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Use of Exogenous Fibrolytic Enzymes to Improve Feed Utilization by Ruminants^1,2

K. A. Beauchemin^*,3, D. Colombatto^*, D. P. Morgavi and W. Z. Yang^*

^* Agriculture and Agri-Food Canada, Lethbridge, AB, Canada T1J 4B1 and and INRA, Centre Clermont-Theix, Unité de Recherche sur les Herbivores 63122 Saint-Genès-Champanelle, France

	Abstract

Top Abstract Introduction Implications Literature Cited

 
Research has demonstrated that supplementing dairy cow and feedlot cattle diets with fiber-degrading enzymes has significant potential to improve feed utilization and animal performance. Ruminant feed enzyme additives, primarily xylanases and cellulases, are concentrated extracts resulting from bacterial or fungal fermentations that have specific enzymatic activities. Improvements in animal performance due to the use of enzyme additives can be attributed mainly to improvements in ruminal fiber digestion resulting in increased digestible energy intake. Animal responses are greatest when fiber digestion is compromised and when energy is the first-limiting nutrient in the diet. When viewed across a variety of enzyme products and experimental conditions, the response to feed enzymes by ruminants has been variable. This variation can be attributed to experimental conditions in which energy is not the limiting nutrient, as well as to the activities and characteristics of the enzymes supplied, under- or over-supplementation of enzyme activity, and inappropriate method of providing the enzyme product to the animal. A limited number of ruminant enzyme products are now commercially available, and this list of products is expected to grow. However, random addition of enzymes to diets without consideration for specific situations and substrate targets will only discourage or delay on-farm adoption of enzyme technology. Although much progress has been made in advancing enzyme technology for ruminants, considerable research is still required to reduce the variability of response. With increasing consumer concern about the use of growth promoters and antibiotics in livestock production, and the magnitude of increased animal performance obtainable using feed enzymes, there is no doubt that these products will play an increasingly important role in the future. This paper reviews the research on enzyme selection, the animal responses to feed enzymes, and the mechanisms by which these products improve nutrient utilization.

Key Words: Cellulase • Cellulose Digestion • Digestion • Enzymes • Fiber • Ruminants

	Introduction

Top Abstract Introduction Implications Literature Cited

 
Over the years, significant improvements in forage cell wall digestibility have been achieved through forage breeding programs and agronomic advances. Despite these improvements, forage digestibility continues to limit the intake of available energy by ruminants, and correspondingly, contributes to excessive nutrient excretion by livestock. The use of exogenous fibrolytic enzymes holds promise as a means of increasing forage utilization and improving the productive efficiency of ruminants.

Recent studies have shown that adding exogenous fibrolytic enzymes to ruminant diets increases milk production (Nussio et al., 1997; Lewis et al., 1999; Rode et al., 1999; Schingoethe et al., 1999; Kung et al., 2000; Yang et al., 2000) and ADG (Beauchemin et al., 1995; 1997; 1999b; Iwaasa et al., 1997; McAllister et al., 1999) in some cases. These increases in animal performance are due to increases in feed digestion. Numerous studies have reported increased digestion of DM and fiber measured in situ, in vitro (Nakashima et al., 1988; Feng et al., 1996; Hristov et al., 1996; Yang et al., 1999; Colombatto, 2000; Colombatto et al., 2002c), or in vivo (Feng et al., 1996; Krause et al., 1998; Rode et al., 1999; Yang et al., 1999; Beauchemin et al., 2000; Kung et al., 2000). However, not all studies report improved animal performance due to the use of exogenous enzymes (Higginbotham et al., 1996; Pritchard et al., 1996; ZoBell et al., 2000), and viewed across a variety of enzyme products and experimental conditions the response to feed enzymes by ruminants has been variable. This article reviews the research on the selection of enzymes for use in ruminant diets, the animal responses to feed enzymes and the potential sources that contribute to the variability in response, and the mechanisms by which exogenous fibrolytic enzymes improve nutrient utilization.

Feed Enzymes for Ruminants

Commercial enzymes used in the livestock feed industry are products of microbial fermentation. Feed enzymes are produced by a batch fermentation process, beginning with a seed culture and growth media (Cowan, 1994). Once the fermentation is complete, the enzyme protein is separated from the fermentation residues and source organism. Although the source organisms are, in many cases, similar among enzyme products, the types and activity of enzymes produced can vary widely depending on the strain selected and the growth substrate and culture conditions used (Considine and Coughlan, 1989; Gashe, 1992; Lee et al., 1998).

Compared to the fermentation extract, these enzyme products are relatively concentrated and purified, containing specific, controlled enzyme activities. They usually do not contain live cells. Enzyme products for ruminant diets are of fungal (mostly Trichoderma longibrachiatum, Aspergillus niger, A. oryzae) and bacterial (mostly Bacillus spp.) origin (Pendleton, 2000). Furthermore, most of the commercially available enzyme products that have been evaluated as ruminant feed additives are produced for nonfeed applications; cellulases and xylanases are used extensively in the food, pulp and paper, textile, fuel, and chemical industries (Bhat and Hazlewood, 2001). Several fibrolytic enzyme products evaluated as feed additives in ruminant diets were originally developed as silage additives (Feng et al., 1996).

In addition to these relatively pure sources of enzymes, crude fermentation products and some nonbacterial direct-fed microbials (DFM) are also marketed, at least partly or implicitly, based on their residual enzymic content (Muirhead, 2001). In this case, the enzymes, as well as the entire medium, are recovered complete with metabolites and fermentation substances. Most nonbacterial DFM consist of A. oryzae fermentation extract, Saccharomyces cerevisiae cultures, or both (Martin, 2000). In comparison to concentrated feed enzyme products, these crude products contain relatively little (<5%) enzyme activity. There is no minimal level of enzyme activity required for products to be registered as feed enzymes, which adds tremendous confusion in the marketplace. Consequently, it can be difficult to distinguish commercially between "true" enzyme products and products with trace levels of activity. The scope of this paper is limited to concentrated fermentation products that have specific, controlled enzyme activities.

Enzyme Activities Involved in Cell Wall Digestion

The focus of most enzyme-related research for ruminants has been on plant cell wall degrading enzymes. Cellulose and hemicellulose, the major structural polysaccharides in plants (Van Soest, 1994), are converted to soluble sugars by enzymes collectively referred to as cellulases and hemicellulases. The types of cellulases and hemicellulases can differ substantially among commercial enzyme products, and differences in the relative proportions and activities of these individual enzymes may have an impact on the efficacy of cell wall degradation by these products. In addition to fiber-degrading enzymes, these products also have secondary enzyme activities, including amylases, proteases, and pectinases.

Cellulose is hydrolyzed through a complex process involving cellulases, and numerous specific enzymes contribute to cellulase activity. The major enzymes involved in cellulose hydrolysis are endocellulase (endoglucanase, endo-ß-1,4-glucanase, carboxymethyl cellulase or ß-1,4-glucan glucanohydrolase; E.C. 3.2.1.4), exocellulase (exoglucanase, exo-ß-1,4-glucanase, cellulose ß-1,4-cellobiosidase; E.C. 3.2.1.91), and ß-glucosidase (cellobiase or glucohydrolase, E.C. 3.2.1.21). In general, endoglucanases hydrolyze cellulose chains at random to produce cellulose oligomers of varying degrees of polymerization; exoglucanases hydrolyze the cellulose chain from the nonreducing end, producing cellobiose, and ß-glucosidases hydrolyze short-chain cellulose oligomers and cellobiose to glucose.

The main enzymes involved in degrading the xylan core polymer to soluble sugars are xylanases (EC 3.2.1.8) and ß-1,4 xylosidase (3.2.1.37) (Bhat and Hazlewood, 2001). The xylanases include endoxylanases, which yield xylooligomers and ß-1,4-xylosidases, which in turn yield xylose. Other hemicellulase enzymes involved primarily in the digestion of side chains include ß-mannosidase (3.2.1.25), -L-arabinofuranosidase (3.2.1.55), -D-glucuronidase (3.2.1.139), -D-galactosidase (3.2.1.22), acetyl xylan esterases (3.1.1.72), and ferulic acid esterase (3.1.1.73) (White et al., 1993; Bhat and Hazlewood, 2001).

Fiber-degrading enzyme activities are generally determined by measuring the rate of release of reducing sugars from pure substrates, with enzyme units expressed as the quantity of reducing sugars released per unit of time per unit of enzyme. Reducing sugars, which include monosaccharides and free sugar ends in oligosaccharides, can be measured colorimetrically using the Nelson/Somogyi copper method (Somogyi, 1952) or the dinitrosalicyclic acid method (Miller, 1959).

The most commonly used substrate for measuring cellulase activity is carboxymethyl cellulose, which measures endo-ß-1,4-glucanase activity (Wood and Bhat, 1988). Exoglucanase activity can be measured using crystalline cellulose preparations, such as Avicel. ß-glucosidase activity is determined by measuring the release of glucose from cellobiose, or the release of p-nitrophenol from p-nitrophenyl-ß-D-glucoside (Bhat and Hazlewood, 2001).

Xylanase activity is most commonly measured by determining the release of reducing sugars from prepared xylan, such as oat spelt or birchwood xylan. Xylanases are specific for the internal ß-1,4 linkages within the xylan backbone, and are generally considered endoxylanases (Bhat and Hazlewood, 2001). Endoxylanases can be considered to be debranching or nondebranching based on their ability to release arabinose in addition to hydrolyzing the main chain of xylan. ß-xylosidase activity can be determined by using p-nitrophenyl derivatives.

Enzyme activity measurements must be conducted under conditions closely defined with respect to temperature, pH, ionic strength, substrate concentration, and substrate type, since all of these factors will affect the activity of an enzyme. Enzyme activities of commercial enzyme products are typically measured at the manufacturers’ recommended optima. A temperature of approximately 60°C and a pH between 4 and 5 are the optimal conditions for most commercial enzymes (Coughlan, 1985). However, the optimal temperature and pH for assessing enzyme activity are not representative of the conditions in the rumen, which are closer to a pH of 6.0 to 6.7 and 39°C (Van Soest, 1994). Thus, the activities quoted for commercial enzyme products are considerably higher than for those that would be measured at a pH and temperature similar to that of the rumen. Furthermore, because the conditions of the assays and method of expressing enzyme activity vary among manufacturers, it is difficult to compare enzyme products or predict the efficacy of the product in ruminant diets.

Further details of the enzymology of cellulases and xylanases are provided by Ghose (1987), Wood and Bhat (1988), Bhat and Hazlewood (2001), and McCleary (2001).

Animal Responses to Feed Enzymes

The use of exogenous fiber-degrading enzyme additives for ruminants was first examined in the 1960s, as reviewed by Beauchemin and Rode (1996). Many of these early enzyme products were poorly characterized, animal responses were variable, and little to no effort was made to design these products specifically for ruminants. In the last decade, researchers have reexamined the potential use of exogenous enzymes for ruminants due to higher feed costs, lower costs of enzyme production, and the availability of more active and better defined enzyme preparations. However, descriptions of the enzyme products in most research papers are still generic at best. The formulation of enzyme products for ruminants has evolved over time, but it is difficult to follow these changes based on the information provided in the published literature. For example, there is no way of knowing whether an enzyme product from a particular enzyme supplier is the same from study to study. Typically, the enzyme products evaluated for ruminants are blends of various cellulases and xylanases that were originally produced and marketed for other uses. Thus, the components of a ruminant enzyme product can change over time due to improvements in production and strain selections of the enzymes driven by other markets. Also, ruminant enzyme products have been reformulated as new information became available.

Beef Cattle

Several recent studies have examined the use of exogenous enzyme products in high-forage diets fed to growing cattle (Beauchemin et al., 1995; Michal et al., 1996; Pritchard et al., 1996; McAllister et al., 1999; Wang et al., 1999; ZoBell et al., 2000). There is evidence that adding fibrolytic enzymes to forage diets can improve fiber digestibility (Beauchemin et al., 1995; Feng et al., 1996), but whether increased digestibility improves performance of cattle may depend on the physiological status of the cattle and the conditions of the experiment.

The results of adding fibrolytic enzymes to high-grain diets have been surprisingly more consistent than those for high-forage diets. Applying an enzyme product (Xylanase B, Biovance Technologies Inc., Omaha, NE) to a 95% barley-based diet improved feed efficiency by 6 to 12%, depending upon the level of enzyme addition (Beauchemin et al., 1997; 1999b; Iwaasa et al., 1997) (Table 1). Increased feed efficiency was due to an increase in diet digestibility (Iwaasa et al., 1997). Similarly, Krause et al. (1998) reported a 28% increase in ADF digestibility using a similar enzyme product added to a high-concentrate diet. Using another enzyme product (Finnfeeds Int. Ltd., Marlborough, U.K.), McAllister et al. (1999) reported that treating both the forage (ryegrass silage; 30% of the diet) and grain (barley, 70% of the diet) portions of the diet with 3.5 L/t of DM increased ADG by 10% (DM basis). However, ZoBell et al. (2000) reported no effect when what appears to be the same enzyme product was added to a high-grain barley-based feedlot finishing diet containing 17% forage (DM basis).

Dairy Cattle

A number of studies have examined the effects of fibrolytic exogenous enzymes on digestibility and milk production in dairy cows (Stokes and Zheng, 1995; Higginbotham et al., 1996; Nussio et al., 1997; Zheng and Stokes, 1997; Beauchemin et al., 1999a, 2000; Lewis et al., 1999; Nussio et al., 1997; Rode et al., 1999; Schingoethe et al., 1999; Yang et al., 1999, 2000; Kung et al., 2000; Phipps et al., 2000a,b; Bowman, 2001; Sutton et al., 2001; K. A. Beauchemin, L. M. Rode, B. I. Farr, J. A. Shelford, J. Baah, and G. F. Hartnell, unpublished data). Across 20 studies and 41 treatments, the average increase in DMI was 1.0 ± 1.3 kg/d, and the average increase in milk yield was 1.1 ± 1.5 kg/d (3.4% ± 4.7). Thus, when viewed across a variety of enzyme products and experimental conditions, the response is positive, but the variability is also high.

In some studies, the response to enzymes has been substantial. For example, Lewis et al. (1999) treated forage with a cellulase/xylanase mixture (FinnFeeds Int.; supplying 1 mL/kg of total mixed ration [TMR], DM basis) and observed that cows in early lactation produced 6.3 kg/d (16%) more milk (Table 2). However, higher and lower levels of the same enzyme product were less effective. Rode et al. (1999) applied an enzyme product (Promote, Biovance Technologies Inc., Omaha, NE) to the concentrate portion of a diet (supplying 1.3 g/kg of TMR on a DM basis) and observed a 3.6 kg/d (10%; P = 0.11) increase in milk production for cows in early lactation (Table 2). Yang et al. (2000) added an enzyme mixture (Biovance Technol, Omaha, NE) to the concentrate, and cows in early lactation produced 2 kg/d (5.9%) more milk (Table 2). However, there was no response when the same enzyme was added to the TMR.

Understanding the Variability in Animal Response

In general, results with beef cattle and dairy cows indicate a positive response to enzymes, but the results are variable. Although this variability may be viewed as an indication that feed enzyme additives are not a suitable technology for ruminants, we believe that much of the variability can be attributed to factors such as enzyme type, level of supplementation, method of enzyme application, and the energy balance of the test animals.

Enzyme Activity

There is increasing evidence that plant cell wall-degrading enzymes stimulate fiber digestion in the rumen (Feng et al., 1996; Yang et al., 1999). The response has been shown to be due to enzymatic activities (Nsereko et al., 1999), but the key enzymes involved have not been identified. Identification of the important enzyme activities would provide a rationale for designing more effective enzyme products for ruminants.

Wallace et al. (2001) used six enzyme products to examine the relationship between enzyme activities and in vitro gas production using grass and corn silage. A significant positive correlation was reported between cellulase activity and gas production from grass silage. In a companion study, it was observed that preparations relatively high in cellulase activity increased the rate of gas production from corn silage compared with the control (no added enzyme). In contrast, products with relatively high xylanase activity did not increase gas production when glucanase activity was low. Based on the results of this study, one may assume that it would be possible to improve the effectiveness of enzyme preparations by increasing cellulase activity. However, it should be noted that the levels of enzymes used in these studies were 20 to 40 times higher than the levels normally (0.5 to 2 mg/g of TMR DM) used as ruminant feed additives. The authors were unable to document any positive effects of exogenous enzymes on rate of gas production when using lower enzyme levels, which may have been a reflection of the techniques used. Caution must be applied when extrapolating from in vitro studies where relatively high levels of enzyme addition are used.

Colombatto et al. (2002a) used 23 commercial enzyme products to determine the relationship between enzyme activity and the in vitro degradation of feeds. The enzyme products were assayed for 16 different activities using pH and temperature conditions similar to those of the rumen (39°C and pH 6.0), and the level of enzyme product added per unit of feed was similar to that used in vivo (1.5 mg/g of DM). For incubations performed in buffer without ruminal fluid, there was a strong relationship between the release of reducing sugars from alfalfa hay or corn silage and the biochemical characteristics of the enzyme products. Protein content of the enzyme product explained about 60% of the variation in reducing sugars released, indicating that concentrated products with higher protein content were most effective. This can be explained by the fact that the same amount of each enzyme product was used even though the enzyme activity varied tremendously among products. ß-glucanase activity explained a further 24% of the variation in reducing sugars released from alfalfa hay, whereas endoglucanase, exoglucanase, ß-glucosidase, xylanase, and amylase explained a further 37% of the variation for corn silage. Thus, for alfalfa hay, about 84% of the variation, and for corn silage, about 97% of the variation, was explained by these factors.

The same enzyme products were evaluated for their effects on in vitro DM degradation (Colombatto et al., 2002b). Five of the 23 products significantly improved the 18-h degradation of alfalfa hay, and nine of the 23 products improved the degradation of corn silage. There was a significant relationship between xylanase activity and feed digestion. However, the relationship was positive with alfalfa hay, but negative with corn silage. This negative relationship observed for corn silage is perplexing and may not be one of "cause and effect." In addition, it should be noted that whereas the correlations between xylanase activity and digestion were significant, the xylanase activity alone explained less than 30% of the variation in degradation. No other significant relationship was observed between enzyme activities and feed degradation.

Similar observations have been reported in vivo. For example, Kung et al. (2000) compared two different enzyme products with similar cellulase and xylanase activities in the diets of lactating dairy cows. Only one of the two enzyme products resulted in an increase in milk yield.

Although further study is warranted, these results indicate that it may not be possible to predict the potential of increasing cell wall digestion in the rumen using exogenous feed enzymes based only on their biochemical characterization. This observation is not surprising because enzyme activities are measured on model substrates that do not represent the complexity of plant cell wall material. Enzyme assays are based on the initial rate of reaction with the substrate and do not relate to overall enzyme persistency. Furthermore, the conditions of the assays may not represent those of the rumen. Although enzyme activity units are important for quality control and to ensure that the customer is actually getting the product they expect, it must be recognized that these activity units bear little relationship to the efficacy of the product as a ruminant feed additive.

Enzyme Level

Some of the variability associated with the use of exogenous enzyme products in ruminant diets is due to supplementation with insufficient or excessive enzyme activity. In vivo responses to enzyme addition are typically nonlinear (Beauchemin et al., 1995; Kung et al., 2000), and it is possible to over-supplement. For example, Kung et al. (2000) offered forage (60% corn silage and 40% lucerne hay; DM basis) treated with increasing levels (0, 1, 2.5 mL/kg of TMR) of an enzyme product (FinnFeeds Int.) to cows. Cows fed the low level of enzyme tended (P < 0.10) to produce more milk (39.5 kg/d) than those fed the control diet (37.0 kg/d) or those fed the high level of enzyme (36.2 kg/d). Nonlinear responses have also been reported for growing beef cattle (Beauchemin et al., 1995). In that study, ADG of cattle fed alfalfa hay increased by 24 to 30% with lower levels of added enzyme (0.25 to 1 mL/kg of DM) as a result of increased intake of digestible DM, but higher levels of enzyme (2 and 4 mL/kg of DM) were not effective. With timothy hay, a high level (4 mL/kg of DM) of exogenous enzymes increased ADG of cattle by 36% as a result of a 17% increase in ADF digestibility and a 14% increase in digestible DM intake.

These studies demonstrate that high levels of enzyme addition can be less effective than low levels, and the optimal level of enzyme supplementation may depend on the diet. Lack of response to low levels of enzyme addition may indicate an insufficient supply of enzyme activity; however, the rationale for reduced efficacy of added enzymes at high levels of incorporation is not clear. Nsereko et al. (2002) reported a quadratic response in total bacterial numbers in ruminal fluid with increasing levels of an enzyme product from Trichoderma longibrachiatum added to a dairy cow diet. The authors speculated that application of a moderate level of enzyme to ruminant feeds caused some beneficial disruption of the surface structure of the feed either before or after ingestion. When excess enzyme was applied, the beneficial disruption of the feed surface structure may have diminished because the excess exogenous enzyme attached to feed may have restricted microbial attachment and limited digestion of feed.

Enzyme-Feed Specificity

The array of enzyme activities required to improve fiber digestion varies according to the composition of the feed. The principle of feed-enzyme specificity is illustrated in a study conducted in our lab (D. Colombatto, K. A. Beauchemin, and D. P. Morgavi; unpublished data) in which 26 enzyme products were used. The products were characterized biochemically, and then assessed in vitro in the presence of rumen fluid to test their ability to influence DM degradation of alfalfa hay or corn silage at 18 h of incubation. The enzyme products that effectively increased degradation were different for both forages (Table 3). Of the candidates that increased degradation of alfalfa, only enzyme E was also effective for corn silage (ranked 10th). These data clearly indicate the importance of matching the enzyme product to the forage.

Method of Providing Enzyme to Animals

Applying fibrolytic exogenous enzymes in a liquid form onto feeds prior to consumption can have a positive effect on animal performance (Rode et al., 1999; Schingoethe et al., 1999; Kung et al., 2000; Yang et al., 2000). In contrast, infusion of enzymes into the rumen has not been effective (Lewis et al., 1996; McAllister et al., 1999; Sutton et al., 2001). The close association of enzymes with feed may enable some form of preingestive attack of the enzymes upon the plant fiber and/or enhance binding of the enzymes to the feed, thereby increasing the resistance of the enzymes to proteolysis in the rumen.

There is apparently little or no requirement for a reaction phase or incubation time between treatment and feeding of forages. Lewis et al. (1996) observed an increase in total-tract NDF digestibility when an enzyme solution was applied to dry hay prior to feeding, but there was no difference between applying the enzyme immediately before feeding and a 24-h incubation period. In vitro studies have reported similar results (Colombatto, 2000).

Enzymes have been applied to TMR (Higginbotham et al., 1996; Beauchemin et al., 1999; Phipps et al., 2000b; Yang et al., 2000), hay (Beauchemin et al., 1995; Lewis et al., 1996; Yang et al., 1999), ensiled forages (Beauchemin et al., 1995; Phipps et al., 2000a), concentrate (Rode et al., 1999; Phipps et al., 2000b; Yang et al., 2000), supplement (Bowman, 2001), or premix (Bowman, 2001). Exogenous enzymes may be expected to be more effective when applied to high-moisture feeds (such as silages) compared to dry feeds because of the higher moisture content. The requirement for water in the hydrolysis of soluble sugars from complex polymers is a fundamental biochemical principle. Furthermore, silage pH values are usually at, or around, the optimal pH for most fungal enzymes. However, in practice, some exogenous enzymes are more effective when applied in a liquid form to dry forage as opposed to wet forage. Feng et al. (1996) applied an enzyme solution directly to grass and observed no effect when added to fresh or wilted forage; however, when it was applied to dried grass, enzymes increased DM and fiber digestibility. Similarly, Yang et al. (2000) reported increased milk production and digestibility of the diet when enzymes were added to the concentrate portion of a dairy cow diet, but not when they were added directly to TMR. In contrast, Phipps et al. (2000b) reported no difference between adding an enzyme product to concentrate or TMR, but the enzyme product used in that study was not effective. The reduced efficacy of exogenous enzymes applied to ensiled feeds may be due to inhibitory compounds in fermented feeds. Nsereko et al. (2000) reported the presence of compounds in whole-crop barley silage that inhibited endo-1,4-ß-xylanase activity of an enzyme product from T. longibrachiatum by 23 to 50%, although there was no effect on cellulase activity. Also, the application of exogenous enzymes to silages can accelerate their aerobic deterioration. The growth of the ephiphytic microbiota is stimulated by soluble sugars released by enzyme treatment, which could lead to a decrease of the silage feed value if the time elapsed between enzyme application and consumption is sufficiently long (Wang et al., 2002).

Yang et al. (1999) observed no difference between applying an enzyme product to dry forage or to both dry forage and concentrate. Others have also found that adding enzyme to concentrate to be effective (Beauchemin et al., 1997; Iwaasa et al., 1997; Rode et al., 1999; Yang et al., 2000). Bowman (2001) examined the effects of adding an enzyme product (Promote N.E.T., Agribrands International, St. Louis, MO) to various proportions of a TMR fed to dairy cows. The enzyme product was added to the concentrate portion of the TMR (45% of the TMR), to the supplement (4% of the TMR), or to the premix (0.2% of TMR). Diets with enzymes delivered the same quantity of enzyme per cow daily. Total-tract digestibility of NDF was increased from 44.3 to 55.6% (25%) when enzyme was added to concentrate, but the other treatments had no effect. When the same diets were evaluated in vitro, DM degradation of the TMR at 12 h of incubation was increased by 15% when enzymes were added to the concentrate portion, and by 17% when added to the premix portion, of the diet. At 48 h of incubation, only the treatment with enzyme added to a premix had higher DM digestibility compared to the control. Reasons for reduced response in vivo when enzymes are applied to a smaller portion of the diet are unclear. Beauchemin et al. (1999b) suggested that enzymes should be applied to a large portion of the diet to increase the chances that enzymes endure in the rumen. Adding enzyme to a small portion of the diet may allow rapid passage of enzyme from the rumen, lessening the enzyme effect in the rumen. Rapid passage of the enzyme is not an issue in vitro. Thus, in vitro batch culture assays used as a bioassay to predict the effects of exogenous enzymes on digestibility may not accurately predict variations in the in vivo response caused by method of providing the enzyme to the animal.

Level of Animal Productivity

Animal responses to exogenous enzymes are expected to be greatest in situations in which fiber digestion is compromised and when energy is the first-limiting nutrient in the diet. High-producing dairy cows and growing cattle require high levels of available energy to meet the demands of milk or meat production. It is not uncommon for feed intake of dairy cows to exceed four times the level of intake required for maintenance. In these commercial feeding situations, fiber digestion is often compromised due to low ruminal pH and rapid transit time through the rumen. The NRC (1989) assumes a 4% reduction in digestibility for each multiple increase in feed intake over maintenance. Thus, a dairy cow diet with a potential digestibility of 77% would have an actual digestibility of 68% when fed to high-producing dairy cows. More recently, the NRC (2001) recognized that the decrease in digestibility at high levels of intake is not constant. For high-quality diets, the decrease in digestibility is even greater than previously estimated.

Enzymes help bridge the gap between potential and actual performance of the animal. This concept is illustrated in a study in which dairy cows and sheep were fed a TMR with and without exogenous enzymes (Yang et al., 2000). When measured in dairy cows, the total-tract digestibility of the control diet was 63.9% for DM and 31.8% for ADF. As a result of lower intake as a percentage of BW, digestibility of the control diet was higher in sheep; total-tract digestion was 77.1% for DM and 49.8% for ADF. Use of an enzyme product improved digestibility of the diet when evaluated in dairy cows, but not in sheep. These results indicate that exogenous enzymes improve feed digestion when the potential digestibility of the diet is not attained because digestion is compromised. It is this "loss" in digestible energy that is captured with the use of feed enzymes.

Existing enzyme technology is not likely to benefit ruminants fed at maintenance; rather, the greatest responses will be for ruminants fed for maximal productivity. Similarly, the response to exogenous enzymes is greater for dairy cows in early lactation than for those in later lactation (Nussio et al., 1997; Schingoethe et al., 1999).

Mode of Action: An Integrated Hypothesis

In light of the exceptionally high starch- and fiber-digesting capacity of the rumen, it is difficult to explain why treatment of grain or forage with enzymes prior to consumption further improves feed utilization. It is clear that the mode of action for exogenous enzymes improving digestion of plant cell wall is complex. Based on our experience and the available information, we have developed a putative hypothesis that we feel accounts for the most critical factors that explain the observed increases in fiber and DM digestion.

Exogenous enzymes in the rumen are generally more stable than previously thought (Hristov et al., 1998; Morgavi et al., 2000b, 2001), particularly when applied to feed prior to ingestion. Application of enzymes to feed enhances the binding of the enzyme with the substrate, which increases the resistance of the enzymes to proteolysis and prolongs their residence time within the rumen. In the rumen, the close association between digestive bacteria and feed particles concentrates digestive enzymes close to their specific substrates. However, some ensiled feeds contain compounds that are inhibitory to xylanases (Nsereko et al., 2000); therefore, applying enzymes to dry feeds decreases the variability in response.

Applying enzymes to feed also provides a slow-release mechanism for enzymes in the rumen (Beauchemin et al., 1999a). Thus, the greater the proportion of the diet treated with enzymes, the greater the chances that enzymes endure in the rumen. Without this stable feed-enzyme complex, the enzymes are solubilized in ruminal fluid and flow rapidly from the rumen.

There is evidence for preconsumptive effects of exogenous enzymes causing the release of soluble carbohydrates (Hristov et al., 1996), and in some cases, partial solubilization of NDF and ADF (Gwayumba and Christensen, 1997; Krause et al., 1998). Nsereko et al. (2000) demonstrated compelling evidence that applying enzymes to feed causes structural changes to occur, thereby making feed more amenable to degradation. Cell wall hydrolysis in the rumen proceeds in an erosive manner (White et al., 1993), and it is well recognized that a major constraint to digestion is the limited colonization and penetration of cellulolytic microbes and their hydrolytic enzymes onto the exposed surfaces of feed particles.

It is most likely that the major portion of the positive production responses resulting from the use of enzyme additives is due to ruminal effects. Adding exogenous enzymes to the diet increases the hydrolytic capacity of the rumen mainly due to increased bacterial attachment (Yang et al., 1999; Morgavi et al., 2000c; Wang et al., 2001), stimulation of rumen microbial populations (Wang et al., 2001; Nsereko et al., 2002), and synergistic effects with hydrolases of ruminal microorganisms (Morgavi et al., 2000a). The net effect is increased enzymatic activity within the rumen, which enhances digestibility of the total diet fed. Thus, improvements in digestibility are not limited to the dietary component to which the enzymes are applied, which explains why fibrolytic enzymes can be effective when added to the concentrate portion of a diet. Increased hydrolytic capacity of the rumen can also lead to an increase in digestibility of the nonfiber carbohydrate fraction, in addition to increasing digestibility of the fiber components of a diet, which explains why fibrolytic enzymes can be effective in high-concentrate diets.

Exogenous enzymes appear to survive in the small intestine for a time sufficient to have an effect on target substrates (Morgavi et al., 2001). However, postruminal effects of exogenous enzymes on digestion are likely only a significant factor when enzymes are infused into the rumen (Hristov et al., 2000) or added to feed in a manner that allows for easy solubilization from feed and rapid passage from the rumen (Beauchemin et al., 1999). Such may be the case when enzymes are applied to wet feeds prior to feeding or when offered as a concentrated premix. Also, the relatively short retention times through the lower gastrointestinal tract means that, postruminally, enzymes are only likely to be effective against the least recalcitrant fraction of dietary fiber. Significant quantities of this easily digestible fiber will only be passing out of the rumen in abnormal circumstances such as extreme ruminal acidosis.

	Implications

Top Abstract Introduction Implications Literature Cited

 
Adding exogenous fibrolytic enzymes to dairy cow and feedlot cattle diets can potentially improve cell wall digestion and the efficiency of feed utilization by ruminants. Positive responses in milk production and growth rate have been observed for cattle fed some enzyme products, although results have been inconsistent. Some of the variation can be attributed to product formulation, under- or over-supplementation of enzyme activity, inappropriate method of providing the enzyme product to the animal, and the level of productivity of the test animal. Research is needed to understand the mode of action of these products so that on-farm efficacy of ruminant enzyme technology can be assured. With increasing consumer concern about the use of growth promoters and antibiotics in ruminant production, and the magnitude of increased animal performance obtainable using feed enzymes, these products could play an important role in future ruminant production systems.

	Footnotes

 
¹ LRC Contribution No. 387 02051.

² Invited presentation given at the Joint 2002 ADSA, ASAS, and the Canadian Soc. Anim. Sci. Mtg., Québec City, Québec, Canada.

³ Correspondance: Box 3000 (phone: 403-317-2235; fax: 403-317-2182; E-mail: beauchemin{at}agr.gc.ca).

Received for publication June 6, 2002. Accepted for publication September 12, 2002.

	Literature Cited

Top Abstract Introduction Implications Literature Cited

 


Beauchemin, K. A., L. M. Rode, and V. J. H. Sewalt. 1995. Fibrolytic enzymes increase fiber digestibility and growth rate of steers fed dry forages. Can. J. Anim. Sci. 75:641–644.

Beauchemin, K. A., and L. M. Rode. 1996. Use of feed enzymes in ruminant nutrition. Page 103 in Animal Science Research and Development–Meeting Future Challenges. L. M. Rode, ed. Ministry of Supply and Services Canada, Ottawa, ON.

Beauchemin, K. A., S. D. M. Jones, L. M. Rode, and V. J. H. Sewalt. 1997. Effects of fibrolytic enzyme in corn or barley diets on performance and carcass characteristics of feedlot cattle. Can. J. Anim. Sci. 77:645–653.

Beauchemin, K. A., W. Z. Yang, and L. M. Rode. 1999a. Effects of grain source and enzyme additive on site and extent of nutrient digestion in dairy cows. J. Dairy Sci. 82:378–390.[Abstract]

Beauchemin, K. A., L. M. Rode, and D. Karren. 1999b. Use of feed enzymes in feedlot finishing diets. Can. J. Anim. Sci. 79:243–246.

Beauchemin, K. A., L. M. Rode, M. Maekawa, D. P. Morgavi, and R. Kampen. 2000. Evaluation of a non-starch polysaccharidase feed enzyme in dairy cow diets. J. Dairy Sci. 83:543–553.[Abstract]

Bhat, M. K., and G. P. Hazlewood 2001. Enzymology and other characteristics of cellulases and xylanases. Page 11 in Enzymes in Farm Animal Nutrition. M. Bedford and G. Partridge, ed. CABI Publishing, Oxon, U.K.

Bowman, G. R. 2001. Digestion, ruminal pH, salivation, and feeding behaviour of lactating dairy cows fed a diet supplemented with fibrolytic enzymes. M.S. Thesis, Univ. of British Colombia, Vancouver, Canada.

Colombatto, D. 2000. Use of enzymes to improve fibre utilization in ruminants. A biochemical and in vitro rumen degradation assessment. Ph.D. Diss., Univ. of Reading, U.K.

Colombatto, D., D. P. Morgavi, A. F. Furtado, and K. A. Beauchemin. 2002a. Screening of fibrolytic enzymes as feed additives for ruminants: Can the effect of enzyme additives on in vitro fermentations be predicted by enzyme activities and feed hydrolysis? J. Dairy Sci. 85:355 (Abstr.) (In press).[Abstract]

Colombatto, D., D. P. Morgavi, A. F. Furtado, and K. A. Beauchemin. 2002b. Screening of fibrolytic enzymes as additives for ruminant diets: relationship between enzyme activities and the in vitro degradation of enzyme-treated forages. Page 210 in Proc. Br. Soc. Anim. Sci Annu. Mtg. Penicuik, U.K.

Colombatto, D., F. L. Mould, M. K. Bhat, and E. Owen. 2002c. The effect of fibrolytic enzyme application on rate and extent of alfalfa stem fermentation, assessed in vitro. Page 209 in Proc. Br. Soc. Anim. Sci. Annu. Mtg. Penicuik, U.K.

Considine, P. J., and M. P. Coughlan. 1989. Production of carbohydrate-hydrolyzing enzyme blends by solid-state fermentation. Pages 273–281 in Enzyme Systems for Lignocellulose Degradation. M. P. Coughlan, ed. Elsevier Applied Science, New York.

Coughlan, M. P. 1985. The properties of fungal and bacterial cellulases with comment on their production and application. Biotechnol. Genet. Eng. Rev. 3:39–109.

Cowan, W. D. 1994. Factors affecting the manufacture, distribution, application and overall quality of enzymes in poultry feeds. Pages 175–184 in Joint Proc. 2nd Int. Roundtable on Anim. Feed Biotechnol.—Probiotics, and Workshop on Anim. Feed Enzymes, Ottawa.

Feng, P., C. W. Hunt, G. T. Pritchard, and W. E. Julien. 1996. Effect of enzyme preparations on in situ and in vitro degradation and in vivo digestive characteristics of mature cool-season grass forage in beef steers. J. Anim. Sci. 74:1349–1357.[Abstract]

Gashe, B. A. 1992. Cellulase production and activity by Trichoderma sp. A-001. J. Appl. Bacteriol. 73:79–82.

Ghose, T. K. 1987. Measurement of cellulase activity. Pure Appl. Chem. 59:257–268.

Gwayumba, W., and D. A. Christensen. 1997. The effect of fibrolytic enzymes on protein and carbohydrate degradation fractions in forages. Can. J. Anim. Sci. 77:541–542. (Abstr.)

Higginbotham, G. E., E. J. dePetters, S. L. Berry, and A. Ahmadi. 1996. Effect of adding a cell wall degrading enzyme to a total mixed ration for lactating dairy cows. Prof. Anim. Sci. 12:81–85.

Hristov, A. N., L. M. Rode, K. A. Beauchemin, and R. L. Wuerfel. 1996. Effect of a commercial enzyme preparation on barley silage in vitro and in sacco dry matter degradability. Pages 282–284 in Proc. Western Section, Am. Soc. Anim. Sci.

Hristov, A. N., T. A. McAllister, and K.-J. Cheng. 1998. Stability of exogenous polysaccharide-degrading enzyme in the rumen. Anim. Feed Sci. Technol. 76:161–168.

Hristov, A. N., T. A. McAllister, and K.-J. Cheng. 2000. Intraruminal supplementation with increasing levels of exogenous polysaccharides-degrading enzymes: Effects on nutrient digestion in cattle fed a barley grain diet. J. Anim. Sci. 78:477–487.[Abstract/Free Full Text]

Iwaasa, A. D., L. M. Rode, K. A. Beauchemin, and S. Eivemark. 1997. Effect of fibrolytic enzymes in barley-based diets on performance of feedlot cattle and in vitro gas production. Joint Rowett Res. Inst.—Inst. Natl. de Recherche Agronomique Rumen Microbiol. Symp., Aberdeen, Scotland, Poster 39.

Krause, M., K. A. Beauchemin, L. M. Rode, B. I. Farr, and P. Nørgaard. 1998. Fibrolytic enzyme treatment of barley grain and source of forage in high-grain diets fed to growing cattle. J. Anim. Sci. 96:1010–1015.

Kung, L., Jr., R. J. Treacher, G. A. Nauman, A. M. Smagala, K. M. Endres, and M. A Cohen. 2000. The effect of treating forages with fibrolytic enzymes on its nutritive value and lactation performance of dairy cows. J. Dairy Sci. 83:115–122.[Abstract]

Lee, B., A. L. Pometto, A. Demici, and P. N. Hinz, 1998. Media evaluation for the production of microbial enzymes. J. Agric. Food Chem. 46:4775–4778.

Lewis, G. E., C. W. Hunt, W. K. Sanchez, R. Treacher, G. T. Pritchard, and P. Feng. 1996. Effect of direct-fed fibrolytic enzymes on the digestive characteristics of a forage-based diet fed to beef steers. J. Anim. Sci. 74:3020–3028.[Abstract]

Lewis, G. E., W. K. Sanchez, C. W. Hunt, M. A. Guy, G. T. Pritchard, B. I. Swanson, and R. J. Treacher. 1999. Effect of direct-fed fibrolytic enzymes on the lactational performance of dairy cows. J. Dairy Sci. 82:611–617.[Abstract]

Martin, S. 2000. Use of fungi and organic acids in animal diets. Page 34 in Direct-Fed Microbial, Enzyme and Forage Additive Compendium. S. Muirhead, ed. The Miller Publishing Co., Minnetonka, MN.

McAllister, T. A., S. J. Oosting, J. D. Popp, Z. Mir, L. J. Yanke, A. N. Hristov, R. J. Treacher, and K.-J. Cheng. 1999. Effect of exogenous enzymes on digestibility of barley silage and growth performance of feedlot cattle. Can. J. Anim. Sci. 79:353–360.

McCleary, B. V. 2001. Analysis of feed enzymes. Pages 85–107 in Enzymes in Farm Animal Nutrition. M. Bedford, and G. Partridge, ed. CABI Publishing, Oxon, U.K.

Michal, J. J., K. A. Johnson, and R. J. Treacher. 1996. The impact of direct fed fibrolytic enzymes on the growth rate and feed efficiency of growing beef steers and heifers. J. Anim. Sci. 74 (Suppl. 1):296. (Abstr.)

Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31:426–428.

Morgavi, D. P., K. A. Beauchemin, V. L. Nsereko, L. M. Rode, A. D. Iwaasa, W. Z. Yang, T. A. McAllister, and Y. Wang. 2000a. Synergy between ruminal fibrolytic enzymes and enzymes from Trichoderma Longibrachiatum. J. Dairy Sci. 83:1310–1321.[Abstract]

Morgavi, D. P., C. J. Newbold, D. E. Beever, and R. J. Wallace. 2000b. Stability and stabilization of potential feed additive enzymes in rumen fluid. Enzyme Microb. Technol. 26:171–177.[Medline]

Morgavi, D. P., V. L. Nsereko, L. M. Rode, K. A. Beauchemin, T. A. McAllister, and Y. Wang. 2000c. A Trichoderma feed enzyme preparation enhances adhesion of Fibrobacter succinogenes to complex substrates but not to pure cellulose. Page 31 in Proc. XXV Conf. Rumen Function, Chicago.

Morgavi, D. P., K. A. Beauchemin, V. L. Nsereko, L. M. Rode, T. A. McAllister, A. D. Iwaasa, Y. Wang, and W. Z. Yang. 2001. Resistance of feed enzymes to proteolytic inactivation by rumen microorganisms and gastrointestinal proteases. J. Anim. Sci. 79:1621–1630.[Abstract/Free Full Text]

Muirhead, S. 2001. Direct-Fed Microbial, Enzyme and Forage Additive Compendium, The Miller Publishing Co., Minnetonka, MN.

Nakashima, Y., E. R. Orskov, P. M. Hotten, K. Ambo, and Y. Takase. 1988. Rumen degradation of straw. 6. Effect of polysaccharidase enzymes on degradation characteristics of rice straw. Anim. Prod. 47:421–427.

NRC. 1989. Page 9 in Nutrient Requirements of Dairy Cattle. 6th ed. Natl. Acad. Press, Washington, DC.

NRC. 2001. Page 16 in Nutrient Requirements of Dairy Cattle. 7th ed. Natl. Acad. Press, Washington, DC.

Nsereko, V. L., R. J. Wallace, L. M. Rode, K. A. Beauchemin, T. A. McAllister, N. McKain, D. P. Morgavi, Y. Wang, A. D. Iwaasa, and W. Z. Yang. 1999. Effects of enzymic and non-enzymic components of a fungal enzyme preparation on digestion of grass silage by rumen microorganisms in vitro. J. Dairy Sci. 82 (Suppl. 1):123. (Abstr.)

Nsereko, V. L., D. P. Morgavi, L. M. Rode, K. A. Beauchemin, and T. A. McAllister. 2000. Effects of fungal enzyme preparations on hydrolysis and subsequent degradation of alfalfa hay fiber by mixed rumen microorganisms in vitro. Anim. Feed Sci. Technol. 88:153–170.[Medline]

Nsereko, V. L., K. A. Beauchemin, D. P. Morgavi, L. M. Rode, A. F. Furtado, T. A. McAllister, A. D. Iwaasa, W. Z. Yang, and Y. Wang. 2002. Effect of a fibrolytic enzyme preparation from Trichoderma longibrachiatum on the rumen microbial population of dairy cows. Can. J. Microbiol. 48:14–20.[Medline]

Nussio, L. G., J. T. Huber, C. B. Theurer, C. B. Nussio, J. Santos, M. Tarazon, R. O. Lima-Filho, B. Riggs, M. Lamoreaux, and R. J. Treacher. 1997. Influence of a cellulase/xylanase complex (C/X) on lactational performance of dairy cows fed alfalfa hay (AH) based diets, J. Dairy Sci. 80 (Suppl. 1):220. (Abstr.)[Abstract/Free Full Text]

Pendleton, B. 2000. The regulatory environment. Page 49 in Direct-Fed Microbial, Enzyme and Forage Additive Compendium. S. Muirhea, ed. The Miller Publishing Co., Minnetonka, MN.

Phipps, R. H., J. D. Sutton, D. E. Beever, M. K. Bhat, G. F. Hartnell, J. L. Vicini, and D. L. Hard. 2000a. Evaluation of feed additives in the diet of lactating dairy cows. J. Dairy Sci. 83(Suppl. 1):23. (Abstr.)[Abstract]

Phipps, R. H., J. D. Sutton, D. E. Beever, M. K. Bhat, G. F. Hartnell, J. Vicini, and D. L. Hard. 2000b. Effect of cell-wall degrading enzymes and method of application on feed intake and milk production of Holstein-Friesian dairy cows. J. Dairy Sci 83(Suppl. 1):23. (Abstr.)

Pritchard, G., C. Hunt, A. Allen, and R. Treacher. 1996. Effect of direct-fed fibrolytic enzymes on digestion and growth performance in beef cattle. J. Anim. Sci. 74(Suppl. 1):296. (Abstr.)

Rode, L. M., W. Z. Yang, and K. A Beauchemin. 1999. Fibrolytic enzyme supplements for dairy cows in early lactation. J. Dairy Sci. 82:2121–2126.[Abstract]

Schingoethe, D. J., G. A. Stegeman, and R. J. Treacher. 1999. Response of lactating dairy cows to a cellulase and xylanase enzyme mixture applied to forages at the time of feeding. J. Dairy Sci. 82:996–1003.[Abstract]

Somogyi, M. 1952. Notes on sugar determination. J. Biol. Chem. 195:19–23.[Free Full Text]

Stokes, M. R., and S. Zheng. 1995. The use of carbohydrase enzymes as feed additives for early lactation cows. Page 34 in Proc. 23rd Conf. Rumen Function, Chicago.

Sutton, J. D., R. H. Phipps, D. E. Beever, D. J. Humphries, G. F. Hartnell, and J. L. Vicini. 2001. Comparison of different methods of administration on the effect of fibrolytic enzymes on digestive processes in lactating cows. J. Dairy Sci. 84(Suppl. 1):37. (Abstr.)

Van Soest, P. J. 1994. Nutritional Ecology of the Ruminant. 2nd ed. Cornell Univ. Press, Ithaca, NY.

Wallace, R. J., S. J. A. Wallace, N. McKain, V. L. Nsereko, and G. F. Hartnell. 2001. Influence of supplementary fibrolytic enzymes on the fermentation of corn and grass silages by mixed ruminal microorganisms in vitro. J. Anim. Sci. 79:1905–1916.[Abstract/Free Full Text]

Wang, Y., T. A. McAllister, R. E. Wilde, J. Baah, K. A. Beauchemin, L. M. Rode, J. A. Shelford, and K.-J. Cheng. 1999. Effects of monensin, exogenous fibrolytic enzymes and Tween 80 on performance of feedlot cattle. Can J. Anim. Sci. 79:587. (Abstr.)

Wang, Y., T. A. McAllister, L. M. Rode, K. A. Beauchemin, D. P. Morgavi, V. L. Nsereko, A. D. Iwaasa, and W. Yang. 2001. Effects of an exogenous enzyme preparation on microbial protein synthesis, enzyme activity and attachment to feed in the Rumen Simulation Technique (Rusitec). Br. J. Nutr. 85:325–332.[Medline]

Wang, Y., T. A. McAllister, L. M. Rode, K. A. Beauchemin, D. P. Morgavi, V. L. Nsereko, A. D. Iwaasa, and W. Yang, 2002. Effect of exogenous fibrolytic enzymes on epiphytic microbial populations and in vitro silage digestion. J. Sci. Food Agric. 82:760–768.

White, B. A., R. I. Mackie, and K. C. Doerner. 1993. Enzymatic hydrolysis of forage cell walls. Pages 455–484 in Forage Cell Wall Structure and Digestibility. H. G. Jung, D. R. Buxton, R. D. Hatfield, and J. Ralph, ed. Am. Soc. Agron., Crop Sci. Soc. Am., Soil Sci. Soc. Am., Madison, WI.

Wood, T. M., and K. M. Bhat. 1988. Methods for measuring cellulase activities. Page 87 in Methods in Enzymology. Vol. 160. W. A. Wood and S. T. Kellogg, ed. Academic Press Inc., New York.

Yang, W. Z., K. A. Beauchemin, and L. M Rode. 1999. Effects of enzyme feed additives on extent of digestion and milk production of lactating dairy cows. J. Dairy Sci. 82:391–403.[Abstract]

Yang, W. Z., K. A. Beauchemin, and L. M. Rode. 2000. A comparison of methods of adding fibrolytic enzymes to lactating cow diets. J. Dairy Sci. 83:2512–2520.[Abstract]

Zheng, S., and M. R. Stokes. 1997. Effects of fibrolytic enzymes on feed stability and performance of lactating cows. J. Dairy Sci. 80(Suppl. 1):278. (Abstr.)

ZoBell, D. R., R. D. Weidmeier, K. C. Olson, and R. J. Treacher. 2000. The effect of an exogenous enzyme treatment on production and carcass characteristics of growing and finishing steers. Anim. Feed Sci. Technol. 87:279–285.

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