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Effect of fibrolytic enzyme preparation from Trichoderma longibrachiatum on

the rumen microbial population of dairy cows

Article  in  Canadian Journal of Microbiology · January 2002

DOI: 10.1139/w01-131 · Source: PubMed

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14

Effect of a fibrolytic enzyme preparation from

Trichoderma longibrachiatum on the rumen
microbial population of dairy cows1
V.L. Nsereko, K.A. Beauchemin, D.P. Morgavi, L.M. Rode, A.F. Furtado,
T.A. McAllister, A.D. Iwaasa, W.Z. Yang, and Y. Wang

Abstract: The effects of supplementing a dairy cow diet with incremental levels of a fibrolytic enzyme preparation
(preparation B) from Trichoderma longibrachiatum on the rumen microbial population were investigated. Two cows fit-
ted with rumen cannulae were each fed a diet containing barley-based concentrate (52%), maize silage (29%), and
chopped alfalfa hay (19%), supplemented with 0, 1, 2, 5, or 10 L of preparation B per tonne of dry matter (DM).
Preparation B stimulated numbers of total viable bacteria in a quadratic manner (P < 0.05), to approximately 230, 330,
390, and 250% at 1, 2, 5, and 10 L·t–1 DM, respectively. Preparation B increased the numbers of cellobiose-utilizing
(P < 0.01), xylanolytic (P < 0.05), and amylolytic bacteria (P < 0.05), but had no effect (P > 0.05) on numbers of
cellulolytic bacteria. However, when bacterial numbers enumerated on each substrate were expressed as a proportion of
total viable bacterial numbers, only cellobiose utilizers were stimulated, and this stimulation was limited to the 1 L·t–1 DM
level of preparation B (P < 0.05). The results of this study demonstrate that the inclusion of an exogenous fibrolytic
enzyme preparation in dairy cow diets increased the numbers of rumen bacteria that utilize hemicelluloses and second-
ary products of cellulose digestion.

Key words: rumen, fibrolytic enzymes, cellulase, xylanase, cellulolytic, xylanolytic.

Résumé : Nous avons évalué chez la vache laitière, les effets sur la population microbienne du rumen d’un ajout à la
diète de doses croissantes d’un supplément, soit une préparation enzymatique fibrolytique (préparation B) provenant du
Trichoderma longibrachiatum. Deux vaches porteuses d’une canule reliée au rumen ont été chacune nourries avec un
concentré d’orge (52 %), d’ensilage de maïs (29 %) et de luzerne hachée (19 %) additionné de 0, 1, 2, 5 ou 10 L de
préparation B par tonne de matière sèche (DM). Cette préparation B a fait augmenter le nombre total des bactéries
viables de quatre façons (P < 0,05), soit de 230, 330, 390 et 250 % à 1, 2, 5 et 10 L·t–1 DM respectivement. Cette
préparation B a causé une augmentation des bactéries amylolytiques (P < 0,05), xylanolytiques (P < 0,05) et des
bactéries utilisatrices du cellobiose (P < 0,01), mais cette préparation n’a pas eu d’effet (P < 0,05) sur le nombre des
bactéries cellulolytiques. Par contre, lorsque les décomptes bactériens obtenus avec chacun des substrats étaient
rapportés comme une proportion des nombres totaux des bactéries viables, seules les bactéries utilisant le cellobiose
étaient stimulées et cette stimulation était uniquement obervée avec 1 L·t–1 DM de préparation B (P < 0,05). Les
résultats de cette étude démontrent que l’incorporation d’une préparation enzymatique fibrolytique dans le régime
alimentaire des vaches laitières fait augmenter le nombre des bactéries du rumen qui utilisent les hémicelluloses et les
produits dérivés de la digestion de la cellulose.

Mots clés : rumen, enzymes fibrolytiques, cellulase, xylanase, cellulolytique, xylanolytique.

[Traduit par la Rédaction] 20

Introduction Nsereko et al. plementing diets of dairy cows and feedlot cattle with fiber-
degrading enzymes has significant potential to improve feed
The use of feed enzymes in ruminant diets is a technology utilization and animal performance (Beauchemin et al. 1995;
in development. Recent research has demonstrated that sup- Rode et al. 1999; Schingoethe et al. 1999; Kung et al. 2000).

Received 20 December 2000. Revision received 24 October 2001. Accepted 26 October 2001. Published on the NRC Research
Press Web site at http://cjm.nrc.ca on 4 January 2002.
V.L. Nsereko,2 K.A. Beauchemin,3 D.P. Morgavi,4 L.M. Rode,5 A.F. Furtado, T.A. McAllister, A.D. Iwaasa,6 W.Z. Yang, and
Y. Wang. Agriculture and Agri-Food Canada, Research Center, Box 3000, Lethbridge, AB T1J 4B1, Canada.
1
Lethbridge Research Centre Contribution No.: 3879968.
2
Present address: Pioneer Hi-bred International Inc., 7300 NW 62nd Avenue, Johnston, IA 50131-1004, U.S.A.
3
Corresponding author (e-mail: beauchemin@em.agr.ca).
4
Present address: INRA, Centre Clermont-Theix, Unité de Recherche sur les Herbivores, 63122 Saint-Genès-Champanelle.
5
Present address: 3302 Beauvais Place, Lethbridge, AB T1K 3J1, Canada.
6
Present address: Agriculture and Agri-Food Canada, Box 1030, Swift Current, SK S9H 3X2, Canada.

Can. J. Microbiol. 48: 14–20 (2002) DOI: 10.1139/W01-131 © 2002 NRC Canada

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Nsereko et al.
A recent review by Beauchemin et al. (2001) reported that
across 16 dairy cow studies, the average increase in dry mat-
ter (DM) intake due to enzyme supplementation was
1.6 kg·day–1, and the average increase in milk yield was
1.3 kg·day–1 (4.2%). However, when viewed across all en-
zyme products and all experimental conditions, the variabil-
ity in production responses to feed enzymes has been
variable. A limited number of ruminant enzyme products are
now commercially available in North America, and this list
of products is expected to grow (Beauchemin et al. 2001).
Research is needed to understand the mode of action of en-
zyme products so that on-farm efficacy can be assured.
The mechanism whereby fibrolytic enzymes improve ani-
mal performance appears to be complex and continues to be
a major focus of the research presently being conducted with
these additives. Researchers have shown that exogenous en-
zymes enhance fiber degradation by ruminal microorganisms
in vitro (Hristov et al. 1996; Feng et al. 1996), in situ (Feng
et al. 1996; Lewis et al. 1996), and in vivo (Yang et al.
1999). Increased feed digestion due to enzyme supplement-
ation is likely due to a number of contributing factors. In-
creased feed digestion is likely not simply the result of
supplemental enzymic activity, because the contribution of
added exogenous enzymes to total ruminal activity is rela-
tively small (Beauchemin et al. 2001). Morgavi et al. (2000)
demonstrated synergism between exogenous enzymes and
ruminal enzymes such that the net combined hydrolytic ef-
fect in the rumen was much greater than that estimated from
the individual activities. Wang et al. (2001) reported that en-
zyme supplementation increased numbers of nonfibrolytic
and fibrolytic bacteria in a batch culture system using rumen
fluid. Stimulation of rumen microbial numbers through the
use of enzymes could result in greater microbial biomass,
which would provide more total polysaccharidase activity to
digest feedstuffs. Consistent with this hypothesis, Yang et al.
(1999) reported that enzyme supplementation of dairy cow
diets increased feed digestion in the rumen and the flow of
microbial protein from the rumen.
The objective of the present study was to investigate the
effects of supplementing a dairy cow diet with a fibrolytic
enzyme preparation from Trichoderma longibrachiatum on
the concentration and distribution of rumen microorganisms.
This study was part of a larger research initiative to obtain
more information on the mechanisms by which exogenous
fungal enzymes improve fiber digestion by ruminants.
Materials and methods
Animals and sampling
The cows used in this study were cared for in accordance
with the guidelines established by the Canadian Council on
Animal Care (1993). The cows used in this study were part
of a larger feeding trial to examine the effects of adding in-
cremental levels of a novel exogenous feed enzyme prepara-
tion on digestion and milk production of lactating dairy
cows. The cows weighed approximately 700 kg, consumed
23 kg·day–1 of feed DM, and produced 30 kg·day–1 of milk.
Two lactating dairy cows fitted with rumen cannulae were
each fed a total mixed ration (TMR) containing barley-based
concentrate (52%), maize silage (29%), and chopped alfalfa
hay (19%). The diet was formulated to provide sufficient en-
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15
ergy, protein, minerals, and vitamins to sustain 35 kg·day–1
of milk production. The composition of the diet is shown in
Table 1.
An experimental enzyme formulation from T. longi-
brachiatum (preparation B) obtained from Monsanto Co. (St.
Louis, Mo.) containing mainly cellulase and xylanase activi-
ties was used. The appropriate amount of concentrated en-
zyme product was diluted into 15 L of water and then added
to a tonne of barley concentrate, at the time of manufactur-
ing. The levels of enzyme added to the final TMR were
equivalent to 0, 1, 2, 5, or 10 L of preparation B per tonne DM.
The experiment consisted of five experimental periods,
each comprised 4 weeks. During each experimental period,
each cow was fed one of the diets, such that by the end of
the experiment both cows had received each diet once. Ani-
mals were fed twice daily at 06:00 h and 15:00 h.
Sampling was done during the last week of the experi-
mental period to allow sufficient time for the rumen micro-
bial population to adapt to the diet. At 05:00 h (14 h after
feeding) and 10:30 h (4.5 h after feeding) approximately
250 g of whole rumen contents were withdrawn from the
bottom of the dorsal rumen sac, the bottom of the ventral ru-
men sac, and the solid mat of feed particles, and these sam-
ples were mixed to form a composite sample for each cow.
Rumen microorganisms were enumerated 4.5 and 14 h after
feeding, because microbial populations are known to exhibit
diurnal variation (Leedle et al. 1982).
Sample processing
All chemicals used were purchased from Sigma Chemical
Co. (St. Louis, Mo.). For the determination of microbial
concentrations, rumen contents were processed using a
method similar to that described by Stewart et al. (1997).
Briefly, 400 g of the composite whole rumen contents from
each cow was strained through two layers of cheesecloth,
and the filtrate and residual solids were combined (1:1, w/w)
and homogenized in a Waring blender for 1 min, under a
stream of oxygen-free CO2. The homogenate was then
strained through four layers of cheesecloth and the strained
rumen fluid (SRF) thoroughly mixed under a stream of CO2.
The samples were transferred to the laboratory in a sealed
Thermos flask. An additional 5 mL of SRF was preserved
using 5 mL of methyl green formalin – saline solution (1:1,
v/v) for protozoa enumeration as described by Ogimoto and
Imai (1981). These protozoa samples were stored at room
temperature and protected from light until counting.
Microbiology
The sterile anaerobic culturing procedures described by
Hungate (1966) were used at all times. With the exception of
the cellulolytic population, all microbial subgroups were
enumerated using the roll tube method (Hungate 1969) on
the basic rumen fluid-containing medium of Scott and
Dehority (1965), with agar (18 mg·mL–1), following incuba-
tion at 39°C for 48 h. For enumeration of the total viable
bacteria population, anaerobic serial dilutions (10–6–10–9) of
rumen inocula were prepared as described by Bryant and
Burkey (1953) using mineral solutions and resazurin from
medium 10, described by Caldwell and Bryant (1966). Each
dilution was inoculated in triplicate into separate roll tubes con-
taining cellobiose, xylan, starch, and glucose (0.5 mg·mL–1
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16 Can. J. Microbiol. Vol. 48, 2002

Table 1. Diet composition (dry matter basis). time after it was established that the interaction between
–1 enzyme level and sampling time was not statistically signifi-
Diet composition g·(kg DM)
cant for any of the variables measured. Contrasts were used
Constituent to determine the linear and quadratic effects of enzyme level
Forage and to compare combinations of enzyme levels to the con-
Maize silage 290 trol. Statistical significance was declared at P < 0.05 and
Chopped alfalfa hay 190 trends discussed at P < 0.10. Differences between enzyme
Concentrate* levels were tested when the main effect was significant, us-
Steam rolled barley grain 324 ing the PDIFF option of SAS (1989).
Wet beet molasses 3.21
Canola oil 6.91
Blood meal 1.65 Results
Maize gluten meal 62
There were no interactions between rumen sampling time
Soybean meal 93
(4.5 versus 14 h after feeding) and enzyme level (P > 0.05)
Calcium carbonate 8.45
for any of the microbial populations enumerated. Thus, only
Dicalcium phosphate 4.4
the main effects of enzyme level and sampling time are pre-
Monosodium phosphate 0.86
sented (Table 2). Duration from the last feeding (4.5 versus
Mineral–vitamin premix† 12.8
14 h) had no effect (P > 0.05) on the concentration of any of
Pellet binder 1.66
the microbial populations enumerated in the present study.
Composition
However, with the exception of protozoa, all microbial popu-
Crude protein 189
lations measured were numerically higher 4.5 h after feed-
Degradable intake protein 112
ing. The most notable increases were for the total viable
Neutral detergent fiber 320
(P = 0.06), xylanolytic (P = 0.13), and amylolytic (P = 0.10)
Net energy for lactation 1.70 bacterial populations.
(Mcal·(kg DM)–1)
Preparation B increased numbers of total viable rumen
*All ingredients except barley grain were pelleted. bacteria in a quadratic manner (P < 0.05; Table 2). These in-
†
Contained 89.5% NaCl, 2.2% ZnSO4·H2O, 2.5% creases were to approximately 230, 330, 390, and 250%, (all
MnSO4·4H2O, and 0.8% CuSO4·5H2O; 95 mg/kg
CoSO4·6H2O, 89 mg/kg Na2SeO3, 4.8% Dynamate® P < 0.05) at the 1, 2, 5, and 10 L·t–1 DM levels of prepara-
((Pitman Moore, Inc, Mundelein, Ill.) 22% S, 18% tion B, respectively. Contrasts of total viable bacteria num-
K, 11% Mg, 5 mg/kg Pb, and 1000 mg/kg Fe); bers confirmed that when compared with the control, the
10 000 IU/g vitamin A, 1250 IU/g vitamin D, and stimulatory effect was greatest at 2 and 5 L·t–1 DM concen-
10 IU/g vitamin E.
trations (P < 0.01) and lowest at 10 L·t–1 DM (P < 0.05). In-
creasing concentrations of preparation B had a negative
each). Fungal zoospore numbers were determined in tripli- linear effect (P < 0.05) on fungal zoospore numbers. Inter-
cate on serial dilutions (10–1–10–4) of the samples using roll estingly, supplementation with preparation B resulted in a
tubes containing the same substrates and media that were convex quadratic effect on protozoa numbers (P < 0.05) and
used for enumerating total bacteria, but with the following had no effect (P > 0.05) on the concentration of cellulolytic
antibiotics added: chloramphenicol, streptomycin, ampicil- bacteria.
lin, and tetracycline, each included at 100 µg·mL–1 media. Overall, the concentration of xylanolytic and amylolytic
For the determination of the concentration of cellobiose-, bacteria (Table 2) in the rumen of cows was not statistically
starch-, and xylan-utilizing bacteria, anaerobic serial dilu- (P > 0.05) affected by enzyme supplementation; however,
tions (10–6–10–9) of rumen inocula were each inoculated in numerical differences were evident, with preparation B tend-
triplicate into the media (Scott and Dehority 1965) contain- ing to increase numbers of both subgroups at all concentra-
ing only the corresponding energy substrates at 5 mg·mL–1. tions. Contrasts between the control and a combination of all
Cellulolytic bacteria were enumerated following a 14-day concentrations of preparation B revealed that preparation B
incubation at 39°C in triplicate tubes with each of the dilu- increased numbers of both xylanolytic and amylolytic bacte-
tions. The most probable number (MPN) procedure de- ria (P < 0.05). In addition, there was evidence of a linear in-
scribed by Garthright (1998), with filter paper (Whatman crease (P = 0.06) in numbers of xylanolytic bacteria, with
No. 1) as the sole substrate and with media prepared without increasing concentrations of preparation B.
soluble carbohydrate (Scott and Dehority 1965), was used. The cellobiose-utilizing population was markedly stimu-
Protozoa were counted with the aid of a hemocytometer lated by preparation B (Table 2). When compared with the
chamber. Duplicate preparations of each sample were control, contrasts revealed that a combination of all levels of
counted, and if either value differed from the average by preparation B increased (P < 0.01) numbers of cellobiose-
more than 10%, the counts were repeated. utilizing bacteria. Preparation B increased numbers of
cellobiose utilizers (P < 0.05) at the 1, 2, and 10 L levels,
Statistical analyses and numbers of this microbial subgroup were numerically
Means for all variables were analysed using the General higher (P > 0.05) at the 5 L·t–1 DM level of supplement-
Linear Model of SAS (1989). Statistical analyses of micro- ation.
bial counts were performed on transformed (log10) data. The When numbers of bacterial subgroups were expressed as a
statistical model accounted for enzyme level and sampling proportion of total viable bacterial numbers (Figs. 1 and 2),

© 2002 NRC Canada

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Nsereko et al. 17

Table 2. Effects of preparation B (L·t–1 DM) on numbers (log10 mL–1) of rumen microorganisms.
Bacteria
Total Cellobiose-
viable utilizing Xylanolytic Amylolytic Cellulolytic Fungi Protozoa
Enzyme level
0 8.90 8.75b 8.82 8.91 7.51 3.31 5.90
1 9.26a 9.27a 9.10 9.29 7.14 3.53 5.83
2 9.35a 9.26a 9.05 9.26 7.92 3.37 5.80
5 9.38a 9.08ab 9.11 9.16 7.45 3.38 5.69
10 9.26a 9.15a 9.18 9.24 7.66 3.07 6.10
SE 0.272 0.297 0.195 0.219 0.403 0.230 0.203
Linear effect NS NS NS NS NS * NS
Quadratic effect * NS NS NS NS NS *
Contrasts
0 vs. 1, 2, 5 ** ** * * NS NS NS
1, 2 vs. 5, 10 NS NS NS NS NS * NS
0 vs. 1, 2, 5, 10 ** ** * * NS NS NS
Sampling timea
4.5 h 9.33 9.17 9.13 9.26 7.60 3.38 5.86
14 h 9.13 9.04 8.98 9.09 7.46 3.28 5.87
SE 0.314 0.200 0.195 0.272 0.213 0.159 0.011
Significance † NS NS † NS NS NS
Note: Means within the same column not followed by the same letter differ at P < 0.05. NS, P > 0.10; †, P < 0.10; *, P < 0.05; **, P < 0.01; SE,
standard error of the mean.
a
Hours after feeding.

only cellobiose-utilizing bacteria (Fig. 2) were stimulated by
preparation B, and this stimulation was limited to the 1 L·t–1
DM level of supplementation (P < 0.05). In contrast, the
proportions of xylanolytic and amylolytic bacteria (Fig. 1)
were consistently lower (P < 0.05) than those of the control,
decreasing with increasing concentrations of preparation B
until the 5 L·t–1 DM level, then increasing at the 10 L·t–1
DM level. By the difference in trends of total viable bacteria
(Fig. 3) and populations of bacteria subgroups (Figs. 1 and
2), it can be deduced that most of the increases in numbers
of total viable bacteria at the 2 and 5 L·t–1 DM levels were
not accounted for by populations enumerated on cellobiose,
xylan, or starch.
Discussion
Mean numbers of cellobiose-utilizing, xylanolytic, amylo-
lytic, and cellulolytic bacteria at 4.5 h after feeding accounted
for 76, 71, 93, and 2.8% of the total viable bacteria popula-
tion, respectively. Similarly, mean numbers of cellobiose-uti-
lizing, xylanolytic, amylolytic, and cellulolytic bacteria at
14 h after feeding accounted for 85, 76, 94, and 4.2% of the
total viable bacteria population, respectively. These propor-
tions of bacteria subgroups were in general agreement with
those reported by Leedle et al. (1982) for steers fed either a
high forage or high grain diet at maintenance.
Numbers of total viable, xylanolytic, amylolytic, and
cellulolytic bacteria as well as fungi were numerically higher
4.5 h after feeding when compared with 14 h after feeding.
In contrast, Leedle et al. (1982) reported for cattle fed at
maintenance that carbohydrate-utilizing subgroups of bacte-
ria were lowest 2 h after feeding and highest 14 h after feed-
ing. The difference between these two studies may relate to
the level of feeding of the animals and availability of sub-
strate. The lower numbers of carbohydrate-utilizing bacteria
observed at 14 h after feeding in the present study may re-
flect a depletion of nutrients, accompanied by a correspond-
ing reduction in growth rate (Warner 1966). Because the
proportions of the bacteria subgroups measured were gener-
ally lower 4.5 h after feeding (Fig. 4), it would appear that
most of the additional viable bacteria measured at 4.5 h
when compared with those measured at 14 h utilized sub-
strates other than those associated with plant fiber.
Cross-substrate utilization probably contributed to the rel-
atively higher numbers of single substrate populations when
compared with the total viable population. Many species of
rumen bacteria utilize a range of substrates. For example,
Prevotella ruminocola, a species prevalent in diets similar to
that used in the present experiment (Stewart et al. 1997),
will grow on both starch and xylan.
Interpretation of the quadratic response to preparation B
supplementation, in numbers of total viable bacteria (Ta-
ble 2), is open to speculation, and there are no published re-
ports of similar responses in rumen microbial numbers.
However, a quadratic response to enzyme supplementation
in average daily weight gain and fiber digestion in steers fed
alfalfa hay is evident from the data of Beauchemin et al.
(1995). It is unlikely that high concentrations of preparation
B were toxic to rumen microorganisms, since in all cases,
they tended to reduce the extent of stimulation when com-
pared with the lower concentrations, but the high concentra-
tions did not have a negative effect on microbial numbers
when compared with the control. We speculate that the ap-
plication of a moderate level of preparation B to ruminant
feeds causes some beneficial disruption to the surface struc-
ture of the feed either before or after ingestion. With this in

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18 Can. J. Microbiol. Vol. 48, 2002

Fig. 1. Effects of supplementing a dairy cow diet with incremen- Fig. 2. Effects of supplementing a dairy cow diet with incremen-
tal levels of a fibrolytic enzyme preparation on the concentration tal levels of a fibrolytic enzyme preparation on the concentration
of xylanolytic and amylolytic bacteria in rumen fluid, expressed of cellobiose-utilizing bacteria in rumen fluid, expressed as a
as a proportion of the number of total viable rumen bacteria. proportion of the number of total viable rumen bacteria.

Fig. 3. Effects of supplementing a dairy cow diet with incremental

levels of a fibrolytic enzyme preparation on the concentration of
total viable bacteria in rumen fluid.
mind, it is plausible that when an excess of the enzyme
preparation is applied, the beneficial disruption of the feed
surface structure can diminish, perhaps because an excess of
exogenous enzyme attached to the feed may restrict micro-
bial attachment, and hence, limit digestion of the feed.
Relative to numbers of total viable bacteria, numbers of
polysaccharide-utilizing bacteria (xylanolytic and amylo-
lytic) in cows fed diets supplemented with preparation B
were similar to or lower than those observed in cows fed the
control diet. In contrast, total viable bacteria counts were
consistently higher in cows fed the control diet (Table 2).
Glucose-utilizing bacteria were not enumerated in the pres-
ent study. However, since glucose was included in the media
used to enumerate total viable bacteria, and glucose-utilizing
bacteria have been reported to comprise up to 86% of total
viable bacteria (Leedle et al. 1982), it can be inferred from
the relative trends in microbial subgroups and total viable
bacteria that glucose and, to a lesser extent, cellobiose were
responsible for most of the stimulation in total viable bacte-
ria.
When numbers of bacteria subgroups were expressed as a
proportion of total viable bacteria, only the cellobiose-
utilizing population was stimulated by preparation B (1 L·t–1
DM). These observations suggest that the main effects of feed were removed by enzymic action, allowing the bacteria
preparation B on the microbial population were on the sec- to colonize plant fiber and out-compete the fungi. The bio-
ondary fermenting bacteria. This conclusion supports obser- logical reasons for the convex quadratic effect of preparation
vations by others (Feng et al. 1996) that feed enzyme B supplementation on protozoal numbers are unclear; how-
preparations increase the rate and not the extent of fiber deg- ever, it is possible that fungal enzyme preparations act as
radation in the rumen. defaunating agents. This would explain why some research-
Joblin et al. (1989) reported that rumen fungi were able to ers reported increases in microbial numbers (Fondevila et al.
gain access to plant polysaccharides that were unavailable to 1990; Oellermann et al. 1990; Varel et al. 1993) and why the
rumen bacteria. It is possible that at high levels of prepara- efficiency of microbial protein synthesis and the quantity of
tion B, some of the many structural barriers to digestion of microbial protein entering the duodenum also increase

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Nsereko et al.
Fig. 4. Effects of sampling time on numbers of specific carbohy-
drate-utilizing bacteria, expressed as a proportion of the number
of total viable rumen bacteria.
(Yang et al. 1999) when fungal extracts are fed to rumi-
nants. Alternatively, the shifts in bacterial populations ob-
served with enzyme supplementation may have resulted in a
bacterial population less suitable for protozoa predation.
Newbold et al. (1996) has shown that rumen protozoa are
discriminating in the bacterial species they engulf. Lower
protozoal numbers may partially explain the increased num-
ber of bacteria. Reduced predation would allow bacterial
populations to increase, particularly for free-floating second-
ary fermenting bacteria.
A number of recent studies have reported increased feed
digestion due to supplementing ruminant diets with exoge-
nous fibrolytic enzymes (Feng et al. 1996; Rode et al. 1999;
Yang et al. 1999). Furthermore, Nsereko et al. (1999) clearly
demonstrated that enzymic activity, rather than nonenzymic
factors present in the preparation, was responsible for en-
hancing digestion. It is reasonable to conclude that the in-
creases in numbers of rumen bacteria with supplementation
of diets with preparation B observed in the present study
were the result of increased feed digestion due to enzymic
factors.
Conclusions
Supplementing dairy cow diets with an exogenous
fibrolytic enzyme preparation stimulated total viable rumen
bacteria numbers. In particular, cellobiose-utilizing bacteria
and by inference glucose utilizers were stimulated, but ef-
fects on the fibrolytic population were negligible. A qua-
dratic effect of the level of enzyme supplementation on
numbers of total viable bacteria was evident, indicating that
it is possible to over-supplement.
The study indicates that increased fiber digestion due to
enzyme supplementation of ruminant diets may be due, at
least in part, to increased microbial numbers, and thus, to an
increased capacity of the rumen to digest feed. These results
also imply that enzyme supplementation may increase the
quantity of microbial protein available to the animal.
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Acknowledgements
This work was funded in part by Monsanto Co. and by
Agriculture and Agri-Food Canada through the Matching In-
vestment Initiative Program. David Erickson, Leslie
McDowell, Linda Neill, and Cindy Barkley are thanked for
their technical assistance.
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