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ANIMAL NUTRITION |
* Departamento de Producción Animal, Universidad de León, 24071, Spain; and and
Universidad Nacional de Colombia, Sede Medellín, Facultad de Ciencias Agropecuarias, 1027, Medellín, Colombia
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: cellulase • ruminal fermentation • microbial protein synthesis • Rusitec
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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, 2004). The majority of these experiments were designed with the expectation that a fibrolytic enzyme should increase the degradability of feed in the rumen (McAllister et al., 2001; Phipps et al., 2002). However, a lack of effects, or even negative effects, has been observed (Nussio et al., 1997; McAllister et al., 2001). These inconsistencies could be attributed to differences in the crude enzyme preparations, the type of diets fed to the animals, and the enzyme application methods (Beauchemin et al., 2003). Exogenous fibrolytic enzymes might enhance the attachment of ruminal microorganisms or improve their access to the cell wall matrix and, by doing so, accelerate the rate of fiber digestion (Nsereko et al., 2000). Many of the studies with enzyme-treated forages have been conducted with commercially available enzymes, but limited data involving the application of enzymes or their mixtures are available. In addition, the effects of exogenous fibrolytic enzymes on ruminal microbial growth and methane production have not been investigated in depth.
The objective of this study was to evaluate the effects of 2 cellulases from fungal origin and a 1:1 mixture of both on microbial growth and ruminal fermentation of a high-forage substrate in Rusitec fermenters.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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. The inoculum (liquid and solid) was obtained from 4 ruminally fistulated sheep that were fed a 70:30 grass hay:concentrate (air-dried basis) diet. Sheep were managed according to the protocols approved by the León University Institutional Animal Care and Use Committee.
Solid and liquid fermentation inocula were collected from sheep immediately before feeding and transferred to the fermenters within 30 min (Carro et al., 1992). Flow through the fermenters was maintained by continuous infusion of McDougall (1948) artificial saliva (pH = 8.4) at a rate of 550 mL/d (dilution rate of 3.82%/h). On d 8, a dose of 2.2 mg of 15N (98% enriched 15NH4Cl; Tracer SA, Madrid, Spain) was added into each fermenter to instantaneously label the ammonia-N pool. From d 8 to 12, a solution of 15NH4Cl was added to the artificial saliva at a rate of 4.0 mg of 15N/L.
Each fermenter received 20 g of substrate DM daily, which was placed into nylon bags (100-µm pore size). The substrate consisted of 70% grass hay and 30% concentrate (DM basis) and contained 92.8% OM, 13.1% CP, 50.7% NDF, and 24.8% ADF (DM basis). The concentrate was based on barley, corn, soybean meal, and a mineral-vitamin premix in the proportions of 50:31:16:3 (air-dried basis). Grass hay was chopped (approximately 0.5-cm pieces), and the concentrate was ground through a 4-mm sieve. Both feed components were weighed independently and were carefully mixed before applying the experimental treatments.
The 4 experimental treatments were as follows: 1) no exogenous enzyme (control), 2) cellulase produced by Trichoderma longibrachiatum (TRI), 3) cellulase produced by Aspergillus niger (ASP), and 4) a 1:1 mixture of both enzymes (MIX). The enzyme treatments were selected based on the results obtained in previous in vitro studies (Giraldo et al., 2004). Both enzymes were powdered preparations from Fluka Chemicals (Seelze, Germany), and their endoglucanase (EC 3.2.1.4.) and exoglucanase (EC 3.2.1.91) activities were determined before use (Colombatto and Beauchemin, 2003).
Solutions (1%) of medium-viscosity carboxymethyl-cellulose (Sigma-Aldrich Química SA, Madrid, Spain) and Avicel PH-101 (Fluka Chemicals) were used as substrates for determination of endoglucanase and exoglucanase activity, respectively. The enzymes were dissolved in distilled water (2.5 mg/mL), and 50 µL was incubated in triplicate with 1 mL of substrate and 1 mL of buffer (sodium phosphate 1 mM, pH = 6.5) at 39°C for exactly 15 and 120 min for endoglucanase and exoglucanase determination, respectively. The enzymatic reaction was terminated by adding 3 mL of 3,5-dinitrosalicylic acid solution reagent and then placing the tubes into boiling water for 5 min. Absorbance was read at 540 nm against glucose standards (from 0 to 2 g/L) processed under identical conditions. Tubes for exoglucanase determination were centrifuged (1,000 x g, 10 min), and the absorbance of the supernatant was read at 540 nm (Ultrospec 500 pro, Amersham Biosciences, Munich, Germany). Tubes containing only buffer, buffer plus substrate, and buffer plus enzyme were also incubated in triplicate to correct for substrate autolysis and sugars present in the enzymes (Colombatto et al., 2003b). At pH 6.5 and 39°C, 1 mg of the cellulase from T. longibrachiatum liberated 1.53 and 0.040 µmol of glucose per minute from carboxymethylcellulose and Avicel PH-101, respectively. Under the same conditions, 1 mg of the cellulase from A. niger liberated 0.567 µmol of glucose per minute from carboxymethylcellulose, but no exoglucanase activity was detected.
All enzyme treatments were applied at a dose of 30 endoglucanase units per gram of substrate DM, with 1 endoglucanase unit being defined as the amount of enzyme required to release 1 µmol of glucose per minute from carboxymethylcellulose at 39°C and pH 6.5. The enzymes were dissolved daily in a sodium phosphate buffer solution (1 mM, pH = 6.5), and the solution (containing 30 endoglucanase units/mL) was carefully applied directly onto the substrate (1 mL/g of DM) using a manual sprayer. After spraying, the substrate was kept at room temperature (21 to 23°C) for 24 h before being placed into nylon bags and placed into the fermenters. This pretreatment of substrate with enzymes was selected, because previous studies (Wang et al., 2001; Giraldo et al., 2004) showed that an enzyme-feed interaction before incubation with ruminal fluid can enhance the beneficial effects of the enzymes on ruminal fermentation. Substrate for the control fermenters was sprayed with the corresponding amount of buffer solution without added enzyme.
Experimental Procedure and Sampling
Two identical 17-d incubation trials were carried out independently, and the treatments were assigned randomly within each trial so that 2 fermenters received each of the treatments; each treatment was, therefore, conducted in quadruplicate. After 10 d of adaptation, microbial growth was measured on d 11 and 12. During these days, 5 mL of saturated HgCl2 was added to the overflow flasks, which were held at 4°C in a water bath to impede microbial growth. For each fermenter, the daily liquid effluent was collected, mixed, and homogenized in a blender at low speed for 1 min. One sample (about 300 mL) was frozen and lyophilized for determination of DM, nonammonia N (NAN), and 15N enrichment. Approximately 500 mL of effluent was used for isolation of liquid-associated microbial pellets (LAM) by differential centrifugation (Ranilla and Carro, 2003). The contents of the nylon bags removed on d 11 and 12 were used to determine the growth of solid-associated microorganisms (SAM). About 20% of the solid content was frozen and lyophilized for determination of DM, NAN, and 15N enrichment. Solid-associated microorganism pellets were isolated after treating the remaining solid content with a saline solution (0.85% NaCl) of 0.1% methylcellulose (wt/vol) at 38°C for 15 min with continuous shaking. The residue was then filtered through 2 layers of nylon cloth (40-µm pore size), resuspended in a cold (4°C) saline solution of 0.1% methylcellulose, and chilled at 4°C for 24 h. The filtrate obtained each day was mixed and used to isolate SAM by differential centrifugation, as described by Ranilla and Carro (2003). Substrate was also analyzed for its natural 15N content, and this value was used for background correction before 15N infusion.
On d 11, four milliliters of fermenter fluid was taken before the addition of feed and immediately frozen at –80°C for determination of endoglucanase and exoglucanase activities. On d 13, 14, and 15, the pH of each fermenter fluid was determined immediately before the addition of feed and collection of the following samples. Liquid effluent was collected daily in flasks containing a solution of 3.66 M H2SO4 to maintain pH values below 2. Then, 1 mL of effluent was added to 1 mL of deproteinizing solution (10% of metaphosphoric acid and 0.06% crotonic acid; wt/vol) for VFA determination, and 5 mL of effluent was stored at –20°C for ammonia-N determination. One nylon bag from each fermenter was collected daily, washed twice with 40 mL of fermenter liquid, and then washed in the cold rinse cycle (20 min) of a washing machine. The DM apparent disappearance (DMAD) after 48 h of incubation was calculated from the loss in weight after oven-drying at 60°C for 48 h, and the residues were analyzed for NDF and ADF to estimate NDF and ADF disappearance (NDFD and ADFD, respectively).
On d 16 and 17, the daily amount of substrate administered to each fermenter was distributed into 3 nylon bags, 1 containing 10 g of DM and 2 containing 5 g of DM each. One bag containing 5 g was removed after 6 h of incubation and the other after 24 h, whereas the 10 g of DM bag remained for 48 h in the fermenters. The nylon bags were washed as described above, and the residues were analyzed for NDF and ADF. On d 16, four milliliters of the liquid obtained from the first washing of the bags incubated for 6 h was taken and immediately frozen at –80°C for determination of endoglucanase and exoglucanase activities.
Adaptative changes in the microbial population of the fermenters to each enzyme treatment were studied using the fluid from each fermenter as inocula for batch cultures. Final pH and production of VFA were measured as indicators of fermentative activity in batch cultures. The fermentative activity of the fluid was tested against 4 pure substrates (Sigma-Aldrich Química SA): oat spelt xylan (95590), cellulose (53504), pectin (P9185) from citrus peel, and a mixture of starch [40% wheat, 40% corn (54126), and 20% potato starch; 85649]. On the last day of each incubation trial, the 2 nylon bags present in each fermenter were removed, and their contents were emptied and mixed with the effluent. The mixture was homogenized for 30 s with a blender, filtered through 2 layers of nylon cloth (40-µm pore size), and the filtrate was mixed with that obtained from the fermenter belonging to the same enzyme treatment. Then, 880 mL of the final filtrate was mixed with 220 mL of artificial saliva (enriched with 472 mg of NH4Cl and 791 mg of trypticase per liter of saliva), and 50 mL of the final mixture was anaerobically (under continuous flushing with CO2) dispensed into 120-mL serum bottles containing 500 mg of one of the substrates described above. A total of 20 bottles (4 bottles for each substrate and 4 bottles without substrate) were incubated per each enzyme treatment in each incubation trial. The bottles were capped, incubated at 39°C, and gas production was measured after 3 and 6 h of incubation. The bottles were then opened, the pH was immediately measured, and samples for VFA determination were taken, as described above. For bottles containing cellulose, the incubation was for 9 h, and gas production was measured after 6 and 9 h of incubation.
Effects of Cellulases on Diet Chemical Composition
To investigate the effects of enzyme treatments on diet fiber composition, diet samples (500 mg) were weighed into artificial fiber bags (F57 bags; 50 x 40 mm; 25 ± 10 µm pore size; Ankom Technology Corporation, Fairport, NY), and 1 mL of buffer solution containing no enzyme (control) or 15 endoglucanase units of the corresponding enzyme solution was added into each bag. The bags were heat-sealed and kept at room temperature (21 to 23°C) for 24 h before NDF and ADF analyses were conducted. The complete procedure was repeated 5 times.
Analytical Procedures
Procedures for determination of DM, ash, N, NDF, ADF, VFA, and ammonia-N have been reported by Carro and Miller (1999). An Ankom220 fiber analyzer unit (Ankom Technology Corporation) was used for NDF and ADF analyses. The volume of gas produced was measured with a drum-type gas meter (model TG1, Ritter Apparatebau GmbH, Bochum, Germany), and the concentration of methane was analyzed by GLC (Carro and Miller, 1999). Preparation of samples for 15 N analysis followed the procedures described by Carro and Miller (1999), and analyses of 15N enrichment were performed by isotope ratio mass spectrometry (VG PRISM II, Middlewich, UK) connected in series to a Dumas-style N analyzer (model 1108, Carlo Erba Instruments, Milan, Italy).
For determination of enzymatic activities in ruminal fluid samples, the samples were thawed, and the cells were lysed using a Mini-Beadbeater (BioSpec Products Inc., Bartlesville, OK) to release intracellular enzymes. The treatment consisted of three 60-s pulses at 4°C using 0.5-mm glass beads. Unbroken cell material was removed by centrifugation (10,000 x g, 10 min, 4°C), and the supernatant (200 µL) was used to analyze endoglucanase and exoglucanase activities, as described above.
Calculations and Statistical Analyses
The proportion of digesta NAN (liquid or solid) of microbial origin was estimated for each fermenter by dividing the 15N enrichment (atoms % excess) of the NAN portion of the digesta by the enrichment of the corresponding microbial pellets (LAM or SAM). Daily microbial N production (mg/d, LAM or SAM) was estimated by multiplying the total NAN production in the corresponding digesta (liquid or solid) by the proportion attributed to the microbes. Total daily microbial production was calculated as the sum of the flows of LAM and SAM.
The volume of gas produced in the fermenters (L/d) was corrected for temperature (0°C) and pressure (1.013 x 105 Pa) conditions, and the amount of methane produced (mmol) was calculated by multiplying the gas produced by the methane concentration in the analyzed sample. The amounts of VFA produced in the batch cultures were obtained by subtracting the amounts present initially in the incubation medium from those determined at the end of the incubation period. For each sampling time, the values of gas production were corrected for the amount of gas produced in the bottles without substrate and that were inoculated with the fluid from the corresponding fermenters.
All analyses were conducted using the GLM procedure (SAS Inst. Inc., Cary, NC). Data relative to the fermentation variables were analyzed as a split-plot design, with enzyme treatment and incubation trial as the main-plot treatments and day of sampling as the subplot treatment. The model included enzyme treatment, fermenter nested within enzyme treatment, incubation trial (blocking factor), and day of sampling. Significance of enzyme treatment effects was tested using the variance among fermenters within treatment as the error term. The effects of other factors were tested against the residual error. In the analysis of data relative to microbial growth and enzyme activities in the fermenters, day of sampling was excluded from the model. When a significant effect of treatment (P < 0.05) was detected, differences between means were assessed by LSD test.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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shows that adding exogenous enzymes did not affect ruminal pH before the addition of feed (P = 0.99). All enzyme treatments increased (P < 0.001) DMAD after 6 and 24 h of incubation, but after 48 h of incubation, only TRI tended to increase DMAD (P = 0.09). Compared with control, enzyme treatments increased NDFD and ADFD after 6 h of incubation, but these responses were not noted (P = 0.28 and 0.56, respectively) after 48 h of incubation. These results suggest that the treatment with cellulases stimulated the initial phases of feed degradation, but the effects were reduced as incubation time increased. These findings are in agreement with results of other studies (Yang et al., 1999; Nsereko et al., 2000; Colombatto et al., 2003b), demonstrating that the treatment of feed with fibrolytic enzymes can increase the rate of digestion without increasing the extent of digestion.
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) and in vitro (Dong et al., 1999; Wang et al., 2001; Yang et al., 2002) studies have indicated that treating different feeds with fibrolytic enzymes produced a shift in the molar proportion of VFA. Enzyme preparations can contain sugars that are fermented by ruminal microorganisms, thus increasing VFA production (Colombatto et al., 2003c). To test this possibility, incubations with enzymes and pure substrates were conducted. Samples (100 mg) of a mixture composed of 70% pure cellulose and 30% cornstarch were incubated with 3 mL of a 1:1 mixture of fermenter liquid and the artificial saliva used in the batch cultures. Incubations were conducted in glass tubes (15 mL) provided with a valve to release the gas. Tubes were incubated at 39°C for 24 h, and then VFA concentration in the medium were measured. Four tubes were used for each of the experimental treatments (control, TRI, ASP, and MIX), and enzymes were added at the same rate that they were used in the Rusitec trial. After 24 h of incubation, there were no differences (P = 0.58) among treatments in VFA concentration (176, 176, 173, and 173 mmol/L for control, TRI, ASP, and MIX, respectively; SEM = 2.0). These results indicate that the observed increase in VFA production in our study was not due to fermentation of enzymes themselves.
Compared with control, the treatment of substrate with TRI, ASP, and MIX increased the production of methane, which is consistent with the greater production of acetate observed for these treatments. Acetate production is associated with the release of H2, which can be used by methanogens to form methane (Stewart et al., 1997). Only few studies have investigated the effects of exogenous enzymes on methane production, and results are conflicting. Dong et al. (1999) found that the treatment of grass hay with cellulase and xylanase enzymes increased cellulose digestibility and methane production by 15 and 43%, respectively, in a Rusitec system, but Colombatto et al. (2003a) reported that adding a proteolytic enzyme to a 60:40 forage:concentrate diet increased NDF degradability by 43% without affecting methane production in dual-flow continuous fermenters. In the present experiment, the ratio of methane:VFA (mol:mol) was similar to values of 0.23 to 0.25 reported by Czerkawski and Breckenridge (1979) for the fermentation of forage-based diets in the Rusitec system. Methane:VFA ratio was not affected (P = 0.70) by any enzyme treatment.
Several authors (Nsereko et al., 2000; Wallace et al., 2001) have suggested that exogenous enzymes could increase fiber degradation via hydrolysis of the feed before incubation in vitro. To test this hypothesis, we analyzed the effects of the 24-h pretreatment with cellulases on NDF and ADF content of substrate (Table 2). Compared with buffer-treated substrate, treatment with TRI and MIX reduced the content of NDF (P < 0.001), ADF (P = 0.03 and 0.01 for TRI and MIX, respectively), and hemicellulose (P < 0.001) in the substrate, but no effect was detected for ASP treatment (P = 0.18 to P = 0.55). These results would indicate that the 24-h pretreatment with the cellulase from T. longibrachiatum altered the fiber structure, as has been previously reported (Nsereko et al., 2000; Colombatto et al., 2003a). The decreases in NDF and ADF content of substrate produced by TRI and MIX treatments support the observations that NDFD and ADFD in Rusitec were increased by these treatments relative to control, especially after 6 h of incubation. The more pronounced effects of TRI on chemical composition and ruminal disappearance of feed in comparison with ASP supports the suggestion that evaluating cellulases as feed additives using carboxymethylcellulose as substrate could be inadequate (Wallace et al., 2001). As pointed out by Wallace et al. (2001), the different ability of the 2 cellulases to break down more ordered, crystalline cellulose may explain the differences in the observed response.
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; Beauchemin et al., 2003), because reducing sugars and other released products of hydrolysis can enhance attachment of ruminal microorganisms to feed particles (Cheng and McAllister, 1997). Although sugar release after enzyme treatment was not measured in the present experiment, our results suggest that both TRI and MIX treatments increased microbial colonization of substrate. The proportion of microbial N in the solid residue after 48 h of incubation (Table 3) was greater for TRI (69.0%; P = 0.04) and MIX (74.0%; P = 0.03) treatments than for control (50.0%), but no difference was detected between control and ASP (49.8%; P = 0.99). In agreement with these results, Wang et al. (2001) reported that treating a 50:50 alfalfa hay:barley grain substrate with xylanase increased 15N incorporation into feed particle-associated microbial N after 24 h of incubation in a Rusitec system.
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). One possibility is that plant structural barriers were removed during the pretreatment of substrate with enzymes, thus allowing greater access of microbes to cell content and increasing protein degradation. Greater degradation of substrate protein would also explain the greater isovalerate and valerate productions observed for TRI and MIX treatments compared with control (Table 1), because these branched-chain VFA are produced by the fermentation of AA (Wallace et al., 1997). In addition, the daily flow of dietary NAN was less (P = 0.05) for TRI and MIX than for control, which would reflect a greater degradation of dietary protein for these 2 treatments.
Daily flow of total NAN was not affected (P = 0.38) by the treatment with enzymes, but microbial N flow was greater (P = 0.05) in the fermenters fed the substrate treated with TRI and MIX than in those fed the buffer-treated feed (Table 3). Although no effects (P = 0.80) of any enzyme treatment were detected on SAM flow, both TRI and MIX treatments increased (P = 0.02 and P = 0.03, respectively) the flow of LAM compared with control and ASP. Although the proportion of microbial N in the solid residue after 48 h of incubation was greater for TRI and MIX compared with control, the total amount of SAM was not increased by these treatments, which could be due to the lower amount of substrate residues recovered for these treatments after 48 h of incubation. The greater LAM flow for TRI and MIX treatments indicates that the growth of these microorganisms was stimulated by the treatment of substrate with enzymes. This microbial fraction is composed of microorganisms located in free suspension or loosely associated with feed particles, and therefore they are not expected to ferment structural carbohydrate. Growth of LAM could have been stimulated by an increase on the sugars arising from the enzymatic hydrolysis of the fiber. In addition, greater endoglucanase and exoglucanase activities were detected in the fluid content of fermenters from TRI (P = 0.002) and MIX (P = 0.002 and P = 0.04, respectively) treatments compared with control (Table 4). Enhanced cellulolytic activity produced by the treatment of feed with exogenous fibrolytic enzymes has also been reported in in vitro (Newbold et al., 1991; Wang et al., 2001) and in vivo studies (Yang et al., 1999; Hristov et al., 2000). As stated by Wang et al. (2001), these changes may reflect a shift in the species profile of colonizing bacteria in response to pretreatment of feed with exogenous enzymes. Those authors observed that pretreating feed with an exogenous xylanase for 24 h before the addition of feed to Rusitec fermenters enhanced the xylanase activity of ruminal fluid and the number of cellulolytic bacteria but did not affect total microbial biomass. In contrast, in the present experiment, we observed a greater flow of LAM in the fermenters from TRI and MIX treatments, which could explain the greater endoglucanase and exoglucanase activities in ruminal fluid compared with control. In addition, greater endoglucanase and exoglucanase activities (P < 0.001) in the liquid obtained from washing the 6-h incubated bags were observed in fermenters from TRI and MIX treatments than in those from control (Table 4), which is consistent with the enhanced NDFD and ADFD after 6 h of incubation and the greater amount of SAB found for these treatments.
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), there were no differences (P = 0.15 to P = 0.41) between control fermenters and those fed the substrate treated with enzymes on final pH after 6 h of incubation (on average 6.63, 6.91, 6.83, and 6.56 for starch, cellulose, xylan, and pectin, respectively; SEM values 0.014, 0.020, 0.007, and 0.018, respectively). Batch cultures inoculated with ruminal fluid from fermenters fed enzyme-treated substrate produced greater (P 
0.001) amounts of gas at all sampling times and with all pure substrates than those inoculated with fluid from control fermenters, with TRI treatment showing the greatest values (P < 0.05) for starch and cellulose. Greater VFA production (P < 0.001) was also observed with the 4 substrates for batch cultures inoculated with fluid from enzyme-treated fermenters compared with control, and MIX treatment produced the greatest values for starch and cellulose. Increased gas and VFA production would indicate a greater fermentative activity in the fluid from TRI and MIX fermenters, and this observation is in agreement with the increased LAM flow and endoglucanase and exoglucanase activities observed in these fermenters.
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Footnotes |
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2 Corresponding author: mdcart{at}unileon.es
Received for publication May 17, 2006. Accepted for publication April 24, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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-bromoethanesulfonate and monensin on fermentation in a rumen simulation (Rusitec) system. Can. J. Anim. Sci. 79:491–498.This article has been cited by other articles:
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  L. A. Giraldo, M. L. Tejido, M. J. Ranilla, S. Ramos, and M. D. Carro Influence of direct-fed fibrolytic enzymes on diet digestibility and ruminal activity in sheep fed a grass hay-based diet J Anim Sci, July 1, 2008; 86(7): 1617 - 1623. [Abstract] [Full Text] [PDF]
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