effect of barley supplement on microbial fibrolytic enzyme activities and cell wall degradation rate...

J Sci Food Agric 1996,72,235-242

Effect of Barley Supplement on Microbial Fibrolytic Enzyme Activities and Cell Wall Degradation Rate in the Rumen Pierre Nozikre, Jean Michel Besle, Cecile Martin and Brigitte Michalet-Doreau* Station de Recherches sur la Nutrition des Herbivores, INRA, Theix, 63122 Saint Genes-Champanelle, France (Received 9 October 1995; revised version received 11 March 1996; accepted 16 May 1996)

Abstract: Three ruminally cannulated dry cows were used in a Latin square design to investigate the relationship between microbial fibrolytic enzyme activities and in succo cell wall degradation of two gramineous hays, in which cell wall content ranged from 510 g kg-' DM for the regrowth to 687 g kg-' DM for the late harvested hay. Animals were fed twice daily a diet consisting of wheat straw, cocksfoot hay and ground barley in the ratios 10 : 90 : 0 (w/w), 10 : 60 : 30 (w/w) and 10 : 30 : 60 (w/w). For each diet and each hay, the in succo degradation of cell wall polysaccharides and phenolic acids was determined. After 2 h and 23 h incubation time in the rumen, pH was measured in the bags, and fibrolytic activities (xylanase, avicelase, fl-glycosidases) of the microbial population colonising hays in succo were measured. Supplementation above 30% barley decreased the degradation rate of the cell wall polysaccharides, concomitantly with a decrease in polysaccharidase activities. The decrease in degradation rate was more marked for the regrowth than for the late harvested hay, for hemicelluloses than for cellulose and for ferulic than for p-coumaric acid. These differences did not appear to be related to microbial activities, which were similar between hays and between polysaccharidases, but rather to differences in accessibility of cell wall components to microbial enzymes, related to the composition of the forage and the cell wall architecture.

Key words : ruminal solid-adherent microorganisms, enzyme activity, cell wall degradation, barley supplement, lignification.

INTRODUCTION (Huhtanen 1991; Nozibre et a1 1995), and more precisely on the nature of the cell wall polysaccharides

The supplementation of forage diets with readily fer- (Wedekind et a1 1986; Kennedy and Bunting 1992), but mentable carbohydrates is known to depress ruminal mechanisms involved in these associative effects fibre digestion (reviews of Sutton (1979) and Tamminga between forage and concentrate are still not well (1993). At a constant feeding level, this depression is mainly due to a decrease in degradation rate, particu- The aim of this work was therefore to investigate the late passage rate being only slightly modified (Poore et variations in fibrolytic microbial enzymes activities a1 1990; Kennedy and Bunting 1992). This depression in induced by increasing level of barley in the diet, and fibre degradation rate has been related to a decrease in their effect on fibre degradation rate as a function of the the fibrolytic activity of microorganisms (Silva et a1 nature of the cell wall polysaccharides of forages. Bio- 1987; Huhtanen and Khalili 1992; Martin and chemically, lignin and possibly also phenolic acid con- Michalet-Doreau 1995). The amplitude of depression in tents may determine the accessibility of cell wall fibre degradation rate varies depending on the forage polysaccharides to microbial enzymes. To understand

the mechanisms involved in the modifications of cell * To whom correspondence should be addressed. wall degradation under changes in fibrolytic activity, it

J Sci Food Agric 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain

known.

235

236 P Noziere et a1

is therefore relevant to study the degradation rate of both carbohydrates and esterified phenolic acids.

MATERIALS AND METHODS

Animals and feeding

Three adult non-lactating Jersey cows fitted with ruminal cannula (110 mm id) were housed in an air- conditioned room and used in a 3 x 3 Latin square experimental design. They were fed successively three diets (Table 1) consisting of cocksfoot hay (Dactylis glomerata), wheat straw and pelleted ground barley (3- mm screen) at ratios of 90 : 10 : 0 (Bo), 60 : 10 : 30 (B30) and 30:10:60) (B60), on a DM basis. Intake was restricted to 7 kg DM day-'. The animals received their ration in two equal portions at 08:OO h and 16:OO h, and had free access to water and mineral- vitamin blocks. A six-week adaptation period was used before each measurement period.

In sacco degradation

Two grass hays, in which cell wall content differed widely, were studied (Table 1): a six-week old vegetative regrowth of cocksfoot, and a natural grasslands hay mown during the first vegetation cycle when in full ear emergence.

The hays were ground to pass through a 3-mm screen. Ground material (3 g) was placed into polyester bags (ANKOM Co, Fairport, New York, USA; internal dimensions: 5 cm x 9 cm; pore size: 50 pm) and incubated on two consecutive days in the rumen of each cow for 2, 4, 8, 16, 23, 48 and 72 h. After removal from the rumen, the bags were washed in cold water using a

household washing machine for five rinsing cycles of 5 min. The bags were then dried at 80°C for 48 h.

The hays and bag residues were analysed in duplicate for NDF, ADF and ADL by the H,SO, procedure according to Goering and Van Soest (1970). The fractions NDF-ADF and ADF-ADL were considered respectively as hemicelluloses and cellulose fractions. Degradation parameters of these different cell wall components were obtained by fitting (NLIN procedure of SAS 1985) the kinetics of in S ~ C C O degradation to an exponential model with lag time (Dhanoa 1988). To measure the variations in degradability due only to modifications in microbial activity, the degradability was calculated from kinetics by fixing particle outflow rate at 0.04 h-' (review of Poncet et al 1995).

Esterified ferulic and p-coumaric acids were analysed in duplicate on NDF residues from initial and fer- mented hays after 8, 16 and 23 h incubation time in the rumen, as described by Hartley et al (1990). NDF (10 mg) was hydrolysed in 10 ml 1 M NaOH at 20°C for 17 h, under nitrogen, with agitation and in the dark. After acidification (pH 1) with 6 M HC1, anisic acid was added as an internal standard. Phenolic acids were extracted in CH,Cl, , vacuum-evaporated and solu- bilised in 2 ml methanol. They were then analysed by reverse-phase HPLC according to Mosoni et al (1994). The phenolic acid contents were corrected by their respective extraction coefficients in CH2CI, measured from standard phenolic acids solutions.

pH and solid-associated microorganisms fibrolytic activity

Only the solid-associated microbial population, which form most of the total rumen microbial population in

TABLE 1 Chemical composition of diets and hays" studied in S ~ C C O

Item Diets Hays

B, B,, B,, Regrowth Late harvested

Chemical composition (g kg- DM)

Organic matter Crude protein Neutral-detergent fibre Acid-detergent fibre Acid-detergent lignin Starch

Esterified phenolic acids (g kg-' NDF)

p-Coumaric Ferulic

887 880 873 154 141 129 548 440 331 347 265 186 73 55 37 - 164 321

888 172 510 320 70 -

1.96 2.55

885 76

687 40 1

67 -

3.48 2.96

~~~~~~~~~ ~ ~

a Regrowth : cocksfoot, second cycle, six weeks; late harvested : natural grasslands, first cycle, full ear emergence.

In sacco cell wall degradation and microbial fibrolytic activity 237

terms of mass (Craig et a1 1987) and enzyme activity (Martin et a1 1993), was considered. In order to study the relationship between microbial fibrolytic activity and in sacco cell wall degradation rate, the activity was measured in the microbial population attached to forage particles in bags, this activity being variable depending on the quality of the forage incubated (Bhat et al 1988). Bags similar to those used for in succo degradation were incubated on two consecutive days in the rumen of each cow for 2 and 23 h, the amplitude of variations in fibrolytic activity being maximal between these two incubation times (Williams et a1 1989).

Immediately after removal from the rumen, one bag of each hay was used for pH measurements, according to the method described by Lindberg et a1 (1984). Bags were turned inside out. The unstrained feed residues were collected in 5 ml distilled water, and pH was immediately measured in triplicate with a combination microelectrode (Ingold-U 402 M 3).

The other bags were used for fibrolytic activity measurements of solid-associated microorganisms (SAM). They were squeezed under a CO, stream and washed by manual shaking with a pre-warmed (39°C) anaerobic salt solution at pH 6.5 (Coleman 1978) to remove the non-adherent microorganisms from particles. Washed bag residues containing SAM were then submitted to the enzyme extraction treatment under anaerobic conditions. Samples were ground in liquid nitrogen, and 5 g (fresh weight) were suspended in 25 ml of pre-cooled (4°C) anaerobic buffer containing 0.025 M 2-(N-morp- holino) ethane sulphonic acid (MES) and 0.001 M DL- dithiothreitol (DTT). Simultaneously, dry matter in samples before suspension in the buffer was determined. The microorganisms present in the suspension were dis- rupted by ultrasonic disintegration (Labsonic U, B Braun Biotech Inc, Bethlehem, PA, USA) at 4°C for four 30-s periods (20 kHz, 60 W) with 30-s intervals, after freezing and defrosting of the samples to increase the eficiency of extraction (Nozibre and Michalet- Doreau 1994). Particulate matter was removed by cen- trifugation (15000 x g for 15 min at 4°C). The supernatant, containing the released soluble proteins, was used as enzyme preparation. It was stored at -80°C under a CO, headspace in capped tubes before assay conducted in the 2 months following enzyme extraction.

The fibrolytic activity of the SAM was assessed by determining xylanase, /?-D-galactosidase and /?-D-xylosidase activities for hemicellulolytic activity, and avicelase, /?-D-cellobiosidase and /?-D-glucosidase activities for cellulolytic activity. Polysaccharidase (xylanase and avicelase) activities were assayed by measuring the amount of reducing sugars released from purified sub- strates (birchwood-xylan, Sigma X-0502; Avicel, Macherey Nagel 81629) after incubation of substrate with enzyme preparation at 39°C for 60 min. Xylan (2 mg ml- ') and Avicel (10 mg m1-I) were prepared in

MES DTT buffer pH 6.5. Incubation mixtures consist- ed of 0.1 ml enzyme preparation with 1.0 ml substrate (xylan assay) or 0-5 ml enzyme preparation with 1.5 ml substrate (Avicel assay). The reaction was stopped by heating at 100°C for 5 min. The amount of reducing sugars released was quantified spectrophoto- metrically at 410 nm, using the p-hydroxybenzoic acid hydrazide method (Lcver 1977) with xylose or glucose as standards for xylan and Avicel assays, respectively. Similarly, /?-glycosidase activities were determined by measuring the amount of p-nitrophenol released from the appropriate p-nitrophenyl glycoside (Sigma, p-nitrophenyl-/?-D-cellobioside, N-5759; -galactopyranoside, N-1252; -xylopyranoside N-1232; -glucopyranoside, N-7006; 5 mmol litre-' in MES DTT buffer pH 6.5) after incubation of 1.0 ml substrate with 0.1 ml enzyme preparation at 39°C for 45 min. The reaction was stopped by addition of 1.1 ml glycine-NaOH solution (0.4mol glycine litre-', pH 10.8). The amount of p - nitrophenol released was quantified spectrophotometri- cally at 420 nm. Control assays were performed simultaneously on substrate and enzyme preparation to adjust for spontaneous substrate breakdown or formation of non-specific products. Activities were expressed as pmol of reducing sugars (polysaccharidases) or mmol of p-nitrophenol (/?-glycosidases) released in 1 h by the enzymes extracted from 1 g (DM) of feed residue.

Statistical analyses

Data were tested by an analysis of variance using the GLM procedure of SAS (1985). As animals were adult, dry, non-pregnant, restricted fed and maintained in con- trolled environment, period effect has been assumed to be negligible. Therefore, enzyme activities and pH were analysed as a factorial model with three main factors, incubation time, diet, hay into bags and their inter- actions. Data of carbohydrates and phenolic acids degradation were analysed as a factorial model with two main factors, diet, hay into bags and their interaction. In each analysis, individual animal served as the experimental unit. The means were separated by Duncan's test (1955) when the F-test was significant (P < 0.05).

RESULTS

I n sacco degradation

Barley supplementation induced a nonlinear decrease (P -= 0.001) in cell wall polysaccharides degradability (Table 2). This decrease was quite low (less than 3 percentage units) between 0 and 30% barley in the diet. Between 30 and 60% barley, the decrease was more marked for hemicelluloses (9.5 percentage units on

238 P Nozidre et a1

TABLE 2 Effect of barley supplement and of nature of hay" on in succo degradation of hemicelluloses (NDF-ADF) and cellulose (ADF-

ADL)

Item Regrowth Late harvested SE Effectb

BO B30 B60 BO B30 B60 Hay Diet Hay x Diet

NDF-ADF NS

Degradation rate (% h- l ) 8.1 7.6 3.3 5.2 5.2 2.8 0.9 *** *** NS Degradability ( O h ) 45.6 42.8 31.2 37.3 34.5 27.2 3.3 ** ***

ADF-ADL NS Degradability (YO) 53.2 51.4 46.5 28.4 27.3 20.6 2.7 *** **

Degradation rate (Yo h- l ) 8.0 6.8 3.7 4.0 2.9 3.4 0.5 *** *** ***

Regrowth : cocksfoot, second cycle, six weeks; late harvested : natural grasslands, first cycle, full ear emergence. NS, non-significant; ** significant at P < 0.01; *** significant at P < 0.001.

average for the two hays) than for cellulose (5.8 percentage units). The depressing effect of barley on the degradability of hemicelluloses was essentially due to an abrupt decrease in degradation rate (P c 0.001) beyond 30% barley in the diet. The extent of decrease in degradation rate of hemicelluloses was more marked for the regrowth (4.3% h- ') than for the late harvested hay (2.4% h- I). Similarly, the decrease in cellulose degradability was due to a nonlinear decrease in degradation rate for the regrowth (1.2% h-' between 0 and 30% and 3.1% h-' between 30 and 60% barley in the diet). However, for the late harvested hay, the cellulose degradation rate remained constant (P > 0.05) and very low (3.4% h-'). The decrease in cellulose degradability between 30 and 60% barley in the diet was mainly due to an increase in the lag time from 5.6 to 11.7 h.

Barley supplementation induced a decrease in disappearance of phenolic acids (Table 3). This depressing effect was not linear. It was more marked between 30 and 60% barley supplementation, for the regrowth than for the late harvested hay, and for ferulic than for p- coumaric acid. After 23 h incubation time in the rumen, the reduction in ferulic acid disappearance between the high-forage diet and the high-concentrate diet was more

marked for the regrowth (from 75.3 to 41.7%) than for the late harvested hay (from 50.0 to 30.8%). In the same way, p-coumaric disappearance changed from 55.6 to 29.0% for the regrowth, but remained constant (P > 0.05) and low (27%) for the late harvested hay.

pH and SAM hydrolytic activities

The pH inside the bags was independent of the nature of the hay incubated in the bags (Table 4), and was not significantly modified by the incubation time in the rumen (P > 0.05). On average for the two hays and the three diets, pH was 6.35 and 6.26 after 2 and 23 h incubation time respectively.

Barley supplementation induced an acidification inside the bags (P c 0401). On average for the two hays, pH fell from 6.57 to 6.29 or 6.07 when the level of barley in the diet increased from 0 to 30 or 60%, respectively.

Enzyme activities of SAM were higher (P < 0.001) after 23 than after 2 h incubation time (data not shown). On average for the three diets and the two hays, total activities at 23 and 2 h incubation time were respec-

TABLE 3 Effect of barley supplement and of nature of hay" on in sacco disappearance (%) of esterified phenolic acids

Phenolic Incubation Regrowth Late harvested SE Effectb acids times

(4 BO B 3 0 B60 BO B30 B60 Hay Diet Hay x Diet ~~~ ~ ~~~ ~ ~ ~

p-Coumaric 8 0.0 4.4 0.8 7.6 8.1 1.8 3.5 * NS NS 16 39.5 32.4 22.4 7.7 13.6 0.0 4.5 *** ** NS

55.6 57-5 29.0 25.7 31.1 24.5 4.1 *** *** *** 23

Ferulic 8 19.0 13.8 3.3 19.6 10.7 5.8 4.5 NS *** NS 43.9 33.1 27-1 30.1 27.2 0.0 4.1 *** *** ** 16 ** 23 75.3 67.0 41.7 50.0 45.5 30.8 3.3 *** ***

" Regrowth : cocksfoot, second cycle, six weeks; late harvested : natural grasslands, first cycle, full ear emergence. NS, non-significant; * significant at P < 0.05; ** significant at P < 0.01; *** significant at P < 0.001.

In sacco cell wall degradation and microbial jibrolytic activity 239

TABLE 4 Effect of barley supplement and of nature of hay" on pH inside the bags

Incubation Regrowth Late harvested S E Effectb times (4 BO B 3 0 B60 BO B30 B60 Hay Diet Time Hay x Hay x Diet x

Diet Time Time

NS *** NS NS NS NS 2 6.44 6-37 6.11 6-52 6.42 6.24 o.21 23 6.64 6.19 5.78 6.66 6.16 6.16

' Regrowth : cocksfoot, second cycle, six weeks; late harvested : natural grasslands, first cycle, full ear emergence. NS, non-significant; *** significant at P < 0.001.

tively 208 vs 23 pmol reducing sugars g-' DM h-', 26.8 vs 3.5 and 9.7 vs 4.9 mmol p-nitrophenol g-' DM h- for xylanase, B-D-xylosidase and fl-D-galactosidase, 12.1 vs 4.3 pmol reducing sugars g-' DM h-', 4.8 vs 2.4 and 18-1 vs 8.7 mmol p-nitrophenol g-' DM h-' for avicelase, B-D-cellobiosidase and B-D-glucosidase. Also, the same trends concerning the effects of both diet and quality of hay on enzyme activities were observed at 2 and 23 h, but these effects were more marked for the longer incubation time. Thus, only activities extracted from 23 h bag residues are reported in this paper.

Irrespective of the diet, enzyme activities in SAM were similar (P > 0.05) for the two hays (Table 5), except for the poorly expressed fl-D-galactosidase which was higher for the regrowth than for the late harvested hay (11.6 vs 7.7 mmol p-nitrophenol g-' DM h-' on average for the three diets).

Barley supplementation induced large modifications in SAM enzyme activities that varied according to the enzyme, polysaccharidase or glycosidase. Irrespective of the hay, polysaccharidase activities were similar for diets Bo and B,, (P > 0.05), and were threefold lower

(P < 0.01) with the high-concentrate diet. A high linear correlation, similar for the regrowth and the late harvested hay, was observed between xylanase and avicelase activity (R2 = 0.88). All /h-glycosidase activities were increased (50% in mean) by a moderate level of supplementation, and a lower decrease compared to polysaccharidases was measured between 30 and 60% barley in the diet.

DISCUSSION

Measurement of fibrolytic enzyme activity in the solid- associated microbial population has been shown to be a sensitive method to investigate changes in rumen flora and their activities that result from changes in the rumen environment (Silva et a f 1987; Huhtanen and Khalili 1992; Martin and Michalet-Doreau 1995). There, this method was developed to investigate the mechanisms involved in the associative effects between forage and concentrate, as a function of the level of starch in the diet and the quality of forage cell walls.

TABLE 5 Effect of barley supplement and of nature of hap on polysaccharidases and B-D-glycosidases activities (g-' DM h-I) in

solid-adherent microorganisms (SAM) isolated from hays bag residues after 23 h incubation time in the rumen

Enzymes Regrowth Late harvested SE Effectb

B O B30 B60 BO B 3 0 B60 Hay Diet Hay x Diet

Poly saccharidases (pmol reducing sugars)

Xylanase 25 1 23 1 69 274 301 120 80 NS ** NS Avicelase 15.7 14.3 5.9 13.5 17.5 5.2 5.0 NS ** NS

B-D-Gl ycosidases (mmol p-nitrophenol)

B-D-Cellobiosidase 4.7 7.3 4.8 3.5 5.2 3.3 2.1 NS NS NS NS B-D-Galactosidase 9.6 15.7 9.5 6.1 11.1 5.9 2.1 ** **

B-D-Xylosidase 21.7 38.4 18.7 18.1 43.2 20.5 8.8 NS ** NS B-D-Glucosidase 18.1 27.9 13.3 13.1 24.3 11.7 3.2 NS *** NS

Regrowth : cocksfoot, second cycle, s ix weeks; late harvested : natural grasslands, first cycle, full ear emergence. NS, non-significant; ** significant at P < 0.01; *** significant at P < 0.001.

240 4 P Nozikre et a1

Increase in the proportion of barley in the diet induced a non-linear decrease in polysaccharidase activities in SAM. This decrease was similar for xylanase and avicelase, suggesting, in agreement with Stewart and Bryant (1988), that most of the cellulolytic microbial population is also able to degrade hemicelluloses. Modifications of B-glycosidase activities were different: when the level of concentrate was moderate (30%), these activities were stimulated, and between 30 and 60% barley in the diet, the depressing effect was less marked than for the polysaccharidases. These variations in SAM enzyme activities may be the result of a shift of the microbial population and/or activity away from cell wall degradation towards starch utilisation.

Unlike polysaccharidases, which are presumably restricted to microorganisms intimately involved in fibre degradation, B-D-glycosidases are widely represent- ed in the ruminal microbial population (Stewart and Bryant 1988). The peak of SAM fl-glycosidase activities observed with 30% barley in the diet may thus be related to increased colonisation of forages with a lower fibrolytic/non-fibrolytic microorganisms ratio in the adherent population, as observed in situ (Silva et al 1987) and in oitro (Firkins et a1 1991).

The acidification and the soluble carbohydrates released from degradation of high amounts of starch may inhibit the attachment of microorganisms to feed particles (Pel1 and Schofield 1993). The drop in pH inside the bags to a value near 6.00 for high-concentrate diet may be responsible for a marked decrease in the proportion of fibrolytic microorganisms, due to the high susceptibility of the major fibrolytic rumen bacteria to acidification (Russell and Dombrowski 1980; Slyter 1986). Competition between fibrolytic and amylolytic bacteria for nitrogen and other 'necessary nutrients (Firkins et al 1991), and shifts in substrate utilisation by the non-obligate fibrolytic bacteria to the more readily fermentable carbohydrates (Huang and Forsberg 1990) may also be involved in the depression in the fibrolytic potential within the SAM. Substrate preference may be related to catabolite regulation involving end-product

l o T . 1 a.

feedback mechanisms (Huang and Forsberg 1990; Teather and Ohmiya 1991). The B-glycosidase activities were depressed to a lesser extent than were polysaccharidases. This could be similarly explained by the wider representation of microorganisms able to degrade oligosaccharides within the ruminal microbial population (Stewart and Bryant 1988).

The barley supplementation induced a depression in polysaccharidase activities in SAM, along with a decrease in cell wall polysaccharide degradation rate, except when this degradation rate was already very low. A nonlinear relationship (R2 > 0.95) was observed between the polysaccharide degradation rate and the corresponding polysaccharidase activity (Fig 1). The other enzymes, which break down oligosaccharides, have no specificity for the fibre-digesting population and therefore would not be expected to correlate with fibrolytic activity. For the diets including 0 or 30% barley, a marked variability in polysaccharidases activities without significant variations in degradation rate was observed between animals. Thus, the microbial fibrolytic activity did not seem to limit the degradation rate of cell wall components with these high-forage feeding conditions. On the other hand, the decrease in fibrolytic activity caused by high-concentrate supplement induced an important decrease of degradation rate. Similarly, with high-concentrate supplementation, Silva et a1 (1987), Huhtanen and Khalili (1992) and Martin and Michalet-Doreau (1995) observed a high decrease in fibrolytic activity of microorganisms together with a decrease in forage degradation rate, compared with high-forage diet. Although modifications in SAM activities were similar for both hemicellulase and cellulase, and for both regrowth and late harvested hays in our trial, the decrease in forage degradation rate was more marked for hemicelluloses than for cellulose, and for the regrowth than for the late harvested hay. Thus, although the decrease in the degradation rate of cell wall polysaccharides can be related to a decrease in enzyme activities in SAM, the differences between forages and between polysaccharides in response to

0 100 200 300 400 500

xylanase activity

O Y 0 5 10 I5 20 25

avicelase activity

Fig 1. Adjustment of the relationship between polysaccharidase activities (pmol reducing sugars g- DM h- I) in solid-associated microorganisms and in sacco degradation rate (% h-l) of cell wall polysaccharides for 0 , the regrowth and A, the first cycle late

harvested hay.

In sacco cell wall degradation and microbial Jibrolytic actioity 24 1

barley supplementation may not be attributed to differences of microbial activities. Intrinsic characteristics of the forage cell walls may be implicated and would therefore limit the effect of modifications in microbial activity on their degradation rate.

Ferulate esters were more rapidly degraded than p- coumarate esters. This agrees with previous studies both in uiuo or in oitro (review of Besle et a1 1994). According to Borneman and Akin (1990), feruloyl esterases are more active than p-coumaroyl esterases in several bacteria and fungi. In addition, ferulic acid linked to hydro- phylic arabinoxylans is probably more accessible to microbial enzymes than p-coumaric acid, which is linked to the hydrophobic lignin.

The depressive effect of barley on phenolic acid disappearance was stronger for the regrowth than for the late harvested hay and for ferulic than for p-coumaric acid. These results are similar to those observed for polysaccharide degradation rates. Hence for all cell wall components, the effect of barley supplement on degradation may depend not only on the extent of decrease in microbial activity but also on the accessibility of cell wall components to the enzymes.

Lignin acts as a physical barrier that limits the pen- etration of the enzymes in the cell walls and thus their accessibility for cell wall components (Chesson 1993; Jung and Deetz 1993). In agreement with Jarrige (1963), lignin content was lower in the first vegetation cycle late harvested hay than in the regrowth (98 vs 137 g kg-' NDF, respectively). Hence the amounts of lignin cannot explain the differences in response between the two hays. Conversely, the cell wall fraction from the late harvested hay contained more esterified p-coumaric acid than that from the regrowth. This may be related to differences in grass species, but variations in p- coumaric content between several temperate grasses at similar stages are small (Hartley 1984). It is more probably related to greater stemfleaves and greater thick-/ thin-cell wall tissue ratios (Jung and Castler 1990; Grabber and Jung 1991) in the first vegetation cycle late harvested hay than in the cocksfoot regrowth, mostly composed of leaves. Thus the higher p-coumaric content in the late harvested hay reflects the presence of struc- tures less accessible to microbial enzymes than in the regrowth. This would thus explain why the decrease in microbial activity had less impact on the late harvested hay than on the regrowth. Also, if p-coumaric content is related to the syringyl proportion in lignin deposited in the secondary wall, as suggested Ralph et a1 (1994) for maize, it would reflect an effect of lignin composition or structure on degradation.

CONCLUSION

The relationship between SAM polysaccharidase activity and cell-wall polysaccharide degradation rate shows

the existence of a threshold activity value above which variations in microbial activity no longer induce variations in degradation rate. However, with high barley supplement, decrease in degradation rate appears closely related to variations in polysaccharidase activities. The differences in response between forage or cell wall components are evidently not related to microbial aspects, but rather to intrinsic characteristics of the forage. The impact of variations in microbial activity on cell wall degradation may be greater for the more accessible components.

ACKNOWLEDGEMENTS

This research was supported by UCAAB 50%. The authors wish to thank R Bergeault, M A Bernard- Vialhe, M Dematteo, L Genestoux, L L'Hotelier and M P Maillot for their precious help.

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effect of barley supplement on microbial fibrolytic enzyme activities and cell wall degradation rate...

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