digestibility trials
TRANSCRIPT
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DIGESTIBILITY
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Apparent v. true digestibility
True digestibility involves correction for endogenous losses,
apparent digestion does not.
Endogenous losses
– Include:
• Sloughed off intestinal cells
• Digestive juices (enzymes)
• Microbial matter
– Quantified by measuring fecal output of fasted animals
– Can be 9.8 to 12.9 % DMI
– Should they be quantified?
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In vivo digestibility methods
Direct or total/complete collection
Difference method
Regression method
Indirect method
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1. Total collection
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In vivo digestibility trials in
metabolism crates
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In vivo digestibility trials in pens
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Total collection
calculations
Digestibility (g/kg) =
Nutrient in feed - Nutrient in feces x 1000
Nutrient in feed
Dry matter digestibility (DMD, g/kg) =
DM in feed - DM in feces x 1000
DM in feed
Organic matter digestibility (OMD, g/kg) =
OM in feed - OM in feces x 1000
OM in feed
Can be expressed as a proportion, % or g/kg
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Digestibility indices that estimate
energy valueDigestible organic matter content (DOMD) (g/kg DM)
= OM in feed - OM in feces x 1000
DM in feed
TDN = DCP + DCF + DNFE + DEE(2.25)
– DCP= Digestible Crude Protein
– DCF= Digestible Crude Fiber
– DNFE= Digestible Nitrogen-Free Extract
– DEE= Digestible Ether Extract (2.25)
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2. Difference method
Allows digy calculation for 2 feeds fed simultaneously
Assumptions
– No interaction b/w the digy of the feeds
– Must know digy & fecal DM output (DMO) of base
feed
Test feed DMD =
Test feed DMI – (Fecal DMO- Base feed DMO)
Test feed DMI
Cons
– Assumptions may be invalid
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3. Regression method
Schneider & Flatt (1975)
Also allows digy. estimation for two feeds
– Feed different ratios of the two feeds
– Estimate digy of each of the ratios
– Fit regression of test feed inclusion vs. digy
– Extrapolate to estimate digy of test feed.
Cons
– Considerable expense and labor for estimating digy of one feed.
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Regression method
20 40 60 80 100
200
400
600
800
Test feed digy.
Base feed digy.
% inclusion of test feed in ration
DM
D (
g/k
g)
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Digy trial issues
Changeover designs
– necessary if period effects are an issue e.g.
• Animal physiological changes
• Forage physiological changes
Adaptation period
– Necessary to adapt the animals to
• New feed (microbial population changes)
• Strange equipment
• Strange housing
– 6 – 14 day period is the norm
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Marker digestibility trials
Particularly useful for grazing animals
Procedure
– Add indigestible marker to feed eg chromic oxide
– Measure concentration in feed & feces
– Estimate disappearance of marker from gut.
E.g. if a feed contains 1% Cr2O3 & feces contains 2%
Cr2O3, diet digestibility = 50%
– Since Cr3O2 conc. has doubled, 50% of DM must have
been digested
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Marker trials contd.
For the digy of a specific nutrient,
must also know the % nutrient in feed & feces
%Nutrient = 100 – 100 x % indicatorfeed X % nutrientfeces
Digestibility % indicatorfeces % nutrientfeed
Homework:
If lambs are fed a bahia grass diet containing 7%
protein & 1% chromic oxide, and their feces contains
5% CP and 2% chromic oxide. Calculate CP digy.
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Marker digestibility
Pros
– Total feces collection not necessary
– Total intake determination not necessary
– Easier, less labor
Cons
– Representative sampling essential
– Accurate estimation of nutrient or marker conc. essential
– Assumes complete excretion of marker hence Recovery of marker determines accuracy of digy
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Marker types
External
– Chromic oxide
– Dysporium
– Polyamide
Can contaminate
forage
Internal
– Lignin
– AIA
– ADF
– n-alkanes
Easier, less labor
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Marker issues
Difficulty of mixing marker with forages
– Dose cows instead- ( s handling)
Marker migration
– Must not affect feed digy
External markers may contaminate forage
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Problems with in vivo
experimentsAnimal trials are:
– Expensive
– Protracted
– Laborious
– Public concerns
– Animal stress ???
Must estimate nutritive value with less animal
dependent techniques
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Ideal in vitro methods should be:
– Rapid (one step) & routinely practicable
– Accurate
– Cheap & not laborious
– Repeatable & robust
– Biologically meaningful
– Broad-based (apply to all forage types)
– Handle large nos. of samples
– Laboratory-based
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Rumen fluid –pepsin in vitro
digestibility (IVOMD)
•Developed by Tilley & Terry
(1967)
•Measures apparent digy in rumen
fluid (48 h) and acid pepsin (48 h)
•Gives accurate predictions of in
vivo digy for most forages
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Prediction of silage OMD in vivo from
different methods (g/kg DM)
Method r2 RSD
KMnO4 lignin 21.8 54.6
ADF 32.1 50.9
NDF 45.7 45.5
(M) ADF 55.8 40.9
IVOMD 74.1 33.6
(Givens et al., 1989)
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Rumen fluid problems
Variation in Inoculum composition & activity due to
– Host animal diet
– Animal species
– Collection time
– Processing (blending vs. filtration)
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Rumen fluid problems
Analytical issues
– Maintenance of anaerobic media; optimal pH, temp
– High viscosity hinders filtration
– Offensive odors
– Hygiene – (Prevent pathogen infection)
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Relationship between in vivo and
in vitro DOMD of wheat silage (g/kg DM)
r2 =0.24
530 580 630 680
Rumen fluid-pepsin DOMD
530
550
570
590
610
630
650
670
690
In v
ivo D
OM
D
(Adesogan et al. 1998)
Year One Year Two
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Rumen fluid technique -
problemsStandards needed to correct for variability in rumen
fluid composition & activity
Disregards / inappropriately represents:
– Ruminal outflow (uses a batch process)
– Digests maillard product not digested in vivo
– Associative effects between feeds
– Endogenous secretions
– Post abomasal digestion
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Alternatives to Tilley & Terry
1. Rumen fluid – Neutral detergent (Van Soest, 1967)
– More akin to true digestibility
– Gives higher digy. values
– Still requires rumen fluid
2. Feces
– Gives lower digestibility estimates
3. Enzyme- based assays
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Prediction of DMD in vivo from in vitro
fecal liquor DMD
Spp. of feces donor r2 range
Ovine 0.33 – 0.98
Bovine 0.77 – 0.97
Equine 0.90
Caprine 0.96-0.97
(Ohmed et al., 2001)
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Cell-free enzyme in vitro digestibility
Examples of procedures used:
1. Cellulase
2. Neutral detergent- cellulase
3. Neutral detergent-cellulase +gammanase
4. Pepsin cellulase
Amylase pre-treatment important for starch-rich feeds
Gammanase for oil-rich feeds
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Relationships between DMD in vivo and
enzyme predicted DMD
Method R2
Cellulase 0.83
Neutral detergent cellulase 0.94
Acid pepsin – cellulase 0.88
Rumen fluid 0.83
(Bughara & Sleper, 1986)
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Prediction of in vivo OMD of
forages from different methods
Method r RSD (%) AE(+)
ND + cellulase 0.90 3.3 0.9
Pepsin + cellulase 0.94 2.6 0.3
(McLeod & Minson, 1982)
Higher analytical error with ND – cellulase technique
may outweigh shorter processing time
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Method r2 RSD
ND + cellulase 76.6 27.1
Pepsin + cellulase 75.9 28.8
Rumen fluid-pepsin 67.0 33.2
(M) ADF 66.9 33.3
(Givens et al., 1990)
Poorer relationships found for autumn grass (r2 = 13- 20)
Prediction of in vivo OMD of spring
grass from different methods
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Effect of enzyme source on cellulase
activity
% DM solubilized
Fungi Herbage Cellulose paper
Trichoderma spp. 57 69
Basidiomycete 48 20
Aspergillus niger 45 10
Rhizopus spp. 35 7
(Jones & Hayward, 1975)
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14C-Casein hydrolysis (mg/ml)
0.0 10 20Time (h)
0.00.25
0.5 Co-culture
S. bovis
S. ruminantium
Commercial enzymes don’t fully simulate microbial
activity of mixed rumen microbes
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Enzyme method problems
Equations are species-specific
Represent effect of a few enzymes
Variability in enzyme activity
– Due to enzyme source & batch
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The ANKOM equipment
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Ankom digestibility validation
Prediction of tube true DOMD from bag true DOMD
y = 0.99x + 3.61
r2
= 0.93; rsd=2.93
50
60
70
80
50 55 60 65 70 75 80 85bag
tub
e
Prediction of tube app. DOMD from bag app. DOMD
y = 0.87x + 4.25
r2
= 0.83; rsd = 4.04
40
50
60
70
80
40 50 60 70 80bag
tub
e
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ANKOM pros & cons
Pros
– Simplifies filtration, incubation and mixing
– Uses a batch process (& ash-free bags)
Cons
– Bag pore size may allow excess outflow or restrict
microbial colonization
– Bag material & pore size may affect results
• Monofilamentous cloth – precise aperture
• Multifilamentous cloth – pore size affected by stresses
e.g. dacron
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In vitro digestibility summary
Pros
– Predicts in vivo digy more accurately than NDF or
lignin
– Handles several samples & are biologically
meaningful
Cons
– May require fistulated animals
– Labor intensive & protracted
– Plagued by variability in composition & activity of
inoculum/enzyme
– Doesn’t indicate the kinetics of digestion
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Chapters 6 – 8 In: D.I. Givens, E. Owen, R.F.E. Axford and H.M. Omed (Editors) 2000,
Forage Evaluation in Ruminant Nutrition. CABI Publishing, Wallingford, UK, pp. 113-
134.
Adesogan, A.T, Givens D.I. and Owen. E. Measuring chemical composition and nutritive
value in forages. Field and Laboratory methods for grassland and animal production
research. CABI Publishing. P 263
Tilley, J.M.A. and Terry, R.A., 1963. A two stage technique for the in vitro digestion of
forage crops. Journal of the British Grassland Society, 18: 104-111.
Van Soest, P.J., Wine, R.H. and Moore, L.A., 1966. Estimation of the true digestibility of
forages by the in vitro digestion of cell walls. Proceedings of , The Xth International
Grassland Congress, Helsinki. Finish Grassland Association., pp 438-441.
Vogel, K.P., Pedersen, J.F., Masterson, S.D. and Toy, J.J., 1999. Evaluation of a filter bag
system for NDF, ADF, and IVDMD forage analysis. Crop Science, 39: 276-279.
Wilman, D. and Adesogan, A., 2000. A comparison of filter bag methods with conventional
tube methods of determining the in vitro digestibility of forages. Animal Feed Science and
Technology, 84: 33-47.
Digestibility references