J. Anim. Sci. 2002. 80:3016-3020
© 2002 American Society of Animal Science
Effect of exogenous fibrolytic enzyme on ruminal fermentation and digestibility of alfalfa and rye-grass hay fed to lambs
J. M. Pinos-Rodríguez*,1,
S. S. González
,
G. D. Mendoza,
R. Bárcena,
M. A. Cobos,
A. Hernández and
M. E. Ortega
* Instituto de Investigación de Zonas Desérticas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México 78377 and
and
Colegio de Postgraduados, Montecillo, Estado de México 56230, México
1 Correspondence:
Phone: 444 8222130; fax: 444 8222718; E-mail:
jpinos{at}uaslp.mx.
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Abstract
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This experiment was carried out to study the effect of a directly fed exogenous fibrolytic enzyme on intake and digestion of DM, OM, protein, NDF, ADF, and hemicellulose of alfalfa and ryegrass hay by sheep. Four diets were randomly assigned to four ruminally cannulated lambs using a 4 x 4 Latin square design, repeated in time, with a factorial arrangement (2 x 2) of diets: 1) alfalfa hay; 2) alfalfa hay + exogenous fibrolytic enzymes (enzyme); 3) ryegrass hay; and 4) ryegrass hay + enzyme. Lambs consumed more DM and OM from alfalfa than from ryegrass hay (P < 0.001). The ADF intake was not different between the hays, but NDF intake was lower for alfalfa (P < 0.001). For both hays, the enzyme increased intake of DM (P < 0.01), as well as OM and CP (P < 0.05); however, NDF and ADF intake were not changed. Alfalfa hay had higher apparent digestibility of DM, OM, and CP (P < 0.001), but lower digestibility for NDF, ADF, and hemicellulose. The enzyme increased apparent digestibility of CP, hemicellulose (P < 0.05), and NDF (P < 0.10) for alfalfa. Also, for both hays, the enzyme improved N balance because lambs retained more N (P < 0.05). The enzyme increased (P < 0.05) total VFA concentration (3 and 6 h) for both hays. Results from this trial indicate that directly fed exogenous fibrolytic enzymes may change ruminal fermentation, intake, and digestibility of forages with different nutritive value.
Key Words: Alfalfa • Digestibility • Enzymes • Intake • Lolium • Ruminants
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Introduction
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Digestion of plant cell walls, which is carried out by ruminal microorganisms, provides a large amount of energy for ruminants. However, intake of highly digestible forage with a large amount of nutrients is critical to reach optimal production levels. Nutrients of forage cell walls are degraded to several metabolites, such as VFA, by ruminal bacteria, protozoa, and fungi. Forage digestibility has been increased by several biotechnological products: ionophores (Zinn et al., 1994), directly fed microbials (Wohlt et al., 1998), and enzymes (Beauchemin et al., 1995). Fibrolytic enzymes isolated from fungi fermentation cultures have been utilized to improve forage digestibility (McAllister et al., 1999). However, direct addition of these enzymes to ruminants has received little attention (Zinn and Salinas, 1999). Our hypothesis was that these enzymes may have similar effects when applied to forages that differ in cell wall structure (e.g., legumes or grass). Therefore, the objective of this research was to evaluate the effect of direct addition of exogenous fibrolytic enzymes on ruminal variables and digestibility of alfalfa (Medicago sativa var. Moapa) and ryegrass (Lolium perenne var. American tetraploid) hay.
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Material and Methods
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These experiments were conducted under the supervision and approval of the Academic Committee of the Animal Science Department, according to the regulations established by the Animal Protection Law, enacted by the Estado de México. Four ruminally cannulated "Criollo" lambs (BW 44 ± 5.8 kg) were assigned to one of four diets in a Latin square 4 x 4 (lambs and period). Experimentals periods lasted 17 d. Within each period, the first 12 d were used for stabilization (e.g., adaptation to diets) and the last 5 d were used for sample collection and the in situ disappeareance trial. Diets were: 1) alfalfa hay; 2) alfalfa hay + exogenous fibrolytic enzymes (enzyme); 3) ryegrass hay; and 4) ryegrass hay + enzyme. Enzyme (5 g; Fibrozyme, Alltech Inc., Nicholasville, KY) was placed daily through the ruminal cannula with the morning feeding (0800). Forages and water were offered ad libitum at 0800 and 1600. Alfalfa and ryegrass (locally grown at our research center; Montecillo, Edo. de México) were cut at 28 and 42 d after a uniformity cut and then sun cured. Both hays were cut close to 7.5-cm particle size. Forages, enzyme product (Table 1
), orts, and feces were collected daily for 5 d, sampled, and analyzed for DM, OM, CP, ash (AOAC, 1990), NDF, ADF (Van Soest et al., 1991), and hemicellulose (NDF-ADF). Data were used to measure apparent in vivo digestibility of DM, OM, CP, NDF, ADF, and hemicellulose. Nitrogen balance was calculated using N values of feed, feces, and urine (Harris, 1970). In situ disappearance was performed according to Vanzant et al. (1998). Bags with alfalfa or ryegrass hays (3 g DM) were placed inside the rumen with the morning feeding (0800), and sampling was done at 0, 3, 6, 12, 24, 48, and 72 h. Disappearance of DM, NDF, ADF, and hemicellulose were used to assess in situ digestibility.
Ruminal fluid samples were taken at 3, 6, and 12 h after the morning feeding; pH was measured immediately and samples were frozen. Later, VFA (Erwin et al., 1961) and ammonia N (McCullough, 1967) were determined in those samples. Data were analyzed using GLM procedures of SAS (SAS Inst., Inc., Cary, NC).
Data were analyzed as a 4 x 4 (period x animal) Latin square, repeated on time, with a factorial arrangement of diets (2 x 2; alfalfa or ryegrass, without or with enzyme); main effects and interaction (type of forage and enzyme level) were analyzed. DMI, apparent in vivo digestibility, and in situ disappearence data were analyzed using GLM (SAS Inst., Inc.); means were compared using a Tukey test. A repeated-measures analysis was used for acetate, propionate and butyrate percentage, total VFA, pH and N-NH3 data, in order to detect time x forage x enzyme interaction (SAS Inst. Inc.).
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Results and Discussion
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Intake of DM and OM were higher (P < 0.001) for alfalfa hay than for ryegrass hay, but NDF intake was lower (P < 0.001) for alfalfa than for ryegrass (Table 2). This seems to be related to the larger amount of NDF found for ryegrass (64.2%; Table 1
). Legumes have a lower total content of cell walls, hemicellulose, and lignin than grass. Dewayne (1993) pointed out that intake of legumes, compared to grass, is higher because they have less of a cell wall and lower resistance to ruminal degradation. Enzyme only increased DM (P < 0.01) and OM (P < 0.05) intake (Table 2) and tended to increase NDF intake (P < 0.1). Feng et al. (1996) reported that intake of DM was increased by fibrolytic enzyme with dry, but not fresh, forages. However, fibrolytic enzyme sprinkled on forages (Lewis et al., 1996; Krause et al., 1998), directly fed to the animal (Lewis et al., 1996), or added to the feed (Rode et al., 1999) did not change DM intake.
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Table 2. Effect of enzyme on intake, in vivo digestibility (%), and N balance (g) for alfalfa and ryegrass hay in lambsa
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Alfalfa hay showed higher (P < 0.001) in vivo digestibility for DM, OM, and CP, and higher N retention, whereas NDF and hemicellulose digestibility were higher for ryegrass hay (Table 2). Oba and Allen (1999) reviewed several studies and found greater NDF digestibility for grasses than legumes due to the fact that NDF filling effect in the rumen might be less for legumes because of greater particle fragility and shorter rumen retention time compared with grass. For alfalfa hay, enzyme increased in vivo digestibility of CP and hemicellulose (P < 0.05) and tended to increase NDF (P < 0.10). Even when digestibility was not improved with enzymes, intake of NDF was increased (P < 0.07). These results do not agree with those reported by Oba and Allen (1999), who found a higher DMI for cows fed forages with high NDF digestibility when comparisons were made within a forage family, but found lower DMI for cows fed forages with high NDF digestibility when comparisons were made across forage families. Forages with high NDF digestibility might increase DMI when physical fill limitations to feed intake exist. More rapid hydrolysis of NDF might allow more rapid disappearance of NDF from the rumen because of increased rate of digestion or passage (Oba and Allen, 1999).
Nitrogen intake, fecal N and N retention were increased (P < 0.05) by enzyme addition in both forages. However, CP digestibility increased for alfalfa but decreased for grass (Table 2). According to McAllister et al. (2001), fibrolytic enzymes seem to have some proteolitic activities as they facilitate degradation of cell wall-bound proteins. Also cellulase has been shown to increase protein degradation of forages in vitro by making proteins more available to proteolytic enzymes (Kohn and Allen, 1992). Increases in NDF digestibility by fibrolytic enzymes have been reported for silages, barley straw (Krause et al., 1998), and forages (Lewis et al., 1996). Rode et al. (1999) found that fibrolytic enzymes significantly increased DM, OM, NDF, ADF, and CP digestibility in dairy cows.
Alfalfa hay in situ disappearance was higher (P < 0.05) for DM at all sampling periods, and for hemicellulose during the first 12 h, whereas NDF (3, 48, and 72 h) and ADF (3, 24, 48, and 72 h) disappearance was larger (P < 0.05) for ryegrass hay (Table 3). Extent of digestion is larger for DM, but lower for cell walls in legumes; however, for ad libitum intake, rate of cell wall digestion is similar between legumes and grasses (Galyean and Goetsch, 1993). For both forages, enzyme increased (P < 0.01) in situ disappearance of DM (3, 6, and 24 h), hemicellulose (3 and 72 h; Table 3), NDF (3, 6, 12, and 24 h), and ADF (3, 6, 12, 24, and 72 h; Table 3). Fibrolytic enzymes sprinkled on forages have increased digestibility of DM (Feng et al., 1996) and NDF (Feng et al., 1996; Lewis et al., 1996). The ruminal digestibility of CP and NDF has also been increased by fibrolytic enzymes (Zinn and Salinas, 1999).
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Table 3. Effect of enzyme on DM, NDF, ADF, and hemicellulose in situ disappearance (%) for alfalfa and ryegrass hay in lambsa
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Previous studies have shown some differences on the effect of fibrolytic enzymes for in vivo and in situ digestibilities. Oba and Allen (1999) found that in vitro or in situ digestibility of NDF is a better indicator of forage to enhance DMI than in vivo digestibility. Measurement of in vivo NDF digestibility is confounded by different retention times in the rumen, which can be affected by differences in DMI. In addition, exposure to acidic conditions in the small intestine and fermentation in the large intestine may reduce differences observed for in vitro or in situ fermentation. Therefore, in vitro or in situ NDF disappearance is an important measure of forage quality and should be distinguished from in vivo NDF digestibility (Oba and Allen, 1999).
Ruminal pH was significantly higher (3, 6, and 12 h postprandial) for ryegrass, but NH3-N concentration had a significant increase for alfalfa at 3, 6, and 12 h. Enzyme had no effect on both variables, but there were significant interactions at 3 h for pH and at 3 and 6 h for NH3-N concentration (Table 4). However, these changes do not seem to have biological significance since pH and NH3-N values were within the normal range for ruminal activity (Van Soest, 1982). Total VFA and propionate concentrations were significantly higher for alfalfa, but acetate was increased by ryegrass. Response to enzyme was inconsistent, except for an interaction at 3-h postprandial for acetate, propionate, and butyrate (Table 4). Lewis et al. (1996) reported that fibrolytic enzymes have decreased ruminal pH and increased total VFA in steers, without changing fiber digestibility. Results from our study show that enzyme did not change pH, but in situ digestibility of DM and cell wall fractions was increased. Diets with a larger amount of NDF usually increase ruminal pH and activity of cellulolytic bacteria (Owens and Goetsch, 1988). Therefore, ryegrass hay induced a higher concentration of acetate and reduced propionate. Dawson and Tricarico (1999) reported that fibrolytic enzymes with xilanasic activity increased total VFA, but there were no changes when enzymes with cellulolytic activity were used. This result agrees with Tricarico et al. (1998), who found no changes in ruminal bacteria and protozoa when using fibrolytic enzymes.
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Table 4. Effect of enzyme on pH, NH3-N, acetate, propionate, butyrate, and total VFA concentration in ruminal fluid of lambs fed alfalfa or ryegrass haya
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Implications
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Results from this study show that an exogenous fibrolytic enzyme may improve apparent digestibility of neutral detergent fiber for alfalfa, but not for ryegrass. However, this enzyme tended to increase ruminal disappearance for both forages. Since intake was improved with enzymes, total digestible nutrients were increased. Also, this enzyme may change concentration and proportion of volatile fatty acids during the first 3 h after feeding. This enzyme may contribute to degradation of cellulose, hemicellulose, and protein in forages. Therefore, ruminant productivity may be improved. However, the effect of an exogenous fibrolytic enzyme seems to be related to substrate (forage species) and rumen environment.
Received for publication March 6, 2002.
Accepted for publication July 10, 2002.
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Abstract
Introduction
