.0762 0.0000 — — — 3.2890 0.1446 -0.0045 -0.0167 5.5421 10.3171 7.8961 4.1056 0.2467 0.2832 1.5715

5 228 Su80Pe0 12 0.1161 0.0000 0.0761 0.0000 — — — 1.8435 0.1069 -0.0043 -0.0103 6.4867 10.5460 7.7475 3.9220 1.6484 0.2935 1.0079

5 229 Su120Pe0 12 0.1162 0.0000 0.1143 0.0000 — — — 2.9671 -0.0715 -0.0105 -0.0448 1.3085 16.7546 17.2815 4.4697 2.0710 0.2374 1.3760

5 230 Su120Pe0 12 0.1160 0.0000 0.1141 0.0000 — — — 2.4053 0.0889 0.0050 -0.0448 0.6423 16.8806 17.3322 4.6052 2.2031 0.2351 1.3606

5 231 Su0Pe40 12 0.1157 0.0000 0.0000 0.0435 — — — 1.6035 -0.0728 0.2901 -0.0448 0.0000 12.9993 3.8352 0.4453 0.0337 0.3072 1.5109

5 232 Su0Pe40 12 0.1160 0.0000 0.0000 0.0435 — — — 0.5599 -0.0596 -0.0105 -0.0448 0.0000 12.7620 3.9415 0.4677 0.0210 0.3692 1.7831

5 233 Su0Pe91 12 0.1159 0.0000 0.0000 0.0869 — — — 1.5274 0.3471 0.0249 -0.0448 0.0000 22.0745 5.4488 0.4059 -0.1325 0.2484 1.4453

5 234 Su0Pe91 12 0.1161 0.0000 0.0000 0.0869 — — — 0.7198 0.2066 0.0060 -0.0132 0.0000 22.1397 5.8420 0.4188 0.0770 0.2818 1.6346

5 235 Su0Pe120 12 0.1158 0.0000 0.0000 0.1304 — — — 1.6874 -0.0556 0.0137 -0.0265 0.0000 30.1229 7.8576 0.6569 -0.0579 0.3400 2.0017

5 236 Su0Pe120 12 0.1161 0.0000 0.0000 0.1304 — — — 0.9598 0.2216 0.0018 -0.0448 0.0000 28.0870 — 1.2883 -0.1663 — 1.1671

5 237 Su40Pe91 12 0.1161 0.0000 0.0379 0.0869 — — — 3.7708 0.1776 0.0179 -0.0262 0.0000 25.9713 10.2684 2.7998 0.5275 0.3906 2.0446

5 238 Su40Pe91 12 0.1161 0.0000 0.0384 0.0869 — — — 2.0834 0.2421 0.0297 -0.0239 0.0000 25.5617 10.5668 2.6850 0.7556 0.3719 2.0065

5 239 Su80Pe40 12 0.1158 0.0000 0.0763 0.0435 — — — 4.3307 -0.0341 -0.0105 0.3351 0.6884 17.7427 10.6031 4.9626 2.1433 0.2476 1.4418

5 240 Su80Pe40 12 0.1160 0.0000 0.0764 0.0435 — — — 1.5274 0.1333 0.0121 -0.0448 0.4771 17.6797 10.5947 4.8937 2.1439 0.2282 1.2889

5 241 BL 16 0.0000 0.0000 0.0000 0.0000 7.44 — — — — — — — — — — — — —

5 242 BL 16 0.0000 0.0000 0.0000 0.0000 7.40 — — — — — — — — — — — — —

5 243 NDF 16 0.1161 0.0000 0.0000 0.0000 7.28 70.2522 — — — — — — — — — — — —

5 244 NDF 16 0.1158 0.0000 0.0000 0.0000 7.31 70.5141 — — — — — — — — — — — —

5 245 Su40Pe0 16 0.1159 0.0000 0.0381 0.0000 7.22 69.5925 — — — — — — — — — — — —

5 246 Su40Pe0 16 0.1160 0.0000 0.0381 0.0000 7.23 70.5693 — — — — — — — — — — — —

5 247 Su80Pe0 16 0.1160 0.0000 0.0763 0.0000 7.18 70.4830 — — — — — — — — — — — —

5 248 Su80Pe0 16 0.1156 0.0000 0.0761 0.0000 7.16 70.5452 — — — — — — — — — — — —

5 249 Su120Pe0 16 0.1160 0.0000 0.1145 0.0000 7.02 71.6901 — — — — — — — — — — — —

260



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 250 Su120Pe0 16 0.1157 0.0000 0.1143 0.0000 7.03 72.1286 — — — — — — — — — — — —

5 251 Su0Pe40 16 0.1158 0.0000 0.0000 0.0435 7.23 73.7958 — — — — — — — — — — — —

5 252 Su0Pe40 16 0.1162 0.0000 0.0000 0.0435 7.23 72.6901 — — — — — — — — — — — —

5 253 Su0Pe91 16 0.1160 0.0000 0.0000 0.0869 7.14 75.2250 — — — — — — — — — — — —

5 254 Su0Pe91 16 0.1157 0.0000 0.0000 0.0869 7.14 75.3266 — — — — — — — — — — — —

5 255 Su0Pe120 16 0.1161 0.0000 0.0000 0.1304 7.01 75.5071 — — — — — — — — — — — —

5 256 Su0Pe120 16 0.1162 0.0000 0.0000 0.1304 7.05 77.3381 — — — — — — — — — — — —

5 257 Su40Pe91 16 0.1158 0.0000 0.0382 0.0869 7.06 77.4230 — — — — — — — — — — — —

5 258 Su40Pe91 16 0.1158 0.0000 0.0380 0.0869 7.09 76.3866 — — — — — — — — — — — —

5 259 Su80Pe40 16 0.1161 0.0000 0.0763 0.0435 7.13 73.5258 — — — — — — — — — — — —

5 260 Su80Pe40 16 0.1157 0.0000 0.0762 0.0435 7.10 73.8573 — — — — — — — — — — — —

5 263 NDF 16 0.1161 0.0000 0.0000 0.0000 — — 3.2536 — — — — — — — — — — —

5 264 NDF 16 0.1161 0.0000 0.0000 0.0000 — — 3.8395 — — — — — — — — — — —

5 265 Su40Pe0 16 0.1161 0.0000 0.0384 0.0000 — — 9.7613 — — — — — — — — — — —

5 266 Su40Pe0 16 0.1159 0.0000 0.0385 0.0000 — — 9.5687 — — — — — — — — — — —

5 267 Su80Pe0 16 0.1162 0.0000 0.0763 0.0000 — — 11.0317 — — — — — — — — — — —

5 268 Su80Pe0 16 0.1159 0.0000 0.0765 0.0000 — — 12.4988 — — — — — — — — — — —

5 269 Su120Pe0 16 0.1161 0.0000 0.1141 0.0000 — — 13.5131 — — — — — — — — — — —

5 270 Su120Pe0 16 0.1161 0.0000 0.1143 0.0000 — — 13.7014 — — — — — — — — — — —

5 271 Su0Pe40 16 0.1159 0.0000 0.0000 0.0435 — — 11.1315 — — — — — — — — — — —

5 272 Su0Pe40 16 0.1158 0.0000 0.0000 0.0435 — — 11.8176 — — — — — — — — — — —

5 273 Su0Pe91 16 0.1160 0.0000 0.0000 0.0869 — — 18.2118 — — — — — — — — — — —

5 274 Su0Pe91 16 0.1159 0.0000 0.0000 0.0869 — — 19.0931 — — — — — — — — — — —

5 275 Su0Pe120 16 0.1157 0.0000 0.0000 0.1304 — — 26.7585 — — — — — — — — — — —

5 276 Su0Pe120 16 0.1161 0.0000 0.0000 0.1304 — — 26.7534 — — — — — — — — — — —

5 277 Su40Pe91 16 0.1162 0.0000 0.0382 0.0869 — — 25.8744 — — — — — — — — — — —

5 278 Su40Pe91 16 0.1159 0.0000 0.0381 0.0869 — — 24.7073 — — — — — — — — — — —

5 279 Su80Pe40 16 0.1161 0.0000 0.0760 0.0435 — — 20.1159 — — — — — — — — — — —

261



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 280 Su80Pe40 16 0.1161 0.0000 0.0759 0.0435 — — 20.6056 — — — — — — — — — — —

5 281 BL 16 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.5552 2.3598

5 282 BL 16 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.5202 2.5005

5 283 NDF 16 0.1158 0.0000 0.0000 0.0000 — — — 0.6038 -0.0135 -0.0002 0.0090 0.0000 5.3258 2.3532 0.5885 0.1841 0.6047 2.4185

5 284 NDF 16 0.1161 0.0000 0.0000 0.0000 — — — -0.0361 -0.0135 -0.0062 -0.0119 0.0000 4.2602 1.9627 0.3006 0.0565 0.5171 2.0678

5 285 Su40Pe0 16 0.1157 0.0000 0.0384 0.0000 — — — 1.9673 0.0133 0.0017 0.0059 0.0000 4.5070 4.9767 2.7315 0.9582 0.4917 2.5394

5 286 Su40Pe0 16 0.1158 0.0000 0.0380 0.0000 — — — 0.8457 0.0161 -0.0054 0.0035 0.0000 7.1412 4.7649 3.8287 1.2432 0.7022 1.6063

5 287 Su80Pe0 16 0.1160 0.0000 0.0761 0.0000 — — — 2.1273 0.0124 0.0119 0.0094 0.0000 4.1263 5.6438 3.7912 2.4468 0.5551 1.0619

5 288 Su80Pe0 16 0.1162 0.0000 0.0762 0.0000 — — — 1.0875 0.0081 -0.0137 0.0261 0.0000 6.5679 6.1886 4.5055 2.6350 0.5752 2.0540

5 289 Su120Pe0 16 0.1160 0.0000 0.1142 0.0000 — — — 1.8874 -0.0135 0.0030 0.0091 0.0000 17.4017 18.9180 4.5390 2.9071 0.2819 1.7132

5 290 Su120Pe0 16 0.1157 0.0000 0.1140 0.0000 — — — 1.0875 -0.0135 -0.0089 0.0235 0.0530 18.3950 18.8909 4.2647 2.4696 0.5035 2.1064

5 291 Su0Pe40 16 0.1160 0.0000 0.0000 0.0435 — — — 0.2838 0.0142 -0.0137 0.0096 0.0000 13.2372 3.0775 0.4282 0.1879 0.6094 2.0408

5 292 Su0Pe40 16 0.1162 0.0000 0.0000 0.0435 — — — 0.0439 0.0185 -0.0137 -0.0002 0.0000 14.4238 4.3200 0.4744 0.1492 0.4179 1.9485

5 293 Su0Pe91 16 0.1161 0.0000 0.0000 0.0869 — — — 0.2877 -0.0021 -0.0137 0.0066 0.0000 23.3430 6.1044 -0.5915 -0.4266 0.5144 2.0023

5 294 Su0Pe91 16 0.1162 0.0000 0.0000 0.0869 — — — 0.6038 -0.0017 -0.0137 -0.0119 0.0000 23.7744 5.7164 1.0155 0.1903 0.6188 2.3247

5 295 Su0Pe120 16 0.1162 0.0000 0.0000 0.1304 — — — 0.6077 0.0035 -0.0137 -0.0119 0.0345 33.5515 8.0912 1.2789 0.3178 0.4418 2.2015

5 296 Su0Pe120 16 0.1159 0.0000 0.0000 0.1304 — — — 0.5277 -0.0087 -0.0137 0.0011 0.0000 35.3608 7.9381 1.3903 0.3048 0.7269 2.2299

5 297 Su40Pe91 16 0.1157 0.0000 0.0383 0.0869 — — — 1.6474 -0.0135 -0.0137 0.0017 0.0505 29.0331 11.3325 2.8826 0.9997 0.6820 2.1474

5 298 Su40Pe91 16 0.1157 0.0000 0.0380 0.0869 — — — 1.4874 -0.0135 -0.0137 -0.0119 0.0000 26.8697 11.0296 2.9699 1.0149 0.5024 2.1746

5 299 Su80Pe40 16 0.1161 0.0000 0.0764 0.0435 — — — 1.9712 -0.0135 -0.0137 -0.0119 0.0456 11.3352 8.4806 4.9486 2.5842 0.2748 2.0620

5 300 Su80Pe40 16 0.1158 0.0000 0.0762 0.0435 — — — 1.0914 -0.0135 -0.0137 -0.0119 0.0000 21.4306 13.1439 5.0480 2.8185 0.6195 2.3376

5 301 BL 20 0.0000 0.0000 0.0000 0.0000 7.44 — — — — — — — — — — — — —

5 302 BL 20 0.0000 0.0000 0.0000 0.0000 7.42 — — — — — — — — — — — — —

5 303 NDF 20 0.1159 0.0000 0.0000 0.0000 7.28 69.8082 — — — — — — — — — — — —

5 304 NDF 20 0.1160 0.0000 0.0000 0.0000 7.31 66.4739 — — — — — — — — — — — —

5 305 Su40Pe0 20 0.1158 0.0000 0.0379 0.0000 7.25 66.0664 — — — — — — — — — — — —

5 306 Su40Pe0 20 0.1159 0.0000 0.0384 0.0000 7.26 67.9098 — — — — — — — — — — — —

5 307 Su80Pe0 20 0.1161 0.0000 0.0762 0.0000 7.21 67.4525 — — — — — — — — — — — —

262



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 308 Su80Pe0 20 0.1159 0.0000 0.0765 0.0000 7.22 69.4631 — — — — — — — — — — — —

5 309 Su120Pe0 20 0.1162 0.0000 0.1140 0.0000 7.08 68.7737 — — — — — — — — — — — —

5 310 Su120Pe0 20 0.1158 0.0000 0.1143 0.0000 7.08 67.2755 — — — — — — — — — — — —

5 311 Su0Pe40 20 0.1157 0.0000 0.0000 0.0435 7.28 69.7517 — — — — — — — — — — — —

5 312 Su0Pe40 20 0.1159 0.0000 0.0000 0.0435 7.27 68.3413 — — — — — — — — — — — —

5 313 Su0Pe91 20 0.1160 0.0000 0.0000 0.0869 7.22 70.4399 — — — — — — — — — — — —

5 314 Su0Pe91 20 0.1161 0.0000 0.0000 0.0869 7.22 70.2953 — — — — — — — — — — — —

5 315 Su0Pe120 20 0.1162 0.0000 0.0000 0.1304 7.13 70.9255 — — — — — — — — — — — —

5 316 Su0Pe120 20 0.1159 0.0000 0.0000 0.1304 7.12 71.7929 — — — — — — — — — — — —

5 317 Su40Pe91 20 0.1162 0.0000 0.0385 0.0869 7.12 71.3559 — — — — — — — — — — — —

5 318 Su40Pe91 20 0.1161 0.0000 0.0383 0.0869 7.14 70.9845 — — — — — — — — — — — —

5 319 Su80Pe40 20 0.1161 0.0000 0.0759 0.0435 7.17 69.4338 — — — — — — — — — — — —

5 320 Su80Pe40 20 0.1157 0.0000 0.0759 0.0435 7.16 69.5788 — — — — — — — — — — — —

5 323 NDF 20 0.1161 0.0000 0.0000 0.0000 — — 2.7166 — — — — — — — — — — —

5 324 NDF 20 0.1160 0.0000 0.0000 0.0000 — — 1.7451 — — — — — — — — — — —

5 325 Su40Pe0 20 0.1159 0.0000 0.0385 0.0000 — — 7.3715 — — — — — — — — — — —

5 326 Su40Pe0 20 0.1157 0.0000 0.0382 0.0000 — — 7.5809 — — — — — — — — — — —

5 327 Su80Pe0 20 0.1161 0.0000 0.0765 0.0000 — — 10.3942 — — — — — — — — — — —

5 328 Su80Pe0 20 0.1161 0.0000 0.0762 0.0000 — — 10.6951 — — — — — — — — — — —

5 329 Su120Pe0 20 0.1157 0.0000 0.1141 0.0000 — — 11.8178 — — — — — — — — — — —

5 330 Su120Pe0 20 0.1157 0.0000 0.1144 0.0000 — — 11.8073 — — — — — — — — — — —

5 331 Su0Pe40 20 0.1159 0.0000 0.0000 0.0435 — — 9.0320 — — — — — — — — — — —

5 332 Su0Pe40 20 0.1156 0.0000 0.0000 0.0435 — — 8.7469 — — — — — — — — — — —

5 333 Su0Pe91 20 0.1157 0.0000 0.0000 0.0869 — — 6.5498 — — — — — — — — — — —

5 334 Su0Pe91 20 0.1161 0.0000 0.0000 0.0869 — — 16.0123 — — — — — — — — — — —

5 335 Su0Pe120 20 0.1157 0.0000 0.0000 0.1304 — — 23.8778 — — — — — — — — — — —

5 336 Su0Pe120 20 0.1160 0.0000 0.0000 0.1304 — — 22.7022 — — — — — — — — — — —

5 337 Su40Pe91 20 0.1160 0.0000 0.0381 0.0869 — — 21.8254 — — — — — — — — — — —

263



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 338 Su40Pe91 20 0.1158 0.0000 0.0384 0.0869 — — 20.6523 — — — — — — — — — — —

5 339 Su80Pe40 20 0.1161 0.0000 0.0761 0.0435 — — 19.2845 — — — — — — — — — — —

5 340 Su80Pe40 20 0.1160 0.0000 0.0763 0.0435 — — 19.4783 — — — — — — — — — — —

5 341 BL 20 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.9895 3.0867

5 342 BL 20 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.9223 3.5115

5 343 NDF 20 0.1157 0.0000 0.0000 0.0000 — — — 1.6006 -0.0277 -0.0003 -0.0031 0.0000 3.9038 3.0896 0.5860 -0.0278 0.9064 1.6532

5 344 NDF 20 0.1161 0.0000 0.0000 0.0000 — — — 0.1648 -0.0278 -0.0003 0.0018 0.0000 4.8013 3.2627 0.7532 -0.8880 0.9237 3.1158

5 345 Su40Pe0 20 0.1161 0.0000 0.0381 0.0000 — — — 1.2046 -0.0414 0.0044 -0.0031 0.0000 10.1834 6.5084 4.1578 1.4469 0.8911 3.0658

5 346 Su40Pe0 20 0.1161 0.0000 0.0383 0.0000 — — — 1.1246 -0.0409 -0.0003 -0.0031 0.0000 11.2840 6.7544 4.3736 -0.5208 0.9467 3.0395

5 347 Su80Pe0 20 0.1161 0.0000 0.0765 0.0000 — — — 1.4465 -0.0528 -0.0003 -0.0031 0.0000 5.5374 7.7733 7.0270 3.7158 0.6679 2.8891

5 348 Su80Pe0 20 0.1158 0.0000 0.0764 0.0000 — — — 1.0446 -0.0528 -0.0003 -0.0031 0.0000 7.2164 8.0818 7.5889 4.0452 0.7767 1.5545

5 349 Su120Pe0 20 0.1159 0.0000 0.1140 0.0000 — — — 0.9685 -0.0528 -0.0003 -0.0031 0.0000 16.8451 16.8669 6.1522 3.9205 0.6593 2.4276

5 350 Su120Pe0 20 0.1160 0.0000 0.1139 0.0000 — — — 1.7684 -0.0528 0.0034 0.0068 0.0000 18.9851 18.2461 6.5207 3.7946 0.7677 1.0840

5 351 Su0Pe40 20 0.1161 0.0000 0.0000 0.0435 — — — 0.8027 -0.0528 0.0069 0.0013 0.0000 17.6468 5.0202 2.0930 0.2338 1.1171 2.9312

5 352 Su0Pe40 20 0.1160 0.0000 0.0000 0.0435 — — — 0.7247 -0.0343 -0.0003 -0.0031 0.0000 10.2351 3.7695 -0.6104 -0.9666 0.7509 2.3035

5 353 Su0Pe91 20 0.1159 0.0000 0.0000 0.0869 — — — 0.8847 -0.0528 -0.0003 -0.0031 0.0000 — 6.8080 1.8233 0.3861 0.6918 2.8441

5 354 Su0Pe91 20 0.1158 0.0000 0.0000 0.0869 — — — 1.4445 -0.0528 0.0036 -0.0015 0.0000 39.8922 9.3503 2.1591 0.2469 0.8856 2.3937

5 355 Su0Pe120 20 0.1158 0.0000 0.0000 0.1304 — — — 0.6447 -0.0528 0.0252 0.0033 0.0310 37.0219 10.4613 2.1907 0.4088 0.5779 2.3735

5 356 Su0Pe120 20 0.1159 0.0000 0.0000 0.1304 — — — 1.2065 -0.0528 0.0058 -0.0011 0.0000 40.8068 9.4939 0.6488 0.0415 0.8183 2.2074

5 357 Su40Pe91 20 0.1160 0.0000 0.0380 0.0869 — — — 0.9685 -0.0528 0.0060 0.0071 0.0000 26.5911 11.3303 3.7793 1.0050 0.5403 2.1949

5 358 Su40Pe91 20 0.1160 0.0000 0.0380 0.0869 — — — 1.6884 -0.0528 0.0248 0.0267 0.0000 27.5159 11.4775 3.7705 1.0655 0.6301 1.6483

5 359 Su80Pe40 20 0.1160 0.0000 0.0760 0.0435 — — — 0.9724 -0.0528 -0.0003 -0.0031 0.0000 19.7911 11.4336 6.4530 3.1876 0.6744 2.3310

5 360 Su80Pe40 20 0.1160 0.0000 0.0759 0.0435 — — — 1.6903 -0.0528 0.0161 0.0124 0.0000 21.0168 10.2252 6.9114 3.5313 0.7905 2.6522

5 361 BL 24 0.0000 0.0000 0.0000 0.0000 7.45 — — — — — — — — — — — — —

5 362 BL 24 0.0000 0.0000 0.0000 0.0000 7.41 — — — — — — — — — — — — —

5 363 NDF 24 0.1160 0.0000 0.0000 0.0000 7.24 61.9906 — — — — — — — — — — — —

5 364 NDF 24 0.1161 0.0000 0.0000 0.0000 7.29 62.8006 — — — — — — — — — — — —

5 365 Su40Pe0 24 0.1159 0.0000 0.0384 0.0000 7.26 62.8188 — — — — — — — — — — — —

264



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 366 Su40Pe0 24 0.1158 0.0000 0.0385 0.0000 7.26 62.6120 — — — — — — — — — — — —

5 367 Su80Pe0 24 0.1159 0.0000 0.0765 0.0000 7.18 62.7325 — — — — — — — — — — — —

5 368 Su80Pe0 24 0.1160 0.0000 0.0764 0.0000 7.20 62.1630 — — — — — — — — — — — —

5 369 Su120Pe0 24 0.1157 0.0000 0.1144 0.0000 7.04 61.9727 — — — — — — — — — — — —

5 370 Su120Pe0 24 0.1160 0.0000 0.1141 0.0000 7.02 60.8698 — — — — — — — — — — — —

5 371 Su0Pe40 24 0.1158 0.0000 0.0000 0.0435 7.27 64.0801 — — — — — — — — — — — —

5 372 Su0Pe40 24 0.1159 0.0000 0.0000 0.0435 7.30 64.6308 — — — — — — — — — — — —

5 373 Su0Pe91 24 0.1160 0.0000 0.0000 0.0869 7.22 66.4739 — — — — — — — — — — — —

5 374 Su0Pe91 24 0.1161 0.0000 0.0000 0.0869 7.22 64.5235 — — — — — — — — — — — —

5 375 Su0Pe120 24 0.1159 0.0000 0.0000 0.1304 7.12 68.3413 — — — — — — — — — — — —

5 376 Su0Pe120 24 0.1157 0.0000 0.0000 0.1304 7.08 66.4672 — — — — — — — — — — — —

5 377 Su40Pe91 24 0.1158 0.0000 0.0383 0.0869 7.12 65.2028 — — — — — — — — — — — —

5 378 Su40Pe91 24 0.1161 0.0000 0.0383 0.0869 7.13 65.5572 — — — — — — — — — — — —

5 379 Su80Pe40 24 0.1159 0.0000 0.0764 0.0435 7.16 63.6817 — — — — — — — — — — — —

5 380 Su80Pe40 24 0.1160 0.0000 0.0763 0.0435 7.16 63.5425 — — — — — — — — — — — —

5 383 NDF 24 0.1159 0.0000 0.0000 0.0000 — — 0.1388 — — — — — — — — — — —

5 384 NDF 24 0.1159 0.0000 0.0000 0.0000 — — -0.1541 — — — — — — — — — — —

5 385 Su40Pe0 24 0.1157 0.0000 0.0385 0.0000 — — 5.8635 — — — — — — — — — — —

5 386 Su40Pe0 24 0.1161 0.0000 0.0384 0.0000 — — 5.5624 — — — — — — — — — — —

5 387 Su80Pe0 24 0.1160 0.0000 0.0759 0.0000 — — 7.6296 — — — — — — — — — — —

5 388 Su80Pe0 24 0.1160 0.0000 0.0759 0.0000 — — 7.6296 — — — — — — — — — — —

5 389 Su120Pe0 24 0.1161 0.0000 0.1143 0.0000 — — 10.4790 — — — — — — — — — — —

5 390 Su120Pe0 24 0.1160 0.0000 0.1142 0.0000 — — 9.9976 — — — — — — — — — — —

5 391 Su0Pe40 24 0.1159 0.0000 0.0000 0.0435 — — 8.1043 — — — — — — — — — — —

5 392 Su0Pe40 24 0.1158 0.0000 0.0000 0.0435 — — 7.0328 — — — — — — — — — — —

5 393 Su0Pe91 24 0.1159 0.0000 0.0000 0.0869 — — 13.6247 — — — — — — — — — — —

5 394 Su0Pe91 24 0.1159 0.0000 0.0000 0.0869 — — 13.6247 — — — — — — — — — — —

5 395 Su0Pe120 24 0.1158 0.0000 0.0000 0.1304 — — 20.7029 — — — — — — — — — — —

265



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

5 396 Su0Pe120 24 0.1159 0.0000 0.0000 0.1304 — — 19.4322 — — — — — — — — — — —

5 397 Su40Pe91 24 0.1159 0.0000 0.0382 0.0869 — — 19.5306 — — — — — — — — — — —

5 398 Su40Pe91 24 0.1162 0.0000 0.0380 0.0869 — — 19.5295 — — — — — — — — — — —

5 399 Su80Pe40 24 0.1157 0.0000 0.0761 0.0435 — — 16.4092 — — — — — — — — — — —

5 400 Su80Pe40 24 0.1157 0.0000 0.0762 0.0435 — — 14.9431 — — — — — — — — — — —

5 401 BL 24 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 1.0639 3.2376

5 402 BL 24 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.9733 2.9673

5 403 NDF 24 0.1159 0.0000 0.0000 0.0000 — — — 0.2448 -0.0102 -0.0020 0.0043 0.0000 11.7320 4.7104 1.8088 0.7935 1.0126 3.3796

5 404 NDF 24 0.1160 0.0000 0.0000 0.0000 — — — 0.2409 -0.0190 -0.0020 -0.0017 0.0000 9.4529 4.4454 1.2631 0.6104 1.0158 3.3666

5 405 Su40Pe0 24 0.1158 0.0000 0.0382 0.0000 — — — 0.8847 -0.0222 0.0023 0.0045 0.0000 15.3891 7.7187 4.7863 1.7474 0.9882 2.9665

5 406 Su40Pe0 24 0.1157 0.0000 0.0383 0.0000 — — — 0.8008 -0.0222 -0.0020 -0.0017 0.0000 15.9126 8.1166 4.5917 1.7553 0.9859 3.1395

5 407 Su80Pe0 24 0.1160 0.0000 0.0762 0.0000 — — — 1.5265 -0.0222 -0.0020 -0.0017 0.0000 17.3192 9.6477 7.4362 4.2426 0.8324 1.3153

5 408 Su80Pe0 24 0.1162 0.0000 0.0761 0.0000 — — — 0.8847 -0.0207 -0.0020 0.0037 0.0000 18.6704 10.5102 7.9215 4.3388 0.7301 2.8829

5 409 Su120Pe0 24 0.1157 0.0000 0.1141 0.0000 — — — 0.8086 -0.0222 0.0140 -0.0017 0.0000 25.0845 20.1048 6.5311 3.9615 0.5969 2.5984

5 410 Su120Pe0 24 0.1157 0.0000 0.1142 0.0000 — — — 0.8847 -0.0222 0.0069 0.0023 0.0000 25.6307 20.5530 6.4886 3.8233 0.6701 2.5082

5 411 Su0Pe40 24 0.1160 0.0000 0.0000 0.0435 — — — 0.5647 0.0001 -0.0020 -0.0017 0.0000 19.5837 6.1720 1.5629 0.0272 1.0044 2.9839

5 412 Su0Pe40 24 0.1157 0.0000 0.0000 0.0435 — — — 0.7208 -0.0222 0.0025 0.0323 0.0000 20.3637 5.2261 1.8812 0.6688 1.0403 3.1787

5 413 Su0Pe91 24 0.1157 0.0000 0.0000 0.0869 — — — 0.6467 -0.0137 -0.0020 -0.0017 0.0000 30.8589 8.6079 0.2027 0.0372 0.8494 2.7524

5 414 Su0Pe91 24 0.1157 0.0000 0.0000 0.0869 — — — 0.8808 -0.0049 -0.0020 -0.0017 0.0000 30.3577 7.8856 2.0361 0.6156 0.7819 2.7155

5 415 Su0Pe120 24 0.1159 0.0000 0.0000 0.1304 — — — 0.8847 -0.0047 -0.0020 -0.0017 0.0000 44.0461 11.4180 2.7225 0.6887 0.5264 1.7925

5 416 Su0Pe120 24 0.1159 0.0000 0.0000 0.1304 — — — 0.9607 -0.0222 0.0014 -0.0017 0.0000 37.8732 11.0348 2.5177 0.7202 0.6623 2.5828

5 417 Su40Pe91 24 0.1156 0.0000 0.0380 0.0869 — — — 1.0485 -0.0068 -0.0020 -0.0017 0.0000 33.7204 12.8314 4.4293 1.5451 0.8000 2.6516

5 418 Su40Pe91 24 0.1162 0.0000 0.0380 0.0869 — — — 0.6447 -0.0059 -0.0020 -0.0017 0.0000 28.8773 14.3167 2.7728 1.4784 0.7510 2.5694

5 419 Su80Pe40 24 0.1159 0.0000 0.0760 0.0435 — — — 1.2085 -0.0203 -0.0020 -0.0017 0.0000 25.9450 13.4727 7.0872 3.6454 0.7633 1.4507

5 420 Su80Pe40 24 0.1157 0.0000 0.0761 0.0435 — — — 0.7247 -0.0222 -0.0020 0.2191 0.0000 24.6829 12.6227 6.7794 3.6697 0.7759 2.5024

6 1 BL 0 0.0000 0.0000 0.0000 0.0000 7.21 — — — — — — — — — — — — —

6 2 BL 0 0.0000 0.0000 0.0000 0.0000 7.20 — — — — — — — — — — — — —

6 3 NDF 0 0.1159 0.0000 0.0000 0.0000 7.23 95.4362 — — — — — — — — — — — —

266



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 4 NDF 0 0.1162 0.0000 0.0000 0.0000 7.21 94.6822 — — — — — — — — — — — —

6 5 Su40Pe0 0 0.1158 0.0000 0.0380 0.0000 7.11 93.6157 — — — — — — — — — — — —

6 6 Su40Pe0 0 0.1162 0.0000 0.0381 0.0000 7.14 94.2518 — — — — — — — — — — — —

6 7 Su80Pe0 0 0.1159 0.0000 0.0761 0.0000 7.23 94.0556 — — — — — — — — — — — —

6 8 Su80Pe0 0 0.1160 0.0000 0.0763 0.0000 7.24 93.9774 — — — — — — — — — — — —

6 9 Su120Pe0 0 0.1159 0.0000 0.1140 0.0000 7.22 94.0556 — — — — — — — — — — — —

6 10 Su120Pe0 0 0.1158 0.0000 0.1144 0.0000 7.21 93.6157 — — — — — — — — — — — —

6 11 Su0Pe40 0 0.1160 0.0000 0.0000 0.0435 7.13 93.6325 — — — — — — — — — — — —

6 12 Su0Pe40 0 0.1159 0.0000 0.0000 0.0435 7.11 93.9693 — — — — — — — — — — — —

6 13 Su0Pe91 0 0.1160 0.0000 0.0000 0.0869 7.13 93.6325 — — — — — — — — — — — —

6 14 Su0Pe91 0 0.1158 0.0000 0.0000 0.0869 7.10 94.0475 — — — — — — — — — — — —

6 15 Su0Pe120 0 0.1159 0.0000 0.0000 0.1304 7.23 94.5733 — — — — — — — — — — — —

6 16 Su0Pe120 0 0.1160 0.0000 0.0000 0.1304 7.21 95.3569 — — — — — — — — — — — —

6 17 Su40Pe91 0 0.1157 0.0000 0.0380 0.0869 7.19 94.2988 — — — — — — — — — — — —

6 18 Su40Pe91 0 0.1157 0.0000 0.0381 0.0869 7.12 94.4716 — — — — — — — — — — — —

6 19 Su80Pe40 0 0.1161 0.0000 0.0760 0.0435 7.11 93.6409 — — — — — — — — — — — —

6 20 Su80Pe40 0 0.1157 0.0000 0.0762 0.0435 7.13 94.1259 — — — — — — — — — — — —

6 23 NDF 0 0.1160 0.0000 0.0000 0.0000 — — -0.4838 — — — — — — — — — — —

6 24 NDF 0 0.1162 0.0000 0.0000 0.0000 — — 0.4846 — — — — — — — — — — —

6 25 Su40Pe0 0 0.1160 0.0000 0.0380 0.0000 — — -0.3906 — — — — — — — — — — —

6 26 Su40Pe0 0 0.1160 0.0000 0.0380 0.0000 — — 0.3906 — — — — — — — — — — —

6 27 Su80Pe0 0 0.1158 0.0000 0.0760 0.0000 — — -0.5278 — — — — — — — — — — —

6 28 Su80Pe0 0 0.1162 0.0000 0.0763 0.0000 — — 0.5297 — — — — — — — — — — —

6 29 Su120Pe0 0 0.1158 0.0000 0.1145 0.0000 — — 0.5907 — — — — — — — — — — —

6 30 Su120Pe0 0 0.1162 0.0000 0.1145 0.0000 — — -0.5917 — — — — — — — — — — —

6 31 Su0Pe40 0 0.1159 0.0000 0.0000 0.0435 — — 0.0035 — — — — — — — — — — —

6 32 Su0Pe40 0 0.1161 0.0000 0.0000 0.0435 — — -0.0035 — — — — — — — — — — —

6 33 Su0Pe91 0 0.1157 0.0000 0.0000 0.0869 — — -0.1938 — — — — — — — — — — —

267



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 34 Su0Pe91 0 0.1158 0.0000 0.0000 0.0869 — — 0.1939 — — — — — — — — — — —

6 35 Su0Pe120 0 0.1157 0.0000 0.0000 0.1304 — — -0.8789 — — — — — — — — — — —

6 36 Su0Pe120 0 0.1157 0.0000 0.0000 0.1304 — — 0.8789 — — — — — — — — — — —

6 37 Su40Pe91 0 0.1157 0.0000 0.0381 0.0869 — — -0.7273 — — — — — — — — — — —

6 38 Su40Pe91 0 0.1159 0.0000 0.0383 0.0869 — — 0.7285 — — — — — — — — — — —

6 39 Su80Pe40 0 0.1162 0.0000 0.0763 0.0435 — — 0.2407 — — — — — — — — — — —

6 40 Su80Pe40 0 0.1158 0.0000 0.0764 0.0435 — — -0.2404 — — — — — — — — — — —

6 41 BL 0 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.1408 0.5211

6 42 BL 0 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.0000 0.1612

6 43 NDF 0 0.1161 0.0000 0.0000 0.0000 — — — -0.3528 0.0188 0.3684 1.4917 0.0000 1.9073 0.6602 0.0684 0.0855 0.1596 0.1941

6 44 NDF 0 0.1161 0.0000 0.0000 0.0000 — — — 0.1221 0.0000 -0.0013 -0.1122 0.0000 0.3772 -0.4697 0.0238 0.0680 0.1459 0.2746

6 45 Su40Pe0 0 0.1161 0.0000 0.0380 0.0000 — — — -0.0356 5.6222 4.8012 23.5515 0.1050 5.1291 0.0735 0.1085 0.0780 0.1211 0.2589

6 46 Su40Pe0 0 0.1158 0.0000 0.0385 0.0000 — — — 0.9124 5.9353 4.9842 31.1115 0.1426 2.3253 -0.0058 0.0866 -0.1300 0.1080 0.1440

6 47 Su80Pe0 0 0.1160 0.0000 0.0760 0.0000 — — — 0.5182 6.8801 5.6439 69.3286 0.1451 4.2458 0.1318 0.0975 0.0345 0.0997 0.2407

6 48 Su80Pe0 0 0.1158 0.0000 0.0763 0.0000 — — — 1.2277 7.3746 6.0396 71.0498 0.0355 2.0992 -0.0594 -0.0299 0.0513 0.0911 0.3000

6 49 Su120Pe0 0 0.1157 0.0000 0.1141 0.0000 — — — 0.2817 7.7178 6.0973 116.7838 0.1236 1.6032 -0.0093 0.0955 0.0424 0.1320 0.2182

6 50 Su120Pe0 0 0.1159 0.0000 0.1144 0.0000 — — — 1.4643 5.5224 7.1685 99.3389 0.1287 1.7451 0.0445 -0.0225 0.0306 0.0000 0.2404

6 51 Su0Pe40 0 0.1157 0.0000 0.0000 0.0435 — — — -0.9009 0.0206 0.4247 1.6300 0.0000 1.6881 -0.9670 -0.0355 0.0712 0.0979 0.3053

6 52 Su0Pe40 0 0.1161 0.0000 0.0000 0.0435 — — — 0.9912 0.0000 -0.0013 -0.0018 0.0985 2.1043 0.6046 -0.0563 0.0685 0.0558 0.1309

6 53 Su0Pe91 0 0.1159 0.0000 0.0000 0.0869 — — — -0.4278 0.0610 0.4900 1.4456 0.1150 1.7453 0.6765 0.0347 0.1714 0.0562 0.4680

6 54 Su0Pe91 0 0.1161 0.0000 0.0000 0.0869 — — — 0.9124 0.0660 0.4538 1.2471 0.1771 1.7567 0.3496 0.0622 0.0423 0.0000 0.2747

6 55 Su0Pe120 0 0.1158 0.0000 0.0000 0.1304 — — — -0.5067 1.2988 0.8945 1.2351 0.0424 1.0851 -1.2765 -0.2029 0.0286 0.0000 0.1934

6 56 Su0Pe120 0 0.1161 0.0000 0.0000 0.1304 — — — 0.5182 0.5578 0.6642 1.5517 0.1069 1.2959 -0.9696 -0.0543 0.0642 0.0420 0.2430

6 57 Su40Pe91 0 0.1161 0.0000 0.0383 0.0869 — — — -0.1125 6.5666 4.6496 25.8743 0.0557 1.3930 0.1384 0.0147 0.0851 0.0000 0.2875

6 58 Su40Pe91 0 0.1161 0.0000 0.0380 0.0869 — — — 0.5221 6.0143 4.5924 26.3574 0.0301 1.3644 0.1304 0.0032 -0.0743 0.0512 0.2748

6 59 Su80Pe40 0 0.1159 0.0000 0.0760 0.0435 — — — 0.2798 6.8902 5.8267 60.1891 0.1012 1.3798 -0.0290 0.1037 0.0855 0.0584 0.3578

6 60 Su80Pe40 0 0.1161 0.0000 0.0760 0.0435 — — — 0.6009 7.4708 5.3735 40.2955 0.1180 1.8024 0.3003 0.0464 0.0582 0.0736 0.2673

6 61 BL 4 0.0000 0.0000 0.0000 0.0000 7.14 — — — — — — — — — — — — —

268



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 62 BL 4 0.0000 0.0000 0.0000 0.0000 7.09 — — — — — — — — — — — — —

6 63 NDF 4 0.1160 0.0000 0.0000 0.0000 7.16 93.8481 — — — — — — — — — — — —

6 64 NDF 4 0.1160 0.0000 0.0000 0.0000 7.15 93.3308 — — — — — — — — — — — —

6 65 Su40Pe0 4 0.1157 0.0000 0.0385 0.0000 6.99 93.1319 — — — — — — — — — — — —

6 66 Su40Pe0 4 0.1160 0.0000 0.0383 0.0000 6.99 92.8997 — — — — — — — — — — — —

6 67 Su80Pe0 4 0.1161 0.0000 0.0765 0.0000 6.86 92.9948 — — — — — — — — — — — —

6 68 Su80Pe0 4 0.1159 0.0000 0.0764 0.0000 6.85 92.7181 — — — — — — — — — — — —

6 69 Su120Pe0 4 0.1161 0.0000 0.1142 0.0000 6.73 93.3394 — — — — — — — — — — — —

6 70 Su120Pe0 4 0.1161 0.0000 0.1140 0.0000 6.69 93.1671 — — — — — — — — — — — —

6 71 Su0Pe40 4 0.1162 0.0000 0.0000 0.0435 7.14 93.1759 — — — — — — — — — — — —

6 72 Su0Pe40 4 0.1161 0.0000 0.0000 0.0435 7.13 92.5641 — — — — — — — — — — — —

6 73 Su0Pe91 4 0.1160 0.0000 0.0000 0.0869 7.11 93.3308 — — — — — — — — — — — —

6 74 Su0Pe91 4 0.1157 0.0000 0.0000 0.0869 7.11 93.5641 — — — — — — — — — — — —

6 75 Su0Pe120 4 0.1160 0.0000 0.0000 0.1304 7.14 94.1067 — — — — — — — — — — — —

6 76 Su0Pe120 4 0.1159 0.0000 0.0000 0.1304 7.15 93.6673 — — — — — — — — — — — —

6 77 Su40Pe91 4 0.1161 0.0000 0.0385 0.0869 7.02 93.5117 — — — — — — — — — — — —

6 78 Su40Pe91 4 0.1157 0.0000 0.0384 0.0869 7.01 92.8726 — — — — — — — — — — — —

6 79 Su80Pe40 4 0.1158 0.0000 0.0759 0.0435 6.82 92.8816 — — — — — — — — — — — —

6 80 Su80Pe40 4 0.1159 0.0000 0.0762 0.0435 6.86 93.4084 — — — — — — — — — — — —

6 83 NDF 4 0.1158 0.0000 0.0000 0.0000 — — 0.5983 — — — — — — — — — — —

6 84 NDF 4 0.1161 0.0000 0.0000 0.0000 — — 0.5863 — — — — — — — — — — —

6 85 Su40Pe0 4 0.1157 0.0000 0.0385 0.0000 — — 6.1453 — — — — — — — — — — —

6 86 Su40Pe0 4 0.1161 0.0000 0.0380 0.0000 — — 5.7583 — — — — — — — — — — —

6 87 Su80Pe0 4 0.1161 0.0000 0.0760 0.0000 — — 8.2537 — — — — — — — — — — —

6 88 Su80Pe0 4 0.1160 0.0000 0.0765 0.0000 — — 7.5603 — — — — — — — — — — —

6 89 Su120Pe0 4 0.1158 0.0000 0.1140 0.0000 — — 8.6116 — — — — — — — — — — —

6 90 Su120Pe0 4 0.1159 0.0000 0.1143 0.0000 — — 13.2880 — — — — — — — — — — —

6 91 Su0Pe40 4 0.1157 0.0000 0.0000 0.0435 — — 4.2093 — — — — — — — — — — —

269



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 92 Su0Pe40 4 0.1161 0.0000 0.0000 0.0435 — — 3.9025 — — — — — — — — — — —

6 93 Su0Pe91 4 0.1158 0.0000 0.0000 0.0869 — — 3.0258 — — — — — — — — — — —

6 94 Su0Pe91 4 0.1157 0.0000 0.0000 0.0869 — — 4.5910 — — — — — — — — — — —

6 95 Su0Pe120 4 0.1160 0.0000 0.0000 0.1304 — — 4.5830 — — — — — — — — — — —

6 96 Su0Pe120 4 0.1159 0.0000 0.0000 0.1304 — — 3.1204 — — — — — — — — — — —

6 97 Su40Pe91 4 0.1160 0.0000 0.0381 0.0869 — — 7.6640 — — — — — — — — — — —

6 98 Su40Pe91 4 0.1159 0.0000 0.0383 0.0869 — — 8.9311 — — — — — — — — — — —

6 99 Su80Pe40 4 0.1161 0.0000 0.0765 0.0435 — — 16.6432 — — — — — — — — — — —

6 100 Su80Pe40 4 0.1159 0.0000 0.0761 0.0435 — — 14.8029 — — — — — — — — — — —

6 101 BL 4 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.0999 0.4780

6 102 BL 4 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.1253 0.4370

6 103 NDF 4 0.1157 0.0000 0.0000 0.0000 — — — 0.0375 -0.0498 -0.0672 0.2680 0.0000 1.1463 0.6613 -0.1069 -0.1465 0.1295 0.3623

6 104 NDF 4 0.1160 0.0000 0.0000 0.0000 — — — 0.4394 -0.0554 -0.0684 -0.0471 0.0000 0.1554 1.0340 0.0123 -0.2284 0.1244 0.0525

6 105 Su40Pe0 4 0.1161 0.0000 0.0385 0.0000 — — — 2.9602 -0.1308 0.0480 -0.3174 9.9296 1.9036 2.3134 0.1305 -0.0070 0.1307 0.4606

6 106 Su40Pe0 4 0.1157 0.0000 0.0384 0.0000 — — — 2.8910 -0.1218 0.1701 -0.2947 9.8183 2.4603 2.2368 0.1333 -0.0040 0.0898 0.3833

6 107 Su80Pe0 4 0.1161 0.0000 0.0765 0.0000 — — — 3.5928 -0.1852 0.0734 -0.0708 23.5068 2.2042 2.7302 0.2158 -0.0110 0.1008 0.3988

6 108 Su80Pe0 4 0.1161 0.0000 0.0764 0.0000 — — — 3.7582 -0.1772 0.1267 0.0754 21.9255 2.2452 2.7389 0.1390 -0.0437 0.1181 0.4122

6 109 Su120Pe0 4 0.1160 0.0000 0.1141 0.0000 — — — 4.6120 -0.1387 0.4689 0.0309 34.3961 2.0921 4.2874 -0.5833 -0.0933 0.0441 0.4534

6 110 Su120Pe0 4 0.1161 0.0000 0.1143 0.0000 — — — 4.3139 -0.2475 0.1033 0.0851 35.7646 2.2020 2.8964 0.2398 -0.0750 0.0763 0.4561

6 111 Su0Pe40 4 0.1158 0.0000 0.0000 0.0435 — — — 1.3816 -0.0388 0.3382 -0.3555 0.0000 4.8130 2.2678 0.0579 -0.0975 0.0756 0.3352

6 112 Su0Pe40 4 0.1158 0.0000 0.0000 0.0435 — — — 1.7046 -0.0361 0.0719 -0.3105 0.0360 5.3433 2.3773 0.0953 -0.0969 0.0878 0.4613

6 113 Su0Pe91 4 0.1162 0.0000 0.0000 0.0869 — — — 1.5412 -0.0684 0.0193 -0.1759 0.0864 5.0120 3.0702 0.0633 -0.0445 0.0480 0.3438

6 114 Su0Pe91 4 0.1157 0.0000 0.0000 0.0869 — — — 1.7085 0.0145 -0.0756 -0.4988 0.0683 6.6365 1.8295 0.1173 -0.0744 0.1646 0.3676

6 115 Su0Pe120 4 0.1161 0.0000 0.0000 0.1304 — — — 1.3854 -0.2897 -0.0814 -0.2073 0.1820 10.2190 2.0817 0.2158 -0.0022 0.1069 0.4376

6 116 Su0Pe120 4 0.1160 0.0000 0.0000 0.1304 — — — 1.7085 -0.2774 0.1731 -0.5459 0.4523 7.2573 1.6271 0.0561 -0.0491 0.1164 0.3746

6 117 Su40Pe91 4 0.1159 0.0000 0.0383 0.0869 — — — 2.0988 -0.1581 0.1957 -0.4962 11.2512 6.5231 3.5842 0.1791 -0.0562 0.0786 0.5639

6 118 Su40Pe91 4 0.1159 0.0000 0.0384 0.0869 — — — 3.6851 -0.1888 0.1007 -0.4303 11.0867 6.7919 2.2709 0.2304 -0.0576 0.1460 0.6848

6 119 Su80Pe40 4 0.1159 0.0000 0.0765 0.0435 — — — 4.0697 -0.1038 0.3517 -0.3364 23.3577 5.6337 3.4684 0.2106 -0.0241 0.0667 0.3698

270



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 120 Su80Pe40 4 0.1158 0.0000 0.0765 0.0435 — — — 4.1601 -0.2282 0.2151 -0.0622 23.6143 5.2802 3.1898 -0.5441 -0.1212 0.0741 0.1992

6 121 BL 8 0.0000 0.0000 0.0000 0.0000 7.22 — — — — — — — — — — — — —

6 122 BL 8 0.0000 0.0000 0.0000 0.0000 7.22 — — — — — — — — — — — — —

6 123 NDF 8 0.1159 0.0000 0.0000 0.0000 7.19 87.7564 — — — — — — — — — — — —

6 124 NDF 8 0.1162 0.0000 0.0000 0.0000 7.20 87.4519 — — — — — — — — — — — —

6 125 Su40Pe0 8 0.1160 0.0000 0.0382 0.0000 7.07 86.1316 — — — — — — — — — — — —

6 126 Su40Pe0 8 0.1159 0.0000 0.0380 0.0000 7.06 85.1678 — — — — — — — — — — — —

6 127 Su80Pe0 8 0.1160 0.0000 0.0765 0.0000 6.94 86.7351 — — — — — — — — — — — —

6 128 Su80Pe0 8 0.1157 0.0000 0.0761 0.0000 6.96 85.9147 — — — — — — — — — — — —

6 129 Su120Pe0 8 0.1159 0.0000 0.1141 0.0000 6.83 86.2895 — — — — — — — — — — — —

6 130 Su120Pe0 8 0.1159 0.0000 0.1144 0.0000 6.80 86.2032 — — — — — — — — — — — —

6 131 Su0Pe40 8 0.1160 0.0000 0.0000 0.0435 7.11 87.0800 — — — — — — — — — — — —

6 132 Su0Pe40 8 0.1162 0.0000 0.0000 0.0435 7.12 87.3658 — — — — — — — — — — — —

6 133 Su0Pe91 8 0.1157 0.0000 0.0000 0.0869 7.00 88.4213 — — — — — — — — — — — —

6 134 Su0Pe91 8 0.1157 0.0000 0.0000 0.0869 7.04 87.6434 — — — — — — — — — — — —

6 135 Su0Pe120 8 0.1161 0.0000 0.0000 0.1304 6.88 88.4721 — — — — — — — — — — — —

6 136 Su0Pe120 8 0.1160 0.0000 0.0000 0.1304 7.01 88.2870 — — — — — — — — — — — —

6 137 Su40Pe91 8 0.1162 0.0000 0.0382 0.0869 6.96 87.6240 — — — — — — — — — — — —

6 138 Su40Pe91 8 0.1160 0.0000 0.0381 0.0869 6.93 88.2008 — — — — — — — — — — — —

6 139 Su80Pe40 8 0.1157 0.0000 0.0763 0.0435 6.93 86.3469 — — — — — — — — — — — —

6 140 Su80Pe40 8 0.1161 0.0000 0.0761 0.0435 6.86 87.3522 — — — — — — — — — — — —

6 143 NDF 8 0.1162 0.0000 0.0000 0.0000 — — 2.3888 — — — — — — — — — — —

6 144 NDF 8 0.1157 0.0000 0.0000 0.0000 — — 2.2136 — — — — — — — — — — —

6 145 Su40Pe0 8 0.1160 0.0000 0.0380 0.0000 — — 7.0796 — — — — — — — — — — —

6 146 Su40Pe0 8 0.1157 0.0000 0.0382 0.0000 — — 7.0824 — — — — — — — — — — —

6 147 Su80Pe0 8 0.1157 0.0000 0.0763 0.0000 — — 9.7693 — — — — — — — — — — —

6 148 Su80Pe0 8 0.1157 0.0000 0.0759 0.0000 — — 9.8767 — — — — — — — — — — —

6 149 Su120Pe0 8 0.1162 0.0000 0.1142 0.0000 — — 11.4763 — — — — — — — — — — —

271



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 150 Su120Pe0 8 0.1160 0.0000 0.1141 0.0000 — — 12.5585 — — — — — — — — — — —

6 151 Su0Pe40 8 0.1162 0.0000 0.0000 0.0435 — — 7.4633 — — — — — — — — — — —

6 152 Su0Pe40 8 0.1161 0.0000 0.0000 0.0435 — — 7.8574 — — — — — — — — — — —

6 153 Su0Pe91 8 0.1160 0.0000 0.0000 0.0869 — — 14.8845 — — — — — — — — — — —

6 154 Su0Pe91 8 0.1159 0.0000 0.0000 0.0869 — — 15.6686 — — — — — — — — — — —

6 155 Su0Pe120 8 0.1161 0.0000 0.0000 0.1304 — — 23.0854 — — — — — — — — — — —

6 156 Su0Pe120 8 0.1161 0.0000 0.0000 0.1304 — — 21.9136 — — — — — — — — — — —

6 157 Su40Pe91 8 0.1162 0.0000 0.0381 0.0869 — — 19.9147 — — — — — — — — — — —

6 158 Su40Pe91 8 0.1159 0.0000 0.0384 0.0869 — — 20.8910 — — — — — — — — — — —

6 159 Su80Pe40 8 0.1157 0.0000 0.0761 0.0435 — — 14.7589 — — — — — — — — — — —

6 160 Su80Pe40 8 0.1159 0.0000 0.0760 0.0435 — — 14.6589 — — — — — — — — — — —

6 161 BL 8 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.1721 0.5865

6 162 BL 8 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.2596 0.7919

6 163 NDF 8 0.1161 0.0000 0.0000 0.0000 — — — 0.5144 0.0000 0.0026 -0.0121 -0.0283 3.0601 0.6969 0.1679 -0.0488 0.1797 0.5965

6 164 NDF 8 0.1158 0.0000 0.0000 0.0000 — — — 0.6740 0.0000 0.0008 -0.0121 -0.0283 0.3129 0.3846 0.1565 0.0061 0.1702 0.7319

6 165 Su40Pe0 8 0.1162 0.0000 0.0383 0.0000 — — — 2.0949 0.0941 -0.0090 0.0105 6.3507 4.1595 2.8860 0.9723 -0.1342 0.1201 0.7214

6 166 Su40Pe0 8 0.1161 0.0000 0.0383 0.0000 — — — 2.0142 0.0969 -0.0095 0.0008 6.9423 3.0653 2.3606 1.0323 0.1790 0.1600 0.5997

6 167 Su80Pe0 8 0.1157 0.0000 0.0760 0.0000 — — — 3.2813 0.0000 -0.0019 -0.0121 19.0573 8.6821 4.5392 0.7207 0.0217 0.1833 0.6031

6 168 Su80Pe0 8 0.1157 0.0000 0.0760 0.0000 — — — 3.5140 0.0000 -0.0111 -0.0121 19.1200 5.8651 4.6436 0.6874 -0.0103 0.1639 0.5161

6 169 Su120Pe0 8 0.1160 0.0000 0.1145 0.0000 — — — 2.8083 0.0000 -0.0119 -0.0121 31.5153 6.5105 5.4494 0.8228 -0.0137 0.1819 0.4613

6 170 Su120Pe0 8 0.1157 0.0000 0.1141 0.0000 — — — 2.9622 0.0000 -0.0143 -0.0121 30.3986 6.2851 6.6230 1.7427 -0.0046 — 0.7457

6 171 Su0Pe40 8 0.1159 0.0000 0.0000 0.0435 — — — 0.9124 0.1497 -0.0126 0.0086 -0.0283 11.7450 2.9825 0.1358 -0.0684 0.1686 0.5409

6 172 Su0Pe40 8 0.1161 0.0000 0.0000 0.0435 — — — 1.0701 0.1138 -0.0154 0.0075 -0.0283 11.0946 3.6573 0.2782 -0.1240 0.1020 0.5367

6 173 Su0Pe91 8 0.1157 0.0000 0.0000 0.0869 — — — 1.3066 0.1395 -0.0200 -0.0012 -0.0283 15.7136 4.0157 -0.5633 -0.0922 0.0000 0.6492

6 174 Su0Pe91 8 0.1162 0.0000 0.0000 0.0869 — — — 1.5469 0.1372 -0.0205 0.0402 -0.0283 22.9146 4.8910 0.2082 -0.0832 0.0898 0.5675

6 175 Su0Pe120 8 0.1160 0.0000 0.0000 0.1304 — — — 2.4141 0.1574 -0.0017 0.0101 0.0077 30.8652 5.3592 0.3526 -0.1054 0.0660 0.3796

6 176 Su0Pe120 8 0.1159 0.0000 0.0000 0.1304 — — — 2.7333 0.1336 -0.0276 -0.0121 0.0617 27.9459 3.3035 0.2276 -0.1329 0.1581 0.3812

6 177 Su40Pe91 8 0.1158 0.0000 0.0385 0.0869 — — — 2.5718 0.2691 -0.0212 -0.0121 8.7276 29.7667 5.3358 0.6350 -0.0627 0.0820 0.5820

272



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 178 Su40Pe91 8 0.1161 0.0000 0.0385 0.0869 — — — 3.2852 0.2721 -0.0276 0.0444 8.3557 22.4947 6.2988 0.6189 -0.3060 0.0788 0.5473

6 179 Su80Pe40 8 0.1158 0.0000 0.0762 0.0435 — — — 2.7295 0.3490 -0.0204 0.0385 18.9550 17.8584 8.2566 0.8113 -0.0379 0.2014 0.6424

6 180 Su80Pe40 8 0.1162 0.0000 0.0760 0.0435 — — — 3.4467 0.3487 -0.0276 0.0308 17.9908 17.7830 8.3238 0.8150 -0.0205 0.1884 0.6777

6 181 BL 12 0.0000 0.0000 0.0000 0.0000 7.22 — — — — — — — — — — — — —

6 182 BL 12 0.0000 0.0000 0.0000 0.0000 7.23 — — — — — — — — — — — — —

6 183 NDF 12 0.1159 0.0000 0.0000 0.0000 7.22 80.2924 — — — — — — — — — — — —

6 184 NDF 12 0.1158 0.0000 0.0000 0.0000 7.21 80.7911 — — — — — — — — — — — —

6 185 Su40Pe0 12 0.1160 0.0000 0.0382 0.0000 7.10 78.8462 — — — — — — — — — — — —

6 186 Su40Pe0 12 0.1160 0.0000 0.0380 0.0000 7.12 78.5875 — — — — — — — — — — — —

6 187 Su80Pe0 12 0.1159 0.0000 0.0761 0.0000 6.99 80.0335 — — — — — — — — — — — —

6 188 Su80Pe0 12 0.1159 0.0000 0.0763 0.0000 7.01 81.2416 — — — — — — — — — — — —

6 189 Su120Pe0 12 0.1162 0.0000 0.1139 0.0000 6.87 79.8343 — — — — — — — — — — — —

6 190 Su120Pe0 12 0.1161 0.0000 0.1140 0.0000 6.84 80.1590 — — — — — — — — — — — —

6 191 Su0Pe40 12 0.1160 0.0000 0.0000 0.0435 7.13 81.2603 — — — — — — — — — — — —

6 192 Su0Pe40 12 0.1161 0.0000 0.0000 0.0435 7.11 81.7958 — — — — — — — — — — — —

6 193 Su0Pe91 12 0.1162 0.0000 0.0000 0.0869 7.02 84.2241 — — — — — — — — — — — —

6 194 Su0Pe91 12 0.1161 0.0000 0.0000 0.0869 6.98 83.8633 — — — — — — — — — — — —

6 195 Su0Pe120 12 0.1160 0.0000 0.0000 0.1304 6.97 84.6228 — — — — — — — — — — — —

6 196 Su0Pe120 12 0.1162 0.0000 0.0000 0.1304 6.95 84.0519 — — — — — — — — — — — —

6 197 Su40Pe91 12 0.1162 0.0000 0.0382 0.0869 6.97 84.3102 — — — — — — — — — — — —

6 198 Su40Pe91 12 0.1161 0.0000 0.0382 0.0869 6.98 85.2416 — — — — — — — — — — — —

6 199 Su80Pe40 12 0.1160 0.0000 0.0761 0.0435 6.95 81.4327 — — — — — — — — — — — —

6 200 Su80Pe40 12 0.1159 0.0000 0.0761 0.0435 6.90 82.1908 — — — — — — — — — — — —

6 203 NDF 12 0.1160 0.0000 0.0000 0.0000 — — 4.2522 — — — — — — — — — — —

6 204 NDF 12 0.1159 0.0000 0.0000 0.0000 — — 4.1586 — — — — — — — — — — —

6 205 Su40Pe0 12 0.1157 0.0000 0.0383 0.0000 — — 9.6182 — — — — — — — — — — —

6 206 Su40Pe0 12 0.1158 0.0000 0.0383 0.0000 — — 9.6152 — — — — — — — — — — —

6 207 Su80Pe0 12 0.1159 0.0000 0.0763 0.0000 — — 13.1824 — — — — — — — — — — —

273



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 208 Su80Pe0 12 0.1160 0.0000 0.0762 0.0000 — — 13.1825 — — — — — — — — — — —

6 209 Su120Pe0 12 0.1162 0.0000 0.1142 0.0000 — — 16.4565 — — — — — — — — — — —

6 210 Su120Pe0 12 0.1159 0.0000 0.1142 0.0000 — — 16.8550 — — — — — — — — — — —

6 211 Su0Pe40 12 0.1159 0.0000 0.0000 0.0435 — — 10.6961 — — — — — — — — — — —

6 212 Su0Pe40 12 0.1158 0.0000 0.0000 0.0435 — — 10.6996 — — — — — — — — — — —

6 213 Su0Pe91 12 0.1159 0.0000 0.0000 0.0869 — — 15.2780 — — — — — — — — — — —

6 214 Su0Pe91 12 0.1162 0.0000 0.0000 0.0869 — — 15.1718 — — — — — — — — — — —

6 215 Su0Pe120 12 0.1161 0.0000 0.0000 0.1304 — — 22.9878 — — — — — — — — — — —

6 216 Su0Pe120 12 0.1158 0.0000 0.0000 0.1304 — — 21.7249 — — — — — — — — — — —

6 217 Su40Pe91 12 0.1162 0.0000 0.0381 0.0869 — — 21.6724 — — — — — — — — — — —

6 218 Su40Pe91 12 0.1157 0.0000 0.0381 0.0869 — — 21.1951 — — — — — — — — — — —

6 219 Su80Pe40 12 0.1161 0.0000 0.0761 0.0435 — — 18.7528 — — — — — — — — — — —

6 220 Su80Pe40 12 0.1157 0.0000 0.0761 0.0435 — — 16.1260 — — — — — — — — — — —

6 221 BL 12 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3113 1.2277

6 222 BL 12 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.3691 1.2899

6 223 NDF 12 0.1159 0.0000 0.0000 0.0000 — — — 0.6307 0.0000 -0.0042 -0.0226 0.0000 4.1659 1.9186 0.5639 -0.0393 0.2964 1.4567

6 224 NDF 12 0.1157 0.0000 0.0000 0.0000 — — — 0.5557 0.0000 -0.0008 -0.0088 0.0000 6.0380 1.8675 0.5663 0.2608 0.3439 1.3520

6 225 Su40Pe0 12 0.1157 0.0000 0.0385 0.0000 — — — 2.2901 0.0367 0.0012 -0.0117 2.7257 6.6058 4.1020 3.0030 0.8000 0.3440 0.9402

6 226 Su40Pe0 12 0.1157 0.0000 0.0380 0.0000 — — — 1.8998 0.0184 -0.0009 -0.0106 4.0738 9.6175 7.0202 7.6048 2.4586 0.2490 1.5793

6 227 Su80Pe0 12 0.1157 0.0000 0.0764 0.0000 — — — 2.3728 0.0559 0.0056 -0.0268 4.6588 8.5517 6.3999 7.4891 2.2383 0.2456 1.5314

6 228 Su80Pe0 12 0.1157 0.0000 0.0760 0.0000 — — — 3.3227 0.0445 -0.0025 -0.0128 3.0890 8.5978 4.1929 2.7876 0.7588 0.2710 1.9663

6 229 Su120Pe0 12 0.1157 0.0000 0.1144 0.0000 — — — 2.7670 0.0237 0.0104 -0.0313 0.0000 15.6678 14.6374 8.0765 2.7171 0.1991 1.1998

6 230 Su120Pe0 12 0.1158 0.0000 0.1140 0.0000 — — — 3.6419 0.0314 0.0032 -0.0317 0.0000 8.6381 17.8991 7.0769 2.7282 0.4642 1.8066

6 231 Su0Pe40 12 0.1160 0.0000 0.0000 0.0435 — — — 1.0287 0.0873 -0.0138 -0.0405 0.0000 15.6664 0.3398 0.8380 0.1869 0.3172 1.7051

6 232 Su0Pe40 12 0.1162 0.0000 0.0000 0.0435 — — — 2.0536 0.0930 -0.0138 -0.0124 0.0000 15.2128 4.2745 0.8507 0.1413 0.3323 2.2408

6 233 Su0Pe91 12 0.1160 0.0000 0.0000 0.0869 — — — 1.1076 0.0000 -0.0138 -0.0405 0.0000 23.3859 5.8756 0.9553 0.1036 0.8756 2.2867

6 234 Su0Pe91 12 0.1162 0.0000 0.0000 0.0869 — — — 2.1363 0.0000 -0.0138 -0.0117 0.0000 23.4907 5.8393 0.8235 0.0696 0.4618 2.1620

6 235 Su0Pe120 12 0.1157 0.0000 0.0000 0.1304 — — — 1.5806 0.0000 -0.0138 -0.0405 0.0000 34.5383 8.2035 1.2382 -0.2146 0.3774 2.0258

274



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 236 Su0Pe120 12 0.1159 0.0000 0.0000 0.1304 — — — 2.5305 0.0000 -0.0138 -0.0405 0.0000 34.9232 8.8464 1.3094 -0.0460 0.3177 2.0904

6 237 Su40Pe91 12 0.1161 0.0000 0.0382 0.0869 — — — 2.6132 0.0601 -0.0138 -0.0316 0.0481 22.1000 10.2097 3.6963 1.2534 0.2080 1.5935

6 238 Su40Pe91 12 0.1162 0.0000 0.0380 0.0869 — — — 2.6132 0.0370 -0.0138 -0.0213 0.0000 23.8554 8.9629 3.6596 1.0851 0.2953 1.8458

6 239 Su80Pe40 12 0.1157 0.0000 0.0764 0.0435 — — — 2.5382 0.0710 -0.0138 -0.0068 0.0000 17.6364 10.0547 9.3722 3.1126 0.2880 2.1166

6 240 Su80Pe40 12 0.1159 0.0000 0.0764 0.0435 — — — 2.6920 0.0757 -0.0138 0.0085 0.0739 18.4229 10.4462 9.4972 3.2722 0.3644 2.2933

6 241 BL 16 0.0000 0.0000 0.0000 0.0000 7.33 — — — — — — — — — — — — —

6 242 BL 16 0.0000 0.0000 0.0000 0.0000 7.33 — — — — — — — — — — — — —

6 243 NDF 16 0.1159 0.0000 0.0000 0.0000 7.29 69.1610 — — — — — — — — — — — —

6 244 NDF 16 0.1160 0.0000 0.0000 0.0000 7.26 71.0866 — — — — — — — — — — — —

6 245 Su40Pe0 16 0.1159 0.0000 0.0384 0.0000 7.20 70.9731 — — — — — — — — — — — —

6 246 Su40Pe0 16 0.1162 0.0000 0.0381 0.0000 7.20 72.0015 — — — — — — — — — — — —

6 247 Su80Pe0 16 0.1161 0.0000 0.0764 0.0000 7.08 69.9076 — — — — — — — — — — — —

6 248 Su80Pe0 16 0.1161 0.0000 0.0761 0.0000 7.08 69.7353 — — — — — — — — — — — —

6 249 Su120Pe0 16 0.1159 0.0000 0.1145 0.0000 6.88 68.9022 — — — — — — — — — — — —

6 250 Su120Pe0 16 0.1160 0.0000 0.1144 0.0000 6.92 69.5346 — — — — — — — — — — — —

6 251 Su0Pe40 16 0.1161 0.0000 0.0000 0.0435 7.09 73.6119 — — — — — — — — — — — —

6 252 Su0Pe40 16 0.1161 0.0000 0.0000 0.0435 7.18 73.1812 — — — — — — — — — — — —

6 253 Su0Pe91 16 0.1161 0.0000 0.0000 0.0869 7.06 78.4361 — — — — — — — — — — — —

6 254 Su0Pe91 16 0.1157 0.0000 0.0000 0.0869 7.07 77.2282 — — — — — — — — — — — —

6 255 Su0Pe120 16 0.1157 0.0000 0.0000 0.1304 6.97 76.7960 — — — — — — — — — — — —

6 256 Su0Pe120 16 0.1159 0.0000 0.0000 0.1304 6.98 77.7900 — — — — — — — — — — — —

6 257 Su40Pe91 16 0.1162 0.0000 0.0384 0.0869 6.97 78.8875 — — — — — — — — — — — —

6 258 Su40Pe91 16 0.1161 0.0000 0.0385 0.0869 6.99 76.9716 — — — — — — — — — — — —

6 259 Su80Pe40 16 0.1160 0.0000 0.0762 0.0435 6.99 72.7247 — — — — — — — — — — — —

6 260 Su80Pe40 16 0.1159 0.0000 0.0763 0.0435 7.02 73.4755 — — — — — — — — — — — —

6 263 NDF 16 0.1158 0.0000 0.0000 0.0000 — — 5.9203 — — — — — — — — — — —

6 264 NDF 16 0.1159 0.0000 0.0000 0.0000 — — 6.4045 — — — — — — — — — — —

6 265 Su40Pe0 16 0.1160 0.0000 0.0385 0.0000 — — 10.2864 — — — — — — — — — — —

275



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 266 Su40Pe0 16 0.1158 0.0000 0.0385 0.0000 — — 10.8783 — — — — — — — — — — —

6 267 Su80Pe0 16 0.1162 0.0000 0.0762 0.0000 — — 13.7637 — — — — — — — — — — —

6 268 Su80Pe0 16 0.1160 0.0000 0.0764 0.0000 — — 14.6423 — — — — — — — — — — —

6 269 Su120Pe0 16 0.1158 0.0000 0.1145 0.0000 — — 16.1659 — — — — — — — — — — —

6 270 Su120Pe0 16 0.1158 0.0000 0.1144 0.0000 — — 17.0474 — — — — — — — — — — —

6 271 Su0Pe40 16 0.1157 0.0000 0.0000 0.0435 — — 11.7772 — — — — — — — — — — —

6 272 Su0Pe40 16 0.1158 0.0000 0.0000 0.0435 — — 12.7502 — — — — — — — — — — —

6 273 Su0Pe91 16 0.1158 0.0000 0.0000 0.0869 — — 17.9174 — — — — — — — — — — —

6 274 Su0Pe91 16 0.1162 0.0000 0.0000 0.0869 — — 17.5154 — — — — — — — — — — —

6 275 Su0Pe120 16 0.1162 0.0000 0.0000 0.1304 — — 23.6691 — — — — — — — — — — —

6 276 Su0Pe120 16 0.1162 0.0000 0.0000 0.1304 — — 23.9621 — — — — — — — — — — —

6 277 Su40Pe91 16 0.1159 0.0000 0.0383 0.0869 — — 23.8227 — — — — — — — — — — —

6 278 Su40Pe91 16 0.1158 0.0000 0.0381 0.0869 — — 24.8060 — — — — — — — — — — —

6 279 Su80Pe40 16 0.1161 0.0000 0.0762 0.0435 — — 19.4338 — — — — — — — — — — —

6 280 Su80Pe40 16 0.1158 0.0000 0.0759 0.0435 — — 19.4487 — — — — — — — — — — —

6 281 BL 16 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.4693 1.9335

6 282 BL 16 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.5073 2.0227

6 283 NDF 16 0.1161 0.0000 0.0000 0.0000 — — — 1.0672 -0.0102 0.0000 0.0087 0.0000 3.7060 2.5006 0.7190 0.3202 0.4804 2.0470

6 284 NDF 16 0.1160 0.0000 0.0000 0.0000 — — — 0.7538 -0.0127 0.0040 -0.0010 0.0000 3.0757 2.5012 0.6746 0.4551 0.5023 2.5683

6 285 Su40Pe0 16 0.1158 0.0000 0.0385 0.0000 — — — 1.7806 -0.0045 0.0000 -0.0017 0.0562 7.1189 4.7424 3.6216 0.9742 0.5666 2.7501

6 286 Su40Pe0 16 0.1157 0.0000 0.0384 0.0000 — — — 1.7806 0.0037 0.0023 -0.0091 0.0479 5.1177 4.5722 5.8517 1.1941 0.5401 2.7456

6 287 Su80Pe0 16 0.1162 0.0000 0.0759 0.0000 — — — 2.1786 0.0081 0.0159 -0.0004 0.0000 7.5525 7.1890 9.3428 3.6313 0.5234 2.2091

6 288 Su80Pe0 16 0.1158 0.0000 0.0762 0.0000 — — — 1.9402 -0.0172 0.0000 -0.0064 0.0000 9.5395 8.9447 9.3932 3.9453 0.6042 2.8361

6 289 Su120Pe0 16 0.1157 0.0000 0.1144 0.0000 — — — 1.9440 -0.0257 0.0000 -0.0088 0.0000 16.0993 22.7293 5.0973 3.0952 0.4403 1.9425

6 290 Su120Pe0 16 0.1159 0.0000 0.1145 0.0000 — — — 2.0978 -0.0257 0.0064 0.0051 0.0000 17.3309 21.6716 4.5778 3.1942 0.4691 2.4119

6 291 Su0Pe40 16 0.1159 0.0000 0.0000 0.0435 — — — 0.7595 -0.0257 0.0000 -0.0183 0.0000 18.7695 4.7066 1.1273 0.3813 0.3754 2.1923

6 292 Su0Pe40 16 0.1162 0.0000 0.0000 0.0435 — — — 0.8326 -0.0226 0.0466 0.0940 0.0000 15.9287 4.1481 0.9954 0.3181 0.5990 2.7209

6 293 Su0Pe91 16 0.1161 0.0000 0.0000 0.0869 — — — 0.8364 -0.0229 0.0000 -0.0183 0.0000 23.5174 7.3528 1.4197 0.6711 0.5016 2.6105

276



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 294 Su0Pe91 16 0.1157 0.0000 0.0000 0.0869 — — — 1.2287 -0.0243 0.0235 -0.0183 0.0000 23.6623 6.4775 1.3699 0.4226 0.4653 2.5134

6 295 Su0Pe120 16 0.1157 0.0000 0.0000 0.1304 — — — 1.0749 -0.0207 0.0000 -0.0183 0.0000 34.5478 10.0944 1.7842 0.5594 0.4176 2.3634

6 296 Su0Pe120 16 0.1158 0.0000 0.0000 0.1304 — — — 1.3056 -0.0257 0.0000 -0.0183 0.0000 34.5917 9.2818 1.4955 0.3464 0.4364 2.3730

6 297 Su40Pe91 16 0.1159 0.0000 0.0384 0.0869 — — — 1.6287 -0.0253 0.0000 -0.0183 0.0000 25.4712 11.7697 3.7243 1.4300 0.4513 2.3332

6 298 Su40Pe91 16 0.1157 0.0000 0.0381 0.0869 — — — 2.0151 -0.0058 0.0335 -0.0183 0.0000 24.8475 10.9183 3.5834 1.2434 0.4177 1.9477

6 299 Su80Pe40 16 0.1157 0.0000 0.0763 0.0435 — — — 1.6287 -0.0257 0.0177 -0.0183 0.0000 18.3212 14.0515 6.3265 3.0498 0.5420 1.9613

6 300 Su80Pe40 16 0.1160 0.0000 0.0763 0.0435 — — — 2.2517 -0.0257 0.0000 -0.0183 0.0000 19.6108 12.9609 6.4178 2.9890 0.5947 1.8328

6 301 BL 20 0.0000 0.0000 0.0000 0.0000 7.37 — — — — — — — — — — — — —

6 302 BL 20 0.0000 0.0000 0.0000 0.0000 7.38 — — — — — — — — — — — — —

6 303 NDF 20 0.1158 0.0000 0.0000 0.0000 7.32 66.9301 — — — — — — — — — — — —

6 304 NDF 20 0.1157 0.0000 0.0000 0.0000 7.32 65.0843 — — — — — — — — — — — —

6 305 Su40Pe0 20 0.1161 0.0000 0.0384 0.0000 7.17 65.1265 — — — — — — — — — — — —

6 306 Su40Pe0 20 0.1161 0.0000 0.0381 0.0000 7.20 65.6434 — — — — — — — — — — — —

6 307 Su80Pe0 20 0.1162 0.0000 0.0763 0.0000 7.11 64.8142 — — — — — — — — — — — —

6 308 Su80Pe0 20 0.1162 0.0000 0.0764 0.0000 7.12 65.4168 — — — — — — — — — — — —

6 309 Su120Pe0 20 0.1161 0.0000 0.1144 0.0000 6.99 63.9205 — — — — — — — — — — — —

6 310 Su120Pe0 20 0.1162 0.0000 0.1145 0.0000 6.98 65.1585 — — — — — — — — — — — —

6 311 Su0Pe40 20 0.1158 0.0000 0.0000 0.0435 7.19 68.0528 — — — — — — — — — — — —

6 312 Su0Pe40 20 0.1160 0.0000 0.0000 0.0435 7.22 69.3191 — — — — — — — — — — — —

6 313 Su0Pe91 20 0.1160 0.0000 0.0000 0.0869 7.12 74.3197 — — — — — — — — — — — —

6 314 Su0Pe91 20 0.1159 0.0000 0.0000 0.0869 7.09 72.6558 — — — — — — — — — — — —

6 315 Su0Pe120 20 0.1158 0.0000 0.0000 0.1304 6.94 71.3345 — — — — — — — — — — — —

6 316 Su0Pe120 20 0.1157 0.0000 0.0000 0.1304 6.96 72.4311 — — — — — — — — — — — —

6 317 Su40Pe91 20 0.1159 0.0000 0.0384 0.0869 7.03 73.0872 — — — — — — — — — — — —

6 318 Su40Pe91 20 0.1157 0.0000 0.0382 0.0869 7.05 73.2955 — — — — — — — — — — — —

6 319 Su80Pe40 20 0.1159 0.0000 0.0759 0.0435 7.06 67.0470 — — — — — — — — — — — —

6 320 Su80Pe40 20 0.1158 0.0000 0.0761 0.0435 7.06 67.9664 — — — — — — — — — — — —

6 323 NDF 20 0.1161 0.0000 0.0000 0.0000 — — 6.5918 — — — — — — — — — — —

277



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 324 NDF 20 0.1157 0.0000 0.0000 0.0000 — — 5.6313 — — — — — — — — — — —

6 325 Su40Pe0 20 0.1162 0.0000 0.0385 0.0000 — — 11.4521 — — — — — — — — — — —

6 326 Su40Pe0 20 0.1160 0.0000 0.0384 0.0000 — — 9.9966 — — — — — — — — — — —

6 327 Su80Pe0 20 0.1161 0.0000 0.0761 0.0000 — — 13.0849 — — — — — — — — — — —

6 328 Su80Pe0 20 0.1162 0.0000 0.0763 0.0000 — — 13.9565 — — — — — — — — — — —

6 329 Su120Pe0 20 0.1158 0.0000 0.1141 0.0000 — — 15.8838 — — — — — — — — — — —

6 330 Su120Pe0 20 0.1161 0.0000 0.1140 0.0000 — — 16.0740 — — — — — — — — — — —

6 331 Su0Pe40 20 0.1157 0.0000 0.0000 0.0435 — — 12.0701 — — — — — — — — — — —

6 332 Su0Pe40 20 0.1159 0.0000 0.0000 0.0435 — — 2.2982 — — — — — — — — — — —

6 333 Su0Pe91 20 0.1161 0.0000 0.0000 0.0869 — — 23.8655 — — — — — — — — — — —

6 334 Su0Pe91 20 0.1160 0.0000 0.0000 0.0869 — — 16.1540 — — — — — — — — — — —

6 335 Su0Pe120 20 0.1157 0.0000 0.0000 0.1304 — — 23.2895 — — — — — — — — — — —

6 336 Su0Pe120 20 0.1157 0.0000 0.0000 0.1304 — — 23.0942 — — — — — — — — — — —

6 337 Su40Pe91 20 0.1160 0.0000 0.0380 0.0869 — — 20.1167 — — — — — — — — — — —

6 338 Su40Pe91 20 0.1162 0.0000 0.0385 0.0869 — — 21.6633 — — — — — — — — — — —

6 339 Su80Pe40 20 0.1159 0.0000 0.0764 0.0435 — — 17.1877 — — — — — — — — — — —

6 340 Su80Pe40 20 0.1160 0.0000 0.0761 0.0435 — — 18.0717 — — — — — — — — — — —

6 341 BL 20 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — — —

6 342 BL 20 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.6956 4.1962

6 343 NDF 20 0.1162 0.0000 0.0000 0.0000 — — — -0.0317 0.0000 -0.0001 0.0000 0.0000 10.6828 4.3383 1.0894 0.6965 0.8846 2.5849

6 344 NDF 20 0.1161 0.0000 0.0000 0.0000 — — — 0.3548 0.0000 -0.0001 0.0726 0.0000 13.2381 4.1580 1.0772 0.7023 1.0205 2.7638

6 345 Su40Pe0 20 0.1157 0.0000 0.0380 0.0000 — — — 0.9182 0.0000 -0.0001 0.0000 0.0000 14.7493 6.3785 3.8089 1.6119 0.8777 3.9084

6 346 Su40Pe0 20 0.1159 0.0000 0.0380 0.0000 — — — 0.9855 0.0000 -0.0001 0.0224 0.0000 15.1850 6.5280 3.7960 1.0879 0.8169 3.7472

6 347 Su80Pe0 20 0.1161 0.0000 0.0762 0.0000 — — — 1.0758 0.0000 -0.0001 0.0000 0.0000 14.3290 7.5227 7.5998 3.8467 0.7355 1.3261

6 348 Su80Pe0 20 0.1162 0.0000 0.0763 0.0000 — — — 1.3008 0.0000 -0.0001 0.0000 0.0000 12.8920 7.9122 8.7319 4.5540 0.7312 2.3006

6 349 Su120Pe0 20 0.1158 0.0000 0.1144 0.0000 — — — 1.3162 0.0000 -0.0001 0.0000 0.0000 20.7097 18.6952 7.3493 3.9469 0.6104 2.0164

6 350 Su120Pe0 20 0.1161 0.0000 0.1141 0.0000 — — — 1.3835 0.0000 -0.0001 0.0000 0.0000 24.3678 19.5265 6.8585 4.0534 0.6530 2.5928

6 351 Su0Pe40 20 0.1159 0.0000 0.0000 0.0435 — — — 0.2836 0.0105 -0.0001 0.0000 0.0000 22.8490 5.9916 1.5388 0.7129 0.6961 2.3553

278



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 352 Su0Pe40 20 0.1160 0.0000 0.0000 0.0435 — — — 0.5913 0.1257 -0.0001 0.0578 0.0000 17.8918 5.9724 1.4725 0.2116 0.8007 3.0154

6 353 Su0Pe91 20 0.1161 0.0000 0.0000 0.0869 — — — 0.4451 0.0000 0.0150 0.0367 0.0000 26.9217 7.4495 1.5556 0.2543 0.6208 1.9846

6 354 Su0Pe91 20 0.1159 0.0000 0.0000 0.0869 — — — 0.9066 0.0000 0.2696 0.0000 0.0000 29.1881 5.9280 1.5599 0.3894 0.6876 3.2782

6 355 Su0Pe120 20 0.1158 0.0000 0.0000 0.1304 — — — 0.3663 0.0000 -0.0001 0.0000 0.0000 44.9759 9.5854 1.8154 0.4108 0.8379 2.5071

6 356 Su0Pe120 20 0.1161 0.0000 0.0000 0.1304 — — — 0.9066 0.0000 -0.0001 0.1628 0.0000 42.6006 10.9188 1.8190 0.3067 0.5379 2.6400

6 357 Su40Pe91 20 0.1161 0.0000 0.0384 0.0869 — — — 1.3123 0.0000 0.0024 0.0000 0.0000 28.2945 11.4030 5.7432 1.7230 0.4217 1.6494

6 358 Su40Pe91 20 0.1161 0.0000 0.0382 0.0869 — — — 1.2258 0.0011 -0.0001 0.0000 0.0000 33.2081 10.4899 4.0924 1.6458 0.6288 2.3569

6 359 Su80Pe40 20 0.1160 0.0000 0.0761 0.0435 — — — 1.0758 0.0000 -0.0001 0.0000 0.0000 22.6359 13.2116 9.3126 4.2177 0.5396 1.6974

6 360 Su80Pe40 20 0.1161 0.0000 0.0760 0.0435 — — — 1.0681 0.0000 0.0044 0.0000 0.0000 27.9833 11.3772 8.2056 3.8143 0.7453 2.4293

6 361 BL 24 0.0000 0.0000 0.0000 0.0000 7.38 — — — — — — — — — — — — —

6 362 BL 24 0.0000 0.0000 0.0000 0.0000 7.45 — — — — — — — — — — — — —

6 363 NDF 24 0.1159 0.0000 0.0000 0.0000 7.31 61.3950 — — — — — — — — — — — —

6 364 NDF 24 0.1162 0.0000 0.0000 0.0000 7.32 61.7586 — — — — — — — — — — — —

6 365 Su40Pe0 24 0.1161 0.0000 0.0382 0.0000 7.21 60.4315 — — — — — — — — — — — —

6 366 Su40Pe0 24 0.1157 0.0000 0.0380 0.0000 7.22 60.8058 — — — — — — — — — — — —

6 367 Su80Pe0 24 0.1157 0.0000 0.0760 0.0000 7.14 60.3737 — — — — — — — — — — — —

6 368 Su80Pe0 24 0.1158 0.0000 0.0763 0.0000 7.09 60.4961 — — — — — — — — — — — —

6 369 Su120Pe0 24 0.1160 0.0000 0.1139 0.0000 6.99 57.7228 — — — — — — — — — — — —

6 370 Su120Pe0 24 0.1158 0.0000 0.1140 0.0000 7.00 58.5962 — — — — — — — — — — — —

6 371 Su0Pe40 24 0.1160 0.0000 0.0000 0.0435 7.18 62.7234 — — — — — — — — — — — —

6 372 Su0Pe40 24 0.1157 0.0000 0.0000 0.0435 7.21 64.3496 — — — — — — — — — — — —

6 373 Su0Pe91 24 0.1158 0.0000 0.0000 0.0869 7.06 68.7868 — — — — — — — — — — — —

6 374 Su0Pe91 24 0.1161 0.0000 0.0000 0.0869 7.07 67.2371 — — — — — — — — — — — —

6 375 Su0Pe120 24 0.1162 0.0000 0.0000 0.1304 6.95 67.3534 — — — — — — — — — — — —

6 376 Su0Pe120 24 0.1159 0.0000 0.0000 0.1304 6.92 66.4861 — — — — — — — — — — — —

6 377 Su40Pe91 24 0.1162 0.0000 0.0384 0.0869 7.01 67.6117 — — — — — — — — — — — —

6 378 Su40Pe91 24 0.1160 0.0000 0.0382 0.0869 6.97 66.6895 — — — — — — — — — — — —

6 379 Su80Pe40 24 0.1161 0.0000 0.0763 0.0435 7.03 63.3605 — — — — — — — — — — — —

279



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 380 Su80Pe40 24 0.1161 0.0000 0.0763 0.0435 7.02 63.9635 — — — — — — — — — — — —

6 383 NDF 24 0.1158 0.0000 0.0000 0.0000 — — 6.2132 — — — — — — — — — — —

6 384 NDF 24 0.1160 0.0000 0.0000 0.0000 — — 5.3263 — — — — — — — — — — —

6 385 Su40Pe0 24 0.1161 0.0000 0.0384 0.0000 — — 9.9935 — — — — — — — — — — —

6 386 Su40Pe0 24 0.1159 0.0000 0.0381 0.0000 — — 10.8879 — — — — — — — — — — —

6 387 Su80Pe0 24 0.1160 0.0000 0.0765 0.0000 — — 13.7611 — — — — — — — — — — —

6 388 Su80Pe0 24 0.1157 0.0000 0.0760 0.0000 — — 12.9015 — — — — — — — — — — —

6 389 Su120Pe0 24 0.1157 0.0000 0.1140 0.0000 — — 15.4986 — — — — — — — — — — —

6 390 Su120Pe0 24 0.1162 0.0000 0.1142 0.0000 — — 15.4800 — — — — — — — — — — —

6 391 Su0Pe40 24 0.1157 0.0000 0.0000 0.0435 — — 10.6054 — — — — — — — — — — —

6 392 Su0Pe40 24 0.1157 0.0000 0.0000 0.0435 — — 10.8983 — — — — — — — — — — —

6 393 Su0Pe91 24 0.1158 0.0000 0.0000 0.0869 — — 15.7691 — — — — — — — — — — —

6 394 Su0Pe91 24 0.1158 0.0000 0.0000 0.0869 — — 16.2573 — — — — — — — — — — —

6 395 Su0Pe120 24 0.1162 0.0000 0.0000 0.1304 — — 21.5208 — — — — — — — — — — —

6 396 Su0Pe120 24 0.1158 0.0000 0.0000 0.1304 — — 21.5296 — — — — — — — — — — —

6 397 Su40Pe91 24 0.1161 0.0000 0.0380 0.0869 — — 20.7004 — — — — — — — — — — —

6 398 Su40Pe91 24 0.1158 0.0000 0.0384 0.0869 — — 20.7955 — — — — — — — — — — —

6 399 Su80Pe40 24 0.1157 0.0000 0.0760 0.0435 — — 16.3238 — — — — — — — — — — —

6 400 Su80Pe40 24 0.1157 0.0000 0.0760 0.0435 — — 17.0074 — — — — — — — — — — —

6 401 BL 24 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.9868 2.5996

6 402 BL 24 0.0000 0.0000 0.0000 0.0000 — — — — — — — — — — — — 0.9062 2.1794

6 403 NDF 24 0.1157 0.0000 0.0000 0.0000 — — — 0.5124 0.0000 0.0000 0.0284 0.0000 9.7498 4.5971 1.3616 0.3852 0.7974 3.3825

6 404 NDF 24 0.1159 0.0000 0.0000 0.0000 — — — 0.0433 0.0000 0.0000 0.0000 0.0000 5.0666 4.1992 1.1839 0.4530 0.8966 4.0667

6 405 Su40Pe0 24 0.1161 0.0000 0.0383 0.0000 — — — 0.7528 0.0000 0.0000 0.0000 0.0000 16.0149 7.3218 5.1424 1.4615 0.8157 1.1499

6 406 Su40Pe0 24 0.1160 0.0000 0.0384 0.0000 — — — 0.5163 0.0048 0.0000 0.0000 0.0000 14.2523 6.6483 5.6727 1.4271 0.7469 2.4743

6 407 Su80Pe0 24 0.1160 0.0000 0.0761 0.0000 — — — 0.9105 0.0000 0.0000 0.0000 0.0000 16.8377 7.9879 7.3254 3.4724 0.7345 2.6662

6 408 Su80Pe0 24 0.1157 0.0000 0.0762 0.0000 — — — 0.6778 0.0000 0.0000 0.0000 0.0000 17.6765 9.4315 7.9166 3.7672 0.6805 1.2548

6 409 Su120Pe0 24 0.1160 0.0000 0.1140 0.0000 — — — 1.3104 0.0174 0.0000 0.0000 0.0000 21.3960 17.6129 6.4377 3.3767 0.6728 3.1854

280



Sucrose(g DM)

Pectin

(g DM) pH rNDF

(g OM) MCP (mg)

GLY (mg)

rGlc (mg)

rFruc (mg)

rSuc (mg)

Lac (mM)

C2 (mM)

C3 (mM)

C4 (mM)

Val (mM)

Isobut (mM)

Isoval + 2MB (mM)

6 410 Su120Pe0 24 0.1160 0.0000 0.1140 0.0000 — — — 0.6817 0.0000 0.0000 0.1423 0.0000 23.3524 20.1712 6.9254 4.0212 0.7678 2.6097

6 411 Su0Pe40 24 0.1160 0.0000 0.0000 0.0435 — — — 0.5951 0.0223 0.0000 0.0000 0.0000 18.0034 5.8925 1.6828 0.5962 0.8532 3.5592

6 412 Su0Pe40 24 0.1157 0.0000 0.0000 0.0435 — — — 0.4374 0.0279 0.0000 0.0628 0.0000 20.2963 6.1803 1.7063 0.1649 0.8339 3.9977

6 413 Su0Pe91 24 0.1160 0.0000 0.0000 0.0869 — — — 0.7528 0.0000 0.0000 0.0000 0.0000 27.9189 6.9495 1.5914 0.3523 0.6613 0.6547

6 414 Su0Pe91 24 0.1158 0.0000 0.0000 0.0869 — — — 0.5163 0.0271 0.0000 0.0000 0.0000 28.3641 7.9311 1.6915 0.5161 0.5520 2.6438

6 415 Su0Pe120 24 0.1159 0.0000 0.0000 0.1304 — — — 0.7566 0.0000 0.0015 0.2803 0.0000 43.5800 11.5964 2.0047 0.2395 0.5771 —

6 416 Su0Pe120 24 0.1158 0.0000 0.0000 0.1304 — — — 0.6740 0.0146 0.0149 0.2732 0.0000 41.8793 10.7777 2.0275 0.5794 0.7185 4.4170

6 417 Su40Pe91 24 0.1159 0.0000 0.0381 0.0869 — — — 1.0720 0.0000 0.0000 0.0399 0.0000 34.8631 12.4773 4.2575 2.0720 0.8361 2.0349

6 418 Su40Pe91 24 0.1157 0.0000 0.0383 0.0869 — — — 0.7566 0.0000 0.0008 0.0000 0.0000 32.8875 11.1006 3.8586 1.7960 0.7211 1.2441

6 419 Su80Pe40 24 0.1162 0.0000 0.0765 0.0435 — — — 0.9182 0.0183 0.0000 0.0471 0.0000 — — — — — —

6 420 Su80Pe40 24 0.1161 0.0000 0.0764 0.0435 — — — 0.9932 0.0000 0.0041 0.0000 0.0000 48.7170 19.6050 9.6575 5.9540 1.0211 2.82631 Ferm = fermentation; Trt = treatment: Su = sucrose, St = starch, Pe = pectin, 0, 40, 80 or 120 mg nominal hexose equivalent, BL = no substrate; iNDF = isolated bermudagrass neutral detergent residue; rNDF = residual NDF; MCP = microbial crude protein; GLY = microbial glycogen; rSuc = residual sucrose; rGlc = residual glucose; rFruc = residual fructose; Lac = lactate; C2 = acetate; C3 = propionate; C4 = butyrate; Val = valerate; Isobut = isobutyrate; Isoval+2MB = isovalerate + 2-Methylbutyrate

281

LIST OF REFERENCES

Aldrich, J. M., L. D. Muller, G. A. Varga, and L. D. Griel Jr. 1993. Nonstructural carbohydrate and protein effects on rumen fermentation, nutrient flow, and performance of dairy cows. J. Dairy Sci. 76:1091-1105.

Al-Rabbat, M. F., R. L. Baldwin, and W. C. Weir. 1971. Microbial growth dependence on ammonia nitrogen in the bovine rumen: A quantitative study. J. Dairy Sci. 54:1162-1172.

Al-Rabbat, M. F. and D. P. Heaney. 1978. The effects of anhydrous ammonia treatment of wheat straw and steam cooking of aspen wood on their feeding value and on ruminal microbial activity. II. Fermentable energy and microbial growth derived from ammonia nitrogen in the ovine rumen. Can. J. Anim. Sci. 58:453-463.

Ammerman, C. B., P. A. Van Walleghem, A. Z. Palmer, J. W. Carpenter, J. F. Hentges, and L. R. Arrington. 1963. Comparative feeding value of dried citrus pulp and ground corn and cob meal for fattening steers. Mimeograph Report No. AN64-8. Florida Agricultural Experiment Station, University of Florida, Gainesville, FL.

Association of Official Analytic Chemists. 1980. Official Methods of Analysis. 13th ed. AOAC, Arlington, VA.

Araba, A., F. M. Byers, and F. Guessous. 2002. Patterns of rumen fermentation in bulls fed barley/molasses diets. Anim. Feed Sci. Technol. 97:53-64.

Ariza, P., A. Bach, M. D. Stern, and M. B. Hall. 2001. Effects of carbohydrates from citrus pulp and hominy feed on microbial fermentation in continuous culture. J. Anim. Sci. 79:2713-2718.

Arthington, J. D., W. E. Kunkle, and A. M. Martin. 2002. Citrus pulp for cattle. Vet. Clin. North Am. Food Anim. Pract. 18:317-326.

Bach, A., I. K. Yoon, M. D. Stern, H. G. Jung, and H. Chester-Jones. 1999. Effects of type of carbohydrate supplementation to lush pasture on microbial fermentation in continuous culture. J. Dairy Sci. 82:153-160.

Baldwin, R. L. and M. J. Allison. 1983. Rumen metabolism. J. Anim. Sci. 57 (Suppl. 2):461-477.

282

Ben-Ghedalia, D., E. Yosef, J. Miron, and Y. Est. 1989. The effects of starch- and pectin-rich diets on quantitative aspects of digestion in sheep. Anim. Feed Sci. Technol. 24:289-298.

Broderick, G. A., N. D. Luchini, W. J. Radloff, G. A. Varga, and V. A. Ishler. 2002a. Effect of replacing dietary starch with sucrose on milk production in lacating dairy cows. 2000-2001 Research Report. US Dairy Forage Research Center, Madison, WI.

Broderick, G. A., D. R. Mertens, and R. Simons. 2002b. Efficacy of carbohydrate sources for milk production by cows fed diets based on alfalfa silage. J. Dairy Sci. 85:1767-1776.

Broderick, G. A., P. Udén, M. L. Murphy, and A. Lapins. 2004. Sources of variation in rates of in vitro ruminal protein degradation. J. Dairy Sci. 87:1345-1359.

Calsamiglia, S., A. Ferret, and M. Devant. 2002. Effects of pH and pH fluctuations on microbial fermentation and nutrient flow from a dual-flow continuous culture system. J. Dairy Sci. 85:574-579.

Cameron, M. R., T. H. Klusmeyer, G. L. Lynch, J. H. Clark, and D. R. Nelson. 1991. Effects of urea and starch on rumen fermentation, nutrient passage to the duodenum, and performance of cows. J. Dairy Sci. 74:1321-1336.

Casper, D. P., H. A. Maiga, M. J. Brouk, and D. J. Schingoethe. 1999. Synchronization of carbohydrate and protein sources on fermentation and passage rates in dairy cows. J. Dairy Sci. 82:1779-1790.

Chamberlain, D. G., S. Robertson, and J. J. Choung. 1993. Sugars versus starch as supplements to grass silage: Effects on ruminal fermentation and the supply of microbial protein to the small intestine, estimated from the urinary excretion of purine derivatives, in sheep. J. Sci. Food Agric. 63:189-194.

Charmely, E. D., D. M. Veira, G. Butler, L. Aroeira, and H. C. V. Codagnone. 1991. The effect of frequency of feeding and supplementation with sucrose on ruminal fermentation of alfalfa silage given ad libitum or restricted to sheep. Can. J. Anim. Sci. 71:725-737.

Cherney, D. J. R., J. H. Cherney, and L. E. Chase. 2003. Influence of dietary nonfiber carbohydrate concentration and supplementation of sucrose on lactation performance of cows fed Fescue silage. J. Dairy Sci. 86:3983-3991.

Chesson, A. and C. W. Forsberg. 1997. Chapter 8. Polysaccharide degradation by rumen microorganisms. Pages 329-381 in The rumen microbial ecosystem. P. N. Hobson and C. S. Stewart, eds. Chapman & Hall, London, UK.

283

Clark, J. H., T. H. Klusmeyer, and M. R. Cameron. 1992. Microbial protein synthesis and flows of nitrogen fractions to the duodenum of dairy cows. J. Dairy Sci. 75:2304-2323.

Cotta, M. A. 1988. Amylolytic activity of selected species of ruminal bacteria. Appl. Environ. Microbiol. 54:772-776.

Cotta, M. A. and J. B. Russell. 1982. Effect of peptides and amino acids on efficiency of rumen bacterial protein synthesis in continuous culture. J. Dairy Sci. 65:226-234.

Craig, W. M., D. R. Brown, G. A. Broderick, and D. B. Ricker. 1987. Post-prandial compositional changes of fluid- and particle-associated ruminal microorganisms. J. Anim. Sci. 65:1042-1048.

Crawford, R. J. J., W. H. Hoover, and P. H. Knowlton. 1980. Effects of solid and liquid flows on fermentation in continuous culture. I. Dry matter and fiber digestion, VFA production and protozoa numbers. J. Anim. Sci. 51:975-985.

Cruz Soto, R., S. A. Muhammed, C. J. Newbold, C. S. Stewart, and R. J. Wallace. 1994. Influence of peptides, amino acids and urea on microbial activity in the rumen of sheep receiving grass hay and on the growth of rumen bacteria in vitro. Anim. Feed Sci. Technol. 49:151-161.

Czerkawski, J. W. and G. Breckenridge. 1969. Fermentation of various soluble carbohydrates by rumen micro-organisms with particular reference to methane production. Br. J. Nutr. 23:925-937.

Dawson, K. A., M. A. Rasmussen, and M. J. Allison. 1997. Chapter 14. Digestive disorders and nutritional toxicity. Pages 633-660 in The rumen microbial ecosystem. P. N. Hobson and C. S. Stewart, eds. Chapman & Hall, London, UK.

De Veth, M. J. and E. S. Kolver. 2001. Diurnal variation in pH reduces digestion and synthesis of microbial protein when pasture is fermented in continuous culture. J. Dairy Sci. 84:2066-2072.

Delahoy, J. E., L. D. Muller, F. Bargo, T. W. Cassidy, and L. A. Holden. 2003. Supplemental carbohydrate sources for lactating dairy cows on pasture. J. Dairy Sci. 86:906-915.

Dijkstra, J., H. D. S. C. Neal, D. E. Beever, and J. France. 1992. Simulation of nutrient digestion, absorption and outflow in the rumen: model description. J. Nutr. 122:2239-2256.

Erfle, J. D., R. J. Boila, R. M. Teather, S. Mahadevan, and F. D. Sauer. 1982. Effect of pH on fermentation characteristics and protein degradation by rumen microorganisms in vitro. J. Dairy Sci. 65:1457-1464.

284

Fegeros, K., G. Zervas, S. Stamouli, and E. Apostolaki. 1995. Nutritive value of dried citrus pulp and its effect on milk yield and milk composition of lactating ewes. J. Dairy Sci. 78:1116-1121.

Friggens, N., G. C. Emmans, S. Robertson, D. G. Chamberlain, C. T. Whittemore, and J. D. Oldham. 1995. The lactational responses of dairy cows to amount of feed and to the source of carbohydrate energy. J. Dairy Sci. 78:1734-1744.

Goering, H. K. and P. J. Van Soest. 1970. Forage fiber analysis (apparatus, reagents, procedures and some applications). Agric. Handb. No. 379. ARS-USDA, Washington, DC.

Gorosito, A. R., J. B. Russell, and P. J. Van Soest. 1985. Effect of carbon-4 and carbon-5 volatile fatty acids on digestion of plant cell wall in vitro. J. Dairy Sci. 68:840-847.

Gradel, C. M. and B. A. Dehority. 1972. Fermentation of isolated pectin and pectin from intact forages by pure cultures of rumen bacteria. Appl. Microbiol. 23:332-340.

Grant, R. J. 1994. Influence of corn and sorghum starch on the in vitro kinetics of forage fiber digestion. J. Dairy Sci. 77:1563-1569.

Grant, R. J. and D. R. Mertens. 1992. Development of buffer systems for pH control and evaluation of pH effects on fiber digestion in vitro. J. Dairy Sci. 75:1581-1587.

Grant, R. J. and S. J. Weidner. 1992. Digestion kinetics of fiber: Influence of in vitro buffer pH varied within observed physiological range. J. Dairy Sci. 75:1060-1068.

Grasser, L. A., J. G. Fadel, I. Garnett, and E. J. DePeters. 1995. Quantity and economic importance of nine selected by-products used in California dairy rations. J. Dairy Sci. 78:962-971.

Hall, M. B. 1998. Making nutritional sense of non-NDF carbohydrates. Pages A1-A15 in Proc. 9th Annual Florida Ruminant Nutrition Symposium, University of Florida, Gainesville, FL.

Hall, M. B. 2002. Working in sugars and molasses. Pages 146-158 in Proc. 13th Annual Florida Ruminant Nutrition Symposium, University of Florida, Gainesville, FL.

Hall, M. B. and C. Herejk. 2001. Differences in yields of microbial crude protein from in vitro fermentation of carbohydrates. J. Dairy Sci. 84:2486-2493.

Hall, M. B., W. H. Hoover, J. P. Jennings, and T. K. Miller Webster. 1999. A method for partitioning neutral detergent-soluble carbohydrates. J. Sci. Food Agric. 79:2079-2086.

Hatfield, R. D. and P. J. Weimer. 1995. Degradation characteristics of isolated and in situ cell wall lucerne pectic polysaccharides by mixed ruminal microbes. J. Sci. Food Agric. 69:185-196.

285

Heldt, J. S., R. C. Cochran, G. L. Stokka, C. G. Farmer, C. P. Mathis, E. C. Titgemeyer, and T. G. Nagaraja. 1999. Effects of different supplemental sugars and starch fed in combination with degradable intake protein on low-quality forage use by beef steers. J. Anim. Sci. 77:2793-2802.

Henning, P. H., D. G. Steyn, and H. H. Meissner. 1991. The effect of energy and nitrogen supply pattern on rumen bacterial growth in vitro. Anim. Prod. 53:165-175.

Hogan, J. P. 1975. Symposium: Protein and amino acid nutrition in the high producing cow. Quantitative aspects of nitrogen utilization in ruminants. J. Dairy Sci. 58:1164-1177.

Hoover, W. H. 1986. Chemical factors involved in ruminal fiber digestion. J. Dairy Sci. 69:2755-2766.

Hoover, W. H., C. R. Kincaid, G. A. Varga, W. V. Thayne, and L. L. J. Junkins. 1984. Effects of solids and liquid flows on fermentation in continuous cultures. IV. pH and dilution rate. J. Anim. Sci. 58:692-699.

Hoover, W. H. and S. R. Stokes. 1991. Balancing carbohydrates and proteins for optimum rumen microbial yield. J. Dairy Sci. 74:3630-3644.

Huhtanen, P. and H. Khalili. 1992. The effect of sucrose supplements on particle-associated carboxymethylcellulase (EC 3.2.1.4) and xylanase (EC 3.2.1.8) activities in cattle given grass-silage-based diet. Brit. J. Nutr. 67:245-255.

Hume, I. D. 1970. Synthesis of microbial protein in the rumen. III. The effect of dietary protein. Aust. J. Agr. Res. 21:305-314.

Hume, I. D. and D. B. Purser. 1974. Ruminal and post-ruminal protein digestion in sheep fed on subterranean clover harvested at four stages of maturity. Aust. J. Agric. Res. 26:199-208.

Hungate, R. E. 1966. The Rumen and Its Microbes. Academic Press, Inc., New York, NY.

Huntington, G. B. 1997. Starch utilization by ruminants: Basics to the bunk. J. Anim. Sci. 75:852-867.

Jarvis, M. C. 1984. Structure and properties of pectin gels in plant cell walls. Plant, Cell and Environ. 7:153-164.

John, R. A. 1984. Effects of feeding frequency and level of feed intake on chemical composition of ruminal bacteria. J. Agric. Sci. 102:45-57.

Karkalas, J. J. 1985. An improved enzymatic method for the determination of native and modified starch. J. Sci. Food Agric. 36:1019-1027.

286

Kennelly, J. J., B. Robinson, and G. R. Khorasani. 1999. Influence of carbohydrate source and buffer on rumen fermentation characteristics, milk yield, and milk composition in early-lactation Holstein cows. J. Dairy Sci. 82:2486-2496.

Khalili, H. and P. Huhtanen. 1991a. Sucrose supplements in cattle given grass silage-based diets. 1. Digestion of organic matter and nitrogen. Anim. Feed Sci. Technol. 33:247-261.

Khalili, H. and P. Huhtanen. 1991b. Sucrose supplements in cattle given grass silage-based diets. 2. Digestion of cell wall carbohydrates. Anim. Feed Sci. Technol. 33:263-273.

Kunkle, W. E., J. T. Johns, M. H. Poore, and D. B. Herd. 2000. Effects of carbohydrate source, feed additives and feeding systems on response to supplements for cattle fed forage based diets. Pages 39-61 in Proc. 11th Annual Florida Ruminant Nutrition Symposium, University of Florida, Gainesville, FL.

Leedle, J. A. Z., M. P. Bryant, and R. B. Hespell. 1982. Diurnal variation in bacterial numbers and fluid parameters in ruminal contents of animals fed low or high forage diets. Appl. Environ. Microbiol. 44:402-412.

Leiva, E., M. B. Hall, and H. H. Van Horn. 2000. Performance of dairy cattle fed citrus pulp or corn products as sources of neutral detergent-soluble carbohydrates. J. Dairy Sci. 83:2866-2875.

Lou, J., K. A. Dawson, and H. J. Strobel. 1997. Glycogen formation by the rumen bacterium Prevotella ruminicola. Appl. Environ. Microbiol. 63:1483-1488.

Lykos, T., G. A. Varga, and D. Casper. 1997. Varying degradation rates of total nonstructural carbohydrates: Effects on ruminal fermentation, blood metabolites, and milk production and composition in high producing Holstein cows. J. Dairy Sci. 80:3341-3355.

Mackie, R. I., F. M. C. Gilchrist, A. M. Roberts, P. E. Hannah, and H. M. Schwartz. 1978. Microbiological and chemical changes in the rumen during the stepwise adaptation of sheep to high concentrate diets. J. Agric. Sci. 90:241-254.

Maeng, W. J. and R. L. Baldwin. 1976a. Factors influencing rumen microbial growth rates and yields: Effects of urea and amino acids over time. J. Dairy Sci. 59:643-647.

Maeng, W. J. and R. L. Baldwin. 1976b. Factors influencing rumen microbial growth rates and yields: Effect of amino acid additions to a purified diet with nitrogen from urea. J. Dairy Sci. 59:648-655.

Maeng, W. J., C. J. Van Nevel, R. L. Baldwin, and J. G. Morris. 1976. Rumen microbial growth rates and yields: effect of amino acids and protein. J. Dairy Sci. 59:68-79.

287

Maglione, G. and J. B. Russell. 1997. The adverse effect of ammonia limitation and excess-cellobiose on Fibrobacter succinogenes S85. Appl. Microbiol. Biotechnol. 48:720-725.

Mansfield, H. R., M. D. Stern, and D. E. Otterby. 1994. Effects of beet pulp and animal by-products on milk yield and in vitro fermentation by rumen microorganisms. J. Dairy Sci. 77:205-216.

Marounek, M., S. Bartos, and P. Brezina. 1985. Factors influencing the production of volatile fatty acids from hemicellulose, pectin and starch by mixed culture of rumen microorganisms. Z. Tierphysiol. Tierernahrg. U. Futtermittelkde. 53:50-58.

Marounek, M. and D. Dušková. 1999. Metabolism of pectin in rumen bacteria Butyrivibrio fibrisolvens and Prevotella ruminicola. Letters in Appl. Microbiol. 29:429-433.

Mathison, G. W. and L. P. Milligan. 1971. Nitrogen metabolism in sheep. Br. J. Nutr. 25:351-366.

McAllan, A. B. and R. H. Smith. 1974. Bacterial carbohydrates formed in the rumen and their contribution to digesta entering the duodenum. Brit. J. Nutr. 31:77-88.

McAllister, T. A., H. D. Bae, G. A. Jones, and K. J. Cheng. 1994. Microbial attachment and feed digestion in the rumen. J. Anim. Sci. 72:3004-3018.

McCormick, M. E., D. D. Redfearn, J. D. Ward, and D. C. Blouin. 2001. Effect of protein source and soluble carbohydrate addition on rumen fermentation and lactation performance of Holstein cows. J. Dairy Sci. 84:1686-1697.

McDonald, P., R. A. Edwards, J. F. D. Greenhalgh, and C. A. Morgan. 1995. Animal Nutrition. Addison Wesley Longman, Harlow, UK.

Miron, J., E. Yosef, D. Ben-Ghedalia, L. E. Chase, D. E. Bauman, and R. Solomon. 2002. Digestibility by dairy cows of monosaccharide constituents in total mixed ration containing citrus pulp. J. Dairy Sci. 85:89-94.

Miura, H., M. Horiguchi, and T. Matsumoto. 1980. Nutritional interdependence among bacteria Bacteriodes amylophilus, Megasphera elsdenii, and Ruminococcus albus. Appl. Environ. Microbiol. 40:294-300.

Moloney, A. P., A. A. Almiladi, M. J. Drennan, and P. J. Caffrey. 1994. Rumen and blood variables in steers fed grass silage and rolled barley or sugar cane molasses-based supplements. Anim. Feed Sci. Technol. 50:37-54.

Mould, F. L. and E. R. Ørskov. 1984. Manipulation of rumen fluid pH and its influence on cellulolysis in sacco, dry matter degradation and the rumen microflora of sheep offered either hay or concentrate. Anim. Feed Sci. Technol. 10:1-14.

288

Mould, F. L., E. R. Ørskov, and S. O. Mann. 1984. Associative effects of mixed feeds. I. Effect of type and level of supplementation and the influence on the rumen fluid pH on cellulolysis in vivo and dry matter digestion on various roughages. Anim. Feed Sci. Technol. 10:15-30.

National Research Council. 2001. Nutrient Requirements of Dairy Cattle. 7th Rev. ed. Natl. Acad. Sci., Washington, D.C.

Nocek, J. E. and J. B. Russell. 1988. Protein and energy as an integrated system. Relationship of ruminal protein and carbohydrate availability to microbial synthesis and milk production. J. Dairy Sci. 71:2070-2107.

Nocek, J. E. and S. Tamminga. 1991. Site of digestion of starch in the gastrointestinal tract of dairy cows and its effect on milk yield and composition. J. Dairy Sci. 74:3598-3629.

Nolan, J. V., B. W. Norton, and R. A. Lang. 1976. Further studies of the dynamics of nitrogen metabolism in sheep. Br. J. Nutr. 53:127-147.

Nombekela, S. W. and M. R. Murphy. 1995. Sucrose supplementation and feed intake of dairy cows in early lactation. J. Dairy Sci. 78:880-885.

Oltjen, R. R. 1969. Effects of feeding ruminants non-protein nitrogen as the only nitrogen source. J. Anim. Sci. 28:673-682.

O'Mara, F. P., J. J. Murphy, and M. Rath. 1997a. The effect of replacing dietary beet pulp with wheat treated with sodium hydroxide, ground wheat, or ground corn in lactating cows. J. Dairy Sci. 80:530-540.

O'Mara, F. P., G. K. Stakelum, P. Dillon, J. J. Murphy, and M. Rath. 1997b. Rumen fermentation and nutrient flows for cows fed grass and grass supplemented with molassed beet pulp pellets. J. Dairy Sci. 80:2466-2474.

Ordway, R. S., V. A. Ishler, and G. A. Varga. 2002. Effects of sucrose supplementation on dry matter intake, milk yield, and blood metabolites of periparturient Holstein dairy cows. J. Dairy Sci. 85:879-888.

Owens, F. N. and W. G. Bergen. 1983. Nitrogen metabolism of ruminant animals: Historical perspective, current understanding and future implications. J. Anim. Sci. 57 (Suppl. 2):498-518.

Pilgrim, A. F., F. V. Gray, R. A. Weller, and C. B. Belling. 1970. Synthesis of microbial protein from ammonia in the sheep's rumen and the proportion of dietary nitrogen converted into microbial nitrogen. Brit. J. Nutr. 24:589-598.

Piva, G. and F. Masoero. 1996. Carbohydrates with different ruminal fermentability: starch and fibrous fractions. Zoot. Nutr. Anim. 22:215-229.

289

Piwonka, E. J. and J. L. Firkins. 1993. Effect of glucose on fiber digestion and particle-associated carboxymethylcellulase activity in vitro. J. Dairy Sci. 76:129-139.

Piwonka, E. J. and J. L. Firkins. 1996. Effect of glucose fermentation on fiber digestion by ruminal microorganisms in vitro. J. Dairy Sci. 79:2196-2206.

Piwonka, E. J., J. L. Firkins, and B. L. Hull. 1994. Digestion in the rumen and total tract of forage-based diets with starch of dextrose supplements fed to Holstein heifers. J. Dairy Sci. 77:1570-1579.

Rooke, J. A. and D. G. Amstrong. 1989. The importance of the form of nitrogen on microbial protein synthesis in the rumen of cattle receiving grass silage and continuous intrarumen infusion of sucrose. Brit. J. Nutr. 61:113-121.

Rooney, L. W. and R. L. Pflugfelder. 1986. Factors affecting starch digestibility with special emphasis on sorghum and corn. J. Anim. Sci. 63:1607-1623.

Royes, J. B., W. F. Brown, F. G. Martin, and D. B. Bates. 2001. Source and level of energy supplementation for yearling cattle fed ammoniated hay. J. Anim. Sci. 79:1313-1321.

Russell, J. B. 1992. Glucose toxicity and the inability of Bacteroides ruminicola to regulate glucose transport and utilization. Appl. Environ. Microbiol. 58:2040-2045.

Russell, J. B. and R. L. Baldwin. 1979. Comparison of maintenance energy expenditures and growth yields among several rumen bacteria grown on continuous culture. Appl. Environ. Microbiol. 37:537-543.

Russell, J. B. and D. B. Dombrowski. 1980. Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture. Appl. Environ. Microbiol. 39:604-610.

Russell, J. B. and T. Hino. 1985. Regulation of lactate production in Streptococcus bovis. J. Dairy Sci. 68:1712-1721.

Russell, J. B., J. D. O'connor, D. G. Fox, P. J. Van Soest, and C. J. Sniffen. 1992. A net carbohydrate and protein system for evaluating cattle diets. I. Ruminal fermentation. J. Anim. Sci. 70:3551-3561.

Russell, J. B. and C. J. Sniffen. 1984. Effect of carbon-4 and carbon-5 volatile fatty acids on growth of mixed rumen bacteria in vitro. J. Dairy Sci. 67:987-994.

Russell, J. B. and H. J. Strobel. 1993. Microbial energetics. Pages 165-186 in Quantitative Aspects of Ruminant Digestion and Metabolism. J. M. Forbes and J. France, eds. CAB International, Wallingford, UK.

Russell, J. B. and P. J. Van Soest. 1984. In vitro ruminal fermentation of organic acids common in forage. Appl. Environ. Microbiol. 47:155-159.

290

Russell, J. B. and D. B. Wilson. 1996. Why are ruminal cellulolytic bacteria unable to digest cellulose at low pH? J. Dairy Sci. 79:1503-1509.

Sannes, R. A., M. A. Messman, and D. B. Vagnoni. 2002. Form of rumen-degradable carbohydrate and nitrogen on microbial protein synthesis and protein efficiency of dairy cows. J. Dairy Sci. 85:900-908.

SAS. 1999. The SAS System for Windows, Release 8.02. SAS Institute, Inc., Cary, NC.

Satter, L. D. and L. L. Slyter. 1974. Effect of ammonia concentration on rumen microbial protein production in vitro. Brit. J. Nutr. 32:199-208.

Shi, Y. and P. J. Weimer. 1992. Response surface analysis of the effects of pH and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose-fed continuous culture. Appl. Environ. Microbiol. 58:2583-2591.

Slyter, L. L., R. R. Oltjen, D. L. Kern, and F. C. Blank. 1970. Influence of type and level of grain and diethylstilbestrol on the rumen microbial population of steers fed all concentrate diets. J. Anim. Sci. 31:996-1002.

Smith, W. R., I. Yu, and R. E. Hungate. 1973. Factors affecting cellulosis by Ruminococcus albus. J. Bacteriol. 114:729-737.

Sniffen, C. J., J. D. O'Connor, P. J. Van Soest, D. G. Fox, and J. B. Russell. 1992. A net carbohydrate and protein system for evaluating cattle diets. II. Carbohydrate and protein availability. J. Anim. Sci. 70:3562-3577.

Sniffen, C. J., J. B. Russell, and P. J. Van Soest. 1983. The influence of carbon source, nitrogen source and growth factors on rumen microbial growth. Pages 26-33 in Proc. Cornell Nutrition Conference for Feed Manufacturers, Cornell University, Ithaca, NY.

Solomon, R., L. E. Chase, D. Ben-Ghedalia, and D. E. Bauman. 2000. The effect of nonstructural carbohydrate and addition of full fat extruded soybeans on the concentration of conjugated linoleic acid in the milk fat of dairy cows. J. Dairy Sci. 83:1322-1329.

Stanley, R. W. and E. M. Kesler. 1959. Preparation and some basic properties of cell-free cellulolytic extracts of rumen fluid. J. Dairy Sci. 42:127.

Stern, M. D. 1986. Efficiency of microbial protein synthesis in the rumen. Pages 10-19 in Proc. Cornell Nutrition Conference for Feed Manufacturers, Cornell University, Ithaca, NY.

Stern, M. D., G. A. Varga, J. H. Clark, J. L. Firkins, J. T. Huber, and D. L. Palmquist. 1994. Evaluation of chemical and physical properties of feeds that affect protein metabolism in the rumen. J. Dairy Sci. 77:2762-2786.

291

Stewart, C. S. 1977. Factors affecting the cellulolytic activity of rumen contents. Appl. Environ. Microbiol. 33:497-502.

Stewart, C. S., H. J. Flint, and M. P. Bryant. 1997. Chapter 2. The rumen bacteria. Pages 11-72 in The Rumen Microbial Ecosystem. P. N. Hobson and C. S. Stewart, eds. Chapman & Hall, London, UK.

Storm, E., D. S. Brown, and E. R. Ørskov. 1983. The nutritive value of rumen micro-organisms in ruminants. 3. The digestion of microbial amino and nucleic acids in, and losses of endogenous nitrogen from, the small intestine of sheep. Br. J. Nutr. 50:479-485.

Strobel, H. J. and J. B. Russell. 1986. Effect of pH and energy spilling on bacterial protein synthesis by carbohydrate-limited cultures of mixed rumen bacteria. J. Dairy Sci. 69:2941-2947.

Sutton, J. D. 1979. Rumen function and the utilisation of readily fermentable carbohydrates by dairy cows. Trop. Anim. Prod. 4:1-12.

Terry, R. A., J. M. A. Tilley, and G. E. Outen. 1969. Effect of pH on cellulose digestion under in vitro conditions. J. Sci. Food Agric. 20:317-320.

Thomas, G. J. 1960. Metabolism of the soluble carbohydrates of grasses in the rumen of the sheep. J. Agric. Sci. 54:360-372.

Valk, H., H. W. Klein Poelhuis, and H. J. Wentink. 1990. Effect of fibrous and starchy carbohydrates in concentrates as supplements in a herbage-based diet for high-yielding dairy cows. Neth. J. Agric. Sci. 38:475-486.

Van der Linden, Y., N. O. Van Gylswyk, and H. M. Schwartz. 1984. Influence of supplementation of corn stover with corn grain on the fibrolytic bacteria in the rumen of sheep and their relation to the intake and digestion of fiber. J. Anim. Sci. 59:772-783.

Van Horn, H. H., S. P. Marshall, C. J. Wilcox, P. F. Randel, and J. M. Wing. 1975. Complete rations for dairy cattle. III. Evaluation of protein percent and quality, and citrus pulp-corn substitutions. J. Dairy Sci. 58:1101-1108.

Van Soest, P. J. 1994. Nutritional Ecology of the Ruminant. 2nd ed. Cornell University Press, Ithaca, NY.

Van Soest, P. J. and J. B. Robertson. 1985. Analysis of forages and fibrous foods - A laboratory manual for Animal Science 613. Cornell University, Ithaca, NY.

Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583-3597.

292

Van Vuuren, A. M., C. J. Van der Koelen, and J. Vroons-De Bruin. 1993. Ryegrass versus corn starch or beet pulp fiber diet effects on digestion and intestinal amino acids in dairy cows. J. Dairy Sci. 76:2692-2700.

Varga, G. A., W. H. Hoover, L.L Junkins Jr., and B. J. Shriver. 1988. Effects of urea and isoacids on in vitro fermentation of diets containing formaldehyde-treated or untreated soybean meal. J. Dairy Sci. 71:737-744.

Voelker, J. A. and M. S. Allen. 2003a. Pelleted beet pulp substituted for high-moisture corn: 1. Effects on feed intake, chewing behavior, and milk production of lactating dairy cows. J. Dairy Sci. 86:3542-3552.

Voelker, J. A. and M. S. Allen. 2003b. Pelleted beet pulp substituted for high-moisture corn: 2. Effects on digestion and ruminal digestion kinetics in lactating dairy cows. J. Dairy Sci. 86:3553-3561.

Voelker, J. A. and M. S. Allen. 2003c. Pelleted beet pulp substituted for high-moisture corn: 3. Effects on ruminal fermentation, pH and microbial protein efficiency in lactating dairy cows. J. Dairy Sci. 86:3562-3570.

Wallace, R. J., C. Atasoglu, and C. J. Newbold. 1999. Role of peptides in rumen microbial metabolism. Asian Aust. J. of Anim. Sci. 12:139-147.

Yang, C.-M. J. 2002. Response of forage fiber degradation by ruminal microorganisms to branch-chain volatile fatty acids, amino acids, and dipeptides. J. Dairy Sci. 85:1183-1190.

Zinn, R. and F. N. Owens. 1993. Ruminal escape protein for lightweight feedlot calves. J. Anim. Sci. 71:1677-1687.

293

BIOGRAPHICAL SKETCH

Lucia Holtshausen was born in Molteno, South Africa. She completed her

undergraduate studies in animal sciences and agricultural economics at Stellenbosch

University near Cape Town. Following her graduation in 1994, she joined a student

trainee program with The Ohio State University, through which she came to the United

States and worked in a variety of positions at a dairy operation in Bell, FL, including

conducting a research project for Eli Lily. Upon completion of her internship, she

returned to Stellenbosch University in June 1996 to work as a laboratory technician in the

Animal Science Laboratory while completing both an honor's degree and master's degree

in animal sciences. Her master's project involved examining nitrogen metabolism in veal

calves. She received the opportunity to fulfill her long-term goal of obtaining a PhD in

the United States when she was offered a research assistantship to study carbohydrate

nutrition at the University of Florida. Following graduation she is planning to complete a

post-doctoral position at Stellenbosch University and then will seek a career in academia,

teaching students about dairy cattle nutrition and management.

effect of nonfiber carbohydrates on product yield...

Documents