Enhancing in vitro degradation of alfalfa hay and corn silage using feed enzymes.

Abstract

A series of in vitro fermentation experiments was performed to assess the effects of 4 feed enzyme products (FE) that varied in enzymatic activities on the degradation of alfalfa hay and corn silage. The FE contained a range of endoglucanase, exoglucanase, xylanase, and protease activities, and a range of dose rates (DR) was used. The objective of the study was to identify effective formulations and optimum DR, and to establish if combining FE would further improve fiber degradation. For alfalfa hay, quadratic increases in gas production and degradation of dry matter (DM) and fiber were observed for all FE, with maximum responses at low to medium DR. For corn silage, none of the FE increased gas production or DM degradation, but all FE increased NDF degradation, with optimum DR in the low to medium range. The proteolytic enzyme papain improved fiber degradation of alfalfa hay and corn silage in a manner similar to that observed for polysaccharidase FE. Among the polysaccharidase FE, added activities of endoglucanase and exoglucanase were positively correlated with improvement in neutral detergent fiber (NDF) degradability of corn silage, whereas only added endoglucanase activity tended to be correlated with improvement in NDF degradability of alfalfa hay. Combining effective polysaccharidase FE further improved fiber degradation of both forages, with greater improvements for corn silage. Combining polysaccharidase and proteolytic FE further improved NDF degradation of corn silage, but not alfalfa hay. Combination treatments generally resulted in additive effects with increases in fiber degradation equal to the sum of the improvements for the individual enzyme components. Improved fiber degradation of corn silage was associated with decreased acetate to propionate ratios. Enzyme products that improve in vitro degradation of forages may have the potential to improve lactational performance of dairy cows.

Full text

Page 1

Utah State University
DigitalCommons@USU
ADVS Faculty Publications
Animal, Dairy & Veterinary Sciences, Department
of
1-1-2007
Enhancing In Vitro Degradation of Alfalfa Hay and
Corn Silage Using Feed Enzymes
Jong-Su Eun
Utah State University
K. A. Beauchemin
This Article is brought to you for free and open access by the Animal, Dairy
& Veterinary Sciences, Department of at DigitalCommons@USU. It has
been accepted for inclusion in ADVS Faculty Publications by an authorized
administrator of DigitalCommons@USU. For more information, please
contactdigitalcommons@usu.edu.
Take a 1 Minute Survey-http://www.surveymonkey.com/s/
BTVT6FR
Recommended Citation
Eun, J.-S., and K. A. Beauchemin. 2007. Enhancing In Vitro Degradation of Alfalfa Hay and Corn Silage Using Feed Enzymes Journal
of Dairy Science 90: 2839-2851.

Page 2

J. Dairy Sci. 90:2839–2851
doi:10.3168/jds.2006-820
© American Dairy Science Association, 2007.
Enhancing In Vitro Degradation of Alfalfa Hay and Corn Silage1
Using Feed Enzymes
J.-S. Eun and K. A. Beauchemin1
Agriculture and Agri-Food Canada, Research Centre, Lethbridge, Alberta, T1J 4B1, Canada
ABSTRACT
A series of in vitro fermentation experiments was
performed to assess the effects of 4 feed enzyme prod-
ucts (FE) that varied in enzymatic activities on the
degradation of alfalfa hay and corn silage. The FE con-
tained a range of endoglucanase, exoglucanase, xyla-
nase, and protease activities, and a range of dose rates
(DR)wasused.Theobjectiveofthestudywastoidentify
effective formulations and optimum DR, and to estab-
lish if combining FE would further improve fiber degra-
dation. For alfalfa hay, quadratic increases in gas pro-
duction and degradation of dry matter (DM) and fiber
were observed for all FE, with maximum responses at
low to medium DR. For corn silage, none of the FE
increased gas production or DM degradation, but all
FE increased NDF degradation, with optimum DR in
the low to medium range. The proteolytic enzyme pa-
pain improved fiber degradation of alfalfa hay and corn
silage in a manner similar to that observed for polysac-
charidase FE. Among the polysaccharidase FE, added
activitiesofendoglucanaseandexoglucanasewereposi-
tively correlated with improvement in neutral deter-
gent fiber (NDF) degradability of corn silage, whereas
only added endoglucanase activity tended to be corre-
lated with improvement in NDF degradability of alfalfa
hay. Combining effective polysaccharidase FE further
improvedfiberdegradationofbothforages,withgreater
improvements for corn silage. Combining polysacchari-
dase and proteolytic FE further improved NDF degra-
dation of corn silage, but not alfalfa hay. Combination
treatments generally resulted in additive effects with
increases in fiber degradation equal to the sum of the
improvements for the individual enzyme components.
Improved fiber degradation of corn silage was associ-
ated with decreased acetate to propionate ratios. En-
zyme products that improve in vitro degradation of for-
ages may have the potential to improve lactational per-
formance of dairy cows.
Received December 6, 2006.
Accepted February 14, 2007.
1Contribution number 38707013.
2Corresponding author: beauchemink@agr.gc.ca
2839
Key words: alfalfa hay, corn silage, feed enzymes, fi-
ber degradation
INTRODUCTION
Supplementing ruminant diets with feed enzymes
(FE) to improve forage utilization has attracted grow-
ing attention (Beauchemin et al., 2003). Products that
contain polysaccharidases have been shown to increase
fiber digestion in some (Rode et al., 1999; Bowman et
al., 2002), but not all (Knowlton et al., 2002; Sutton
et al., 2003), feeding studies. Increased ruminal fiber
digestion is expected to increase DMI and milk produc-
tion of dairy cows. Using numerous forage species rang-
ing in NDF digestibility (24 to 87%), Oba and Allen
(1999) reported that a 1-percentage unit increase in
NDF digestibility (measured in vitro or in situ) was
associated with a 0.25-kg/d increase in 4% FCM yield
and a 0.17-kg/d increase in DMI.
Ideal enzyme formulations and effective dose rates
(DR) need to be identified using in vitro methods before
FE products can be used cost effectively in commercial
dairy production. The structure of plant cell walls is
complex (Wilson and Mertens, 1995) and ruminal mi-
croorganisms produce numerous enzymatic activities
that hydrolyze the plant cell wall to its constituent
monomeric components. Exogenous FE are thought to
improvefiberdegradationintherumenbyactingsyner-
gistically with the rumen microflora (Morgavi et al.,
2000),therebyincreasingthehydrolyticcapacitywithin
the rumen environment (Beauchemin et al., 2004). The
major activities involved are cellulases and xylanases,
which degrade cellulose and hemicellulose, respec-
tively,withsynergyoccurringbetweenthese2activities
(Bhat and Hazlewood, 2001). A previous study showed
a positive relationship between added endoglucanase
activityandimprovementininvitroNDFdegradability
from alfalfa hay and corn silage (Eun et al., 2007). In
addition to these key enzymes, Colombatto et al.
(2003a) indicated that proteases improved the in vitro
degradation of alfalfa hay.
The objective of this study was to evaluate the poten-
tialofvariousFEproductsdifferinginenzymaticactivi-
ties to improve in vitro degradation of alfalfa hay or

Page 3

EUN AND BEAUCHEMIN
2840
Table 1. Protein concentration and enzymatic activities of the feed enzyme products used in experiments
1 and 21
Protein
concentration,
mg/g
Enzymatic activity3
Feed
enzyme2
Endoglucanase XylanaseExoglucanaseProtease
FF
FS
FT
P
6251,405 ± 69.5
1,099 ± 51.7
1,613 ± 139.5
0
12,990 ± 1,611.7
5,785 ± 174.8
955 ± 108.0
0
38 ± 5.0
40 ± 4.5
79 ± 3.0
9.1 ± 2.48
0.1 ± 0.05
0.2 ± 0.10
0
63 ± 0.9
1,212
875
522
1Data are for the concentrated enzyme solutions.
2Four developmental feed enzyme products (FF, FS, FT, and P) from Dyadic International Inc. (Jupiter,
FL) were used. FF, FS, and FT were polysaccharidases from a strain of Trichoderma longibrachiatum (for
FF and FT) or Penicillum funiculosum (for FS), whereas P was a proteolytic enzyme originating from papaya
(papain; EC 3.4.22.2).
3Substrates were (1% in 0.1 M citrate phosphate buffer, pH 6.0) medium-viscosity carboxymethylcellulose
for endoglucanse, birchwood xylan for xylanase, and Sigmacell 50 for exoglucanase activities. Endoglucanase
and exoglucanase or xylanase activity was expressed as nanomoles of glucose or xylose released per minute
per milligram; protease activity was expressed as milligrams of azocasein hydrolyzed per minute per gram.
corn silage. A range of doses was used for each product
to determine optimum DR. We hypothesized that im-
provementsinfiberdegradabilitywouldbeproportional
to endoglucanase or protease activity (or both) supple-
mented. Feed enzyme products that improved forage
degradation in the first experiment were combined to
determine whether combination products, particularly
those containing proteases, further improved in vitro
fiber degradation.
MATERIALS AND METHODS
Forages and Enzyme Products
Two forage substrates were used in the study. The
alfalfa hay was of moderate quality and had the follow-
ing chemical composition (DM basis): 17.3% CP, 49.9%
NDF, and 35.4% ADF. The corn silage contained (DM
basis): 6.6% CP,43.8% NDF, and 21.7% ADF.The same
batches of alfalfa hay and corn silage were used
throughout the study. Fresh alfalfa hay and freeze-
dried corn silage were milled to pass a 1-mm screen
using a Wiley mill (standard model 4; Arthur H.
Thomas Co., Philadelphia, PA) and stored for use in
the in vitro incubations.
Four developmental FE products (FF, FS, FT, and
P) from Dyadic International Inc. (Jupiter, FL) were
used (Table 1). Enzyme products FF, FS, and FT were
polysaccharidases from a strain of Trichoderma longi-
brachiatum (for FF and FT) or Penicillum funiculosum
(for FS), whereas P was a proteolytic enzyme originat-
ing from papaya (papain; EC 3.4.22.2). These source
organisms are acceptable for use in animal feeds in
North America (AAFCO, 2002; CFIA, 2005). All of the
FE products were in powder form.
Journal of Dairy Science Vol. 90 No. 6, 2007
Experiment 1: Identifying Potential Candidate
FE and Their Optimum DR for Alfalfa Hay
and Corn Silage
Experiment 1 was undertaken to identify the promis-
ing enzyme candidates and their optimum DR. The in
vitro procedures used in this series of incubations were
the same as those described by Eun et al. (2006) for
screening exogenous enzymes for their effectiveness in
increasing forage degradability. The incubations were
performed in separate runs for alfalfa hay and corn
silage. Each run consisted of a 24-h in vitro batch cul-
ture fermentation with treatments applied in a 4 (FE)
× 5 (DR) factorial design.
For the incubations, approximately 0.7 g (DM) of
ground alfalfa hay or corn silage was weighed into ace-
tone-washed and preweighed filter bags (F57; Ankom
Technology, Macedon, NY). Exactly 0.5 g of each en-
zyme powder was solubilized using 25 mL of water, and
8.75, 17.5, 26.25, or 35.0 ?L of the diluted enzyme was
added to the forage in the bags to achieve a DR of 0.25,
0.5, 0.75, or 1.0 mg of concentrated enzyme product per
g of forage DM. In addition, substrate without enzyme
was included (DR of 0) as a control. The bags were heat-
sealed and placed in gas-tight culture vials (125-mL
capacity,WheatonScienceProducts,Millville,NJ)with
4 replications. Because the enzymes were applied to
the feed within the bag using a pipette, uniform distri-
bution of the enzymes on the feed may not have been
achieved. Thus, 3 h after adding the enzymes, 36 mL
of anaerobic buffer medium, prepared as outlined by
Hall et al. (1998) with pH 6.0, was added to each vial.
The vials were gently shaken to disperse the enzymes.
The vials were then stored at 20°C for 17 h. The incuba-
tion with buffer, but without ruminal fluid, was used

Page 4

IMPROVING FIBER DEGRADATION USING ENZYMES
2841
to ensure adequate interaction time between the forage
substrate and the exogenous enzymes.
Ruminal fluid was collected 4 h after the morning
feeding (1100h) from2 ruminallycannulated, lactating
Holstein cows fed a TMR composed of barley silage
(46.6%), chopped alfalfa hay (4.5%), rolled corn grain
(6.8%), and concentrate (42.1%) on a DM basis. The
diet consumed was formulated to meet the nutrient
requirements of a dairy cow in early lactation. To pre-
pare the ruminal fluid, ruminal contents were obtained
from various locations within the rumen, composited,
and strained through polyester material (PeCAP, pore
size 355 ?m; B. & S. H. Thompson, Ville Mont-Royal,
Quebec, Canada) under a stream of oxygen-free CO2.
The strained ruminal fluid (pH of 6.2 and 6.0 in alfalfa
hay and corn silage incubations, respectively) was im-
mediatelytransferredtothelaboratoryinasealedflask
and kept at 39°C in a water bath. The inoculum was
dispensed (7.0 mL per vial) into the culture vials, which
had been warmed to 39°C in an incubator and flushed
with oxygen-free CO2. Each vial was sealed with a 14-
mm butyl rubber stopper plus aluminum crimp cap
immediately after loading and the vials were then
storedat39°C inanincubator.Negative controls(rumi-
nal fluid plus buffer alone or ruminal fluid plus buffer
and enzyme product without substrate) were also incu-
bated using 4 replications. These controls were used
to correct for gas release and fermentation residues
resulting directly from the inoculum or the enzyme
product itself. The incubation was terminated at 24 h.
Then, the vials were placed in a refrigerator at 4°C for
2 h to stop fermentation. Headspace gas production
(GP)producedduringsubstratefermentationwasmea-
sured at 2, 6, 12, 18, and 24 h of incubation using the
procedure reported by Mauricio et al. (1999). The vials
were handled in the same order during the entire pro-
cess to ensure that the time interval (6 h) between
procedures was the same for each vial.
At the end of the incubation, the bags were removed
from the vials and washed under cold tap water until
excess water ran clear. The bags were dried at 55°C for
24 h, and degradability of DM was determined by the
loss of DM. The contents of the bags were retained for
subsequent analysis of fiber content. Profiles of VFA
weremeasuredusing5mLofthefermentationcontents
added to 1 mL of 25% meta-phosphoric acid. The fer-
mentation samples were stored frozen at −40°C until
analyzed.
Experiment 2: Effects of Combining Enzyme
Treatments on In Vitro Fermentation
of Alfalfa Hay and Corn Silage
The aim of experiment 2 was to confirm the efficacy
ofselectedenzymesfromexperiment1andtodetermine
Journal of Dairy Science Vol. 90 No. 6, 2007
whether combining these treatments further improved
their effectiveness. A completely randomized design
was conducted in a single run. From experiment 1 with
alfalfa hay, FF, FT, and P at 0.25, 0.75, and 0.25 mg/
g of DM, respectively, were selected as single enzyme
treatments. Two combination treatments were pro-
duced by combining each of the polysaccharidase treat-
ments with the proteolytic enzyme treatment (i.e., FF
+ P and FT + P). A third combination treatment was
producedby combiningthe2polysaccharidases (i.e.,FF
+FT).EnzymeproductFSwasnotusedinexperiment2
with alfalfa hay due to its lack of effect when used with
alfalfa hay in experiment 1. From experiment 1 with
corn silage, FF, FS, FT, and P at 0.25, 0.75, 0.75, and
0.5 mg/g of DM, respectively, were chosen as single
enzyme treatments. Three combination treatments
were produced by combining each polysaccharidase
with the protease (i.e., FF + P, FS + P, and FT + P). A
fourth combination of FF and FT (FF + FT) was made,
similar to the combination used for alfalfa hay. A sum-
mary of the enzyme treatments and DR used in experi-
ment 2 is given in Table 2.
Theincubationswereconductedusingalfalfahayand
corn silage as previously described for experiment 1.
For combination treatments, component enzyme treat-
ments were added separately to the forage substrates
at the respective DR chosen. The strained ruminal fluid
used in experiment 2 had a pH of 6.3.
Chemical Analyses
The amount of protein present in the enzyme prod-
ucts was determined using the Bio-Rad DC protein de-
termination kit (Bio-Rad Laboratories, Hercules, CA),
with BSA as the standard according to the procedure
described by Colombatto et al. (2003a). The enzyme
products were analyzed for their endoglucanase (EC
3.2.1.4), exoglucanase (EC 3.2.1.91), and xylanase (EC
3.2.1.8) activities according to the procedures reported
by Nelson (1944), Somogyi (1952), and Bailey et al.
(1992) using medium-viscosity carboxymethylcellulose,
Sigmacell 50, and birchwood xylan (1% in 0.1 M citrate
phosphate buffer, pH 6.0), respectively, as substrates
(all obtained from Sigma Chemicals, St. Louis, MO).
Birchwood rather than oat spelt xylan was used as the
substrate for determining xylanase activity because of
its low turbidity at 1% concentration and its extended
range of linearity during the reaction (Bailey et al.,
1992). The assay conditions were 39°C and pH 6.0 to
reflect ruminal conditions. Suitably diluted enzyme (50
?L) and substrate solutions (450 ?L) were incubated
with the substrates for 5 min, and endoglucanase and
exoglucanase or xylanase activity was expressed as na-
nomoles of glucose or xylose released per minute per

Page 5

EUN AND BEAUCHEMIN
2842
Table 2. Enzyme treatments and dose rates used in experiment 2
Enzyme
treatment1
SubstrateDose rate
Alfalfa hayFF
FT
FF + FT
P
FF + P
FT + P
FF
FT
FF + FT
FS
P
FF + P
FT + P
FS + P
FF at 0.25 mg/g of DM
FT at 0.75 mg/g of DM
FF at 0.25 mg/g of DM + FT at 0.75 mg/g of DM
P at 0.25 mg/g of DM
FF at 0.25 mg/g of DM + P at 0.25 mg/g of DM
FT at 0.75 mg/g of DM + P at 0.25 mg/g of DM
FF at 0.25 mg/g of DM
FT at 0.75 mg/g of DM
FF at 0.25 mg/g of DM + FT at 0.75 mg/g of DM
FS at 0.75 mg/g of DM
P at 0.5 mg/g of DM
FF at 0.25 mg/g of DM + P at 0.5 mg/g of DM
FT at 0.75 mg/g of DM + P at 0.5 mg/g of DM
FS at 0.75 mg/g of DM + P at 0.5 mg/g of DM
Corn silage
1Four developmental feed enzyme products (FF, FS, FT, and P) from Dyadic International Inc. (Jupiter,
FL) were used. FF, FS, and FT were polysaccharidases from a strain of Trichoderma longibrachiatum (for
FF and FT) or Penicillum funiculosum (for FS), whereas P was a proteolytic enzyme originating from papaya
(papain; EC 3.4.22.2).
milligram, respectively. Protease activity was assayed
using azocasein (lot 25H7125, Sigma Chemical) in 0.1
M citrate phosphate buffer (pH 6.8) as a substrate in
a similar manner as used by Brock et al. (1982) and
Eun and Beauchemin (2005a). Protease activity was
expressed as milligrams of azocasein hydrolyzed per
minute per gram.
The alfalfa hay and corn silage were analyzed for DM
(method 930.15) and N (method 990.03) according to
AOAC (1995). The NDF and ADF, both inclusive of
residual ash, were determined according to Hall et al.
(1998) with the method modified for use with an An-
kom200Fiber Analyzer (Ankom Technology). Heat sta-
ble α-amylase and sodium sulfite were used in the
NDF analysis.
The VFA were quantified using a gas chromatograph
(model 5890, Hewlett-Packard Lab, Palo Alto, CA) with
a capillary column (30 m × 0.32 mm i.d., 1 ?m phase
thickness, Zebron ZB-FAAP, Phenomenex, Torrance,
CA), and flame-ionization detection. The oven tempera-
ture was 170°C held for 4 min, which was then in-
creased by 5°C/min to 185°C, and then by 3°C/min to
220°C, and held at this temperature for 1 min. The
injector temperature was 225°C, the detector tempera-
ture was 250°C, and the carrier gas was helium.
Statistical Analyses
All the statistical analyses were conducted using the
MIXED procedures (SAS Institute, 2001). Data from
experiment 1 were analyzed separately by forage sub-
strate as a completely randomized design with FE, DR,
and the FE × DR interaction included in the model
as fixed effects. Orthogonal polynomial contrasts were
performed to determine linear and quadratic effects
Journal of Dairy Science Vol. 90 No. 6, 2007
of DR. Cubic and quartic effects were not examined,
because they could not be interpreted biologically. The
relationship between added endoglucanase, exoglu-
canse, or xylanase activities and improvement of NDF
degradability was determined among polysaccharidase
FEbylinearregressionusingthePROCREGprocedure
of SAS. Data for experiment 2 were also analyzed as a
completely randomized design according to substrate
with treatment as a fixed effect in the model. Differ-
ences between control (no added enzyme) and enzyme
treatments were detected using the Dunnett adjust-
mentoption.Toevaluatethebenefitsofthecombination
treatments in experiment 2, the actual response was
compared with the individual responses obtained for
the control and each of the single component enzymes,
as well as to the overall calculated response for the
combination. The calculated response was determined
by summing the response for the control and the incre-
mental response to each of the component enzymes.
These treatment means were compared using a pro-
tected (P < 0.05) LSD test from a model that included
treatmentasafixedeffect.Theresponsetothecombina-
tion treatment was said to be additive when the ob-
served response was similar to the calculated response,
and synergistic when the observed response exceeded
the calculated response. Least squares means are re-
ported throughout, and significance was declared at P
< 0.05.
RESULTS AND DISCUSSION
The DR used in this study were half of those used in
a previous study that examined formulation of rumi-
nant FE (Eun et al., 2007). The lower DR range was
used because these FE had higher enzymatic activities

Page 6

IMPROVING FIBER DEGRADATION USING ENZYMES
2843
Table3.Influenceoffeedenzymes(FE)onthecumulativegasproduction(mL/gofOM)andthedegradability
(%) of DM and NDF from alfalfa hay during in vitro fermentation (experiment 1, n = 4)
Dose rate3
Significance of effect5
Item1
FE2
Mean0 0.250.50.75 1.0 SE4
FEDRFE × DR
GPFF
FS
FT
P
SE6
FF
FS
FT
P
SE6
FF
FS
FT
P
SE6
102b
93c
100b
119a
100ef
100e
100ef
100g
105e
88f
93f
118ef
103e
93ef
95f
119ef
96f
99e
107e
126e
105e
93ef
107e
114f
2.9
P < 0.01Q (P = 0.03)
Q (P < 0.01)
Q (P = 0.02)
Q (P = 0.05)
P < 0.01
1.7
44.9b
41.6d
43.7bc
46.3a
0.51
22.0a
19.1c
20.7b
21.8a
0.44
DMD
43.0f
43.0e
43.0f
43.0f
46.0e
41.2ef
41.5f
47.6e
46.2e
40.5f
41.2f
46.6e
43.2f
43.2e
46.0e
46.4e
44.3ef
41.5ef
46.2e
44.7f
0.69
P < 0.01 Q (P < 0.01)
Q (P < 0.01)
Q (P < 0.01)
Q (P = 0.05)
P < 0.01
NDFD
19.7fg
19.7ef
19.7f
19.7g
22.5e
18.2f
19.2fg
23.5e
22.7e
18.5f
18.0f
22.1ef
20.8ef
20.3e
22.3e
21.6f
22.0e
19.5ef
23.2e
20.1g
0.58
P < 0.01 Q (P < 0.01)
Q (P < 0.01)
Q (P = 0.02)
Q (P = 0.05)
P < 0.01
a–dMeans within a column for FE that do not have a common superscript differ at P < 0.05.
e–gMeans within a row for dose rates of 0 to 1.0 mg/g of DM that do not have a common superscript differ
at P < 0.05.
1All items were measured at 24 h of incubation. GP = gas production; DMD = DM degradability; NDFD =
NDF degradability.
2Four developmental FE products (FF, FS, FT, and P) from Dyadic International Inc. (Jupiter, FL) were
used. FF, FS, and FT were polysaccharidases from a strain of Trichoderma longibrachiatum (for FF and
FT)orPenicillumfuniculosum(forFS),whereasPwasaproteolyticenzymeoriginatingfrompapaya(papain;
EC 3.4.22.2).
3Dose rate as mg/g of DM forage substrate; Mean = mean for individual FE across dose rates except dose
rate of 0; 0 = control without added FE.
4SE for FE × DR.
5DR = dose rate; Q = quadratic effect of DR; FE × DR = interaction between FE and DR.
6SE for pooled mean of FE excluding the dose rate of 0.
per unit of product than those used previously (Table
1). Enzymes FF, FS, and FT had high endoglucanase
and xylanase activities, moderate exoglucanase activ-
ity, and negligible protease activity. Enzyme P con-
tained mainly protease activity with no endoglucanase
or xylanase and little exoglucanse activity.
Experiment 1
Foralfalfahay,theeffectofeachofthe4FEdepended
upon the DR used, as evidenced by significant FE × DR
interactions (Table 3). A quadratic response to DR was
observed for all FE, but the optimum DR varied among
FE. Some FE affected both GP and degradability of DM
andfiber,whereasothersonlyaffectedfiberdegradabil-
ity. In this study, optimum DR was considered to be the
minimumdoserequiredtoelicitthegreatestsignificant
increase in degradability of fiber (NDF or ADF) com-
pared withthe control.Fiber degradability,rather than
GP and DM degradability, was selected to ensure that
improvements due to FE supplementation lead to an
increase in fiber utilization.
None of the polysaccharidases (FF, FS, or FT) in-
creased GP from alfalfa hay compared with the control,
Journal of Dairy Science Vol. 90 No. 6, 2007
whereas the lowest DR of P(0.25 mg/g of DM) increased
GP by 18% compared with control. The optimum DR of
FE for improving degradability of NDF was 0.25 mg/g
of DM for FF and P, and 0.75 mg/g of DM for FT.
Relative improvements in the degradability of NDF
were 14% for FF, 19% for P, and 13% for FT. Enzyme
FS did not increase GP or degradability at any DR.
For corn silage, none of FE evaluated affected GP
or DM degradation regardless of DR used (Table 4).
However, the FE produced variable effects on NDF de-
gradabilityacrossDR,indicatedbytheFE×DRinterac-
tion. Optimum DR for increasing NDF degradability
was 0.25 mg/g of DM forFF, 0.75 mg/g of DM for FS and
FT, and 0.5mg/g of DM for P.Relative improvements in
NDF degradability at these DR were 14, 26, 54, and
17% for FF, FS, FT, and P, respectively.
Summarizing the data for all polysaccharidase FE
and DR, improvement in NDF degradability of alfalfa
hay was positively correlated with added endogluca-
nase activity (r = 0.51, P = 0.09; Figure 1), but not with
added xylanase activity (P = 0.37). A similar relation-
ship was observed for corn silage; improvement in NDF
degradability was positively correlated with endogluca-
nase activity (r = 0.66, P = 0.02), but not with xylanase

Page 7

EUN AND BEAUCHEMIN
2844
Table4.Influenceoffeedenzymes(FE)onthecumulativegasproduction(mL/gofOM)andthedegradability
(%) of DM and NDF from corn silage during in vitro fermentation (experiment 1, n = 4)
Dose rate3
Significance of effect5
Item1
FE2
Mean0 0.250.50.751.0SE4
FEDRFE × DR
GPFF
FS
FT
P
SE6
FF
FS
FT
P
SE6
FF
FS
FT
P
SE6
114
120
116
117
112
112
112
112
119
118
114
115
112
119
115
121
113
117
116
115
113
126
117
117
3.4NSNS NS
1.8
42.2b
43.4ab
44.6a
42.2b
0.48
13.3b
13.9b
15.7a
11.6c
0.52
DMD
42.8
42.8
42.8
42.8
42.8
42.3
43.8
41.3
42.2
42.1
44.4
44.0
41.1
44.1
45.2
42.4
42.9
45.0
44.9
41.2
0.85
P < 0.01NS NS
NDFD
11.5f
11.5f
11.5g
11.5f
13.1e
13.6f
12.8g
10.8gf
13.8e
12.4f
14.5f
13.5e
11.8f
14.5e
17.7e
12.6ef
14.5e
15.3e
17.4e
9.4g
0.73
P < 0.01L (P < 0.01)
Q (P < 0.01)
L (P = 0.04)
Q (P = 0.05)
P < 0.01
a–cMeans within a column for FE that do not have a common superscript differ at P < 0.05.
e–gMeans within a row for dose rates of 0 to 1.0 mg/g of DM that do not have a common superscript differ
at P < 0.05.
1All items were measured at 24 h of incubation. GP = gas production; DMD = DM degradability; NDFD =
NDF degradability.
2Four developmental FE products (FF, FS, FT, and P) from Dyadic International Inc. (Jupiter, FL) were
used. FF, FS, and FT were polysaccharidases from a strain of Trichoderma longibrachiatum (for FF and
FT)orPenicillumfuniculosum(forFS),whereasPwasaproteolyticenzymeoriginatingfrompapaya(papain;
EC 3.4.22.2).
3Dose rate as mg/g of DM forage substrate; Mean = mean for individual FE across dose rates except dose
rate of 0; 0 = control without added FE.
4SE for FE × DR.
5DR = dose rate; NS = nonsignificant (P > 0.05); L = linear effect of DR; Q = quadratic effect of DR; FE
× DR = interaction between FE and DR.
6SE for pooled mean of FE excluding the dose rate of 0.
activity (P = 0.36). In addition, added exoglucanase ac-
tivity was positively associated with improvement in
NDF degradability for corn silage (r = 0.85, P < 0.01;
Figure 1), but not alfalfa hay. Combining endogluca-
nase and exoglucanase in the model explained 87% of
the variation (P < 0.01) in the improvement in NDF
degradability of corn silage due to the addition of the
enzymatic activities.
Cellulose is hydrolyzed through a complex process
involving cellulases. In general, endoglucanases hy-
drolyze cellulose chains at random to produce cellulose
oligomers of varying degree of polymerization, whereas
exoglucanases hydrolyze the cellulose chain from the
nonreducingend,producingcellobiose(BhatandHazle-
wood, 2001). Thus, it is not surprising that endogluca-
nasewasshowninthisstudytobelinkedtoNDFdegra-
dability of both forages. The incremental effect of exog-
lucanase on NDF degradability of corn silage indicates
that the ideal enzyme formulations for these 2 forages
differ as a result of their differences in chemical compo-
sition. One of the main characteristics of exoglucanases
is that they act on cellulose chains in a progressive
manner. They progress along the polymer chain while
Journal of Dairy Science Vol. 90 No. 6, 2007
releasing cellobiose in a recurrent fashion (Tomme et
al., 1996; Reverbel-Leroy et al., 1997), resulting in thin-
ning of crystalline cellulose (Boisset et al., 2000). The
versatility of exoglucanases may be important for the
degradation of more recalcitrant fiber, such as the corn
silage used in this study.
Therelationshipbetweenaddedendoglucanaseactiv-
ity and improvement in NDF degradability from alfalfa
hay and corn silage observed in this study supports the
findings of previous studies with other FE products.
Wallace et al. (2001) suggested that endoglucanase ac-
tivity was rate limiting in the fermentation of corn si-
lage. Similarly, we previously reported a strong rela-
tionship (r = 0.77, P < 0.001) between added endogluca-
nase activity and improvement in NDF degradability
from alfalfa hay and corn silage (Eun et al., 2007). In
that study, the added activity of endoglucanase aver-
aged 224 ± 144.3 (n = 15) nmol of glucose released/min,
whereas in the present study the activity was 858 ±
428.1 (n = 12) nmol of glucose released/min. The much
higher endoglucanase levels used in this study may
havecontributedtotheweakerrelationshipbetweenits
activity level and improvement in NDF degradability.

Page 8

IMPROVING FIBER DEGRADATION USING ENZYMES
2845
Figure 1. The relationship between added endoglucanase or exoglucanase activity (nmol of glucose released/min) and improvement in
NDF degradability (NDFD) for alfalfa hay and corn silage in experiment 1 due to the addition of exogenous feed enzymes (n = 12): FF (▲),
FS (?), and FT (?). Improvement in NDFD (%) is calculated as [(Ye − Ycont)/Ycont] × 100, where Ye is the observed NDFD with enzyme
addition and Ycont is the mean NDFD for control incubations. The root mean square error (RMSE) for the improvement of NDFD with
added endoglucanase activity for alfalfa hay is 8.71. The RMSE for the improvement in NDFD with added endoglucanase and exoglucanase
activities for corn silage are 12.49 and 8.80, respectively.
Journal of Dairy Science Vol. 90 No. 6, 2007

Page 9

EUN AND BEAUCHEMIN
2846
Table 5. Influence of adding single or combination feed enzymes (FE) on cumulative gas production (GP)
and 24-h degradability of alfalfa hay measured in vitro (experiment 2, n = 4)
GP (mL/g of OM)Degradability (%)
Dose rate
(mg/g of DM)
Treatment1
12 h 24 hDMNDFADF
Control
FF
FT
FF + FT
P
FF + P
FT + P
SE
0
0.25
0.75
0.25 + 0.75
0.25
0.25 + 0.25
0.75 + 0.25
58.8
61.0
64.3a
62.7b
59.4
60.2
60.0
1.01
99
102
107
109b
103
114b
108
45.5
46.9
48.4b
50.0a
46.0
47.9b
49.0a
0.69
22.5
24.4
27.7a
28.8a
25.0b
25.7b
27.5a
0.62
13.6
13.9
19.6a
20.7a
15.3
15.6
18.5a
0.86 3.4
a,bDifferent from the control within columns at P < 0.01 and P < 0.05, respectively.
1Developmental FE products (FF, FT, and P) from Dyadic International Inc. (Jupiter, FL) were used. FF
and FT were polysaccharidases from a strain of Trichoderma longibrachiatum, whereas P was a proteolytic
enzyme originating from papaya (papain; EC 3.4.22.2). Control was alfalfa hay without added enzymes.
The linear relationship between individual enzy-
maticactivities(i.e., endoglucanaseorexoglucanaseac-
tivities) and NDF degradability presented in Figure 1
differs from the quadratic responses in NDF degrada-
bility observed with increasing DR of FE presented in
Tables 3 and 4. This apparent discrepancy is likely
the result of the interrelationships among the various
enzymatic activities within FE, which cause quadratic
rather than linear responses.
The substantial increase in NDF degradability with
the use of a proteolytic FE supports previous studies
thatusedaproteolyticFEderivedfromBacilluslicheni-
formis (Protex 6L, Genencor International, Rochester,
NY)thatdidnotcontaincellulolyticorxylanolyticactiv-
ities (Colombatto et al., 2003a,b). These studies re-
ported large increases in DM and NDF degradability
of alfalfa hay and TMR with use of a proteolytic FE.
When that same product was fed to dairy cows, total
tract digestibilities of DM, OM, N, NDF, and ADF were
Table 6. Influence of adding single or combination feed enzymes (FE) on cumulative gas production (GP)
and 24-h degradability of corn silage measured in vitro (experiment 2, n = 4)
GP (mL/g of OM)Degradability (%)
Dose rate
(mg/g of DM)
Treatment1
12 h24 h DM NDFADF
Control
FF
FT
FF + FT
FS
P
FF + P
FT + P
FS + P
SE
0
0.25
0.75
0.25 + 0.75
0.75
0.5
0.25 + 0.5
0.75 + 0.5
0.75 + 0.5
54.4
55.5
53.1
59.8b
53.6
52.6
54.8
53.3
54.2
1.42
99
104
106
111
102
109
100
98
100
54.2
52.8
55.8
57.8b
56.0
54.4
52.8
57.7b
56.4
0.93
19.8
19.4
22.3b
26.0a
20.9
22.8b
18.8
24.4a
19.9
0.77
13.9
11.5
16.5
20.4a
14.5
13.3
14.1
17.7b
14.1
1.155.3
a,bDifferent from the control within columns at P < 0.01 and P < 0.05, respectively.
1Four developmental FE products (FF, FS, FT, and P) from Dyadic International Inc. (Jupiter, FL) were
used. FF, FS, and FT were polysaccharidases from a strain of Trichoderma longibrachiatum (for FF and
FT)orPenicillumfuniculosum(forFS),whereasPwasaproteolyticenzymeoriginatingfrompapaya(papain;
EC 3.4.22.2). Control was corn silage without added enzymes.
Journal of Dairy Science Vol. 90 No. 6, 2007
increased (Eun et al., 2005a). It is unclear how prote-
ases positively affect forage degradation; however, Co-
lombatto et al. (2003a) speculated that proteolytic en-
zymes remove some of the cell wall components that
are physical barriers to degradation.
To our knowledge, ours is the first study to examine
theeffectsofpapainonforagefiberdegradation.Papain
is a proteolytic enzyme produced by the tropical fruit
papaya and is used widely as a meat tenderizer. In
ruminant nutrition research, papain has been used to
predict ruminal degradation of feed protein (Toma ´n-
kova ´ and Kopec ˇny ´, 1995; Mirza and Miller, 2005). Pro-
teolytic activity from papain in this study was appar-
ently greater than that from Bacillus used previously
(63 vs. 36 mg of azocasein/min per g; Eun et al., 2005a,
2006).LowlevelsofproteolyticenzymesfromB.licheni-
formis increased DM degradation of alfalfa hay (Co-
lombatto et al., 2003a), but not corn silage (Colombatto
et al., 2003a), alfalfa haylage (Eun and Beauchemin,

Page 10

IMPROVING FIBER DEGRADATION USING ENZYMES
2847
Figure 2. Comparison of single vs. combination enzyme treatments for the degradability of DM, NDF, and ADF of alfalfa hay after 24
h of in vitro fermentation in experiment 2 (n = 4 for each mean). A) Effect of the combination of FF and FT. Calculated values for FF + FT
are the sum of control and increments due to FF and FT enzyme treatments. The SE for DM, NDF, and ADF degradability are 0.70, 0.52,
and 0.88, respectively. B) Effect of the combination of FF and P. Calculated values for FF + P are the sum of control and increments due
to FF and P enzyme treatments. The SE for DM, NDF, and ADF degradability are 0.65, 0.67, and 0.87, respectively. C) Effect of the
combination of FT and P. Calculated values for FT + P are the sum of control and increments due to FT and P enzyme treatments. The
SE for DM, NDF, and ADF degradability are 0.69, 0.61, and 1.06, respectively. Bars within each fraction having a different letter differ (P
< 0.05). Control = alfalfa hay without enzyme treatment.
2005b), or barley silage (McGinn et al., 2004; Eun and
Beauchemin, 2005a). In the present study, papain im-
provedbothalfalfahayandcornsilageNDFdegradabil-
Journal of Dairy Science Vol. 90 No. 6, 2007
ity, indicating that papain may have a wide range of
forage specificity or that higher supplementation rates
areneededforsomeforages.Althoughpapainwaseffec-

Page 11

EUN AND BEAUCHEMIN
2848
Figure 3. Comparison of single vs. combination enzyme treatments on the degradability of DM, NDF, and ADF of corn silage after 24
h of in vitro fermentation in experiment 2 (n = 4 for each mean). A) Effect of the combination of FF and FT. Calculated values for FF + FT
are the sum of control and increments due to FF and FT enzyme treatments. The SE for DM, NDF, and ADF degradability are 1.08, 0.77,
and 0.98, respectively. B) Effect of the combination of FT and P. Calculated values for FT + P are the sum of control and increments due
to FT and P enzyme treatments. The SE for DM, NDF, and ADF degradability are 1.43, 0.81, and 1.23, respectively. Bars within each
fraction having a different letter differ (P < 0.05). Control = corn silage without enzyme treatment.
tive for both alfalfa hay and corn silage, its optimum
DR differed for each forage: 0.25 and 0.5 mg/g of DM,
respectively.AttheseDR,papainimprovedNDFdegra-
dability to a similar extent for each forage.
Experiment 2
For alfalfa hay, the single enzyme treatment FT in-
creased GP at 12 h and FT and P improved degradabil-
ity of NDF, whereas unlike in experiment 1, there was
no effect of FF (Table 5). Relative to the control, all
combination treatments increased GP or DM degrada-
bility, with substantial improvements in NDF and ADF
degradability: 28 and 52% for FF + FT, 14 and 15% for
FF + P, and 22 and 36% for FT + P, respectively. These
improvements generally reflected the additivity of the
responses to the component enzymes (Figure 2A and
Journal of Dairy Science Vol. 90 No. 6, 2007
B), with the exception of FT + P, for which responses
were similar to responses obtained using FT alone (Fig-
ure 2C). Thus, in most cases, P and FF were both im-
proved by combining them with FT, but FT was not
improved by combining it with other enzymes (Figure
2A). The difference in the response may be due to the
higher exoglucanase activity of FT compared with the
other products. In our previous study with alfalfa hay
(Eun and Beauchemin, 2007), we observed that combi-
nation treatments did not increase degradation of al-
falfa hay beyond that of the component enzymes when
endoglucanase and xylanase from single-activity en-
zyme products were combined in a 1:1 ratio. From this
finding, we suggested that there might be an ideal ratio
between the major enzymatic activities to achieve fur-
ther improvement of degradation with combination
treatments. However, the information from the present

Page 12

IMPROVING FIBER DEGRADATION USING ENZYMES
2849
Table 7. Influence of adding single or combination feed enzymes (FE) on the VFA profiles after 24 h of in
vitro fermentation with alfalfa hay (experiment 2, n = 4)
Individual VFA (mol/100 mol)
Dose rate
(mg/g of DM)
Total VFA
(mM)
Treatment1
Acetate (A)Propionate (P)Butyrate A:P
Control
FF
FT
FF + FT
P
FF + P
FT + P
SE
0
0.25
0.75
0.25 + 0.75
0.25
0.25 + 0.25
0.75 + 0.25
71.4
73.3
77.6
83.7a
77.6
76.0
81.2a
3.34
57.1
56.3
57.1
57.6
57.5
56.8
56.9
0.61
22.4
22.7
22.9
23.0a
22.7
22.9
23.1a
0.17
8.60
8.92
8.79
8.69
8.37
8.65
8.70
0.202
2.55
2.48
2.50
2.50
2.54
2.48
2.47
0.042
aDifferent from the control within columns at P < 0.05.
1Developmental FE products (FF, FT, and P) from Dyadic International Inc. (Jupiter, FL) were used. FF
and FT were polysaccharidases from a strain of Trichoderma longibrachiatum, whereas P was a proteolytic
enzyme originating from papaya (papain; EC 3.4.22.2). Control was alfalfa hay without added enzymes.
study suggests that in addition to high endoglucanse
and low xylanase activities, high exoglucanse activity
(asinthecaseofFT)maybebeneficialinanFEformula-
tion for alfalfa forage.
For corn silage, most FE failed to increase GP but
the single enzyme treatments (FT and P) increased
NDF degradability (Table 6). Only the combination
treatmentsthatcontainedFTincreaseddegradabilities
of fiber, with improvements in NDF and ADF of 31 and
47% for FF + FT, and 23 and 27% for FT + P, respec-
tively. In the case of FF + FT, the substantial increase
in fiber degradability exceeded that obtained by the
component enzymes (Figure 3A). Therefore, for this
particular combination of FF + FT, the response was
synergistic. The FF + FT combination was also the only
treatment that increased GP throughout fermentation.
In contrast to the results with alfalfa hay for which
adding P to FT did not improve the response, the effects
of FT + P were additive for corn silage (Figure 3B).
Table 8. Influence of adding single or combination feed enzymes (FE) on the VFA profiles after 24 h of in
vitro fermentation with corn silage (experiment 2, n = 4)
Individual VFA (mol/100 mol)
Dose rate
(mg/g of DM)
Total VFA
(mM)
Treatment1
Acetate (A)Propionate (P)ButyrateA:P
Control
FF
FT
FF + FT
FS
P
FF + P
FT + P
FS + P
SE
0
0.25
0.75
0.25 + 0.75
0.75
0.5
0.25 + 0.5
0.75 + 0.5
0.75 + 0.5
84.0
87.7
76.4
74.9
75.4
76.7
81.7
77.0
79.5
4.49
45.3
43.3
42.2b
42.2b
41.5b
41.6b
42.8
43.6
42.2b
0.83
29.9
31.4a
31.2a
31.3a
31.5a
31.0b
31.4a
30.8b
31.5a
0.26
13.4
13.6
14.3
14.4
14.4
14.6
13.9
13.7
14.1
0.45
1.52
1.38b
1.35b
1.35b
1.32a
1.34a
1.37b
1.42
1.34a
0.035
a,bDifferent from the control within columns at P < 0.01 and P < 0.05, respectively.
1Four developmental FE products (FF, FS, FT, and P) from Dyadic International Inc. (Jupiter, FL) were
used. FF, FS, and FT were polysaccharidases from a strain of Trichoderma longibrachiatum (for FF and
FT)orPenicillumfuniculosum(forFS),whereasPwasaproteolyticenzymeoriginatingfrompapaya(papain;
EC 3.4.22.2). Control was corn silage without added enzymes.
Journal of Dairy Science Vol. 90 No. 6, 2007
The additivity of FF + FT for alfalfa hay can be ex-
plained based on the results of experiment 1, in which
improvementsinNFDdegradationwerecorrelatedpos-
itively to added endoglucanase activity. However, of
particular interest is the synergy between these FE for
corn silage fiber degradation. This synergy could have
been due to the specific cellulases and xylanases within
these 2 products or other secondary enzymes that were
not measured, such as esterases. Although the primary
enzymes involved in xylan degradation are xylanases,
the side-chain components of xylans are removed by
severalenzymesthatincludeacetylesterase,arabinosi-
dase, and glucuronidase (Hespell and Whitehead,
1990). When arabinosidase or xylanase were incubated
individually with alfalfa cell walls, only small amounts
of sugars were released; however, sugar release in-
creased 5- to 10-fold when the enzymes were used to-
gether (Hespell and Whitehead, 1990). Therefore, enzy-
matic synergism for the degradation of the side-chain

Page 13

EUN AND BEAUCHEMIN
2850
components of xylans may account for the increased
fiber degradation observed when FF + FT was added at
an optimum DR. Further work on enzyme formulation
needs to address the possible role of the secondary
enzymes.
Colombatto et al. (2003a) reported that the increase
inDMdegradabilityofalfalfahayusingFEwasdirectly
related to xylanase and protease activities. Therefore,
we expected additive effects on fiber degradation of al-
falfa hay by combining fibrolytic and proteolytic en-
zymes. Grabber et al. (2002) reported that the degrada-
tion of xylans from alfalfa cell walls was severely re-
strictedwhencompared
polysaccharides, probably as a result of cross-linkages
involving lignin. Therefore, xylanase alone might be
ineffectiveifnotaccompaniedbyotherenzymescapable
of cleaving the cross-linkages. Ferulic acid does not ap-
pear to be involved in the interactions between xylans
and lignin in alfalfa (Grabber et al., 2002). Although
the specific mechanisms are not known, it has been
suggested (Jung, 1997) that tyrosine residues could
play a role in the cross-linking of dicotyledonous plants,
which supports the potential role of protease activity
to enhance alfalfa cell wall degradation. However, in
our study, combining polysaccharidase and protease
treatments had neither additive nor synergistic effects
on the degradation of alfalfa hay. In contrast, combina-
tionsofpolysaccharidaseandproteolyticenzymesacted
synergistically in improving corn silage fiber degrada-
bility when an effective polysaccharidase (i.e., FT) was
used (Figure 2B). One possible explanation for the lack
of synergy between the key enzymatic activities for al-
falfa hay fiber degradation may be that a certain combi-
nation of endoglucanase, xylanase, and protease is re-
quired for different forage substrates.
Total VFA production from alfalfa hay was increased
by FF + FT and FT + P, and these combination treat-
ments increased molar proportions of propionate but
did not affect the acetate to propionate ratio (Table 7).
Total VFA production from corn silage was not influ-
encedbyenzymetreatment(Table8).However,enzyme
treatment generally decreased the molar proportion of
acetate and increased the molar proportion of propio-
nate, resulting in decreased acetate to propionate ratio.
The molar proportion of butyrate was not affected by
enzyme treatments.
Changes in VFA proportions corresponded to in-
creased fiber degradation of corn silage. However, the
changes in VFA proportions due to enzyme addition
were relatively small for alfalfa hay. In our previous
experiment (Eun and Beauchemin, 2007), adding exog-
enous fibrolytic enzymes to alfalfa hay resulted in more
propionate and butyrate and less acetate and corres-
ponded to a considerable increase in fiber degradation.
with thatof other
Journal of Dairy Science Vol. 90 No. 6, 2007
The inconsistent effects of enzyme addition on ruminal
fermentation may indicate that changes in VFA compo-
sition depend on the enzyme activities added and the
forage substrates used. The most pronounced changes
in VFA composition in response to enzyme addition
in the current experiment were decreased acetate to
propionate ratio from corn silage fermentation. De-
creased acetate to propionate ratio is another potential
benefit of supplementing diets with FE. Increasing
availability of glucogenic precursors to cows could im-
prove nutrient utilization, particularly for dairy cows
in early lactation when nutrient intake lags behind
nutrient requirement.
CONCLUSIONS
Exogenous enzymes containing endoglucanase, exog-
lucanase, and xylanase or protease activities improved
in vitro degradability of alfalfa hay and corn silage
fiber,and theiroptimumDR varieddepending uponthe
forage.Ingeneral,lowtomediumDRofsomeindividual
polysaccharidase products resulted in substantial in-
creases in NDF degradability (13 to 19% for alfalfa
hay, 14 to 54% for corn silage). The proteolytic enzyme
product papain also improved NDF degradability of
both forages (19 and 17%, respectively). In most cases
combiningpolysaccharidaseswithpolysaccharidasesor
proteolytic products had additive, and sometimes syn-
ergistic, effects on fiber degradation of forages. It is
recommended that the combination treatments FF +
FT and FT + P be further evaluated in a dairy cow
feeding study using diets based on corn silage or al-
falfa forage.
ACKNOWLEDGMENTS
The authors wish to thank Dyadic International Inc.
(Jupiter,FL)forgenerouslysupplyingtheenzymeprod-
ucts used in the study and for financially supporting
the research.
REFERENCES
AAFCO (Association of American Feed Control Officials). 2002. Offi-
cial publication. Page 464. AAFCO, Washington, DC.
AOAC. 1995. Official Methods of Analysis. AOAC, Arlington, VA.
Bailey,M.J.,P.Biely,andK.Poutanen.1992.Interlaboratorytesting
of methods for assay of xylanase activity. J. Biotechnol.
23:257–270.
Bowman, G. R., K. A. Beauchemin, and J. A. Shelford. 2002. The
proportionofthediettowhichfibrolyticenzymesareaddedaffects
nutrient digestion by lactating dairy cows. J. Dairy Sci.
85:3420–3429.
Beauchemin, K. A., D. Colombatto, D. P. Morgavi, and W. Z. Yang.
2003. Use of exogenous fibrolytic enzymes to improve feed utiliza-
tion by ruminants. J. Anim. Sci. 81(E. Suppl. 2):E37–E47.
Beauchemin, K. A., D. Colombatto, D. P. Morgavi, W. Z. Yang, and
L. M. Rode. 2004. Mode of action of exogenous cell wall degrading
enzymes for ruminants. Can. J. Anim. Sci. 84:13–22.

Page 14

IMPROVING FIBER DEGRADATION USING ENZYMES
2851
Bhat, M. K., and G. P. Hazlewood. 2001. Enzymology and other char-
acteristics of cellulases and xylanases. Pages 11–60 in Enzymes
in Farm Animal Nutrition. M. R. Bedford, and G. G. Partridge,
ed. CABI Publishing, Wallingford, UK.
Boisset, C., C. Fraschini, M. S. Lein, B. Henrissat, and H. Chanzy.
2000. Imaging the enzymatic digestion of bacterial cellulose rib-
bons reveals the endo character of the cellobiohydrolase Cel6A
from Humicola insolens and its mode of synergy with cellobiohy-
drolase Cel7A. Appl. Environ. Microbiol. 66:1444–1452.
Brock,F.M.,C.W.Forsberg,andJ.G.Buchanan-Smith.1982.Proteo-
lytic activity of rumen microorganisms and effects of proteinase
inhibitors. Appl. Environ. Microbiol. 44:561–569.
CFIA (Canadian Food Inspection Agency). 2005. Schedule IV, Part
1,ListofApprovedFeedIngredientsforLivestock.CFIA,Ottawa,
Ontario, Canada.
Colombatto,D.,G.Herva ´s,W.Z.Yang,andK.A.Beauchemin.2003b.
Effects of enzyme supplementation of a total mixed ration on
microbial fermentation in continuousculture, maintained at high
and low pH. J. Anim. Sci. 81:2617–2627.
Colombatto, D., D. P. Morgavi, A. F. Furtado, and K. A. Beauchemin.
2003a. Screening of exogenous enzymes for ruminant diet: Rela-
tionshipbetweenbiochemicalcharacteristicsandinvitroruminal
degradation. J. Anim. Sci. 81:2628–2638.
Eun, J.-S., and K. A. Beauchemin. 2005a. Effects of a proteolytic feed
enzyme on intake, digestion, ruminal fermentation, and milk
production. J. Dairy Sci. 88:2140–2153.
Eun, J.-S., and K. A. Beauchemin. 2005b. Exogenous proteolytic en-
zymes improve in vitro degradation of alfalfa hay but not alfalfa
silage. J. Dairy Sci. 88(Suppl. 1):316. (Abstr.)
Eun, J.-S., and K. A. Beauchemin. 2007. Assessment of the efficacy
of varying experimental exogenous fibrolytic enzymes using in
vitro fermentation characteristics. Anim. Feed Sci. Technol.
132:298–315.
Eun, J.-S., K. A. Beauchemin, and H. Schulze. 2006. Use of an in
vitro fermentation bioassay to evaluate improvements in degra-
dation of alfalfa hay due to exogenous feed enzymes. Anim. Feed
Sci. Technol. doi:10.1016/j.anifeedsci.2006.07.003
Eun,J.-S., K.A.Beauchemin,and H.Schulze.2007.Use ofexogenous
fibrolytic enzymes to enhance in vitro fermentation of alfalfa hay
and corn silage. J. Dairy Sci. 90:1440–1451.
Grabber, J. H., M. T. Panciera, and R. D. Hatfield. 2002. Chemical
composition and enzymatic degradability of xylem and nonxylem
walls isolated from alfalfa internodes. J. Agric. Food Chem.
50:2595–2600.
Hall, M. B., A. N. Pell, and L. E. Chase. 1998. Characteristics of
neutral detergent-soluble fiber fermentation by mixed ruminal
microbes. Anim. Feed Sci. Technol. 70:23–39.
Hespell, R. B., and T. R. Whitehead. 1990. Physiology and genetics
of xylan degradation by gastrointestinal tract bacteria. J. Dairy
Sci. 73:3013–3022.
Jung, H. G. 1997. Analysis of forage fiber and cell walls in ruminant
nutrition. J. Nutr. 127:810S–813S.
Knowlton,K.F.,J.M.McKinney,andC.Cobb.2002.Effectofadirect-
fedfibrolyticenzymeformulationonnutrientintake,partitioning,
Journal of Dairy Science Vol. 90 No. 6, 2007
and excretion in early and late lactation Holstein cows. J. Dairy
Sci. 85:3328–3335.
Mauricio, R. M., F. L. Mould, M. S. Dhanoa, E. Owen, K. S. Channa,
andM.K.Theodorou.1999.Asemi-automatedinvitrogasproduc-
tion technique for ruminant feedstuff evaluation. Anim. Feed Sci.
Technol. 79:321–330.
McGinn, S. M., K. A. Beauchemin, T. Coates, and D. Colombatto.
2004. Methane emissions from beef cattle: Effects of monensin,
sunflower oil, enzymes, yeast, and fumaric acid. J. Anim. Sci.
82:3346–3356.
Mirza, M. A., and E. L. Miller. 2005. In vitro degradability of feed
proteins in the rumen: Use of non-rumen proteases. J. Agric. Res.
56:797–801.
Morgavi, D. P., K. A. Beauchemin, V. L. Nsereko, L. M. Rode, A. D.
Iwaasa,W. Z.Yang, T.A. McAllister,and Y.Wang. 2000.Synergy
between ruminal fibrolytic enzymes and enzymes from Tricho-
derma longibrachiatum. J. Dairy Sci. 83:1310–1321.
Nelson, N. A. 1944. A photometric adaptation of the Somogyi method
for the determination of glucose. J. Biol. Chem. 153:375–380.
Oba, M., and M. S. Allen. 1999. Evaluation of the importance of the
digestibility of neutral detergent fiber from forage: Effects on
dry matter intake and milk yield of dairy cows. J. Dairy Sci.
82:589–596.
Reverbel-Leroy, C., S. Pages, A. Belaich, J.-P. Belaich, and C. Tardif.
1997. The processive endocellulase CelF, a major component of
theClostridiumcellulolyticumcellulosome:Purificationandchar-
acterization of the recombinant form. J. Bacteriol. 179:46–52.
Rode, L. M., W. Z. Yang, and K. A. Beauchemin. 1999. Fibrolytic
enzyme supplements for dairy cows in early lactation. J. Dairy
Sci. 82:2121–2126.
SAS Institute. 2001. The SAS System for Microsoft Windows, release
8.2. SAS Institute Inc., Cary, NC.
Somogyi, M. 1952. Notes on sugar determination. J. Biol. Chem.
195:19–23.
Sutton, J. D., R. H. Phipps, D. E. Beever, D. J. Humphries, G. F.
Hartnell, J. L. Vicini, and D. L. Hard. 2003. Effect of method of
application of a fibrolytic enzyme product on digestive processes
and milk production in Holstein-Friesian cows. J. Dairy Sci.
86:546–556.
Toma ´nkova ´, O., and J. Kopec ˇny ´. 1995. Prediction of feed protein
degradation in the rumen with bromelain. Anim. Feed Sci. Tech-
nol. 53:71–80.
Tomme,P.,E.Kwan,N.R.Gilkes,D.G.Kilburn,andR.A.J.Warren.
1996. Characterization of CenC, an enzyme from Cellulomonas
fimi with both endo- and exoglucanase activities. J. Bacteriol.
178:4216–4223.
Wallace, R. J., S. J. A. Wallace, N. McKain, V. L. Nsereko, and G.
F. Hartnell. 2001. Influence of supplementary fibrolytic enzymes
on the fermentation of corn and grass silages by mixed ruminal
microorganisms in vitro. J. Anim. Sci. 79:1905–1916.
Wilson, J. R., and D. R. Mertens. 1995. Cell wall accessibility and
cell structure limitations to microbial digestion of forage. Crop
Sci. 35:251–259.