Mode of action of exogenous cell wall degrading enzymes

for ruminants
K. A. Beauchemin, D. Colombatto1, D. P. Morgavi2, W. Z. Yang, and L. M. Rode3
Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada T1J 4B1. Contribution no (387) 02101,
received 14 November 2002, accepted 21 October 2003.

Beauchemin, K. A., Colombatto, D., Morgavi, D. P., Yang, W. Z. and Rode, L. M. 2004. Mode of action of exogenous cell wall
degrading enzymes for ruminants. Can. J. Anim. Sci. 84: 13–22. Recent studies have shown that adding exogenous fibrolytic
enzymes to ruminant diets can increase milk production of dairy cows and weight gain of growing beef cattle as a result of
Can. J. Anim. Sci. Downloaded from cdnsciencepub.com by 201.34.160.172 on 10/10/23

enhanced feed digestion. While much progress has been made in terms of advancing feed enzyme technology for ruminants, con-
siderable research is still required to develop more effective enzyme products. The mode of action whereby exogenous enzymes
improve digestion of plant cell wall is complex, and there is evidence for numerous potential modes of action suggesting they are
interdependant. A mode of action that accounts for the most critical factors that explain the observed increases in feed digestion
is presented. Adding exogenous enzymes to the diet increases the hydrolytic capacity of the rumen mainly due to increased bac-
terial attachment, stimulation of rumen microbial populations and synergistic effects with hydrolases of ruminal microorganisms.
The net effect is increased enzymic activity within the rumen, which enhances digestibility of the total diet fed. Thus, improve-
ments 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 supplement or grain. The magnitude of the improvements in feed digestibility reported
in some research studies using feed enzymes suggests a viable future for enzyme products in commercial ruminant production sys-
tems. A more complete understanding of the mode of action of these products will allow development of low-cost enzyme prod-
ucts designed specifically to improve feed digestion by ruminants.

Key words: Cattle, digestion, fibre digestion, enzymes, cellulases, hemicellulases

Beauchemin, K. A., Colombatto, D., Morgavi, D. P., Yang, W. Z. et Rode, L. M. 2004. Mode d’action des enzymes attaquant
la paroi cellulosique chez les ruminants. Can. J. Anim. Sci. 84: 13–22. Des études récentes montrent que l’addition d’enzymes
fibrolytiques exogènes à la ration des ruminants accroît la production des vaches laitières et le poids gagné par les bovins de
boucherie à cause d’une meilleure digestion des aliments. Bien que la technologie des enzymes alimentaires destinés aux rumi-
nants ait progressé, il reste encore beaucoup à faire avant qu’on arrive à des produits enzymatiques plus efficaces. La manière dont
les enzymes exogènes facilitent la digestion de la paroi cellulosique est particulièrement complexe et tout indique que les nom-
breux modes d’action potentiels sont interdépendants. Les auteurs proposent un mode d’action qui tient compte des principaux fac-
teurs expliquant la meilleure digestion des aliments observée. L’addition d’enzymes exogènes à la ration accroît la capacité
d’hydrolyse du rumen principalement à cause d’une meilleure fixation des bactéries, d’une stimulation de la microflore du rumen
et de la synergie avec les hydrolases des microorganismes. Il s’ensuit une plus grande activité enzymatique dans le rumen, ce qui
améliore la digestibilité de la ration. Cette meilleure digestibilité ne vient donc pas uniquement de la composante alimentaire à
laquelle s’attaquent les enzymes, ce qui explique l’efficacité des enzymes fibrolytiques même quand on les ajoute à un supplément
ou à du grain. L’accroissement de la digestibilité que rapportent certaines études à la suite de l’utilisation d’enzymes laisse croire
à un avenir viable pour les produits enzymatiques destinés à l’élevage commercial des ruminants. Une analyse plus complète du
mode d’action de tels produits concourra à l’élaboration de composés enzymatiques peu coûteux, spécifiquement conçus pour
améliorer la digestion des aliments du bétail par les ruminants.

Mots clés: Bovins, digestion, digestion de la cellulose, enzymes, cellulases, hémicellulases

Forages are the backbone of commercial ruminant production
systems throughout the world. However, the efficiency of con-
verting forages to meat and milk is limited by digestibility of
forage cell walls. Plant cell walls comprise 40 to 70% of the dry
matter (DM) of typical forages and, even under ideal feeding
1Current address: Departamento de Producción Animal,
Facultad de Agronomía, Universidad de Buenos Aires, Av
San Martín 4453, (C1417DSQ) Buenos Aires, Argentina.
2Current address: INRA, Centre Clermont Theix, Unité de
Recherche sur les Herbivores, 63122 Saint-Genès-
Champanelle, France.3Current address: Rosebud Technology
Development Ltd., Lethbridge, Alberta, Canada T1K 3J5.
13
conditions, cell wall digestibility in the total digestive tract is
still generally less than 65% (Van Soest 1994). Furthermore,
when conditions in the rumen are suboptimal for fibre diges-
tion, as can be the case for cattle fed high grain diets, cell wall
digestion in the total tract may be only 50%, with only 35%
ruminal digestion (Beauchemin et al. 2001a). Over the years,
significant improvements in forage cell wall digestibility have
been achieved through agronomic advances. Despite these
improvements, forage digestibility continues to limit the intake
of available energy by ruminants and correspondingly, con-
tributes to excessive manure excretion by livestock.
Abbreviations: DM, dry matter
 14 CANADIAN JOURNAL OF ANIMAL SCIENCE
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Fig. 1. Synergy between exogenous polysaccharidases and extracted rumen microbial enzymes [adapted from Morgavi et al. (2000a)]. The
graph shows the hydrolysis of corn silage incubated at pH 6.0 in the presence of ruminal and Trichoderma enzymes. Corn silage was prein-
cubated with an experimental feed enzyme preparation (lots no. 214 and 211) at concentrations similar to those used in vivo before the addi-
tion of rumen enzymes from cattle fed a high forage (HF) or low forage (LF) diet. Endogenous rumen enzymes (white bars) or exogenous
feed (black bars) enzymes were incubated separately or together (checked bars). Error bars are SD, n = 3.

The use of supplemental exogenous fibre-degrading
enzymes to improve fibre digestibility and feed utilization
was first examined for ruminants in the 1960s, as reviewed
by Beauchemin and Rode (1996). Unfortunately, many of
these early enzyme products were poorly characterized, ani-
mal responses were variable, and little or no effort was made
to design these products specifically for ruminants. At that
time, production of exogenous enzymes was expensive, as
fermentation systems were poorly defined and the proper
isolates and tools of molecular biology were not available to
lower the cost of enzyme production. Increased feed costs,
together with lower fermentation costs and the availability
of more active and better defined enzyme products, prompt-
ed researchers in the 1990s to re-examine the potential use
of exogenous enzymes in ruminant production. Recent stud-
ies have shown that, in some cases, adding exogenous
fibrolytic enzymes to ruminant diets increases milk produc-
tion of dairy cows and weight gain of growing beef cattle, as
recently reviewed by Beauchemin et al. (2001b, 2003a). To
a large extent, increases in animal performance have been
attributed to increases in feed digestion. However, not all
studies report improved animal performance due to the use
of exogenous enzymes, and viewed across a variety of
enzyme products and experimental conditions the response
to feed enzymes by ruminants has been variable. While
much progress has been made in terms of using existing
enzyme products in ruminant diets, considerable research is
still required to produce enzyme products specifically
designed for ruminants. An understanding of mode of action
is necessary to ensure effective and consistent results in
vivo. This review presents current information on mode of
action of exogenous enzymes in ruminant diets that account
for observed increases in feed digestion.
MODE OF ACTION
The scope of this paper is limited to concentrated fermenta-
tion products that have specific, controlled plant cell wall
degrading enzymes. Although some crude fermentation
extracts and direct-fed microbials are also marketed, at least
partly or implicitly, based on their residual enzymic content
(Muirhead 2000/2001), the mode of action of these products
is likely substantially different than that of concentrated
enzyme products.
Increases in animal performance due to the use of feed
enzymes have been attributed to increases in feed digestion.
Numerous studies have reported increased digestion of DM
and fibre measured in situ or in vitro (Nakashima et al.
1988; Feng et al. 1996; Hristov et al. 1996; Yang et al. 1999;
Colombatto 2000; Colombatto et al. 2002) 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). These
increases in feed digestion are somewhat unexpected given
the exceptionally high starch and fibre digesting capacity of
the rumen. Recent studies have begun to elucidate some of
the critical factors involved in the observed increases in feed
digestion, but mode of action continues to be a major focus
of the research presently being conducted with these addi-
tives.
Pre-ingestive Effects
Exogenous enzymes are most effective when applied to feed
prior to ingestion by cattle, as explained by Beauchemin

Table 1. Effects of supplementing diets fed to dairy cows in early lac-
tation or sheep fed at maintenance with an enzyme mixture (from
Yang et al. 2000)
Dairy Cows Sheep
Item Control Enzyme Control Enzyme
Dry matter intake (kg d–1) 19.4 19.8 1.40 1.48
DM digestibility (%) 63.9b 66.6a 77.1 76.8
NDF digestibility (%) 42.6 44.3 57.7 56.5
ADF digestibility (%) 31.8 33.7 49.8 48.1
a, b Means within a class of livestock differ (P < 0.05).
et al. (2003a). However, some ensiled feeds contain com-
pounds that are inhibitory to xylanases (Nsereko et al.
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2000b), therefore, applying enzymes to dry feeds decreases
the variability in response. Because enzymes are most effec-
tive when applied to feed, it is not unreasonable to assume
that the mode of action is through some form of pre-inges-
tive attack of the enzymes upon the plant fibre. The adsorp-
tion of enzyme onto the substrate is an important
prerequisite for hydrolysis (Pell and Schofield 1993).
Applying exogenous enzymes directly to feed causes a
release of reducing sugars (Hristov et al. 1996), and in some
cases, partial solubilization of neutral detergent fibre and
acid detergent fibre (Gwayumba and Christensen 1997;
Krause et al. 1998). Forsberg et al. (2000) postulated that
this release of soluble sugars would provide sufficient addi-
tional available carbohydrates to encourage rapid microbial
growth, shortening the lag time required for microbial colo-
nization. However, the quantity of sugars liberated repre-
sents only a minute portion of the total carbohydrate present
in the diet. Thus, the significance of a pre-ingestive release
of reducing sugars is unclear.
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. There
is evidence that applying fibrolytic enzymes to feed prior to
feeding alters the structure of the feed, thereby making it
more amenable to degradation. Nsereko et al. (2000a)
applied an enzyme product to alfalfa hay that was then auto-
claved to inactivate enzyme activities and washed to remove
any products of hydrolysis. Thus, the design of the experi-
ment eliminated the possibility of chemotactic mechanisms
or synergy between microbial enzymes and exogenous
enzymes. In vitro neutral detergent fibre digestion was high-
er at 12 and 48 h for treated hay than non-treated hay and
generally this effect was enhanced by a longer pre-incuba-
tion with enzymes. Thus, the effects of exogenous enzymes
on digestion were observed in the absence of active
enzymes and soluble hydrolysis products, suggesting that
exogenous enzymes caused structural changes to the forages
that improved digestion. While the study by Nsereko et al.
(2000a) demonstrates compelling evidence that structural
changes to the substrate are an integral component of the
mode of action of feed enzymes in improving digestion, this
explanation does not account for improved dietary fibre
digestion when exogenous enzymes are applied to the con-
BEAUCHEMIN ET AL. — ENZYME MODE OF ACTION 15
centrate (low fibre) portion of the diet (Rode et al. 1999;
Yang et al. 2000).
Another important reason for applying enzymes to feed
prior to ingestion is to enhance binding of the enzyme to the
feed, thereby increasing the resistance of the enzymes to
proteolysis in the rumen. Enzymes applied to feed prior to
ingestion are particularly stable; the presence of substrate is
known to increase enzyme resistance to proteolytic inacti-
vation (Fontes et al. 1995).
There is also evidence that the proportion of the diet to
which enzymes are added must be maximized to ensure a
beneficial response in vivo. Bowman et al. (2002a) reported
that enzymes were more effective when added to rolled
grain, which comprised 45% of the diet, compared with
enzymes added to a finely ground premix, which comprised
0.2% of the diet (DM basis). It was hypothesized that the
greater the proportion of the diet treated with enzymes, the
greater the chances that enzymes endure in the rumen. Their
hypothesis was supported by an in vitro study using the
same treatments showing 15 and 17% increases in DM
digestibility at 12 h of incubation, when enzymes were
added to the grain and premix portions of the diet, respec-
tively (Bowman et al. 2002b). Thus, having the enzyme
delivered in a highly concentrated form did not hinder the
product in vitro, but residence time was not a factor in batch
culture. In vivo, residence time of premix in the rumen may
have been reduced if the premix flowed from the rumen
with the liquid phase rather than with the solids, thereby
reducing potential effects.
Ruminal Effects
Stability of Exogenous Enzymes
Previously it was assumed that the proteolytic activity in the
rumen ecosystem would rapidly inactivate unprotected
enzyme feed additives (Chesson 1994; Graham and Balnave
1995). This assumption was based in part on earlier work
from Kopecny et al. (1987) reporting the rapid inactivation
of a Trichoderma viride cellulase preparation by rumen bac-
terial proteases. However, more recent studies have shown
that exogenous enzymes in the rumen are generally more
stable than previously thought (Hristov et al. 1998a;
Morgavi et al. 2000b; 2001), particularly when added to
feed pre-ingestion (Fontes et al. 1995).
The resistance of feed enzyme products to rumen inacti-
vation has been tested in vitro and in vivo. Both Hristov et
al. (1998a) and Morgavi et al. (2001) observed the cellulase
(EC 3.2.1.4, carboxymethyl cellulose) and xylanase [EC
3.2.1.8; endo-β-(1,4)-xylanase] activities of some enzyme
products to be remarkably stable with little or no decline in
enzymic activity when incubated in rumen fluid for up to 6
h. However, Morgavi et al. (2000b) reported that β-glucosi-
dase (EC 3.2.1.21) and β-xylosidase (EC 3.2.1.37, xylan
1,4-β-xylosidase) activities were much more labile than cel-
lulose and xylanase, with most of the activity destroyed
within an hour of incubation with ruminal fluid.
The stability of xylanases and cellulases in the rumen
may be related to their glycosylation, which may protect
them from inactivation from temperature and proteases, as
discussed by Fontes et al. (1995). Many xylanases and cel-
 16 CANADIAN JOURNAL OF ANIMAL SCIENCE
A)
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5 µm
B)
5 µm
Fig 2. Scanning electron micrograph taken after 24 h of incubation
showing differences in corn fibre colonization by Fibrobacter suc-
cinogenes between control (A) and exogenous enzyme treatment
(B) [adapted from Morgavi et al. (2000c)].
lulases from bacteria and fungal sources are glygosylated,
which may protect them from inactivation from temperature
and proteases. The survival of exogenous enzyme activities
in the rumen may also depend upon the proteolytic environ-
ment of the host animal, which can be variable (Falconer
and Wallace 1998). For example, stability of exogenous
enzymes varied depending upon the donor cow, and ruminal
fluid obtained from cows before feeding inactivated poly-
saccharidases to a greater extent than ruminal fluid taken
after feeding (Morgavi et al. 2001).
These studies indicate that exogenous enzymes are gener-
ally stable in the rumen, although there can be differences
depending on the source organism, the enzyme activity, and
whether the enzyme product is applied to feed. Exogenous
enzymes are likely to be more susceptible to gastrointestinal
proteases in the abomasum and intestines than to ruminal
proteases (Inborr and Grönlund 1993; Hristov et al. 1998b;
Morgavi et al. 2001). Even so, it is likely that exogenous
enzymes survive for a considerable amount of time in the
small intestine and they probably maintain activity against
target substrates in this environment.
Direct Hydrolysis
Various studies have shown that exogenous enzymes
enhanced rate of in situ or in vitro digestion measured using
short-term incubations (Nakashima et al. 1988; Feng et al.
1996; Hristov et al. 1996; Yang et al. 1999; Colombatto
2000; Colombatto et al. 2002). However, most studies have
not found exogenous enzymes to improve the extent of in
situ or in vitro digestion (Nakashima et al. 1988; Feng et al.
1996; Hristov et al. 1996; Iwaasa et al. 1999; Yang et al.
1999; Colombatto 2000). These results suggest that enzyme
additives only degrade substrates that would be naturally
digested by the rumen microflora.
Supplementing the diet with exogenous enzymes is
expected to increase the total enzyme activity within the
rumen. Thus, it is possible that exogenous enzymes improve
digestion by increasing the hydrolytic capacity within the
rumen environment. This hypothesis is difficult to verify
because of the complexity of measuring the enzyme activi-
ty within the rumen. Based on the enzymic activity of rumi-
nal fluid and the amount of enzyme product consumed daily,
Beauchemin and Rode (1996) calculated that adding exoge-
nous enzymes to feed could potentially increase cellulase
activity in the rumen by up to 15%. Similarly, Wallace et al.
(2001) calculated that an enzyme product added to the diet
at 1.5 mg g–1 would increase xylanase (measured using oat
spelt xylan) activity by 5% and cellulase (measured using
carboxymethyl cellulose) activity by 15%. Both authors
assumed no synergism between the activities of endogenous
and exogenous enzymes, and that the exogenous enzymes
were completely stable in rumen fluid. Furthermore,
Wallace et al. (2001) reported that glycosidases are present
at high concentrations in feed. Therefore, exogenous gly-
cosidases are not expected to have any measurable effect on
total activity in the rumen.
Several studies have been conducted to measure the
increase in enzymic activities of ruminal contents due to
exogenous enzymes. Hristov et al. (1998b) administered
two enzyme products through the rumen cannula of a dairy
cow and reported that cellulase activity (measured using
carboxymethyl cellulose) increased by 169 and 198%,
xylanase activity (measured using oat spelt xylan) increased
by 49 and 375%, and β-glucanase activity (measured using
barley β-glucan) increased by 61 and 107%, for the two
products, respectively. The activities peaked about 1.5 h
after the enzymes were dosed. In a second study, Hristov et

al. (2000) infused an enzyme product into the rumen of cat-
tle and measured an 11% increase in cellulase activity (mea-
sured using carboxymethyl cellulose) and a 32% increase in
xylanase activity (measured using oat spelt xylan) of rumi-
nal fluid. However, the enzyme application rates used in
those studies (100 g d–1 equal to 12.5 mg g–1) were about 10
times higher than typically used in feeding studies. Wang et
al. (2001) applied a concentrated enzyme product to feed
such that the added xylanase activity (measured using oat
spelt xylan) was about 30 to 50 times higher than the level
normally used in feeding trials. Incubating the enzyme-
treated feed in the Rumen Simulation Technique (Rusitec)
doubled the xylanase activity in ruminal fluid and increased
the feed-particle associated xylanase activity by about 70%.
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Colombatto et al. (2003a) used pure substrates (Avicel and
xylan) incubated in vitro with exogenous enzymes (0.5–2.5
µL g–1) and reported that in the initial 6 h of incubation,
xylanase, cellulase and β-glucosidase activity of the ruminal
fluid increased on average by 85%. Although the application
rate of the enzyme product used in that study was similar to
levels typically used in feeding studies, the substrates were
artificial, which may account for the high enzyme activities
in the fluid fraction.
From these studies it appears that the extent to which
adding exogenous enzymes to the diet increases the
hydrolytic capacity of the rumen depends upon the amount
of enzyme applied to the feed. At the levels typically used in
feeding studies, the direct increase in enzymic activity
expected may be less than 15%, which would not complete-
ly account for the increases in fibre digestion observed in
vivo (Feng et al. 1996; Yang et al. 1999). Thus, the direct
ruminal hydrolytic effect of exogenous enzymes on improv-
ing cell wall digestion is likely minor, at the levels of exoge-
nous enzymes typically used in feeding studies.
Synergism with Ruminal Microorganisms
The actual increase in hydrolytic capacity in the rumen due
to exogenous enzymes may be greater than calculated from
microbial and exogenous enzyme activity due to synergism
(Morgavi et al. 2000a). Synergy between exogenous
enzymes and rumen microbial enzymes can be defined as
the enhanced effect of these two entities acting cooperative-
ly. The net effect is the increase in enzymic activity that
exceeds the additive effects of each of the individual com-
ponents. Synergy among cellulases and xylanases has been
extensively documented by enzymologists (Bhat and
Hazlewood 2001). Synergy among microorganisms in the
rumen, including crossfeeding of hydrolysis products, uti-
lization of end products, and production of an essential
nutrient, is well documented (Dehority 1993). Furthermore,
synergy in the solubilization of crystalline cellulose between
cellulase components from Trichoderma and the cellulo-
some-type complex of the rumen anaerobic fungus
Neocallimastix has been reported (Wood et al. 1994). Thus,
synergy between exogenous feed enzymes and microbial
enzymes may not be surprising.
Morgavi et al. (2000a) demonstrated substantial syner-
gism between exogenous enzymes and ruminal enzymes
such that the net combined hydrolytic effect in the rumen
BEAUCHEMIN ET AL. — ENZYME MODE OF ACTION 17
was much greater than estimated from the individual activi-
ties (Fig. 1). When enzymes from Trichoderma longi-
brachiatum were combined with ruminal enzymes extracted
from cattle receiving high fibre or high concentrate diets,
hydrolysis of soluble cellulose and xylan increased by up to
35 and 100%, respectively. Hydrolysis of corn silage, a
more complex natural plant substrate, also increased by
40%. Measurement of individual solubilized monosaccha-
rides revealed that the synergistic effect occurred between
enzymic activities attacking the same substrate at the same
hydrolysis site. For example, exogenous xylanase did not
stimulate glucose release, and exogenous cellulase did not
stimulate xylose release when combined with endogenous
ruminal enzymes.
The concept of synergy implies that the multiplicity of
enzymes and total enzymic activity in the rumen are
enhanced by the addition of exogenous enzymes. The sig-
nificance of enhanced enzymic activity to the mode of
action of exogenous enzymes in unclear, but cannot be dis-
counted. There is some debate whether the rumen popula-
tion and its degradative ability limit digestibility of fibre.
For example, there is ample evidence to indicate that in vitro
fibre digestion follows first-order kinetics of digestion with
respect to cellulose concentration or available surface area
(Weimer 1998). This process implies that the amount of
accessible cellulose, rather than the microbial population
itself, limits digestion. Nevertheless, Wallace et al. (2001)
reported that cellulase activity [measured as endo-(β-1,4)-
glucanase] limited rate of fermentation of corn and grass
silage in the rumen. In that study, increasing the enzymic
activities in ruminal fluid using exogenous enzymes
increased rate of forage digestion. Thus, it is possible that
enhanced enzymic activity due to synergy of exogenous
enzymes and rumen bacterial enzymes enhances fibre diges-
tion in the rumen.
Sub-optimal Ruminal Conditions for Fibre Digestion
Conditions in the rumen of intensively reared ruminants are
often suboptimal for growth of fibrolytic bacteria. Typically,
diets fed to dairy cows (Yang et al. 2001) and feedlot cattle
(Beauchemin et al. 2003b) are highly fermentable causing
ruminal pH to remain below optimum (approximately pH 5.9;
Weimer 1998) for fibre digestion for extended periods each
day. However, some exogenous enzymes commonly
employed as additives are optimally active at lower pH than
ruminal enzymes. Thus, it has been speculated that their addi-
tion might be of greater benefit when pH conditions are sub-
optimal for fibrolytic ruminal bacteria.
Yang et al. (2002) used a dual-flow continuous culture
system to evaluate the effects of pH and exogenous enzyme
addition. These authors reported an interaction between
ruminal pH and fibre digestion; the increase (8%) in
digestibility of neutral detergent fibre due to enzyme sup-
plementation was lower when mean fermenter pH was 5.6
compared to when pH was over 6.0 (18% increase). These
results indicate that exogenous enzyme improved fibre
digestion during low rumen pH conditions, but their effec-
tiveness was reduced compared to during higher rumen pH
conditions. Colombatto et al. (2003b) used the same contin-
 18 CANADIAN JOURNAL OF ANIMAL SCIENCE
uous culture system maintained at high (pH 6.0 to 6.6) or
low (pH 5.4 to 6.0) pH. Enzyme addition increased neutral
detergent fibre degradability by 43% and 25% at high and
low pH, respectively, largely due to an increase in hemicel-
lulose degradation. Thus, exogenous enzymes can help
improve fibre digestion when conditional are sub-optimal,
but exogenous enzymes are not expected to fully overcome
the limits to digestion imposed by low rumen pH.
Exogenous enzyme are expected to be more effective for
high producing animals compared to those fed a mainte-
nance level of energy intake, as explained by Beauchemin
et al. (2003a). Digestibility is reduced as the level of ener-
gy intake increases due to low ruminal pH and rapid transit
time through the rumen (National Research Council 2001).
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Because enzymes improve the rate of digestion, they help
maintain high digestibility despite high levels of feed
intake. This concept is illustrated in a study in which dairy
cows and sheep were fed diets 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
dry matter and 31.8% for acid detergent fibre (Table 1).
Due to lower intake as a percentage of body weight,
digestibility of the control diet was higher in sheep: total
tract digestion was 77.1% for dry matter and 49.8% for acid
detergent fibre. 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.
Bacterial Attachment and Colonization
There is some evidence that exogenous enzymes stimulate
the attachment of rumen microbes to plant fibre (Morgavi et
al. 2000c), which may partially explain how small quantities
of enzymes can improve rate of fibre degradation.
Attachment of rumen microorganisms to some components
of forage fibre, such as mesophyll and phloem tissue, is
essential for the digestion of feedstuffs in the rumen (Cheng
et al. 1991; Weimer 1993). Adherence of rumen bacteria to
fibre places the enzyme-system in close proximity to the
substrate, and disrupts the hydrogen bonding within the cel-
lulose matrix (White et al. 1993).
The mechanism by which fibrolytic enzymes stimulate
the attachment of rumen bacteria to plant cell walls remains
unknown. It is possible that applying enzymes to feed caus-
es the release of soluble sugars from feed, thereby increas-
ing the chemotactic attraction and eventual attachment of
fibrolytic rumen bacteria to the plant surface. However, for
enzyme-treated feeds that were washed to remove soluble
sugars, adherence of F. succinogenes was still stimulated
indicating that other mechanisms might be more relevant
(Morgavi et al. 2000c). The application of enzymes to feed
may also increase attachment by increasing the “roughness”
of the feed surface making it more suitable for microbial
colonization (Morgavi et al. 2000c). Feed enzymes may
weaken the surface of the feed thereby removing some of
the physical barriers that impede microbial attachment.
Treating alfalfa hay with exogenous enzymes prior to
ingestion increased bacterial colonization and in situ DM
disappearance of forage between 3 h and 24 h of ruminal
incubation (Yang et al. 1999). These responses were sup-
ported by concurrent increases of fibre digestibility in the
rumen and in the total gastrointestinal tract. Similarly, Wang
et al. (2001) reported that supplemental exogenous enzymes
increased microbial colonization of ruminal microbes to
feed and increased feed particle-associated enzyme activity,
also observed by Colombatto et al. (2003a). Morgavi et al.
(2000c) reported that low levels of Trichoderma longi-
brachiatum enzymes increased the colonization of
Fibrobacter succinogenes to corn silage and alfalfa hay
(Fig. 2).
While low levels of exogenous enzymes have been shown
to increase bacterial adhesion to substrate, there is also evi-
dence that when high levels of exogenous enzymes are used,
the enzymes compete with the rumen population for cellu-
lose’s binding sites available on feeds (Morgavi et al.
2000c). This process could explain the lack of response usu-
ally reported when enzymes are used at high doses.
While there is evidence that exogenous feed enzymes
increase bacterial attachment to feed, the significance of this
mode of action must be weighed in terms of the imperfect
relationship between colonization and digestion. Although
colonization is necessary for digestion of certain plant tis-
sues, the adhesion process is not thought to limit the overall
rate of digestion (Weimer 1993). Thus, the significance of
enhanced adhesion due to exogenous enzymes is unclear.
Stimulation of Rumen Microbial Populations
There is some evidence that adding feed enzymes to the diet
indirectly increases numbers of non-fibrolytic, as well as
fibrolytic, bacteria in the rumen. This effect may be similar
to that reported for direct-fed microbials and fermentation
extracts (Varel et al. 1993; Newbold 1995). Nsereko et al.
(2002) added various levels of an enzyme product from
Trichoderma longibrachiatum to the diet of dairy cows and
reported an increase in total viable rumen bacteria. Most of
this increase was accounted for by increases in bacterial
species that use cellobiose or glucose. Interestingly, the
increase in bacterial numbers disappeared when higher lev-
els of enzyme supplementation were used. This quadratic
response may explain the non-linear dose responses typical-
ly observed in vivo (Beauchemin et al. 1995; Lewis et al.
1999; Kung et al. 2000).
The stimulation of cellobiose- and glucose-utilizing bac-
teria may indicate an increased availability of substrate as a
result of improved cell wall digestion. Wang et al. (2001)
used the Rumen Simulation Technique (Rusitec) and
observed that applying enzyme onto feed increased cellu-
lolytic bacteria 10-fold.
These studies indicate that exogenous enzymes may
increase bacterial numbers. However, it must be recognized
that this information is based on only a couple of studies in
which rumen bacteria were presented as viable cell counts,
a quantitative method that can severely underestimate cell
counts. There is a need to further investigate the effects of
exogenous enzymes on the rumen microbial population, and

the significance of increased bacterial numbers. Several
studies have shown that concentration of cellulolytic bacte-
ria is not a limiting factor in the digestion of cellulose in the
rumen (Dehority and Tirabasso 1998; Krause et al. 2001).
However, in those studies, digestibility was measured at 24
to 30 h, so the detection of any increase in the rate of degra-
dation in the first few hours of digestion was missed.
Furthermore, the experimental conditions used, e.g., high
fibre diets, sheep at 80% ad libitum, are not comparable to
the conditions at which positive effects of exogenous
enzymes are observed.
An increase in cellulolytic microorganisms may acceler-
ate the digestion of newly ingested feedstuffs, and may
amplify the synergy between ruminal enzymes and exoge-
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nous enzymes. Furthermore, stimulation of total microbial
numbers due to the use of enzymes can result in greater
microbial biomass. This increase may explain the reported
increased flow of microbial protein to the duodenum of cat-
tle fed enzyme-treated feed (Yang et al. 1999). Increased
microbial flow would have a significant impact on the sup-
ply of metabolizable protein to the animal, which would
benefit animals fed for high levels of production.
Post-ruminal Effects
It is generally agreed that the primary mode by which feed
enzymes function to improve animal performance in non-
ruminant animals is by reducing the viscosity of intestinal
digesta (Bedford 1995; Choct 2001). The viscous nature of
the non-starch polysaccharides in grains is associated with
altered intestinal transit time, modification of the intestinal
mucosa, and changes in hormonal regulation due to varied
rate of nutrient absorption. The mechanism of action of feed
enzymes in non-ruminant diets raises the possibility that
enzyme-mediated reductions in viscosity could improve
nutrient absorption in the small intestine of cattle. However,
intestinal viscosity in cattle is only between 1 and 2 cPoise
(Mir et al. 2000) whereas intestinal viscosity in poultry may
exceed 400 cPoise (Bedford 1993). Improved growth per-
formance in poultry supplemented with enzymes is usually
associated with 10-fold reductions in intestinal viscosity
(Bedford 1995). Consequently, it is difficult to comprehend
how the relatively modest declines in intestinal viscosity
observed in ruminants supplemented with high levels of
enzymes results in a substantial improvement in nutrient
absorption in the small intestine.
It must also be questioned whether supplementing rumi-
nant diets with exogenous enzymes increases the polysac-
charidase activity within the small intestine. Hristov et al.
(1998b) showed that abomasal infusion of exogenous
enzymes did not successfully supply cellulases and amy-
lases to the intestine probably because of their limited resis-
tance to low pH and pepsin proteolysis (Morgavi et al.
2001). However, xylanase activity in the duodenum
increased 12- to 30-fold. Despite the increased level of
xylanase activity in the duodenum, total tract digestibility
was not increased. However, in studies with dairy cows fed
barley grain diets, improvements in total tract digestion
were attributed, at least partly, to an improvement in the
digestibility of fibre and starch in the lower tract
BEAUCHEMIN ET AL. — ENZYME MODE OF ACTION 19
(Beauchemin et al. 1999a). Hydrolysis of complex carbohy-
drates by exogenous enzymes in the small intestine and sub-
sequent absorption of released sugars would offer energetic
and nitrogen balance benefits to the animal that would not
be accessible if these substrates remained undigested or
were fermented by microbial populations residing in the
large intestine. It is possible that exogenous enzymes work
synergistically with the microbes even in the large intestine.
This raises the possibility that exogenous enzymes may
even still be active in faeces and could aid in reducing over-
all manure output from cattle. However, we have been
unable to measure any increases in the fibrolytic enzyme
activity in manure when feeding exogenous enzymes, even
at 10-times the recommended level of supplementation
(Nsereko, Beauchemin, and Rode; unpublished data).
An Integrated Mode of Action
It is clear that the mode of action for exogenous enzymes
improving digestion of plant cell wall is complex, and there
is a need for additional research in this area. The literature
suggests numerous potential modes of action that are likely
interdependent. Based on the available information, we pro-
pose the following integrated mode of action that we feel
accounts for the most critical factors that explain the
observed increases in feed digestion.
Enzymes are most effective when applied in a liquid form to
feeds prior to ingestion. Preconsumptive effects of exogenous
enzymes help remove structural barriers that limit the microbial
digestion of feed in the rumen and cause the release of soluble
carbohydrates. An additional rationale for applying enzymes to
feeds pre-feeding is to increase their stability in the rumen.
Application of enzymes to feed enhances the binding of the
enzyme with the substrate, which may increase the resistance
of the enzymes to proteolysis.
It is most likely that the major portion of the positive pro-
duction responses resulting from the use of enzyme supple-
ments is due to ruminal effects. Adding exogenous enzymes
to the diet increases the hydrolytic capacity of the rumen
mainly due to increased bacterial attachment, stimulation of
rumen microbial populations and synergistic effects with
hydrolases of ruminal microorganisms. The net effect is
increased enzymic activity within the rumen, which
enhances digestibility of the total diet fed. Thus, improve-
ments in digestibility are not limited to the dietary compo-
nent to which the enzymes are applied. Increased hydrolytic
capacity of the rumen can also lead to an increase in
digestibility of the non-fibre carbohydrate fraction, in addi-
tion to increasing digestibility of the fibre, which explains
why fibrolytic enzymes can be effective in high concentrate
diets (Beauchemin et al. 1999b).
Exogenous enzymes appear to survive in the small intes-
tine for sufficient time to have an effect on target substrates.
However, post-ruminal effects of exogenous enzymes on
digestion are likely only a significant factor when enzymes
are infused into the rumen, or added to feed in a manner that
allows for easy solubilization from feed and rapid passage
from the rumen. Such may be the case when enzymes are
applied to wet feeds prior to feeding or when offered as a
concentrated premix.
 20 CANADIAN JOURNAL OF ANIMAL SCIENCE
CONCLUSION
There is an increasing body of evidence to suggest that
exogenous polysaccharide enzymes can improve feed diges-
tion and weight gain or milk production of cattle. However,
the mechanism whereby exogenous enzymes improve
digestion of plant cell wall is complex with many interrelat-
ed factors. There is a need to continue to elucidate these
interrelated factors because a more complete understanding
of the mode of action will allow development of enzyme
products designed specifically to improve feed digestion by
ruminants. The availability of low-cost, effective enzyme
products for ruminants will allow these products to play an
important role in future ruminant production
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