Enzymes to Enhance Utilization of

Enzymes to Enhance Utilization of Feed in Dairy Cows

Lyle M. Rode and Karen A. Beauchemin

Research Center, Agriculture and Agri-Food Canada, Box 3000, Lethbridge, AB T1J 4B1, Canada

# Take Home Messages

Fibrolytic enzymes can be a highly effective means of increasing milk production and diet digestibility in dairy cows consuming a wide range of feedstuffs.

Enzymes with similar in vitro activity can differ greatly in their effectiveness when fed to cattle.

Method of application is critical to successful use of enzyme technology.

Feed enzymes are more effective when a liquid preparation is sprayed on feed than when included in the diet in a dry formulation.

Feed enzymes are more effective when applied to dry forage or concentrates than when applied to wet feeds.

# Introduction

Enzymes are commonly used to improve the nutritive value of feeds for non-ruminants, particularly in broiler diets, and as silage additives, but they have not been routinely used in adult ruminant diets. Fibrolytic activity within the rumen environment is normally very high, and presumably not easily increased by a simple addition of exogenous enzyme products. Furthermore, it is usually assumed that because exogenous enzymes are soluble, they cannot survive proteolysis in the rumen. In the 1960's, a number of studies with cattle and sheep showed that feed enzymes substantially improved feed digestibility and animal performance, as recently reviewed (1). However, other studies reported no effects, and even negative responses. Given the relatively high cost of feed enzymes compared to other feed additives, the inconsistency of responses, and the potential of improving animal performance using other emerging technologies, the concept of using enzymes in ruminant diets was abandoned at that time. High costs of livestock production and consumer concern over the use of Achemical@ and antibiotic feed additives, combined with the availability of newer enzyme preparations has prompted a renewed interest in the potential of feed enzymes for ruminants.

# What are Feed Enzymes?

Enzymes are naturally occurring proteins that catalyze chemical reactions in biological systems. Enzymes are involved in the digestion of all complex feed molecules into their smaller base chemical constituents (e.g. glucose, amino acids) in both bacteria and the host ruminant. Without enzymes, feed would remain undigested by the animal. There are over 50 registered products in Canada for use in ruminant diets that contain enzymes. The major active ingredients in most of these products are direct-fed microbials (DFMs), microbial extracts or vitamin/mineral preparations. For the most part, the fibrolytic enzyme activities in these products are low and their modes of action fall outside the scope of this presentation. The information contained in this article pertains to products with high fibrolytic enzyme activity and is not necessarily applicable to DFMs with low enzyme activity.

Enzyme Activity

Enzymes are classified broadly by the substrate upon which they react, and by their specificity. Commercial enzyme products are fermentation extracts of bacterial (Bacillus sp.) or fungal (Trichoderma and Aspergillus spp.) origin and contain a unique array of enzymatic activities. For example, an enzyme preparation with predominant xylanase activity, may also have significant protease, cellulase, pectinase, beta-glucanase, or other enzymatic activities. Enzyme activity can be assayed using an in vitro method by measuring production of end-products (i.e., reducing sugars, amino acids or peptides) per unit time, using a specified substrate, under defined conditions. These substrates are often purified or modified to simplify measurement of activity. Companies that market enzyme products use a wide range of enzyme assays and subsequent activity units. While these activity units are important to ensure that the customer is actually getting the product they expect, it is must be realized that these activity units bear little relationship to the efficacy as a ruminant feed additive. For example, we compared commercial enzyme preparations at the same activity units (carboxymethyl cellulase; CMCase) and observed a ten-fold difference in the enzymes= ability to digest alfalfa hay. Furthermore, assaying enzyme activity subsequent to the enzyme=s incorporation into a diet is exceedingly difficult, although newer approaches may overcome some of these difficulties (12). This makes it difficult to compare the relative effectiveness of enzyme mixtures without conducting a biological assay or feeding trial. Typical methods used to evaluate feeds such as in vitro and in sacco assays can be used to screen enzymes in relation to different substrates, but in our experience, the results obtained often do not concur with results from feeding trials.

Inclusion rate of exogenous enzymes in ruminant diets is usually in the range of .01 to 1% of the diet, contributing about 10 to 100-times higher fibrolytic activity per gram of feed than when silage additives are used. Based on the estimated average fibrolytic activity normally present in the fluid fraction in the rumen (R. J. Wallace and C. J. Newbold, personal communication), we estimate that supplemental enzymes may contribute up to 15% of the total fibrolytic activity. However, the activity of commercial enzymes is measured at optimal pH, which generally differs from that of rumen fluid. Thus, once ingested, exogenous enzymes likely contribute considerably less fibrolytic activity than calculated. Furthermore, fibrolytic enzyme activity associated with particulate matter is about 2 to 10-times higher than for ruminal fluid (17). Thus, the contribution of exogenous enzymes to the fibrolytic activity is difficult to estimate, but is likely low relative to that of the rumen.

Stability in the rumen is critical for enzymes to be effective as feed additives. Considerable variation exists among fibrolytic enzymes in their ability to maintain activity in the rumen environment. Some enzymes lose their activity rapidly when incubated in ruminal fluid whereas other enzymes show little or no loss in activity even after 12 hours of ruminal incubation. Feed enzyme supplements may impart their effect on feed digestibility in the small intestine as well as the rumen. Therefore stability is very important if these enzymes are to remain active in the intestine as well as the rumen. Xylanases have been shown to be much more stable in the rumen than cellulases (8). This may be due to the relatively larger and more complex structure of cellulases compared to xylanases.

# Factors Affecting Enzyme Effectiveness

More than thirty years ago, various studies showed significant improvements in average daily gain (ADG) and feed conversion ratio (FCR) of cattle by supplementing diets with feed enzymes containing amylolytic, proteolytic, and cellulolytic activities (3, 14, 11). Improvements in animal performance were due to increased dry matter and fiber digestibility (7, 13, 15). However, other studies showed that exogenous enzymes did not consistently improve animal performance (3, 11), and the mechanism for improved growth was not always confirmed by digestibility trials. Lack of information about the enzyme products used and the method of providing the product to the animal makes it difficult to compare among studies, or to compare the results from early studies to more recent studies. Inconsistent results seem to be caused by a number of factors including diet composition, type of enzyme preparation used, complement of enzyme activities, level of enzyme provided, enzyme stability, and method of application. The focus of our research has been to determine the mode of action of fibrolytic enzyme additives in ruminant diets, so that enzyme preparations can be formulated and applied to produce consistent and optimum responses.

Enzyme - Feed Specificity

Use of exogenous enzymes can be beneficial when the preparation and the feed composition are complementary. We evaluated commercial enzyme preparations in vitro using alfalfa hay or barley silage as a substrate. Effectiveness of enzyme products differed for the two substrates, indicating that an enzyme product that elicits a positive response in one diet may not be effective if evaluated using a different diet. However, a good feed enzyme product should contain a blend of enzymes so that it will be effective in a range of feedstuffs. It is unlikely that an enzyme preparation that is effective in one feed will be completely ineffective in another feed. However, the level of enzyme required to effectively treat feedstuffs will vary with the diet being considered. Feed enzymes have been demonstrated to be effective in a wide range of diets containing alfalfa hay and haylage, barley and corn silage and barley and corn grain.

Enzyme Level

The optimal level of enzyme addition depends on the substrate, indicating the need to determine optimum application rates of enzyme preparations for individual feed substrates (2). For example, with alfalfa hay, in growing cattle, average daily gain (ADG) was increased with lower enzyme levels, but not at higher levels (Table 1).

Compared to the Control diet, ADG was increased by 30% by adding 4X the amount of enzyme. For timothy hay, enzymes added at the highest level (16X) improved ADG by 36% due mainly to a 17% increase in fibe digestibility. This improvement in ADG was not accompanied by an increase in feed intake, and therefore, feed conversion ratio (FCR) improved by 20%. No response in ADG was observed at the low and moderate levels for timothy, in contrast to the observations for alfalfa hay.

Our early studies (2; Table 1) as well as those of others (16) have shown a quadratic response where high levels of enzyme application can cause reduced or even negative responses. However, these are ultra-high levels of enzyme and well above economical levels. Within cost-effective ranges of supplementation, the responses to varying levels of enzyme additions are essentially linear for both dairy and feedlot diets (Table 2).

Table 1. Effects of incremental levels of fibrolytic enzyme additives to alfalfa hay cubes, timothy hay cubes, and barley silage fed to cattle (2).^{a, b}

	Control	1X	2X	4X	8X	16X
Alfalfa hay
ADG, kg/d	1.03a	1.27bc	1.28bc	1.34c	1.19abc	1.12ab
DMI, kg/d	10.2a	10.8a	10.5a	11.7b	10.9a	10.3a
FCR kg DM/kg gain	9.9	9	8.7	8.5	9.6	9.5
Timothy hay
ADG, kg/d	1.21a	1.32a	1.13a	1.24a	1.27a	1.64b
DMI, kg/d	8.8bc	8.3ab	7.5a	9.2bc	8.6bc	9.3c
FCR kg DM/kg gain	7.3b	6.5ab	7.5b	6.3ab	6.8ab	5.9a
Barley silage
ADG, kg/d	1.12	1.15	0.99	1.02	1.12	1.11
DMI, kg/d	7.5ab	8.1b	6.8a	7.8b	7.3ab	7.3ab
FCR kg DM/kg gain	7.1	7	7.2	7.6	6.9	7

^{a Enzyme was obtained from Biovance Technologies Inc. (Omaha, NE) and Genencor (Rochester, NY).
^{b Initial weight of cattle was 289 kg. Cattle were fed for 72 d. Supplements were added to each forage diet to provide a minimum of 12% crude protein, and to supply adequate UIP and minerals.
Table 2. Dry matter intake, milk yield and composition of dairy cows fed diets containing 45% alfalfa cubes treated with Low (1X) or Medium (2X) levels of fiber degrading enzymes (DM basis).}}

Item	Diet
Item	Control	Low Enzyme	Medium enzyme
Dry matter intake, kg/d	20.4	20.7	20.7
Milk yield, kg/d	23.7^b	24.6^ab	25.6^a
4% fat corrected milk, kg/d	22.7^b	23.3^ab	24.6^a
Milk fat content, %	3.79	3.7	3.78
Milk protein content, %	3.4	3.4	3.4
Kg milk/kg dry matter intake	1.2	1.22	1.29
Net revenue, $/cow/day	12.35	12.65	13.22
Enzyme cost, $/cow/day	0	0.1	0.2
Return over enzyme ($/cow/day)¹	0	0.2	0.73
Return over enzyme ($/100 cows/year)	0	7,300	26,645

^{a,b Means with different letter within a row differ ( P < 0.05).
^{1 Net revenue is gross milk revenue minus producer deductions, based on: 1,000 litres fluid quota plus MSQ to cover excess milk and class 1 skimoff; October, 1997 Alberta Dairy Control Board.}}Application Method

Effects of exogenous enzymes are maximized when an aqueous enzyme solution is applied onto dry feed. In our laboratory, we have observed that applying the enzyme to dry feed creates a stable enzyme-feed complex that increases enzyme effectiveness. This stable complex occurs quickly (within hours) and once stabilized onto dry feed, the enzymes are stable and effective for at least several weeks. While it is reasonable to expect that there is an effect of temperature in the development of the enzyme-feed complex, we have observed no difference in efficacy when enzymes are applied to dry barley grain over a temperature range of -30 to +35^BC. However, McAllister (unpublished results) observed a linear improvement for in vitro digestion of barley silage as temperature increased. Whether this difference is due to feedstuff or moisture content is unclear.

Processed forages and grains are stored prior to feeding, providing an ideal opportunity for the use of enzyme products (2). Enzymes can be applied during feed manufacturing, but care must be taken to ensure that the temperature used during processing is within the acceptable range for the particular enzyme preparation used (4). Processing temperatures similar to those used for enzyme-treated poultry feeds are suitable for ruminant feed enzymes as well.

It has been more difficult to obtain positive responses to enzyme supplementation to barley silage than dry hay (2; Table 1). The lack of response in barley silage diets could be due to substrate specificity, method of applying the enzymes, time required for the enzymes to interact with the feed or moisture content of the feed. Feng et al. (5, 6) applied an enzyme solution directly to grass, and observed no effect when added to fresh or wilted hay, but when applied to dried grass, enzymes increased DM and fiber digestibilities. When we applied a low level of a fibrolytic enzyme preparation onto alfalfa silage prior to feeding, no effects on DM digestibility were observed. However, when the enzyme was added to the silage after it had been dehydrated, DM digestibility increased by 2.9% (Beauchemin and Rode, unpublished).

Treacher et al. (16) also reported that the effects of adding enzymes to barley silage diets were variable. A fibrolytic enzyme preparation was sprayed daily onto the silage portion (60% of DM) of a diet of barley silage and barley. Enzyme additives did not improve ADG, although DMI increased at the highest level of addition (Table 3). When the same enzyme preparation was incorporated into a supplement which was added to barley silage at 15% of the dietary DM, such that the application rate in the final diet was equivalent to 2X, 4X, and 7X (Beauchemin and Rode, unpublished data), no effects on ADG, intake or FCR were observed (data not shown). When the same enzyme preparation was sprayed onto a complete diet (equivalent to 21X) of 70% ryegrass/barley silage and 30% barley concentrate (DM basis), ADG increased without affecting intake (Table 3; 16). The authors speculated that treatment of the entire ration, rather than a portion, increases its effectiveness. However, the enzyme level per kilogram of diet was also higher in this last study, and may also indicate that for the product to be effective in silage diets, high levels are needed.

Direct application of enzymes into the ruminal environment is of less benefit than application to the feed prior to feeding. Treacher et al. (16) compared the effects of spraying the enzyme onto forage to adding enzyme directly into the rumen via a cannulae. Digestibilities of DM and fiber were higher when the enzyme was applied onto feed. In fact, direct addition into the rumen can actually decrease DM digestibility (16). This implies that, at least for certain enzyme mixtures, use of a direct-fed product, that has not been previously stabilized onto the feed, will be of little or no benefit.

Table 3. Digestibility of diets containing 45% cubes treated with Low or Medium levels of fiber degrading enzymes (dry matter basis).

Item	Diet
Item	Control	Low Enzyme	Medium enzyme
Neutral detergent fiber digestibility
Rumen	30.7	34.9	36.9
Total digestive tract	38.8^b	41.2^ab	43.6^a
Crude protein
Rumen undegraded intake protein¹, %	50.0^a	47.7^ab	39.7^b
Microbial protein synthesis, g of nitrogen/d	290	273	336
Total nitrogen available for absorption from the intestine, g/d	636	606	609

^{a,b Means with different letter within a row differ ( P < 0.05).
^{1 Bypass protein.}}

Although applying an aqueous solution of enzymes directly to the feed enhances binding of the enzyme with the substrate, the possibility of obtaining a beneficial response by direct feeding of enzymes cannot be ruled out entirely as early studies reported positive effects of providing enzymes in a topdressed supplement (3, 11). However, the amount of enzyme (and cost) will be greater when a dry enzyme preparation is used compared to an aqueous preparation applied prior to feeding.

Predicting Enzyme Effectiveness

The development of a model that accurately predicts in vivo response to feed enzymes has been a difficult challenge in this research. A model that predicts animal performance is necessary as optimum inclusion rates need to be determined for individual enzyme preparations and individual feed types. We have conducted in sacco studies (9, 10) to evaluate enzyme efficacy, however, these results do not always support in vivo results. Ultimately it is the in vivo results that are important, so it is critical that screening assays be relevant. In recent studies, we have found that a gas production technique shows some promise. This will remain one of the most difficult issues as new enzyme products become available.

Ideally, new products should be evaluated based upon their performance from in vivo testing. However, this is expensive and in vitro data will be used to support claims for product efficacy. If products do not live up to expectations, it is possible that other products that are effective will be viewed with skepticism in the marketplace. It will be very important for producers to evaluate the benefit of each product based upon its own merits rather than placing all enzyme products in a single category.

Responses in Dairy Diets

At Lethbridge Research Centre, we have used our knowledge of enzyme type with optimal method of enzyme application. In these studies, the addition of feed enzymes to dairy cow diets has been shown to be cost-effective in a range of diets fed to dairy cows.

Feed enzymes were applied to processed alfalfa cubes with the cubes comprising 45% of the total diet (DM basis). When these diets were fed to dairy cows with ruminal and duodenal cannulae, the enzymes enhanced diet digestibility, and these effects on digestibility improved performance (Table 2 and 3). Two levels of enzyme addition were used. Milk yield was increased by about 1 kg/d for the Low enzyme treatment, and by 2 kg/d for the Medium enzyme level, with no effects of enzyme on feed intake (Table 2). Increased milk yield due to enzymes did not change the fat or protein content of milk. Because the enzymes improved feed digestibility without affecting feed intake, feed efficiency was 2 to 8% higher for cows fed enzyme-treated cubes. Feed efficiency was measured as kilograms of milk yield per kilogram of dry matter consumed.

The increase in milk yield with Low and Medium levels of enzyme represented a 4 and 8% increase in milk yield, respectively. This is a substantial increase in milk yield for surgically prepared cows; effects of feeding these enzyme-treated cubes are expected to be even greater for commercial dairy cows in early lactation. Because cows in early lactation are in negative energy balance, increasing energy availability by treating feeds with enzymes would lead to a direct increase in milk output.

We recently completed a lactation study with cows in early lactation fed diets treated with the same enzyme product. The enzyme was applied to the rolled barley at the time of concentrate preparation. During the first 12 weeks of lactation, cows fed the enzyme-treated diets produced 4 kg/d more milk than cows fed the control diet, yet feed intake was unchanged (Table 4).

The improvement in milk yield due to enzymes, was the direct result of increased feed digestibility rather than a change in feed intake. Fiber digestibility, measured as neutral detergent fiber, increased both in the rumen and in the total digestive tract (Table 3). Because digestion of feed in the rumen increased due to enzymes, protein degradability within the rumen also increased. Thus, the rumen undegradable protein content (ie., bypass protein) was about 10% units lower for the Medium enzyme diet compared with the Control. However, this effect was not entirely negative as the amount of microbial protein synthesized in the rumen increased as a result of more feed digested in the rumen. Thus, the total amount of nitrogen available for absorption from the small intestine was only slightly lower due to the use of enzymes.

Table 4. Effect of enzyme supplementation to rolled barley on dry matter intake and milk production in lactating cows.

Item	Control	Enzyme	Percentage Increase
Feed intake, kg/d	18.7	19	1.6
Body weight, kg	668	636
Body wt change, kg/d	-0.63	-0.6	0
Milk production, kg/d	35.9	39.5	10
Milk fat yield, kg/d	1.35	1.32	-2.2
Milk protein yield, kg/d	1.13	1.19	5.3
DM digestibility, %	60.9	68.9	13.1
NDF digestibility, %	61.5	70.9	15.3
Energy balance, Mcal/d	-3.62	-3.33	0

Conclusions

Until recently, the use of exogenous enzymes for ruminants has been viewed with considerable skepticism. It is evident from both historical and recent studies that these enzyme preparations can be highly effective in enhancing animal performance. However, commercial usage of enzymes in ruminant diets is limited by the variability of responses observed among studies and the relatively high cost of enzymes compared to alternative feed additives.

There is sufficient evidence of highly significant enzyme-feed interactions. Furthermore, enzyme level is critical in eliciting a positive response. The development of newer enzyme preparations that are designed for ruminants and for specific types of feeds, will improve the potential profitability of on-farm use of these enzyme products. Using existing technology, it is currently possible to obtain large increases in animal performance using relatively low levels of enzymes in a range of dairy cow diets. It is evident that exogenous enzymes can be used to elicit an improvement in animal performance when ruminants are fed a range of diets. However, differences in enzyme preparations make direct comparisons among studies difficult. Method of application is critical for optimal efficacy of any new enzyme product. It is clear that the moisture content of the feed will influence efficacy of the enzyme supplementation. Additionally, there is an interaction between the feed source and the enzyme type. Further research is required to clearly elucidate the important factors to consider to reduce the variability associated with using enzymes in ruminant diets. 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, there is no doubt that enzyme technology will play an important role in future ruminant production.

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