J. Anim Sci. 2008. 86:1617-1623. doi:10.2527/jas.2007-0343
© 2008 American Society of Animal Science
Influence of direct-fed fibrolytic enzymes on diet digestibility and ruminal activity in sheep fed a grass hay-based diet1
L. A. Giraldo*,
,
M. L. Tejido*,
M. J. Ranilla*,
S. Ramos* and
M. D. Carro*,2
* Departamento de Producción Animal, Universidad de León, 24071 León, Spain; and
Universidad Nacional de Colombia, Sede Medellín, Facultad de Ciencias Agropecuarias, 2037 Medellín, Colombia
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Abstract
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Six rumen-fistulated Merino sheep were used in a crossover design experiment to evaluate the effects of an exogenous fibrolytic enzyme preparation (12 g/d; ENZ), delivered directly into the rumen, on diet digestibility, ruminal fermentation, and microbial protein synthesis. The enzyme contained endoglucanase and xylanase activities. Sheep were fed a mixed grass hay:concentrate (70:30; DM basis) diet at a daily rate of 46.1 g/kg of BW0.75. Samples of grass hay were incubated in situ in the rumen of each sheep to measure DM and NDF degradation. The supplementation with ENZ did not affect diet digestibility (P = 0.30 to 0.66), urinary excretion of purine derivatives (P = 0.34), ruminal pH (P = 0.46), or concentrations of NH3-N (P = 0.69) and total VFA (P = 0.97). In contrast, molar proportion of propionate were greater (P = 0.001) and acetate:propionate ratio was lower (P < 0.001) in ENZ-supplemented sheep. In addition, ENZ supplementation tended to increase (P = 0.06) numbers of cellulolytic bacteria at 4 h after feeding. Both the ruminally insoluble potentially degradable fraction of grass hay DM and its fractional rate of degradation were increased (P = 0.002 and 0.05, respectively) by ENZ treatment. Supplementation with ENZ also increased (P = 0.01 to 0.02) effective and potential degradability of grass hay DM and NDF. Ruminal fluid endoglucanase and xylanase activities were greater (P < 0.001 and 0.03, respectively) in ENZ-supplemented sheep than in control animals. It was found that ENZ supplementation did not affect either exoglucanase (P = 0.12) or amylase (P = 0.83) activity. The results indicate that supplementing ENZ directly into the rumen increased the fibrolytic activity and stimulated the growth of cellulolytic bacteria without a prefeeding feed-enzyme interaction.
Key Words: enzymatic activity • fibrolytic enzyme • microbial protein synthesis • ruminal fermentation
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INTRODUCTION
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The use of exogenous fibrolytic enzymes as feed additives has been investigated in the last years as a means to improve forage utilization by ruminants (Beauchemin et al., 2003
). Positive effects of supplementing the diet with fibrolytic enzymes have been reported in dairy cows (Zheng et al., 2000; Beauchemin et al., 2003) and beef steers (Beauchemin et al., 1995, 1997), but the use of enzymes in the feeding of small ruminants has received little attention. Forages usually constitute a major portion of the diet in most small ruminant production systems, and any improvement in their nutritive value would increase the productivity of the animals.
The potential of enzymes to increase the performance of lambs (Rojo et al., 2005), ewes (Titi and Lubbadeh, 2004), and goats (Titi and Lubbadeh, 2004) has been previously investigated, but information on the effects of enzymes on ruminal fermentation in small ruminants is limited (Pinos-Rodriguez et al., 2002). Several studies (Hristov et al., 1998, 2000; Nsereko et al., 2002) conducted in cattle have shown that enzyme supplementation can influence ruminal variables, fibrolytic activity of ruminal fluid, and microbial populations, but the effects seem to be dependent on the type of supplemented enzyme. Previous studies from our group (Giraldo et al., 2007a,b) have shown that the treatment of a high-forage diet with an enzyme preparation from Trichoderma longibrachiatum increased the production of VFA, the fibrolytic activity of ruminal fluid, and microbial numbers in Rusitec fermenters.
Our hypothesis was that this fibrolytic enzyme could produce similar effects when it was administered directly into the rumen of sheep. The objective of this study was therefore to evaluate the effects of an exogenous fibrolytic enzyme from T. longibrachiatum on diet digestibility, ruminal variables, fibrolytic activity, and rumen microbial numbers in sheep fed a high-forage diet.
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MATERIALS AND METHODS
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Animals, Diet, and Enzyme
The experimental protocol was approved by the León University Institutional Animal Care and Use Committee.
Six Merino sheep (60.0 ± 4.4 kg of BW) fitted with a permanent ruminal plastic cannula (5-cm i.d.) were used. Sheep were housed in individual pens and had continuous access to fresh water and vitamin-mineral block. Animals were fed a mixed diet of grass hay and concentrate. Ingredients and chemical composition of the diet are shown in Table 1. The diet was formulated according to the NRC (1985) guidelines. The diet was offered to the animals twice daily (0800 and 2000 h) at a rate of 46.1 g of DM/kg of BW0.75, which corresponded to 1.1 times maintenance requirements according to NRC (1985). Refusals and individual feed intakes were recorded daily.
The enzyme preparation was a commercial product (Fibrozyme, Alltech Inc., Nicholasville, KY) from fermentation extracts of Aspergillus niger and T. longibrachiatum. The product was assayed for endoglucanase (EC 3.2.1.4.), exoglucanase (EC 3.2.1.91), xylanase (EC 3.2.1.8.), and amylase (EC 3.2.1.1.) activities following the procedures described by Colombatto and Beauchemin (2003). All activities were measured at pH 6.5 and 39°C to resemble ruminal conditions in sheep fed high-forage diets (Carro et al., 2000). Solutions (10 g/L) of medium-viscosity carboxymethylcellulose (21902, Fluka Chemie GmbH, Steinheim, Germany), Avicel PH-101 (K26942731, Merck, Darmstadt, Germany), oat spelt xylan (X0627, Sigma-Aldrich, Steinheim, Germany), and soluble starch (S9765, Sigma-Aldrich) were used as substrates for determination of endoglucanase, exoglucanase, xylanase, and amylase activity, respectively. For all enzymatic activities, the absorbance was read at 540 nm using a spectrophotometer (Ultrospec 500 pro, Amersham Biosciences, Munich, Germany) against glucose (8337, Merck) or xylose (95729, Fluka Chemie GmbH) standards (from 0 to 2 g/L) processed under identical conditions. 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 enzyme. At pH 6.5 and 39°C, 1 g of the Fibrozyme liberated 148 µmol of glucose from carboxymethylcellulose and 791 µmol of xylose from oat spelt xylan per minute, and no exoglucanase and amylase activities were detected.
Experimental Procedure and Sampling
Treatments consisted of 0 (control) or 12 g/d (ENZ) of enzyme administered in 2 equal portions by direct introduction into the rumen through the cannula immediately before feeding. The sheep were randomly divided into 2 groups of 3, and each group was randomly assigned to treatments in a crossover design. The experiment consisted of two 32-d experimental periods, with a 15-d initial period for diet and enzyme adaptation and a 17-d sampling period.
On d 13, sheep were moved to metabolism cages equipped for quantitative collection of feces and urine separately. After 3 d of adaptation, feces and urine voided by each sheep in 12 h were quantitatively collected for 6 d. Fecal collection did not involve the use of bags or harnesses. An aliquot (10%) of total fecal output was collected each day for digestibility determination and dried to constant weight before analysis. Urine was collected in a solution of 3.6 M H2SO4 to keep the pH below 3. The volume of urine at each sampling time was determined, and a subsample (20%) was taken for each sheep and frozen. Samples of feces and urine were pooled for each sheep to form composite samples. On d 21, sheep were moved again to floor pens.
On d 23 of each period, about 500 g of ruminal contents was taken through the cannula of each sheep 4 h after the morning feeding, strained through 4 layers of cheesecloth, and 1 mL of the fluid was diluted through a series of tubes containing 9.0 mL of anaerobic dilution solution (Dehority, 1969). Using the 10–6 through 10–11 dilution tubes, 1 mL was placed in each of 3 tubes containing the most probable number media (Dehority et al., 1989). Total and cellulolytic bacteria concentrations were determined according to the most probable number procedure (Dehority et al., 1989).
On d 25, ruminal content samples were taken through the cannula of each sheep at 0, 4, and 8 h after the morning feeding. Ruminal content was strained through 4 layers of cheesecloth, the pH of the fluid was immediately measured, 5 mL of fluid was added to 5 mL of deproteinizing solution (100 g of metaphosphoric acid and 0.6 g of crotonic acid per L) for VFA analyses, and 2 mL was added to 2 mL of 0.5 M HCl for NH3-N determination. Additionaly, 5 mL of fluid was immediately frozen at –80°C for determination of enzymatic activities.
The nylon bag technique (Mehrez and Ørskov, 1977) was used to measure ruminal degradation of grass hay over d 27 to 32 of each experimental period. Samples of grass hay (5 g of DM; ground through a 3-mm screen) were incubated in polyester bags in the rumen of each sheep for 0, 3, 6, 9, 15, 24, 48, and 72 h following the procedure described by Ranilla et al. (1997). Two bags were incubated for each incubation time and sheep. The removed bags were washed thoroughly under running cold water for 1 min and were then washed in the cold rinse cycle (20 min) of a washing machine. Dry matter disappearance was measured from the loss in weight after oven drying at 60°C for 48 h, and the residues were analyzed for NDF to estimate the loss of fiber.
Analytical Procedures
Procedures for analysis of DM, N, NDF, ADF, VFA, and NH3-N have been described previously (Carro et al., 2006). Concentration of purine derivatives (allantoin, uric acid, xanthine, and hypoxanthine) was analyzed by HPLC (Balcells et al., 1992), and microbial flow at the duodenum was estimated from the daily excretion of purine derivatives (Balcells et al., 1991).
For determination of enzymatic activities in ruminal fluid samples, cells were lysed using a Mini-BeadBeater (BioSpec Products Inc., Bartlesville, OK) to release intracellular enzymes. Cell material was removed by centrifugation (10,000 x g, 10 min, 4°C), and the supernatant was used to analyze enzymatic activities as described before.
Calculations and Statistical Analyses
The values for disappearance of DM from grass hay were fitted to the exponential model y = a + b (1 –e–ct), as described by Ørskov and McDonald (1979), where y represents the DM disappearance at each incubation time (t), and a, b, and c are constants that represent the soluble rapidly degradable fraction, the insoluble potentially degradable fraction, and the fractional degradation rate for fraction b, respectively. The fitted equation was constrained so that a + b did not exceed 100%, and this value represented the potential degradability of grass hay. The values for NDF disappearance were fitted to the model y = b[1 – e–c(t – lag)], where y is the NDF disappearance at each incubation time (t), b is the potentially degradable fraction, c is the fractional degradation rate for fraction b, and lag is the lag time (h) before degradation commenced. Effective degradability (ED) was estimated in each sheep by using the parameters a, b, c, and lag and assuming a rumen particulate outflow rate (kp) of 3.5%/h according to the following equation: ED = a + [(b x c)/(c +kp)]e(–kp x lag) (France et al., 1990). A lag time value of 0 was assumed for calculation of DM effective degradability.
Data were analyzed as a mixed model using the MIXED procedure (SAS Inst. Inc., Cary, NC). The effect of treatment was considered fixed, and sheep and period effects were considered random. Time-sequence data (pH, VFA, NH3-N, and enzymatic activities in ruminal fluid, and in situ disappearance of grass hay) were analyzed using the same model with repeated measures. Data for each variable were analyzed using compound symmetry, unstructured, and autoregressive covariance structures, and the one that produced the minimum Akaike information criterion was chosen. Mean effects were declared significant at P 
0.05, and P 0.10 values were considered as trends and discussed.
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RESULTS AND DISCUSSION
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As shown in Table 2, ENZ supplementation did not affect either DMI (P = 0.33) or diet digestibilities (P = 0.30 to 0.66). Diet was offered at a restricted level to minimize feed selection and refusals, and therefore differences between groups in diet intake were not expected. Pinos-Rodriguez et al. (2002) administered 5 g/ d of the same enzyme used in this experiment to forage-fed lambs and did not observe any effect (P = 0.17 to 0.90) on diet digestibility. On the contrary, fibrolytic enzymes have been shown to increase diet digestibility in other studies (Lewis et al., 1996; Yang et al., 1999; Kung et al., 2000), although in most of them, the diet was pretreated with enzymes before being fed to animals. A pretreatment of feeds with enzymes before feeding has been reported to enhance the beneficial effects of enzymes on ruminal fermentation (Hristov et al., 1998; Wang et al., 2001; Giraldo et al., 2004). However, the current study was designed to exclude a prefeeding enzyme-feed interaction, to investigate the effects of the enzyme when it was directly administered into the rumen.
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Table 2. Influence of enzymatic treatment on diet DMI and apparent total tract digestibilities of DM, OM, NDF, ADF, and CP in sheep fed a 70:30 grass hay:concentrate diet (n = 6)
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As shown in Table 3, ENZ supplementation increased (P = 0.02 to 0.04) in situ DM disappearance of grass hay at 6, 9, and 48 h of incubation and tended to increase it at 24 h of incubation (P = 0.08). There was greater (P = 0.01 to 0.03) NDF disappearance at 6, 9, 48, and 72 h of incubation in ENZ-fed sheep compared with unsupplemented animals. Lewis et al. (1996) found similar results when steers fed a 70:30 grass hay:barley diet were supplemented with a commercial fibrolytic enzyme. These authors attributed the improved DM and NDF disappearances to enhanced colonization and digestion of the slowly degradable fiber fraction by ruminal microorganisms. Consistent with this hypothesis, Giraldo et al. (2007a, b) observed that the treatment of a high-forage substrate with a cellulase from T. longibrachiatum increased the incorporation of 15N-NH3 into the substrate after 6 h of incubation in Rusitec fermenters and enhanced fiber degradation, which would indicate that enzyme treatment stimulated the initial phases of microbial colonization. Although the diet was not pretreated with the enzyme in the current experiment, the results would indicate that ENZ treatment stimulated fiber degradation.
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Table 3. Influence of enzymatic treatment on DM and NDF disappearance after different times of incubation in the rumen of sheep fed a 70:30 grass hay:concentrate diet (n = 6)
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As shown in Table 4, ENZ supplementation did not affect (P = 0.89) the soluble rapidly degradable fraction (a) of grass hay DM but increased the insoluble potentially degradable fraction (b; P = 0.002) and its fractional rate of degradation (c; P = 0.05). As a consequence, both potential degradability and ED of grass hay DM were greater (P < 0.05) in sheep supplemented with ENZ than in control sheep. Enzyme supplementation also increased (P = 0.02) b fraction and ED of grass hay NDF and tended (P = 0.10) to increase the fractional rate of degradation of fraction b. Similar results have been reported by Pinos-Rodriguez et al. (2002) in lambs receiving the commercial enzyme used in the current study. On the contrary, Hristov et al. (1998, 2000) reported that the supplementation with fibrolytic commercial enzymes increased the soluble fraction and decreased the degradation rate of the diet in heifers fed a barley grain diet and a mixed diet, respectively. An increase of the soluble fraction would be expected if feeds are directly treated with enzymes, because this pretreatment has been shown to start fiber degradation and to reduce the NDF content of different feeds (Hristov et al., 1998; Giraldo et al., 2007a,c).
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Table 4. Influence of enzymatic treatment on in situ ruminal degradation parameters of DM and NDF of grass hay in sheep fed a 70:30 grass hay:concentrate diet (n = 6)
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The greater in situ degradation of grass hay observed in ENZ-treated sheep is in accordance with the enhanced endoglucanase (P < 0.001) and xylanase (P = 0.03) activities observed in their ruminal fluid (Table 5). In contrast, there was no effect of ENZ treatment on exoglucanase (P = 0.12) and amylase (P = 0.83) activities. Enhanced fibrolytic activities in rumen fluid produced by the treatment of feed with exogenous fibrolytic enzymes have been reported in in vitro (Wang et al., 2001; Giraldo et al., 2007a) and in vivo studies (Hristov et al., 2000).
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Table 5. Influence of enzymatic treatment on enzymatic activities and microbial numbers in ruminal fluid from sheep fed a 70:30 grass hay:concentrate diet (n = 6)
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The enzyme preparation used in this experiment presented endoglucanase and xylanase activities, but it seems unlikely that the observed increase in these enzymatic activities in ruminal fluid was exclusively due to a direct effect of the enzyme. Morgavi et al. (2001) incubated 4 different commercial preparations from T. longibrachiatum with ruminal fluid for 6 h and observed that they were relatively stable and resistant to microbial degradation for a time sufficiently long to act in the rumen. Stability in ruminal fluid varied among commercial products and also depended on the enzymatic activity assayed (Morgavi et al., 2001). It would be possible that in the current study ENZ contributed directly to increase endoglucanase and xylanase activities. Based on results from previous experiments conducted by our group (Ranilla et al., 1998; Carro et al., 2000), a ruminal volume of 7 L can be assumed for Merino sheep fed a forage-based diet at a restricted level of intake; thus, daily supplementation of 6 g of ENZ before the morning feeding would represent 0.127 endoglucanase and 0.678 xylanase units/mL of ruminal fluid. Compared with control sheep, enzymatic activity in ENZ-supplemented animals was increased by 0.380, 0.308, and 0.255 endoglucanase and 0.972, 2.77, and 2.68 xylanase units/mL of ruminal fluid at 0, 4, and 8 h after the morning feeding and adding 6 g of enzyme, respectively (Table 5
). These results would indicate that the observed increase in the enzymatic activity of ruminal fluid from ENZ-supplemented sheep was not exclusively due to a direct effect of the exogenous enzyme and that ENZ supplementation stimulated fibrolytic activity of ruminal fluid. In accordance with our results, Morgavi et al. (2000) demonstrated synergism between exogenous enzymes produced by T. longibrachiatum and those produced by rumen microorganisms such that the net combined hydrolytic effect in the rumen was much greater than that estimated from the individual activities.
There were no effects of ENZ treatment on ruminal pH (P = 0.46) and concentrations of NH3-N (P = 0.69) and total VFA (P = 0.97) concentration (Table 6). Several in vivo (Hristov et al., 2000; Pinos-Rodriguez et al., 2002; Beauchemin et al., 2003) and in vitro (Wang et al., 2001; Giraldo et al., 2007a,b) studies have shown that treating different feeds with fibrolytic enzymes produced a shift in the molar proportions of VFA, but changes in rumen fermentation pattern seem to be affected by the characteristics of the diet fed to the animals and the type of supplemented enzyme (Beauchemin et al., 2003; Giraldo et al., 2007c). In the present experiment, molar proportion of propionate was greater (P = 0.001) and acetate:propionate ratio was lower (P < 0.001) in ENZ-supplemented sheep compared with unsupplemented animals.
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Table 6. Influence of enzymatic treatment on pH, concentrations of NH3-N and total VFA, molar proportions of the main VFA, and acetate:propionate ratio in the rumen of sheep fed a 70:30 grass hay:concentrate diet
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Several authors (Hristov et al., 2000; Wang et al., 2001) have suggested that changes in fermentation pattern may reflect concomitant changes in rumen bacterial populations, but only a few studies have investigated this issue. Nsereko et al. (2002) reported that treating the diet of dairy cows with a commercial product from T. longibrachiatum increased the numbers of rumen bacteria that utilize hemicellulose or secondary products of cellulose digestion, and Giraldo et al. (2007a) observed that treating a high-forage substrate with a cellulase produced by T. longibrachiatum increased the numbers of cellulolytic bacteria in Rusitec fermenters. In the current study, ENZ supplementation tended to increase (P = 0.06) numbers of cellulolytic bacteria, but no significant differences (P = 0.24) were detected in total bacterial numbers. Supporting these results, there was no effect of ENZ supplementation on urinary excretion of purine derivatives (P = 0.34) and the estimated microbial N duodenal flow (P = 0.46; Table 7). In agreement with these results, no effects of enzymes on urinary excretion of purine derivatives have been reported in other studies (Hristov et al., 1998; Yang et al., 1999; Hristov et al., 2000).
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Table 7. Influence of enzymatic treatment on urinary excretion of purine derivatives (PD) and estimated microbial N flow at the duodenum in sheep fed a 70:30 grass hay:concentrate diet (n = 6)
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The results of the present study demonstrated that supplementing a fibrolytic enzyme directly into the rumen increased the fibrolytic activity in ruminal fluid without a prefeeding feed-enzyme interaction. Although enzyme supplementation did not affect significantly the concentration of total VFA in ruminal fluid, molar proportions of propionate increased and acetate:propionate ratio decreased, which might indicate a change in ruminal bacterial populations. Further research is needed to determine the effects of fibrolytic enzymes on specific ruminal microbial populations.
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Footnotes
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1 Funding was provided by the Spanish Centro de Investigación Cientifica y Tecnológica (Project AGL2001-0130). L. A. Giraldo gratefully acknowledges receipt of a grant from the Fundación Carolina. 
2 Corresponding author: mdcart{at}unileon.es
Received for publication June 12, 2007.
Accepted for publication February 14, 2008.
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Abstract
INTRODUCTION
