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Journal of Animal Science - Board-Invited Review

BOARD-INVITED REVIEW: Opportunities and challenges in using exogenous enzymes to improve ruminant production

 

This article in JAS

  1. Vol. 92 No. 2, p. 427-442
     
    Received: July 05, 2013
    Accepted: Nov 22, 2013
    Published: November 24, 2014


    1 Corresponding author(s): tim.mcallister@agr.gc.ca
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doi:10.2527/jas.2013-6869
  1. S. J. Meale*†,
  2. K. A. Beauchemin,
  3. A. N. Hristov,
  4. A. V. Chaves* and
  5. T. A. McAllister 1
  1. Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia
    Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB T1J 4B1, Canada
    Department of Animal Science, Pennsylvania State University, University Park 16802

Abstract

The ability of ruminants to convert plant biomass unsuitable for human consumption into meat and milk is of great societal and agricultural importance. However, the efficiency of this process is largely dependent on the digestibility of plant cell walls. Supplementing ruminant diets with exogenous enzymes has the potential to improve plant cell wall digestibility and thus the efficiency of feed utilization. Understanding the complexity of the rumen microbial ecosystem and the nature of its interactions with plant cell walls is the key to using exogenous enzymes to improve feed utilization in ruminants. The variability currently observed in production responses can be attributed to the array of enzyme formulations available, their variable activities, the level of supplementation, mode of delivery, and the diet to which they are applied as well as the productivity level of the host. Although progress on enzyme technologies for ruminants has been made, considerable research is still required if successful formulations are to be developed. Advances in DNA and RNA sequencing and bioinformatic analysis have provided novel insight into the structure and function of rumen microbial populations. Knowledge of the rumen microbial ecosystem and its associated carbohydrases could enhance the likelihood of achieving positive responses to enzyme supplementation. The ability to sequence microbial genomes represents a valuable source of information in terms of the physiology and function of both culturable and unculturable rumen microbial species. The advent of metagenomic, metatranscriptomic, and proteomic techniques will further enhance our understanding of the enzymatic machinery involved in cell wall degradation and provide a holistic view of the microbial community and the complexities of plant cell wall digestion. These technologies should provide new insight into the identification of exogenous enzymes that act synergistically with the rumen microbial populations that ultimately dictate the efficiency of feed digestion.



INTRODUCTION

The efficiency by which ruminants obtain energy from structural plant polysaccharides and in turn produce high quality meat and milk protein is increasingly important if the demands of an expanding human population are to be met. Due to both economic considerations and maintenance of rumen health, forage will almost always be a component of the diet of ruminants (Krause et al., 2003). However, digestibility of forage cell walls ultimately limits nutrient availability as conditions for fiber digestion are often suboptimal in the rumen.

Exogenous enzymes are increasingly considered as a cost-effective means of improving feed efficiency (Krause et al., 2003), yet production responses to exogenous enzymes are still highly variable. Several reviews (Krause et al., 2003; Beauchemin et al., 2004; Beauchemin and Holtshausen, 2011; Tricarico et al., 2008) have been published on the use of exogenous enzymes in ruminants; however, this paper aims to provide an updated perspective on current research and new techniques being used to use these products as a means of improving feed efficiency. Most research has centered on the use of fibrolytic enzymes to increase fiber digestion and thus digestible energy intake, but responses have been variable. Increases in milk (Gado et al., 2009; Klingerman et al., 2009), ADG (Beauchemin et al., 1999; McAllister et al., 1999), and DM and fiber digestion in situ, in vitro (Yang et al., 1999; Hristov et al., 2008), and in vivo (Rode et al., 1999; Beauchemin et al., 2000) have been reported. However, in many cases, the efficiency of growth or milk production in ruminants has not been improved (ZoBell et al., 2000; Eun et al., 2009; Arriola et al., 2011).

Characterization of rumen microbial populations using “-omics” technologies provides insight into the functionality of rumen microbiota (Morgavi et al., 2012) and the construction of gene catalogues through metagenomics and genomic sequencing expands our understanding of the interactions between community members and the carbohydrases they produce. These knowledge advancements are providing new insight into the formulation of exogenous enzymes that act synergistically with the carbohydrases of rumen microbial communities in a manner that enhances the efficiency of plant cell wall digestion.

SOURCES OF ENZYMES

Commercial enzyme products used in the livestock industry are of fungal (Aspergillus oryzae and Trichoderma reesei) and bacterial (Bacillus subtilis, Lactobacillus acidophilus, Lactobacillus plantarum, and Enterococcus faecium spp.) origin (Muirhead, 1996; McAllister et al., 2001). Beginning with a seed culture and growth media, enzymes for the feed industry are produced through microbial fermentation and although the source organisms only constitute a very limited group, the types and activity of enzymes produced can be diverse depending on the strain selected, the substrate they are grown on, and the culture conditions used (Considine and Coughlan, 1989; Gashe, 1992; Lee et al., 1998).

A vast array of enzymes is required to degrade the complex arrangements of structural carbohydrates in plant cell walls (Morgavi et al., 2012). Although commercial enzyme preparations are commonly referred to as cellulases or xylanases, secondary enzyme activities such as amylases, proteases, esterases, or pectinases are invariably present as these preparations seldom consist of a single pure enzyme (McAllister et al., 2001). This diversity is advantageous, as it facilitates targeting of a range of substrates using a single product, yet it complicates the identification of the specific enzymes responsible for any positive responses observed in feed digestion. Degradation of cellulose and hemicellulose alone requires a number of glycosidic hydrolases (Chesson and Forsberg, 1997; Krause et al., 2003) and differences in the relative proportions and activity of individual enzymes affects the overall efficacy of cell wall degradation (McAllister et al., 2001). A common approach is to use an enzyme that may not be suited to a specific feed but instead to formulate enzyme mixtures that are suitable for a range of feed types (Beauchemin et al., 2003). However, this approach of adding enzymes to diets without consideration for specific substrates has contributed to the highly variable results observed when enzymes are used in ruminants, an outcome that has undoubtedly discouraged and delayed the adoption of the technology.

MEASUREMENT OF ENZYME ACTIVITY

The activity of enzymes is assayed by measuring the generation of the product from the biochemical reaction that the enzyme catalyzes over time and is expressed as the amount of product produced per unit of time (McAllister et al., 2001; Beauchemin et al., 2003). In the case of carbohydrases, the production of free sugars is the most common product measured. Measurement of enzyme activities must be conducted under closely regulated conditions as variations in temperature, pH, ionic strength, substrate concentration, and substrate type influence enzyme activity (McAllister et al., 2001). Synthetic substrates can also be used to assay enzyme activity by measuring the release of a dye or chromophore (Higginbotham et al., 1996). However, these conditions fail to replicate those of the digestive tract, nor are the substrates representative of intact feeds that are fed to ruminants (McAllister et al., 2001). Consequently, these assays may have limited relevance to the potential worth of an enzyme as a feed additive for ruminants.

Biological assays using mixed ruminal microorganisms incubated with complex substrates has been one approach to identify enzyme preparations that are more suitable for use in ruminants. After the addition of enzymes, these in vitro incubations measure the digestion of ingredients commonly included in ruminant diets (i.e., grains, hay, silage, or straw) by recording the production of gas that arises from the fermentation process and the digestibility of the feed DM and fiber at a given incubation time. Using this system, several enzyme preparations can be simultaneously screened for their effectiveness with different application methods and rates (Muirhead, 1996; Iwaasa et al., 1998; Morgavi et al., 2000b). However, these systems are not very representative of in vivo conditions (Hristov et al., 2012) and do not account for animal-to-animal variation in the microbial community, making extrapolation of these results to whole animal scenarios challenging. Additionally, these systems do not account for the possible impact of exogenous enzymes on biological parameters such as feed intake, rate of passage, or postruminal digestion of nutrients (McAllister et al., 2001). Even in vitro systems may require more enzyme than what is typically produced by most laboratory-based expression systems. To overcome this problem we have developed a micro-in vitro system that enables the synergistic interactions of milligram quantities of exogenous enzymes with rumen microbial enzymes to be assessed in a reaction mixture as small as 250 μL (Badhan, A., Wang, Y., and McAllister, T.A. [Agriculture and Agri food Canada, AB]; Patton, D., Powlowski, J., and Tsang A. [Centre for Structural and Functional Genomics, Concordia University, QC]). We used the system to identify carbohydrases from thermophilic and anaerobic fungi that could enhance the liberation of sugars from alfalfa hay and barley straw by mixed rumen enzymes. Such mini-assays could prove invaluable for identifying those enzyme activities that merit production scale up for animal experiments and the formulation of efficacious enzyme cocktails.

In vitro screening of exogenous enzymes provides a cost effective, less time consuming means of screening large numbers of products with specific substrates and can be used to predict possible in vivo responses. However, the final assessment of the true value of exogenous enzymes for ruminants in terms of improving feed utilization can only be assessed through the use of animal production trials.

PRODUCTION RESPONSES TO EXOGENOUS ENZYMES

Dairy Cattle

A search of the Commonwealth Agricultural Bureaux International (CABI) database (www.cabdirect.org) and the Journal of Dairy Science ( www.journalofdairyscience.org) for “enzyme” and “dairy” in the title returned 328 and 39 publications, respectively. From these, 28 were selected and are summarized in Table 1. Criteria for this selection were that the study had to be published in English in a refereed journal, be with lactating dairy cows, and have a valid experimental design. “Field” trials where control over cow grouping was not clearly described or if there was potential for confounding factors were excluded from the analysis. Studies judged as having major flaws in experimental design or statistical analysis of the data were also excluded from Table 1. A large portion of the studies were not included as they did not involve the use of lactating cows.


View Full Table | Close Full ViewTable 1.

Summary of exogenous polysaccharide-degrading enzyme effects on production traits and total tract apparent digestibility of nutrients in lactating dairy cows

 
Source1 Experimental design (number of cows) Product/manufacturer Declared primary activities Application level Forage level in basal diet Milk production, kg/d Effects2
DMI Milk yield Milk components Total tract digestibility
Chen et al., 1995 Completely randomized (36) Digest M, Loveland Industries Inc., Greeley, CO Amylase and protease 209 g/t3 34% 34 to 37 - - - ↑CP4
Rode et al., 1999 Completely randomized (20) Pro-Mote5 Xylanase and cellulase 1.3 kg/t TMR6 DM 39% 36 to 40 - - ↓MF ↑DM, OM, NDF, ADF, and CP
Schingoethe et al., 1999 Completely randomized block (50) FinnFeeds Int.7 Cellulose and xylanase 0.7 to 1.5 L/t forage DM 55% 25 to 28 - -8 - NR
Yang et al., 19999 Latin square (4) Pro-Mote5 Cellulase and xylanase 0.5 to 1 g/kg TMR DM 55% 24 to 26 - 10 - ↑OM, and NDF
Beauchemin et al., 2000 Latin square (6) Natugrain 33-L11 β-glucanase, xylanase, and endocellulase 1.22 to 3.67 L/t TMR 45% 30 to 31 - ↑MMP ↑DM, and ↓NDF13
Kung et al., 200014 Completely randomized (30) FinnFeeds Int.7 Cellulase, hemicellulase, and xylanase 2 to 10 L/t fresh forage 50% 33 to 35 and 36 to 39 - ↑ (both experiments) - or ↓MF and MMP NR
Yang et al., 20009 Completely randomized block (43) Biovance Technol. Inc.5 Xylanase 50 mg/kg TMR DM 38% 35 to 37 - - ↑DM
Zheng et al., 2000 Completely randomized block (48) Bovizyme 40117 Cellulase and xylanase 1.25 L/t forage DM 50 to 65% 33 to 37 - - - NR
Bowman et al., 20029 Latin square (8) Promote N.E.T.15 Xylanase and cellulose 1 g/cow per d 55% 29 to 30 - - ↑MF and MMP ↑All4
Knowlton et al., 2002 Switchover (12) Loveland Industries Inc., Greeley, CO Cellulase 204 g/t TMR 45 to 61% 31 to 43 - - - -
Kung et al., 2002 Completely randomized (30) FinnFeeds Int.7 Cellulase and xylanase 10 L/t fresh forage 45% 36 to 39 - - - NR
Sutton et al., 20038 Latin square (4) Biovance Technologies, Inc.5 Xylanase and endoglucanase 2 kg/t TMR DM 57% 34 to 36 - - ↑MMP ↓DM, and OM
Vicini et al., 200314 Completely randomized (257 and 122) FinnFeeds Int.14 and Biovance Technologies Inc.5 Xylanase and endoglucanase 1.25 to 2 L/t TMR DM 43 to 57% 32 to 33 and 28 to 29 - - - NR
DeFrain et al., 200516 Completely randomized block (24) Amaize17 Amylase 0.1% TMR DM 47 to 69% 38 - - - NR
Eun and Beauchemin, 2005 Latin square (8) Protex 6L18 Protease 1.25 mg/kg TMR DM 34 to 60% 41 to 48 ↑MF; and ↓MMP19 ↑All
Tricarico et al., 2005 Latin square (20) Amaize17 Amylase 240 to 720 dextrinizing units/kg TMR DM 55% 29 to 30 NR -8 - NR
Elwakeel et al., 2007 Split plot (24) Saf Agri, Milwaukee, WI β-glucanase and xylanase 15 g/cow per d 37% 43 to 44 - - - NR
Knowlton et al., 2007 Completely randomized (24) Cattle-Ase-P20 Cellulase and phytase 297 g/t TMR DM 37% 37 to 39 - - - -21
Reddish and Kung, 2007 Switchover (24) Alltech Inc.17 Cellulose and xylanase 10 g/cow per d 50% 40 - - - -22
Hristov et al., 2008 Latin square (4) Alltech, Inc.17 Amylase and xylanase 10 g/cow per d 40% 30 - - - ↑DM, OM, and CP
Miller et al., 2008 Completely randomized block (72) Roxazyme G2 Liquid23 Xylanase and endoglucanase 2.15 and 4.30 mL/kg concentrate Pasture and 6.7 kg/d grain supplement 28 to 29 - - - NR
Gado et al., 200924 Completely randomized (20) ZADO24 Protease, amylase, and cellulase 40 g/cow per d 70% 13 to 16 - ↑DM, OM, NDF, and ADF
Klingerman et al., 2009 Latin square (28) Amaize17 and an experimental preparation23 Amylase 0.4 g/kg TMR DM and 0.88 to 4.4 mL/kg 50% 44 to 47 25 - ↑DM, OM, CP, and NDF25
Bernard et al., 2010 Completely randomized (44) Promote N.E.T.-L26 Cellulase 4 g/cow per d 50 to 54% 40 to 42 - - - NR
Peters et al., 2010 Switchover (6) Roxazyme G223 Cellulase and xylanase 6.2 mL/kg TMR DM 50% 26 to 27 - - - -
Holtshausen et al., 2011 Completely randomized (60) AB Vista, Marlborough, UK Xylanase and endoglucanase 0.5 to 1.0 mL/kg DM 52% 38 - - NR
Arriola et al., 2011 Completely randomized block (66) Dyadic International Inc., Jupiter, FL Xylanase, exoglucanase, and endoglucanase 3.4 mg/g TMR DM 52 to 67% 32 to 36 - - - ↑All
Ferraretto et al., 2011 Completely randomized (45) Ronozyme RumiStar CT23 Amylase 300 kilo novo units/kg TMR DM 50% 49.5 to 52.1 - - ↓MMP NR
1In chronological order.
2 = increase; ↓ = decrease; - = no statistically significant effect; NR = not reported; MF = milk fat percentage; MMP = milk protein percentage; All = all nutrients studied.
3Applied to the grain portion of the diet.
4Interaction with grain processing.
5Biovance Technologies Inc., Omaha, NE.
6TMR = total mixed ration.
7FinnFeeds International, Marlborough, Wiltshire, UK.
8Reported increase of 3.5% fat corrected milk.
9These studies investigated enzyme application method.
10For one of the application methods.
11BASF Corporation, Ludwigshafen, Germany.
12Only the low enzyme application level.
13Only the high enzyme application level.
14Two experiments.
15Agribrands International, St. Louis, MO.
16Experimental diets were fed from 21 d before expected calving through 21 d in milk.
17Alltech Inc., Nicholasville, KY.
18Genencor International, Rochester, NY.
19Only low-forage (34%) diet.
20Animal Feed Technologies, Greeley, CO.
21Trends for increased NDF and ADF digestibility.
22Digestion experiment with sheep.
23DSM Nutritional Products Ltd., Basel, Switzerland.
24Molecular Biology Laboratory of the Ain Shams University, Cairo, Egypt. Questionable data; see discussion.
25Milk yield and digestibilities increased only by low level of an experimental amylase enzyme.
26Cargill Animal Nutrition, Minneapolis, MN.

Naturally, the main objective of using exogenous enzymes in dairy cow nutrition is to improve milk production and yield of milk components and as previous reviews (McAllister et al., 2001; Beauchemin et al., 2004; Tricarico et al., 2008) have focused on the dairy-specific effects of exogenous enzymes on ruminal fermentation and intestinal digestion, this review will focus on production effects. Most products tested in dairy cows are described as cellulases and/or xylanases, with proteases and amylases being investigated in fewer instances. It is practically impossible to compare exogenous enzyme preparations on an equal activity basis, as there is a distinct lack of standardization in the methodology used to assess enzyme activities among labs. Even when the same methods are used, it is difficult to standardize enzyme products because they contain multiple activities and can only be standardized for 1 or 2 activities at a time. For example, when 2 products are used to supply the same level of endoglucanase activity, they may supply vastly different levels of xylanase activity. Another common limitation of the enzyme literature is the lack of repeatability of the effects and repeated investigations of a common exogenous enzyme preparation as most are only examined in a single experiment. Experimental cost is perhaps a major reason for this lack of repetition, but the possibility that unfavorable results were not published cannot be excluded. Interestingly, the commercial α-amylase product Amaize (Alltech Inc., Nicholasville, KY) has been examined in 3 studies discussed in this review (DeFrain et al., 2005; Tricarico et al., 2005; Klingerman et al., 2009). All of these studies fed a diet containing either alfalfa hay, alfalfa haylage and maize silage, or a mixed legume hay and maize silage diet and observed no effects on milk yield or composition of milk components. The dose of enzyme and the portion of the diet to which it was applied varied across studies raising questions about the enzymes applicability for use in lactating dairy cows fed current diets.

Few studies have reported positive effects of exogenous enzymes on milk components; for example, Beauchemin et al. (2000) reported a 2% increase in milk true protein with a β-glucanase/xylanase/endoglucanase product. Similarly, Bowman et al. (2002), Sutton et al. (2003), and Eun and Beauchemin (2005) reported increased milk fat or protein. A few instances of increasing milk yield have also been observed. For example, Yang et al. (1999) found milk yield was increased by 1.9 kg/d when an exogenous enzyme (Pro-Mote; Biovance Technologies Inc., Omaha, NE) composed predominantly of cellulase and xylanase activities was applied to hay at 2 g of enzyme mixture/kg. This effect was attributed to a 12% increase in nutrient digestibility. Despite these results, the application of exogenous enzymes to dairy cow diets has shown extremely variable results and largely failed to improve production efficiency. Of the studies compared in this paper, the majority showed no effect on milk yield (DeFrain et al., 2005; Hristov et al., 2008; Bernard et al., 2010; Peters et al., 2010; Ferraretto et al., 2011) or the production of milk components (Yang et al., 2000; Holtshausen et al., 2009; Bernard et al., 2010; Arriola et al., 2011).

A recent study by Holtshausen et al. (2011) screened 5 doses of a fibrolytic enzyme additive (AB Vista, Marlborough, UK) and further assessed its efficacy in situ before the enzyme additive was fed to lactating Holstein dairy cows. The enzyme product improved fat corrected milk (FCM) production efficiency in a dose dependent manner up to 11.3%. Similarly, Arriola et al. (2011) screened varying amounts of a fibrolytic enzyme product in situ before conducting a feeding trial. Milk production efficiency was increased in cows fed this enzyme product with a low-concentrate diet as compared with those fed either an untreated low-concentrate diet or a high-concentrate diet (treated or untreated). Therefore, it is evident that careful attention needs to be paid to the type and dose of enzymes being applied to dairy cattle diets. The use of in vitro screening can help to elucidate appropriate doses and predict production responses when combined with specific substrates. However, it remains difficult to assess the consistency of animal responses to individual enzyme products, as most products are experimental and can vary in activity over time. In many instances the same product formulation is often used in only a limited number of studies of widely differing experimental conditions.

Beef Cattle

Although the first reports of exogenous enzymes improving beef cattle BW gains were over 50 yr ago (Burroughs et al., 1960), adoption of enzyme technology has been slow as the cost of enzymes outweighs that of other additives, such as ionophores, antibiotics, and implants (Beauchemin et al., 2006). We used a similar criterion as described above for dairy production studies to identify beef production studies (CABI and the Journal of Animal Science [ www.journalofanimalscience.org]) with a search for “enzyme” and “beef cattle” in the title returning 383 and 70 titles, respectively. From these, 11 studies were selected and summarized in Table 2. Experimental criteria for selection were the same as for dairy cattle, with studies having to be conducted with growing, backgrounding, or finishing beef cattle. Although responses to exogenous enzymes are expected to be greater in beef cattle fed roughage-based diets as compared with high-grain diets, many exogenous enzyme formulations have shown promising effects in cattle fed high-grain finishing diets, at least when included in barley-based diets (Beauchemin and Holtshausen, 2011). However, these responses are still variable depending on the dosage of the enzyme applied, the time of application in relation to feeding, and the portion of the diet to which they are applied. Application of a mixture of xylanase and cellulase products (Xylanase B [Biovance Technologies Inc., Omaha, NE] and Spezyme CP [Genencor, Rochester, NY]) increased ADG of steers fed alfalfa hay or timothy hay by 30 and 36%, respectively, but had no effect when applied to barley silage (Beauchemin et al., 1995). These positive responses were attributed to an increase in digestible DM intake; however, it was noted that forage type influenced the optimal dose required to elicit these responses (0.25 to 1.0 L/t DM for alfalfa hay versus 4 L/t DM for timothy hay), demonstrating the importance of interactions between dosage, enzyme, and substrate. A subsequent study with steers assessed the same enzyme formulation in high-concentrate diets (95% DM) containing either barley or corn grain (Beauchemin et al., 1997). Application to a barley grain diet improved feed efficiency by 11%, yet performance was unaffected when added to corn. Supplementing a similar exogenous enzyme mixture (FinnFeeds Int. Ltd., Marlborough, UK) increased ADG of steers by 10% when applied to both the grain and forage portions of the diet (McAllister et al., 1999) and resulted in a 28% increase in ADF digestibility (Krause et al., 1998). These studies indicate the application of a xylanase and cellulase enzyme formulation is promising in terms of increasing ADG when applied to either barley grain or forage diets; however, the use of this enzyme formulation is not recommended in diets based on corn grain or barley silage due to its apparent lack of effectiveness with these feeds. Conversely, a study by Tricarico et al. (2007) reported an A. oryzae extract containing α-amylase activity quadratically increased ADG when included in either a cracked corn or high-moisture corn and corn silage diet but had no effect when included with alfalfa hay, cotton seed hulls, or steam-flaked corn. Similarly, DiLorenzo et al. (2011) observed no effects of supplementing an amylase enzyme formulation (600 kilo novo units/kg of dietary DM; RumiStar; DSM Nutritional Products, Inc., Kaiseraugst, Switzerland) to either a dried-rolled corn or steam-flaked corn diet, indicating the need for further investigation of the various enzyme types and their applicability for feeds commonly supplied to beef cattle. A lack of response to amylases may reflect the fact that starch digestion is generally not limited in the rumen, provided that the grains are adequately processed (McAllister and Cheng, 1996).


View Full Table | Close Full ViewTable 2.

Summary of exogenous polysaccharide-degrading enzyme effects on production traits and total tract apparent digestibility of nutrients in beef cattle

 
Source1 Experimental design (number of cows) Product/ manufacturer Declared primary activities Application level Forage level in basal diet Effects2
FCR3 Total tract digestibility
DMI ADG
Beauchemin et al., 1995 Completely randomized (72) Xylanase B4 and Spezyme CP5 Xylanase and cellulase 40 to 316 FPU6/kg DM 91 to 96.7% 7 8 - NR
Lewis et al., 1996 Latin square (5) Grasszyme, FinnFeeds Int.9 Xylanase and cellulase 1.65 mL/kg forage DM 70% - NR NR ↑DM, NDF, and ADF
Beauchemin et al., 1997 Completely randomized block (56) Xylanase B4 and Spezyme CP5 Xylanase and cellulase 4.0 L/t concentrate DM 4.90% - - - NR
Krause et al., 1998 Latin square Pro-Mote4 Xylanase and cellulase 1.5 g/kg DM 5% - NR NR ↑ADF
Beauchemin et al., 1999 Completely randomized block (1,200) Pro-Mote4 Xylanase and cellulase 1.4 L/t DM 7.8% - - NR
McAllister et al., 1999 Completely randomized (98 and 66) FinnFeeds Int.9 Xylanase and cellulase 1.25 to 5.0 L/t DM 70 to 82.5% 10 11 - -12
ZoBell et al., 2000 Completely randomized (32) FinnFeeds Int.9 Xylanase and endoglucanase 15,880 and 5,580 IU/kg TMR13 DM 20 to 65% - - - NR
Balci et al., 2007 Completely randomized (16) Promote N.E.T.14 Xylanase and cellulase 60 g/d Ad libitum wheat straw NR - (in vitro)
Tricarico et al., 2007 Completely randomized block (120, 96, and 56) Amaize15 Amylase 580 to 1,160 DU16/kg DM - 17 - NR
Krueger et al., 2008 Completely randomized (50) Biocellulase A20, Loders Croklaan, Channahon, IL Xylanase and cellulase 16.5 g/t Ad libitum access to hay 18 - - ↑DM, NDF, and CP18
Eun et al., 2009 Completely randomized (60) Fibrozyme15 Endoglucanase, exoglucanase, xylanase, and amylase 1 to 2 g/kg TMR DM 20 to 58% - - - NR
DiLorenzo et al., 2011 Completely randomized block (32) RumiStar19 Amylase 400 kilo novo units/kg DM 5.1% - - - -
Vera et al., 2012 Completely randomized (48) Danisco-Agtech, Waukesha, WI Protease 0.52 g/kg DM TMR 25 to 63.4% 20 - - ↓NDF,11 ↑DM, N, NDF, and ADF11
1In chronological order.
2↑ = increase; ↓ = decrease; - = no statistically significant effect; NR = not reported.
3FCR = feed conversion rate.
4Biovance Technologies Inc., Omaha, NE.
5Genencor, Rochester, NY.
6FPU = filter paper units of cellulase.
7Dependant on forage and application rate (increases seen at alfalfa level 3).
8Dependant on forage and application rate (increases seen at alfalfa level 1, 2, and 3 and at timothy hay level 5).
9Finnfeeds International, Marlborough, Wiltshire, UK.
10Only the greatest amount of enzyme application (5.0 L/t) in the backgrounding study.
11Only in the finishing stage.
12Digestion experiment with sheep.
13TMR = total mixed ration.
14Cargill Animal Nutrition, Minneapolis, MN.
15Alltech Inc., Nicholasville, KY.
16DU = dextrinizing unit.
17Quadratic increase observed in experiment 2 only.
18Dependent on time of enzyme application before feeding.
19DSM Nutritional Products, Inc., Kaiseraugst, Switzerland.
20Only in growing phase.

Recently, the addition of an experimental exogenous proteolytic enzyme (Danisco-Agtech, Waukesha, WI) during the growing phase increased DMI of steers by 14.8%, but an increase in ruminal passage rate reduced NDF digestibility (4.1%) and as a result this increase in DMI was not reflected in improvements in BW gain or feed efficiency nor were any effects observed when this same enzyme was added to a finishing diet (Vera et al., 2012). ZoBell et al. (2000) observed no effects on ADG or feed efficiency when applying the same enzyme product to either a barley-based growing (65:35 forage to concentrate ratio; DM basis) or finishing diet (20:80 forage to concentrate ratio; DM basis) as McAllister et al. (1999), who observed an increase in DMI when this enzyme was applied (0.5 L/t DM) to barley silage as well as increase an in ADG when it was applied at (3.5 L/t total mixed ration [TMR]) to a finishing TMR. Comparatively, Balci et al. (2007) applied Promote N.E.T. (60 g/d; Agribands Int., St. Louis, MO) with cellulase and xylanase activities to a corn and barley diet and observed increases in ADG and feed conversion efficiency. Eun et al. (2009) supplemented both growing and finishing diets with a commercial enzyme product (Fibrozome; Alltech Inc.) and observed no effect on growth performance, despite minor improvements in carcass characteristics. Lewis et al. (1996) applied Grasszyme (FinnFeeds Int. Ltd., Marlborough, UK) to a grass hay and barley diet (70:30) and measured the impact of application time before feeding and the portion of the diet to which the enzyme was applied. No effects on DMI were observed; however, digestibility of DM, NDF, and ADF increased when the enzyme was added to the forage either 24 h before or at the time of feeding. Similarly, Krueger et al. (2008) applied an enzyme mixture (Biocellulase A20; Loders Croklaan, Channahon, IL) to Bermudagrass hay at 3 different stages, immediately after cutting, at bailing, or at feeding and although enzyme treatment at cutting increased DMI, no effect was observed on final live weight, ADG, or G:F, regardless of the time of application. Evidently, most studies have observed inconsistent responses after enzyme supplementation in beef cattle despite the commonality of a barley-based diet, hampering the adoption of this technology on an industry-wide scale. Similarly, the studies that exhibited increases in ADG used different application methods whereby the enzyme was applied to the TMR in the study by McAllister et al. (1999) in the study of Balci et al. (2007), it was only applied to the concentrate portion of the diet, and as these studies were the only reports of positive effects on production performance, it is difficult to discern at this point which method, if either, would best ensure the efficacy of exogenous enzymes in beef cattle.

Small Ruminants

Generally, the application of exogenous enzymes to the diets of small ruminants has had little impact on production performance. Miller et al. (2008) fed a barley-based diet treated with a commercial exogenous enzyme (Roxazyme G2 Liquid; DSM Nutritional Products Pty Ltd, Basel, Switzerland) to Dorset-cross ewe lambs and observed no effects on DMI, ADG, feed conversion, or wool growth. Similarly, no effects were observed on milk yield, milk composition, or DMI when Promote (Agribrands Int., St. Louis, MO) was applied to the diets of lactating Manchega and Lacaune ewes (Flores et al., 2008). Additionally, Rojo et al. (2005) fed exogenous amylases from Bacillus licheniformis and Aspergillus niger (up 2.90 g enzyme/kg DM sorghum; ENMAX, Mexico City, Mexico) and observed no effects on production performance in Suffolk lambs. As such, the majority of exogenous enzyme studies found in sheep and goats have focused on investigating the impact of exogenous enzymes on diet digestibility. In a study by Reddish and Kung (2007) lambs were fed a commercial diet supplemented with an enzyme mixture (4 g/lamb daily; Alltech Inc.); however, no effect on apparent digestibility of DM, ADF, NDF, or N was observed. A study by Avellaneda et al. (2009) fed Suffolk lambs Guinea grass in conjunction with fibrolytic enzymes (3 g/lamb daily; Fibrozyme; Alltech Inc.) and also reported no effects on DMI, ruminal fermentation, or ruminal or total tract digestion. Giraldo et al. (2008) delivered exogenous fibrolytic enzymes (12 g/lamb daily; Fibrozyme; Alltech Inc.) directly into the rumen of fistulated Merino sheep fed a grass–hay concentrate diet (70:30; DM basis) without affecting diet digestibility. By supplementing the enzyme directly into the rumen the prefeeding feed–enzyme interaction was negated, yet the enzymes were able to stimulate fibrolytic activity and the growth of cellulolytic bacteria. Conversely, Bala et al. (2009) applied 2 levels of exogenous enzymes, described as a cellulase and a xylanase (4,000 and 12,500 or 8,000 and 18,750 IU/kg, respectively), to the diets of lactating Beetle-sannen crossbred goats. The greatest amounts of supplementation decreased DMI (g/kg FCM yield) and increased the digestibility of DM, OM, CP, NDF, ADF, and total carbohydrates and improved FCM yield (kg/d) in the last quarter of lactation. These studies suggest that the application of existing exogenous enzymes to lamb diets has limited impact on diet digestibility or growth performance. Additional studies are required to determine if the response to exogenous enzymes in lactating goats is a reflection of their high metabolic demand for milk production.

MODES OF ACTION

Preconsumption Effects

Application of exogenous enzymes before consumption appears to be the most effective when they are applied in a liquid form to dry as opposed to wet forage as there appears to be components in silage that can inhibit exogenous enzymes (Morgavi et al., 2000a; Nsereko et al., 2000; Wallace et al., 2001). Even the low moisture content in dry feeds, such as hay and grain, appears sufficient to enable hydrolysis of carbohydrates from complex polymers (Morgavi et al., 2000b). This release of sugars arises, at least partially, from the solubilization of NDF and ADF (Morgavi et al., 2000b; Morrison and Miron, 2000; Devillard et al., 2004) encouraging rapid microbial growth and reducing the lag time required for microbial colonization (Beauchemin et al., 2004). The type of exogenous enzyme and substrate determines the degree of sugar release, yet this represents only a minute portion of the total carbohydrate present in the diet. As such, it is difficult to attribute production responses solely to the generation of soluble carbohydrates before consumption.

Fibrolytic enzymes that bind to the feed appear to be more active, possibly because of increased resistance to proteolytic inactivation in the rumen (Fontes et al., 1995). Similarly, maximizing the proportion of the diet to which the enzyme is added is considered to increase the chances that the enzymes will remain active in the rumen. Bowman et al. (2002) reported that enzymes were more effective when added to rolled grain, which comprised 45% of the diet, compared with if they were added to a finely ground premix, which comprised 0.2% of the diet (DM basis).

Ruminal Effects

Exogenous enzymes have been shown to be more stable in the rumen environment than originally proposed (Hristov et al., 1998b; Morgavi et al., 2000b). For example, Morgavi et al. (2001) found 4 commercial enzymes remained stable when incubated in ruminal fluid, pepsin, or pancreatin. Enzyme stability in the rumen is considered to be a result of glycosylation and is usually enhanced by adding exogenous enzymes to feed before consumption (Fontes et al., 1995). However, nonglycosylated enzymes may also resist ruminal proteolysis, but their persistence in the rumen may depend on the microbial source from which they were derived (Fontes et al., 1995). Variation in enzyme stability may contribute to the inconsistent production responses observed when enzymes are included in ruminant diets.

Supplementing ruminant diets with exogenous enzymes increases the rate but seldom the extent of feed digestion. This suggests that positive responses to present exogenous enzymes are not a result of these preparations solubilizing substrates that would not be normally digested if retained in the rumen for a sufficient period of time. However, an increase in total enzymatic activity in the rumen can increase ruminal hydrolytic capacity, which can enhance the digestibility of the complete diet rather than just being limited to the specific components targeted by the enzyme (Beauchemin et al., 2004). As such, digestibility of both nonfibrous and fibrous fractions can increase, explaining why fibrolytic enzymes can also be effective in increasing the digestibility of nonfiber fractions in high grain diets (Beauchemin et al., 1999).

Given that exogenous enzymes represent only a fraction of enzyme activity in the rumen combined with the inherent capacity of the ruminal microbiota to digest fiber, it is difficult to attribute an increase in fiber degradation by exogenous enzymes to direct hydrolysis alone (McAllister et al., 2001). A synergistic relationship between exogenous enzymes and rumen microbiota and an increase in bacterial attachment are other likely modes of action of exogenous enzymes in the rumen. Synergism acts to increase the effects of both indigenous ruminal microbes and exogenous enzymes so that the combined response exceeds the additive effects of each individual component (Morgavi et al., 2000a). Identification and supplementation with exogenous enzymes not produced by ruminal microbes could be theorized to further heighten this synergistic response. Current enzyme preparations do not appear to introduce novel enzyme activity into the rumen as they typically increase only the rate and not the extent of cell wall digestion (Wallace et al., 2001; Krause et al., 2003; Jalilvand et al., 2008). Exogenous enzymes can also stimulate the attachment of ruminal microbes to plant fiber (Morgavi et al., 2000a), yet the mechanism by which this occurs is unknown. Reduced amounts of exogenous enzymes have been shown to enhance attachment of ruminal bacteria to fiber resulting in a disruption in the hydrogen bonds within the cellulose matrix (White et al., 1993). However, increased amounts of exogenous enzymes can also compete with the ruminal microbial population for cellulose binding sites on feed (Morgavi et al., 2000b), potentially explaining the lack of or even negative responses observed with the increased amounts of exogenous enzyme supplementation in vivo. For exogenous enzymes to be effective, it is important that they complement and not replace the existing natural enzyme activities produced by ruminal microbes.

Enzymes have also been associated with a reduction in digesta viscosity in poultry (Choct, 2006); if a similar reduction was observed in ruminants, an increase in passage rate through the rumen would be expected, reducing gut fill and subsequently increasing intake. From a production perspective, increasing intake is beneficial. However, if the enzymes are in the fluid phase and there is a rapid flow of digesta through the rumen, they may not be afforded sufficient time to degrade the fibrous portion of the diet before they are largely inactivated by exposure to the low pH and pepsin in the abomasum (Hristov et al., 1998b).

Postruminal Effects

Hristov et al. (1996, 1998a) were the first to report that approximately 30% of xylanases can escape ruminal fermentation and are active in intestinal digesta of ruminants. These findings confirmed previous reports in vitro (Fontes et al., 1995) and studies with pigs (Chesson, 1993; Inborr et al., 1994). Depending on application level, other enzymes may also bypass the rumen and increase polysaccharide-degrading activities in intestinal digesta (Chesson, 1994). Glycosylation confers proteolytic stability to exogenous enzymes (Gorbacheva and Rodionova, 1977), but as demonstrated by Fontes et al. (1995), nonglycosylated enzymes may also resist proteolysis. Hristov et al. (1998a) identified the abomasum as a major barrier to active exogenous enzymes entering the intestine. A follow-up study by Morgavi et al. (2001) confirmed that some exogenous enzymes survive ruminal fermentation and the abomasal environment and may exert activity for a period of time in the small intestine. In general, xylanases are more stable in the rumen and abomasum than cellulases and consequently xylanase activity in the small intestine that is attributable to exogenous enzymes is usually greater than cellulase activity.

The few studies designed to investigate ruminal stability and bypass of exogenous enzymes have clearly shown that some exogenous enzymes are remarkably resistant to microbial proteases, bypass the abomasums, and remain active in the small intestine and have even been shown to linearly increase polysaccharide-degrading activities in feces (Hristov et al., 2000). However, the practical implication of these effects remains unclear. In theory, exogenous enzymes could improve animal performance by not only enhancing ruminal carbohydrate degradability but also by reducing the viscosity of digesta and improving postruminal nutrient absorption. Presently, although postruminal effects can be documented, they are thought to account for a minor component of any positive responses observed with existing enzyme preparations with improvements primarily arising from positive alterations in rumen function.

NEW APPROACHES TO FORMULATING EXOGENOUS ENZYMES

Role of Metagenomics in Enzyme Discovery

Before the development and application of the “-omics” disciplines, culturing isolates in a laboratory was the only method of identifying rumen microbial species. Such cultivation-based techniques applied to the rumen identified approximately 60 to 70 genera and 300 to 400 species of bacteria, protozoa, and fungi (Krause et al., 2007). However, this represents less than 10% of the total microbial species that inhabit this environment (Edwards et al., 2004; Krause et al., 2007), raising the question as to whether the numerically dominant or functionally significant members of the rumen remained unidentified. The diversity and complexity of the rumen ecosystem possess a major challenge in terms of determining the functionality and prevalence of the various carbohydrases produced by ruminal microorganisms over a range of dietary conditions (Morgavi et al., 2012). Although the genomes of 3 of the most highly fibrolytic culturable bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) have been sequenced (Krause et al., 2003), the enzymatic strategies used by each of these species to hydrolyze cellulose are still not well understood. Consequently, culture-independent approaches such as metagenomics, metatranscriptomics, and proteomics have been used to further characterize the carbohydrases produced in the rumen without necessarily attributing their presence to any 1 specific microorganism. With the rapid advancements that have occurred in these sequence- or function-based technologies, an integrated and holistic picture of the metabolic potential and activity of this complex microbial ecosystem is being generated.

Although sequenced-based metagenomics may use various techniques and approaches, the main aim remains to develop a catalogue of all genes present in the rumen (Brulc et al., 2009). A recent study by Hess et al. (2011) assembled 15 microbial genomes that were previously unculturable and identified more than 27,000 putative genes coding for carbohydrases. Functional metagenomics, in this instance, aims to use these catalogues or libraries to identify and isolate specific hydrolytic enzymes involved in structural plant digestion in the rumen (Zhao et al., 2010). Ferrer et al. (2005) identified 9 endogulcanases, 12 esterases, and 1 cyclodextrinases within a metagenomic library derived from the rumen of a dairy cow. The success of this approach in identifying novel enzymes and metabolic pathways, however, is still dependent on the availability of appropriate bioassays and the development of innovative strategies to screen for enzyme activities of interest. Current interest is focusing on the degradation of cellulose and hemicellulose by enzyme members of the glycoside hydrolase family 5 (GH5; Morgavi et al., 2012). This family of glycosyl hydrolyases represents the most abundant cellulases from both cultured (Krause et al., 2003) and uncultured ruminal bacteria (Ferrer et al., 2005). As such, it is not surprising that even though Duan et al. (2009) identified and characterized novel cellulases from the rumen, they were identified as members of the GH5. Recently a metatranscriptomic approach was used by our laboratory (Qi et al., 2011) to examine carbohydrases associated with fungi and protozoa in the rumen of musk oxen, identifying a greater percentage of cellulases per gigabase of sequence than Hess et al. (2011). The application of this technology can be broadened to study carbohydrase complexes in the rumen of a variety of ruminant species over a range of ecological niches. In fact, New Zealand researchers are presently leading an international research group with the objective of collecting samples from ruminants across the globe. This global survey could provide new insight into the carbohydrases that are key to plant cell wall digestion in a variety of species. Identification of carbohydrases that are lacking within ruminal environment could provide the knowledge needed to specifically formulate exogenous enzymes that can fill these deficiencies.

A Focus on Rate-Limiting Enzymes

Lignin is the most recalcitrant of the 3 main heterogeneous polymers in lignocellulose (Himmel et al., 2007; Sanchez, 2009). The formation of ferulate-polysaccharide-lignin complexes that cross-link cell wall polymers is a major factor limiting the rate and extent of enzymatic dissolution of cell walls as it interferes with hydrolysis by preventing the binding of xylanases to their target substrate (Hatfield et al., 1999; Buanafina et al., 2008). However, the cellulolytic capacity of the rumen is not considered to limit cellulose digestion; rather, it is the surface area available for enzyme attachment that limits lignocellulose digestion in the rumen (Weimer et al., 1990). True degradation of lignin is an oxidative process that is primarily performed by aerobic fungi. As the rumen is anaerobic, lignin is not truly degraded, but rather its solubilization is a key step in increasing the amount of cellulose and hemicellulose available for microbial fermentation. Chemical pretreatment with either acidic (e.g., sulfuric acid; Himmel et al., 2007) or alkali solutions (e.g., ammonium hydroxide, sodium hydroxide) aims to increase the susceptibility of cellulose to enzymatic hydrolysis by breaking the ester bonds that link lignin with the plant cell wall (Buanafina et al., 2008). In biorefineries, cellulosic feedstuffs are subjected to pretreatment strategies including thermochemical pretreatment, where dilute sulfuric acid is applied at 140 to 200°C, rendering the cellulose in cell walls more accessible to saccharifying enzymes. Alternatively, the application of alkalines through processes such as ammonia fiber expansion renders cell walls considerably more susceptible to enzyme hydrolysis (Himmel et al., 2007). Such pretreatments can reduce the recalcitrance of cellulose to enzymatic hydrolysis; however, they are costly, require heat or pressurization, and can reduce the availability of fermentable carbohydrate (Wyman, 2007; Weimer et al., 2009).

Carbohydrases have advantages over the use of alkali treatment as chemical treatments can be detrimental to the digestibility of other plant components and denature plant-associated enzymes (Mathew and Abraham, 2004). Ferulic acid esterases, produced by A. oryzae, perform a function similar to that of alkali de-esterification of lignin–hemicellulose linkages in plant cell walls (Tenkanen et al., 1991). Ferulic acid esterases act synergistically with xylanases and pectinases to facilitate access of hydrolyases to the backbone of cell wall polymers. However, the source and type of ferulic acid esterase can influence the amount of ferulic acid released and thus the accessibility of cellulose (Faulds et al., 2003, 2006).

The success of this technology is reliant on its ability to encompass a vast array of dietary and production scenarios. Therefore, it becomes essential to examine variations in ruminal microbial communities across vastly different production environments. The use of the metagenomic and metatranscriptomic procedures described above to identify high activity esterases that could be used to complement existing enzyme products could be one approach to improving the efficacy of these preparations (Fig. 1.). Similarly, the development and refinement of biomass pretreatment strategies that can be used in conjunction with exogenous enzymes to enhance hydrolysis of cellulose in the rumen could further improve the efficacy of enzyme preparations. Effective use of these technologies is considered the most promising way to identify novel enzyme cocktails and thus advance enzyme technology by minimizing the variability currently seen in production responses of ruminants.

Figure 1.
Figure 1.

Stages in the development of novel enzymatic pretreatments to enhance ruminal digestion of structural plant polysaccharides.

 

Conclusion

The use of exogenous enzymes in ruminant diets is still an emerging technology. Although progress has been made, previous strategies have been largely unsuccessful at improving ruminant production or produced highly variable results due to our lack of understanding of the carbohydrase complex and microbial interactions within the rumen ecosystem. Enzymes have been added to diets with the aim of increasing cell wall digestibility and have shown some improvements in milk yield and feed conversion efficiency, yet the true mechanistic effects responsible for these positive outcomes remain elusive. New approaches such as metagenomics, metatranscriptomics, and proteomics are providing novel insights into the structure, interactions, and function of the rumen microbial community to a degree that was previously impossible with lab-based culture techniques. Such approaches have the potential to allow for formulation of exogenous enzymes based on optimal properties for specific ruminal conditions. Ideally, responses to these additives will need to be broad based across a range of diet types as development of a specific exogenous enzyme formulation and dose rate for each diet type and feeding level would introduce a degree of complexity in on-farm application that would likely discourage adoption by producers.

 

References

Footnotes


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