Effects of enzyme application method on in vitro rumen fermentation of tropical forages*

The objective of this study was to investigate the effect of method of delivery of a solution contaning cellulase on the in vitro rumen fermentation (24 h) of three tropical forages. Enzyme was applied to forages either at the time of incubation or 24 h before. Both cellulase treatments increased (P<0.05) acetate, propionate and total VFA production, as well as neutral-detergent fi bre degradability (NDFD) with all forages. NDFD increased signifi cantly (P<0.05) with all forages when 24 h pre-treatment of forage with the enzyme was allowed, and this pre-treatment also tended to increase (P<0.05) gas production with two of the three forages. The results indicate that 24 h pre-treatment of forages with cellulase could increase the effi cacy of this enzyme, but the incubated forage affects the results.


INTRODUCTION
In the last years many studies have explored the possibility of improving the nutritive value of forage for ruminants by using exogenous enzymes.However, results have been highly variable, and these inconsistencies could be attributed to differences in crude enzyme preparations, type of diets fed to the animals and enzyme application methods (Beauchemin et al., 2003).Some studies (Lewis et al., 1997;Hong et al., 2003) have compared methods of enzyme application, but results were variable.The aim of this study was to evaluate the effects of two application methods on the in vitro rumen fermentation of three tropical forages.

MATERIAL AND METHODS
One sample of Kikuyo grass and two samples of Angleton grass were fermented in vitro with buffered rumen fl uid.Samples of 500 mg of each forage were accurately weighed into 120 ml serum bottles.One solution of cellulase from Trichoderma longibrachiatum (Fluka Chemie GmbH) containing 5 international units (IU) per ml was prepared in 0.1 M sodium phosphate buffer (pH=6.5).Two ml of the solution were added directly to each bottle either 24 h before incubation (E24) or just at time of incubation (E0).Bottles dosed 24 h before incubation were kept at room temperature (21-23ºC), and 2 ml of buffer were added to bottles corresponding to control treatment.Rumen fl uid was obtained from four rumen-cannulated Merino sheep fed medium-quality lucerne hay ad libitum.Rumen fl uid was 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.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 for each treatment were included to correct the gas production values for gas release from endogenous substrates and enzyme treatment.After 24 h of incubation total gas production was measured with a calibrated syringe using a pressure transducer; bottles were then uncapped, the pH was immediately measured and one ml of the bottle content was taken for volatile fatty acid (VFA) analyses.The content of the bottles was transferred to previously weighed fi lter crucibles and the residues of incubation were analysed for neutral-detergent fi bre (NDF) to estimate fi bre degradability (NDFD).
Data relative to fermentation parameters were analysed for each forage by ANOVA with enzyme treatment (control, E24 and E0) and rumen inocula as main factors.The sums of squares were further partitioned by orthogonal polynomial contrasts to analyse differences among treatments, and the contrasts were distributed as follows: 1. control vs both enzyme treatments, and 2. E24 vs E0.The GLM procedures of SAS (SAS Inst., Inc., Cary, NC) were used for all statistical analyses.

RESULTS
The 24 h pre-treatment of forages with cellulase decreased (P<0.05) the NDF content of all forages (Table 1).Acid-detergent fi bre content of forages was decreased (P<0.05) by this enzyme pre-treatment with Kikuyo grass and a trend (P<0.10) was observed with Angleton grass-1.
The treatment with cellulase enzyme increased (P<0.05)acetate, propionate and total VFA production, as well as NDFD, with all forages.Gas production was increased (P<0.05) by cellulase treatment with Kikuyo grass and Angleton grass-1 and a trend (P<0.10) to a higher gas production was observed with Angleton grass-2.Enzyme application method did not affect (P>0.05)VFA production with Kikuyo grass and Angleton grass-1, but pre-treatment of Angleton grass-2 with cellulase increased (P<0.05)acetate production.NDFD increased signifi cantly (P<0.05) with all forages when a pre-treatment of forage with the enzyme was allowed, and this pre-treatment also tended to increase (P<0.05)gas production with both Angleton grass forages.

DISCUSSION
The observed effects of 24 h pre-treatment with cellulase on the forages NDF content suggest that an hydrolytic action prior to rumen incubation took place.These results agree with those reported by Nsereko et al. (2000), who studied the effects of 2 h pre-treatment with several fi brolytic enzymes on fi bre concentration of lucerne hay and observed that some of the enzymatic preparations produced a decrease on the NDF content.In agreement with our results, these authors reported that 2 h pre-treatment with several fi brolytic enzymes increased (P<0.05)NDF degradation of lucerne hay at 12 h when compared to 0 h pre-treatment.
Compared to E0 treatment, E24 increased total VFA production by 4.7 and 6.5% for Angleton grass-1 and Angleton grass-2, respectively, but a 3.3% decrease was observed for Kikuyo grass.Hong et al. (2003) obtained no differences in VFA concentrations in the rumen of goats when comparing the effects of 24 h enzyme pre-treatment of a mixed diet with the addition of enzyme to the diet just before feeding the animals, but observed an increase in NDF digestibility when the diet was pre-treated with the enzyme.A lack of effect on rumen VFA concentrations has also been found by Lewis et al. (1996) in steers fed a 70:30 grass hay:barley diet.

CONCLUSIONS
The results seem to indicate that 24 h pre-treatment of forages with a cellulase solution could be more effective in enhancing forage in vitro degradability than the addition of the enzyme to forage just prior to incubation, but the results could be affected by the incubated forage.

Table 2 .
Infl uence of application method of a cellulase preparation on fi nal pH, volatile fatty acid production (VFA; µmol), gas production (ml/500 mg sample) and neutral-detergent fi bre degradability (NDFD; %) after incubating three forages with buffered rumen fl uid for 24 h

Table 1 .
Infl uence of 24 h pre-treatment of tropical forages with a cellulase preparation (E24) on their neutral-detergent fi bre (NDF) and acid-detergent fi bre (ADF) content, g/kg DM