Effect of exogenous fi brolytic enzymes on in vitro rumen fermentation of tropical forages*

The aim of the study was to evaluate the effects of exogenous fi brolytic enzymes on the in vitro ruminal fermentation of three tropical forages (Kikuyo grass 1 and 2 and Angleton grass). Three different enzyme preparations were tested: cellulase (CEL), xylanase (XYL) and a 1:1 mixture cellulase:xylanase (MIX). Dry matter disappearance was increased (P<0.05) by CEL and MIX for Kikuyo grass 1. The treatment of forages with CEL and MIX increased (P<0.05) NDF degradability, except for Kikuyo grass 2. CEL treatment also increased (P<0.05) total VFA production of Kikuyo grass 2 and Angleton grass. The results indicate that the treatment of tropical forages with cellulase stimulate their in vitro ruminal fermentation, but the xylanase enzyme used did not produce any positive effect.


INTRODUCTION
Dietary fi bre is an important energy source for ruminants.However, its digestion in the rumen is slow and incomplete.Preparations of exogenous cell wall-degrading enzymes, such as cellulases and xylanases, have the potential to hydrolyse forage fi bre, thus improving the digestion of some ruminant feedstuffs (Beauchemin et al., 2003).Most of studies have been conducted with enzyme-treated good-quality forages, but data involving application of exogenous enzymes to fi brous feeds are much limited.The objective of this study was to evaluate the effects of exogenous fi brolytic enzymes on the in vitro ruminal fermentation of three tropical forages.

MATERIAL AND METHODS
Samples of three tropical forages (Kikuyo grass 1 and 2 and Angleton grass) were ground through a 1-mm screen and fermented in vitro with buffered rumen fl uid.The chemical composition of forages is given in Table 1.Samples of 500 mg of each forage were accurately weighed into 120-ml serum bottles.Three different enzyme preparations were tested: cellulase from Trichoderma longibrachiatum (CEL; Fluka Chemie GmbH), xylanase from rumen microorganisms (XYL; Megazyme International Ireland Ltd.), and a 1:1 mixture cellulase:xylanase (MIX).Solutions of each enzyme containing 5 IU per ml were prepared in 0.1 M sodium phosphate buffer, pH 6.5.Two ml of the corresponding solution were added directly to each bottle 24 h before starting the incubation, and bottles were kept at room temperature (21-23ºC) until incubation.Two ml of 0.1 M sodium phosphate buffer, pH 6.5 were added to bottles corresponding to control treatment.Rumen fl uid was obtained before the morning feeding from four rumen-cannulated Merino sheep fed medium-quality lucerne hay ad libitum, and mixed with a buffer solution in a proportion 1:4 (v:v) at 39ºC under continuous fl ushing with CO 2 .Bottles were prewarmed (39ºC) prior to the addition of 50 ml of buffered rumen fl uid into each bottle under CO 2 fl ushing.Bottles were sealed with rubber stoppers and aluminium caps and incubated at 39ºC for 24 h.Four incubation runs were performed on different days, so that each treatment was conducted in quadruplicate.In each incubation run, two blanks were included to correct the gas production values for gas release from endogenous substrates and enzyme treatment.Bottles were withdrawn from the incubator 24 h after inoculation and total gas production was measured with a calibrated syringe.Bottles were uncapped and the pH was measured immediately with a pH meter.One ml of the bottle content was added to 1 ml of deproteinizing solution (10% of metaphosphoric acid and 0.06% crotonic acid; w/v) for VFA analysis.The content of the bottle was then transferred to previously weighed fi lter crucibles and the residue of incubation was washed with 50 ml of hot distilled water and dried at 50ºC for 48 h to calculate DM apparent disappearance.Residues were analysed for NDF to estimate fi bre degradability.

FIBROLYTIC ENZYMES AND TROPICAL FORAGES
Data for each forage were analyzed by ANOVA with four treatments (control (C), CEL, MIX and XYL) and rumen inoculum as main factors.The GLM procedures of SAS (SAS Inst., Inc., Cary, NC) were used for all statistical analyses.

RESULTS AND DISCUSSION
The effects of exogenous fi brolytic ezymes on in vitro rumen fermentation of the three forages are shown in Table 2. Final pH was not affected (P>0.05) by added enzymes for any substrate.For Kikuyo grass 1, CEL and MIX treatments increased (P<0.05)gas production, compared to the control and to XYL treatment, but there were no differences (P>0.05) for the other two substrates.144.8 1 treatments, C -control, CEL -cellulase, MIX -cellulose:xylanase mixture (1:1), XYL -xylanase; a,b mean values within a row not sharing a common superscript letter were signifi cantly different, P<0.05 DM disappearance was increased (P<0.05) by CEL and MIX just for Kikuyo grass 1.Similarly, the treatment of forages with CEL and MIX increased (P<0.05)NDFD, compared to the control, except for Kikuyo grass 2. Similar results were obtained by Feng et al. (1992), who reported that treatment of dry GIRALDO L.A. ET AL. grass with fi brolytic enzymes improved in vitro ruminal fi bre digestion.Zinn and Salinas (1999) also showed that a fi brolytic enzyme supplement increased ruminal digestion of diet NDF.However, XYL had no effect (P>0.05) on cell wall degradability of any substrate.
It would be expected that, as degradation of a feedstuff increases, as indicated by DMD and NDFD, there would be a concomitant increase in the end-products of that fermentation (i.e.VFA).Compared to control, the treatment of Kikuyo grass 2 and Angleton grass with CEL increased (P<0.05) total VFA production.In line with our results, Lewis et al. (1996) reported that enzymes sprayed onto a grass hay:barley diet increased VFA production and NDF digestion.However, in the present experiment, XYL and MIX treatments had no effect (P>0.05) on VFA production for any substrate.These results, and those obtained for DMD and NDFD suggest that effectiveness of enzymes varies with the substrate and that the xylanase used in the present experiment did not contribute to ruminal fi brolytic activity.

CONCLUSIONS
Under the conditions of the present experiment, the treatment of three tropical forages with cellulase seemed to stimulate their in vitro ruminal fermentation, but the xylanase used did not produce any positive effect.Although these and other results demonstrate that exogenous fi brolytic enzymes may enhance ruminal utilization of fi brous diets, further study is warranted to investigate specifi c, optimal enzyme-substrate combinations.

Table 1 .
Chemical composition of forages incubated in vitro, g/kg dry matter