| Livestock Research for Rural Development 19 (12) 2007 | Guide for preparation of papers | LRRD News | Citation of this paper |
Batch cultures of mixed rumen microorganisms were used to study the effects of exogenous fibrolytic enzymes on the in vitro fermentation of a substrate composed by a 60:40 mixture of grass hay and concentrate. Three enzymatic preparations were tested: cellulase from Aspergillus niger (CEL), xylanase from Trichoderma viride (XYL), and a 1:1 mixture cellulase:xylanase (MIX). Samples of substrate (500 mg dry matter (DM)) were accurately weighed into 120-ml serum bottles and enzymes were added directly into the bottles at three levels: 0 (control; CON), 15 (-15) and 30 (-30) IU/g DM substrate. Bottles were kept at room temperature for 24 h before incubation with buffered rumen fluid.
All enzymatic treatments, with the exception of CEL-15, increased (P<0.05) CH4 production after 6 h of incubation, but after 24 h of incubation differences (P<0.05) with control were only detected for XIL-15, XIL-30 and MIX-30. Acetate, propionate and butyrate productions at both 6 and 24 h of incubation were increased (P<0.05) by all enzymatic treatments. NH3-N concentrations, pH and gas production at 24 h of incubation were not affected (P>0.05) by the addition of enzymes. The treatment of substrate with XIL-15, MIX-15 and MIX-30 increased (P<0.05) neutral-detergent fibre degradability (61.3, 59.9 and 60.6%, respectively) compared to the control (56.5%).
The results indicate that the xylanase was more effective than the cellulase in stimulating the in vitro fermentation of substrate, but for most of the measured parameters there were no differences (P>0.05) due to the level of enzymes (15 vs. 30 IU/g DM).
Key words: cellulase, digestibility, in-vitro gas production, volatile fatty acids, xylanase
Cellulose and hemicellulose are quantitatively the most important structural carbohydrates present in forages and rumen microorganisms produce enzymes that catalyse their hydrolysis. However, the complex network formed by structural carbohydrates and lignin reduce the digestibility of these carbohydrates and restricts efficient utilization of forages by ruminants. Many attempts have been made to overcome this limitation, and in the last years the use of enzymes has received considerable attention. Results, however, have been highly variable. Exogenous fibrolytic enzymes might enhance attachment, and/or improve access to the cell wall matrix by rumen microorganisms and by doing so, accelerate the rate of digestion (Nsereko et al 2000). The effects of enzymes are influenced by factors such as type and dose of enzyme, type of diets fed to the animals and enzymes application methods (Beauchemin et al 2003). The objective of this study was to evaluate the effects of two doses of three fibrolytic enzymes on the in vitro rumen fermentation of one substrate containing 60% of grass hay.
The substrate (dry matter (DM) basis) was composed of grass hay (60%) and a commercial concentrate (40%) based on barley, maize, soybean meal and a vitamin-mineral mixture. Crude protein, neutral-detergent fibre (NDF) and acid-detergent fibre content of substrate was 151, 470 and 213 g/kg DM, respectively. Samples of 500 mg of ground substrate (1-mm screen) were accurately weighed into 120-ml serum bottles.
Three different enzymes preparations were tested: cellulase from Aspergillus niger (CEL; Cellulase 22178; Fluka Chemie GmbH), xylanase from Trichoderma viride (XYL; Xylanase 95595; Fluka Chemie GmbH), and a 1:1 mixture cellulase:xylanase (MIX). Enzyme preparations were added directly into the bottles at three levels: 0 (control; CON), 15 (-15) and 30 (-30) international units (IU)/g DM substrate. Solutions of each enzyme preparation containing either 3.75 or 7.50 units 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. This pre-treatment of samples with enzymes was selected as previous in vitro studies (Giraldo et al 2004a) have shown that this enzyme-feed interaction increased the efficacy of enzymes. Two ml of 0.1 M sodium phosphate buffer were added to the bottles corresponding to control treatment.
Rumen fluid was obtained from four rumen-cannulated Merino sheep fed medium-quality grass hay ad libitum and supplemented daily with 400 g of concentrate. Rumen contents of each sheep were obtained before the morning feeding, mixed and strained through four layers of cheese-cloth. Particle-free fluid was mixed with the buffer solution of Goering and Van Soest (1970) in a proportion 1:4 (vol:vol) at 39 ºC under continuous flushing with CO2. Fifty ml of buffered rumen fluid were added into each bottle. Bottles were sealed with rubber stoppers and aluminium caps and incubated at 39 ºC for 24 h. Four incubation runs were performed on different days and in each of them two bottles per treatment were included. Two blanks per treatment (bottles without substrate but with the corresponding enzymatic solution added) were included in each incubation run to investigate the effects of enzyme fermentation itself on gas and volatile fatty acids (VFA) production. After 6 hours of incubation, total gas production was measured in all bottles using a pressure transducer and a calibrated syringe, and a gas sample (about 15 ml) from each bottle was stored in an evacuated tube holder (Venoject®, Terumo Europe, Belgium) before analysis for CH4 concentration. (Gas production technique, Theodorou et al 1994),
Two ml of bottle contents were sampled for VFA and NH3-N analyses. Bottles were withdrawn from the incubator 24 h after inoculation, gas production was measured and a gas sample for CH4 determination was taken as before described. Bottles were then uncapped, the pH was immediately measured and the fermentation was stopped by swirling the bottles in ice. Two ml of the bottle content were taken for VFA and NH3-N analyses. Finally, the content of the bottles was transferred to previously weighed filter crucibles, the solid residue was washed with 50 ml of hot distilled water (50ºC) and the crucibles were dried at 50ºC for 48 h to estimate DM apparent degradability (DMAD). Residues were analysed for NDF and acid-detergent fibre (ADF) to estimate fibre degradability (NDFD and ADFD). Procedures for analysis of VFA, CH4 and NH3-N have been described by Carro et al (1999).
The amounts of VFA produced were obtained by subtracting the amounts present initially in the incubation medium from those determined at the end of the incubation period. Data were analysed as an ANOVA with seven enzymatic treatments (CON, CEL-15, CEL-30, XYL-15, XYL-30, MIX-15 and MIX-30) and four rumen inocula as main effects. When a significant effect of the treatment (P<0.05) were detected, differences between means were assessed by the LSD test.
The effects of the treatment with fibrolytic enzymes on in vitro production of CH4 and VFA, and NH3-N concentrations after 6 hours of incubation of substrate samples with rumen mixed microorganisms are shown in Table 1.
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Table 1. Influence of different enzymatic treatments1 on production of gas (ml/500 mg substrate), CH4 (µmol) and volatile fatty acids (VFA; µmol), acetate/propionate (Ac/Pr) and CH4/VFA ratios and NH3-N concentration (mg/l) after in vitro fermentation of a substrate containing 60% of grass hay in batch cultures of mixed rumen microorganisms for 6 h (n=8) |
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|
Item |
CON |
CEL-15 |
CEL-30 |
XYL-15 |
XYL-30 |
MIX-15 |
MIX-30 |
s.e.d.2 |
|
Gas |
44.6 |
45.9 |
44.9 |
47.0 |
46.6 |
47.3 |
45.7 |
1.20 |
|
CH4 |
94.6 a |
105 ab |
109 bc |
128 d |
127 cd |
119 bcd |
122 cd |
6.90 |
|
Total VFA |
1031 a |
1176 b |
1177 b |
1263 c |
1220 bc |
1219 bc |
1198 b |
27.7 |
|
Acetate |
643 a |
726 b |
726 b |
781 c |
761 bc |
755 bc |
742 b |
17.54 |
|
Propionate |
267 a |
316 b |
315 b |
339 c |
326 bc |
330 bc |
318 b |
7.63 |
|
Butyrate |
91.6 a |
99.9 b |
100 bc |
108 c |
102 bc |
101 bc |
102 bc |
4.01 |
|
Other VFA3 |
30.2 a |
33.7 abc |
36.3 c |
34.7 bc |
31.4 ab |
33.1 abc |
35.4 bc |
2.02 |
|
Ac/Pr |
2.41 b |
2.30 a |
2.30 a |
2.30 a |
2.33 a |
2.29 a |
2.33 a |
0.04 |
|
CH4/VFA |
0.091 |
0.090 |
0.092 |
0.101 |
0.105 |
0.097 |
0.101 |
0.0086 |
|
NH3-N |
179 |
184 |
179 |
175 |
178 |
179 |
177 |
4.6 |
|
1 Treatments, CON: control, CEL-15 and CEL-30: cellulase at 15 and 30 IU/g DM substrate, respectively; XYL-15 and XYL-30: xylanase at 15 and 30 IU/g DM substrate, respectively; MIX-15 and MIX-30: cellulase:xylanase mixture (1:1) at 15 and 30 IU/g DM substrate, respectively. 2 standard error of the difference. 3 calculated as the sum of isobutyrate, isovalerate and valerate acids. a, b, c, d : mean values within a row with unlike superscript letters differ (P<0.05) |
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There were no effects (P>0.05) of enzymes either on gas production or on NH3-N concentration. All enzymatic treatments, with the exception of CEL-15, increased (P<0.05) CH4 production. Acetate, propionate and butyrate productions at 6 h of incubation were increased (P<0.05) by all enzymatic treatments and, as a consequence, total VFA production was increased (P<0.05) by 14, 14, 23, 18, 18 and 16% for CEL-15, CEL-30, XYL-15, XYL-30, MIX-15 and MIX-30 treatments, respectively. Compared to the buffer treated substrate, all enzymatic treatments decreased (P<0.05) the acetate/propionate ratio, but had no effect (P>0.05) on the CH4/VFA ratio.
As shown in Table 2, no effects (P>0.05) of any enzymatic treatment were observed on final pH and gas production after 24 h of incubation. In agreement with the results obtained at 6 h of incubation. There was no significant change (P>0.05) in the NH3-N concentration with added enzymes, thus indicating no differences in protein degradability and/or NH3-N incorporation by rumen microorganisms.
|
Table 2. Influence of different enzymatic treatments1 on final pH, production of gas (ml/500 mg substrate), CH4 (µmol) and volatile fatty acids (VFA; µmol), acetate/propionate (Ac/Pr) and CH4/VFA ratios, NH3-N concentration (mg/l), dry-matter apparent degradability (DMAD; %) and neutral- and acid-detergent fibre degradability (NDFD and ADFD, respectively; %) of a substrate containing 60% of grass hay incubated in batch cultures of mixed rumen microorganisms for 24 h (n=8) |
||||||||
|
Item |
CON |
CEL-15 |
CEL-30 |
XIL-15 |
XIL-30 |
MIX-15 |
MIX-30 |
s.e.d.2 |
|
pH |
6.58 |
6.59 |
6.57 |
6.56 |
6.58 |
6.56 |
6.57 |
0.016 |
|
Gas |
113 |
116 |
114 |
116 |
113 |
116 |
111 |
2.8 |
|
CH4 |
387 a |
392 ab |
401 abc |
420 bc |
410 abc |
399 abc |
423 c |
4.4 |
|
Total AGV |
2496 a |
2703 bc |
2692 b |
2762 c |
2697 bc |
2735 bc |
2708 bc |
33.9 |
|
Acetate |
1506 a |
1631 b |
1628 b |
1645 b |
1609 b |
1642 b |
1620 b |
18.7 |
|
Propionate |
591 a |
654 bc |
645 b |
673 c |
648 b |
660 bc |
648 b |
11.6 |
|
Butyrate |
295 a |
309 b |
312 bc |
326 c |
322 bc |
318 bc |
325 c |
7.4 |
|
Other VFA |
103 a |
108 ab |
106 a |
116 c |
117 c |
113 bc |
115 c |
3.5 |
|
Ac/Pr |
2.56 b |
2.50 ab |
2.55 b |
2.46 a |
2.49 ab |
2.49 ab |
2.51 ab |
0.042 |
|
CH4/VFA |
0.155 |
0.145 |
0.149 |
0.152 |
0.151 |
0.145 |
0.156 |
0.0072 |
|
NH3-N |
258 |
262 |
259 |
257 |
266 |
255 |
259 |
11.1 |
|
DMAD |
60.9 a |
62.6 ab |
63.5 ab |
63.0 ab |
63.8 b |
63.8 b |
63.8 b |
1.32 |
|
NDFD |
56.5 a |
58.9 ab |
59.2 ab |
61.3 b |
60.3 b |
59.9 b |
60.6 b |
1.66 |
|
ADFD |
50.1 a |
53.3 ab |
53.4 ab |
56.2 b |
53.5 ab |
54.9 ab |
54.7 ab |
2.50 |
|
1 Treatments, CON: control, CEL-15 and CEL-30: cellulase at 15 and 30 IU/g DM substrate, respectively; XYL-15 and XYL-30: xylanase at 15 and 30 IU/g DM substrate, respectively; MIX-15 and MIX-30: cellulase:xylanase mixture (1:1) at 15 and 30 IU/g DM substrate, respectively. 2 standard error of the difference. 3 calculated as the sum of isobutyrate, isovalerate and valerate acids. a, b, c : mean values within a row with unlike superscript letters differ (P<0.05). |
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Although all enzymatic treatments (with the exception of CEL-15) increased CH4 production after 6 h of incubation, most of the differences disappeared after 24 h and only XYL-15 and MIX-30 treatments increased (P<0.05) CH4 production (by 8.5 and 9.3 %, respectively). However, no effects (P>0.05) of enzymatic treatments on the CH4/VFA ratio were detected. In agreement with the results observed at 6 h of incubation, all enzymatic treatments increased (P<0.05) the production of acetate, propionate and butyrate. Total VFA production was increased by 8.3, 7.9, 10.7, 8.1, 9.6 and 8.5% for CEL-15, CEL-30, XYL-15, XYL-30, MIX-15 and MIX-30 treatments, respectively. These results would indicate that added enzymes stimulated the in vitro fermentation of substrate. The effects of enzymes were more marked at 6 than at 24 h of fermentation, thus indicating that enzymes produced their effects at early stages of fermentation.
The treatment of substrate with CEL-15, CEL-30, XYL-15, XYL-30, MIX-15 and MIX-30 increased NDFD by 4.2, 4.8, 8.5, 6.7, 6.0 and 7.3%, respectively, when compared to CON, but the difference did not reach the significance level (P>0.05) for CEL-15 and CEL-30 treatments. Effects of enzymes on ADFD were only evident for XYL-15, which increased ADFD by 12.2% (P<0.05). For most of the measured parameters, there were no effects (P>0.05) of level of enzyme (15 vs. 30 IU/g DM) and no interaction (P>0.05) enzymatic treatment x level of enzyme was detected.
Compared to the 24-h buffer treated substrate, all enzymatic treatments decreased (P<0.05) substrate NDF content (see Table 3), reductions ranging from 19 to 53 g NDF/kg DM. On the contrary, no effects (P>0.05) of enzymatic treatments on substrate ADF content were detected.
|
Table 3. Influence of 24 h pre-treatment of a substrate containing 60% of grass hay with different enzymatic treatments1 on its neutral- (NDF) and acid-detergent fibre (ADF) content (n=4) |
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|
Item |
CON |
CEL-15 |
CEL-30 |
XIL-15 |
XIL-30 |
MIX-15 |
MIX-30 |
s.e.d.2 |
|
NDF (g/kg DM) |
499 d |
461 ab |
448 ab |
480 c |
466 bc |
446 a |
455 ab |
0.89 |
|
ADF (g/kg DM) |
271 |
270 |
263 |
273 |
271 |
263 |
269 |
0.63 |
|
1 Treatments, CON: control, CEL-15 and CEL-30: cellulase at 15 and 30 IU/g DM substrate, respectively; XYL-15 and XYL-30: xylanase at 15 and 30 IU/g DM substrate, respectively; MIX-15 and MIX-30: cellulase:xylanase mixture (1:1) at 15 and 30 IU/g DM substrate, respectively. 2 standard error of the difference. a, b, c, d : mean values within a row with unlike superscript letters differ (P<0.05) |
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These results would indicate the effects of enzymes were more marked at 6 than at 24 h of fermentation that enzymes produced their effects at early stages of fermentation. Dawson and Tricario (1999) also reported that fibrolytic enzyme effects in vitro were generally larger during the initial stages of degradation.
Previous studies (Wang et al 2001; Giraldo et al 2004a) have shown that a pre-treatment of feed with enzymes before feeding or incubation with ruminal fluid enhanced the beneficial effects of enzymes on ruminal fermentation. As pointed out by Colombatto et al (2003a), some authors have suggested that this could be due to the creation of a stable enzyme-feed complex (Kung et al 2000), but others have indicated the possibility of alteration in the fibre structure, which would stimulate microbial colonization (Newbold 1997; Giraldo et al 2007). In addition, an increase in the initial rate of gas production produced by enzyme treatment of forages has been previously reported (Wallace et al 2001; Colombatto et al 2003b; 2007; Carro et al 2005)
Several authors (Nsereko et al 2000; Wallace et al 2001, Giraldo et al 2004b) have suggested that exogenous enzymes could increase fibre degradation through a hydrolytic action prior to feeding or to incubation with rumen microorganisms. To test this hypothesis we decided to analyse the effects of the 24 h pre-treatment with enzymes on the NDF and ADF content of substrate. These results would indicate that the 24 h pre-treatment with enzymes altered the fibre structure, as previously reported in other studies (Nsereko et al 2000; Colombatto et al 2003c).
The 24 h pre-treatment of a substrate containing 60% of grass hay with fibrolytic enzymes increased in vitro NDF degradation and production of VFA by mixed rumen microorganisms. There were, however, differences between enzymes in their effects, but for all enzymes the effects were more marked at 6 than at 24 h of fermentation.
These results therefore indicate that enzymes can enhance the degradation at early stages of fermentation, probably by altering the fibre structure during the pre-incubation substrate/enzyme interaction and making it more amenable to rumen microorganisms.
Further animal trials are required to test the effect of supplementation of fibrolytic enzymes on the feed intake and performance of ruminant animals.
The authors wish to acknowledge the financial support received from the M.C.Y.T. of Spain (Project AGL2001-0130) and the Excma. Diputación Provincial de León.
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Received 11 March 2007; Accepted 5 October 2007; Published 12 December 2007