Animal Feed Science and Technology 77 (1999) 317±329

Effect of enzyme or microbial treatment of

bermudagrass forages before ensiling on cell
wall composition, end products of silage
fermentation and in situ digestion kinetics
P. Mandebvu1,a,*, J.W. Westa, M.A. Froetschelb,
R.D. Hatfieldc, R.N. Gatesd, G.M. Hilla
a
University of Georgia, Department of Animal and Dairy Science, Tifton, GA 31793-0748, USA
b
University of Georgia, Department of Animal and Dairy Science, Athens, GA 30602, USA
c
U.S. Dairy Forage Research Center, University of Wisconsin, Madison, WI 53706, USA
d
USDA-ARS, Tifton, GA 31793-0748, USA

Received 6 March 1998; accepted 19 August 1998

Abstract

Tifton 85 bermudagrass (T85) and Coastal bermudagrass (CBG) established on adjacent plots
and managed similarly were harvested after 3 or 6 weeks of regrowth and used to investigate the
effects of fibrolytic enzymes or microbial inoculant treatment before ensiling on nutrient
composition and recovery, cell wall chemistry and digestion. Prior to ensiling T85 had higher
concentrations of neutral detergent fiber (NDF) and acid detergent fiber (ADF), similar
concentrations of total lignin, and greater (p < 0.05) in vitro and in situ dry matter (DM) and
NDF disappearances when compared with CBG. Coastal bermudgrass had higher (p < 0.05)
concentrations of acid-insoluble lignin and ether-linked ferulic acid (monomers and dimers), and
lower concentrations of glucose and mannose than T85. Treatment of bermudagrass forages with
microbial inoculant decreased (p < 0.05) concentrations of NDF, hemicellulose, butyrate, lactate,
cell walls and acid-insoluble lignin, and increased (p < 0.05) concentrations of ammonia, total
volatile fatty acids (VFA) and acetate in silages. Treatment of bermudagrass forages with fibrolytic
enzymes had no effect on silage fiber concentration, cell wall carbohydrate fraction or
concentration of p-coumaric and ferulic acids, but increased the concentration of butyrate. Among
silages, T85 had higher (p < 0.05) in vitro and in situ dry matter DM and NDF disappearance and
higher (p < 0.05) potentially digestible fractions and smaller (p < 0.05) indigestible fractions of DM

* Corresponding author. Tel.: +1-518-846-7121; fax: +1-518-846-7774; e-mail: mandep@westelcom.com

1
Present address: Miner Agricultural Research Institute, P.O. Box 100, Chazy, New York 12921, USA.

0377-8401/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 8 ) 0 0 2 4 7 - 8
318 P. Mandebvu et al. / Animal Feed Science and Technology 77 (1999) 317±329

and NDF than CBG. Treatment of bermudagrass forages with fibrolytic enzymes had no effect on in
vitro or in situ DM or NDF disappearance of silages. Treatment of bermudagrass forages with
microbial inoculant increased in situ DM disappearance at 72 h of incubation (p < 0.10) and the
potentially digestible fraction of DM (p < 0.05) of silages. Although treatment of bermudagrass
forages with microbial inoculant had no effect on silage in vitro or in situ NDF disappearance at
48 h of incubation, it increased in situ NDF disappearance at 72 h (p < 0.05) and the potentially
digestible fraction of NDF. It is concluded that the greater cellulose content of cell walls with the
same or less lignin in T85, and the greater concentration of ether-linked ferulic acid in CBG explain
the greater digestibility of T85 when compared with CBG at similar stages of maturity. Treatment
of bermudagrass forage at ensiling with microbial inoculants may have more potential than extracts
of fibrolytic enzymes in improving silage fiber digestion. # 1999 Elsevier Science B.V. All rights
reserved.

Keywords: Bermudagrass; Fibrolytic enzymes; Microbial inoculant; Ferulic acid; Ether linkages; Digestion
kinetics

1. Introduction

Tifton 85 bermudagrass (T85), one of the highest quality and yielding bermudagrass
cultivar released thus far, has been used successfully in diets for growing steers (Hill
et al., 1997) and in total mixed rations for lactating dairy cows (West et al., 1998).
However, the high neutral detergent fiber (NDF) content limits extensive use of T85 in
total mixed rations for lactating dairy cows because of the potential to reduce dry matter
(DM) intake and milk yield. Although treatment of low quality forage with chemicals
such as sodium hydroxide, alkaline hydrogen peroxide, or anhydrous ammonia increases
the rate and extent of DM and fiber digestion (Fahey et al., 1993), the hazardous nature of
these chemicals limits the practical application of chemical treatment of forage by many
farmers. Improvements in extent of DM and fiber digestibility from the addition of lactic
acid producing (LAB) bacteria to forage at ensiling have been small (Harrison et al.,
1994) but encouraging (Ball et al., 1991). This may have been a factor of different plant
species treated as well as differences in enzyme or microbial inoculant products. When
cellulases or microbial inoculants are added to forages prior to ensiling the pH decreases
rapidly, leading to improved ensiling characteristics due to production of readily
fermentable sugars from cellulose hydrolysis (Fahey et al., 1993; Harrison et al., 1994).
When combinations of fibrolytic enzymes and microbial inoculants are used enzymatic
degradation of fiber should provide more sugars to the LAB leading to a more rapid
achievement of a stable pH thus improving the silage preservation characteristics. This is
especially important in grass silages because grasses such as bermudagrass are generally
high in NDF and low in soluble carbohydrates. The objective of this study was to
determine the effect of enzyme and/or microbial treatment at ensiling of T85, an
improved bermudagrass cultivar, and Coastal bermudagrass (CBG), an older and widely
used bermudagrass cultivar, with fibrolytic enzymes or microbial inoculant on cell wall
composition, end products of silage fermentation, and in situ digestion kinetics of DM
and NDF.
 P. Mandebvu et al. / Animal Feed Science and Technology 77 (1999) 317±329 319

2. Materials and methods

2.1. Forages, enzyme and microbial treatment

In 1996, T85 and CBG grown in adjacent plots on Tifton loamy sand soil were mowed
to stage forage growth on 7 August, fertilized (43 kg N/ha) and harvested after 3 weeks or
6 weeks of regrowth (vegetative growth stage, nonflowering stems). Forages were
chopped to about 9 cm in length using a farm forage chopper, mixed in large plastic
barrels and treated with water (control), fibrolytic enzymes, microbial inoculant or
combination of the enzymes and inoculant. The enzyme mixture was composed of
cellulase, /-amylase and hemicellulase, while the microbial inoculant consisted of
selected strains of homofermentative LAB (Lactobacillus plantarum, Lactobacillus
brevis, Pediococcus acidilacti, and Streptococcus diacetylacti) mixed with bacterial
nutrients. The enzyme mixture and microbial inoculant were applied in liquid solution at
the manufacturer's (L.B.J. Pakke, New York) recommended rate of application of 900 and
100 g, respectively, mixed with 94.6 l of distilled water for the treatment of 45.5 t of fresh
forage. Each treated forage was packed tightly using a hydraulic press into pre-weighed
9.5 l plastic silage fermentation buckets and allowed to ferment for 6 weeks. At the end of
the fermentation period samples were either dried at 608C until weight was constant for
DM determination and nutrient analyses or stored frozen for later analysis of volatile
components and pH determination.

2.2. Chemical analyses

Dried forage and silage samples were ground to pass through a 6 mm screen using a
Wiley mill (model 3; Arthur T. Thomas, Philadelphia, PA). The resulting 6 mm sized
samples were then sub-sampled for later in situ digestion. A sub-sample was ground to
pass through a 1 mm screen and analyzed for DM (1008C); ash (5008C); crude protein
(CP) [Kjeldahl N  6.25]; ash-free NDF and acid detergent fiber (ADF) [Van Soest et al.,
1991]; and permanganate lignin and cellulose (Van Soest and Wine, 1968). Hemicellulose
was determined as the difference between ash-free NDF and ADF. Cell walls (CW) were
isolated from samples using the Uppsala method (Theander and Westerlund, 1986;
Theander, 1991) as modified by Hatfield and Weimer (1995). Although NDF is often
used to refer to CW, the CW determined using the Uppsala method contain pectins and
more cell wall protein. The CW were analyzed for acid insoluble lignin (AIL), total
neutral sugars, total uronosyls and neutral sugar composition using a modified Saeman
hydrolysis procedure (Saeman et al., 1963; Hatfield et al., 1994; Hatfield and Weimer,
1995). Ferulates and p-coumarates were analyzed using the method of Ralph et al. (1994).
In vitro DM disappearance (IVDMD) after 48 h of incubation followed by subsequent
acid and pepsin digestion for an additional 48 h (Moore and Mott, 1974) and 48 h in vitro
NDF disappearance were determined. Frozen silage samples were thawed and processed
according to Williams et al. (1995) for determination of pH and free ammonia nitrogen
using a digital pH meter (Ammonia Electrode Instruction Manual, Orion Research,
1990), volatile fatty acids (VFA) using gas chromatography (Supelco, 1975), lactic acid
(Holdeman et al., 1973) and water soluble carbohydrates (WSC) [Dubois et al., 1956].
320 P. Mandebvu et al. / Animal Feed Science and Technology 77 (1999) 317±329

2.3. In situ digestion

Two ruminally cannulated lactating Holstein cows fed a bermudagrass and corn silage
based total mixed ration were used for in situ digestion. Duplicate samples ground to pass
through a 6 mm screen were composited by treatment and 5 g of sample DM weighed
into duplicate 10  20 cm dacron bags (Ankom Technology, New York) and incubated in
the rumen of each cow for 0, 2, 4, 6, 12, 24, 36, 48, and 72 h of incubation (Nocek and
English, 1986; Nocek, 1988). Bags were made from nitrogen-free white polyester
monofilament fabric with 53 micron (10) pore size and allowing a 12.5 mg/cm2 sample
surface area. Heat-sealed bags were grouped into polyester vegetable bags (23  38 cm2,
with 0.5  0.5 cm2 pore size) that were weighted and suspended at the bottom of the
rumen. At the end of each incubation period bags were removed from the rumen,
immersed in ice water for about 15 min to inhibit further microbial action before
processing according to Nocek (1988) and dried at 608C for 48 h in a forced air oven.
Residual DM in the bags after digestion was expressed as a percentage of initial DM in
the bag, and then subtracted from 100 to calculate the percentage DM disappearance.
Digesta samples were ground to pass through a 1 mm screen in a Cyclone sample mill
(UD Corporation, Boulder, Co) and analyzed for NDF content which was expressed as a
fraction of the initial NDF concentration to calculate NDF disappearance.

2.4. Statistical analyses

The study had a 2  2  2  2 factorial arrangement of treatments with bermudagrass

cultivar, stage of maturity, enzyme and microbial inoculant as main variables. Data were
analyzed using the General Linear Models procedures of SAS (1989). In the analysis of in
situ DM and NDF disappearance data, cow was also included as an additional class
variable. The disappearance data were also fitted to the nonlinear model
p ˆ a ‡ b…1 ÿ eÿct † of érskov et al. (1980) to calculate digestion constants, where a is
the readily digested fraction, b represents the slowly digested fraction, a‡b total
potentially digestible fraction, and c is the rate of digestion for the slowly digested
fraction (or potentially digestible fraction for NDF). Lag time of NDF digestion was
calculated using a linear model (Nocek and English, 1986).

3. Results and discussion

3.1. Cell wall composition

Prior to ensiling, T85 had higher concentrations of OM, CP, NDF, ADF and cellulose
than CBG at similar stages of maturity (Table 1), and CBG had higher AIL and ether
linked ferulic acid (monomers and dimers) concentrations and lower concentration of
glucose than T85 (Table 2. The amount of cell walls, p-coumaric acid and ester linked
ferulic acid (monomers and dimers) were similar between the two cultivars (Table 2).
Among silages (Table 3), T85 still had higher (p < 0.05) concentrations of OM, CP,
NDF, ADF and cellulose, and lower (p < 0.05) concentrations of DM, hemicellulose and
 P. Mandebvu et al. / Animal Feed Science and Technology 77 (1999) 317±329 321

Table 1
Nutrient composition of T85 and CBG forages harvested at different maturities

Nutrient T85 Coastal

3 weeks 6 weeks 3 weeks 6 weeks

% of DM
DM 21.9 23.1 25.1 28.5
Organic matter 92.0 91.6 86.1 88.6
Crude protein 18.2 16.4 16.3 14.2
NDFa 68.6 72.3 66.9 68.9
ADFa 32.7 35.0 29.9 29.9
Hemicellulosea 35.9 37.3 37.0 39.0
Cellulosea 28.9 30.8 25.6 26.0
Lignina,b 4.7 5.8 6.6 6.8
a
Analyses are on ash-free basis.
b
Lignin is permanganate lignin.

Table 2
Chemical components of cell walls of T85 and CBG forages harvested at different maturities

Component T85 Coastal

3 weeks 6 weeks 3 weeks 6 weeks

Cell walls (CW), g/kg DM 814.2 852.9 840.9 847.4

(g/kg CW)
Acid insoluble lignin 164.7 174.7 200.5 191.1
Total uronosyls 26.2 24.0 25.7 23.4
Ester p-coumaric acid 9.3 9.3 9.1 8.9
Ester ferulic acid 11.6 10.0 10.6 10.6
Ether p-coumaric acid 1.0 0.7 0.9 1.1
Ether ferulic acid 6.2 4.9 8.1 7.6
Neutral sugars 716.0 703.3 677.2 679.1
Fucose 0.2 0.5 0.4 0.5
Arabinose 48.1 42.9 47.8 47.2
Rhamnose 0.0 0.0 0.0 0.0
Galactose 15.7 13.2 15.8 16.7
Glucose 402.3 394.3 366.6 375.4
Xylose 249.6 251.9 246.3 238.6
Mannose 0.0 0.5 0.4 0.7

lignin than CBG. Silage made from bermudagrass harvested at 6 weeks had higher
(p < 0.05) concentrations of DM, OM, ash-free NDF, ash-free ADF, hemicellulose,
cellulose and lignin, and lower (p < 0.05) concentration of CP than bermudagrass
harvested at 3 weeks due to increase in maturity. Treatment of bermudagrass forages with
fibrolytic enzymes had no effect on silage nutrient composition. Treatment of
bermudagrass forages with microbial inoculant decreased (p < 0.05) concentrations of
NDF and hemicellulose in silages. Treatment of bermudagrass forages with fibrolytic
enzymes or microbial inoculant had no effect on the concentration of cell wall
carbohydrates, p-coumaric acid or ferulic acid.
 322
P. Mandebvu et al. / Animal Feed Science and Technology 77 (1999) 317±329
Table 3
Nutrient composition of T85 and CBG silages (Silage) made from forages harvested at different maturities (Maturity) and treated with an enzyme mixture (Enzyme) or
microbial inoculant (Microbe) and ensiled for 6 weeks

Nutrienta Silage Maturity Enzyme Microbe Interactionf

T85 Coastal SE 3 weeks 6 weeks SE No Yes SE No Yes SE Silage  Enzyme  SE

maturity microbe

Dry matter 22.4e 26.0d 0.1 23.2e 25.2d 0.1 24.2d 24.1d 0.1 24.4d 24.0e 0.1 **
NS 0.1
(% DM)
Organic matter 91.0d 86.6e 0.1 88.1e 89.6d 0.1 88.9d 88.7d 0.1 89.3d 88.3e 0.1 ** **
0.1
Crude protein 19.2d 17.5e 0.1 19.2d 17.5e 0.1 18.4d 18.3d 0.1 18.1e 18.5d 0.1 *
NS 0.1
NDFb 61.9d 60.9e 0.2 60.1e 62.8d 0.2 61.5d 61.3d 0.2 61.8d 61.0e 0.2 **
NS 0.2
ADFb 34.5d 31.9e 0.1 32.6e 33.8d 0.1 33.2d 33.2d 0.1 33.1d 33.3d 0.1 NS NS 0.1
Hemicelluloseb 27.4e 29.0d 0.2 27.5e 28.9d 0.2 28.3d 28.1d 0.2 28.7d 27.7e 0.2 **
NS 0.2
Celluloseb 30.1d 27.4e 0.1 28.4e 29.6d 0.1 29.0d 28.9d 0.1 29.0d 28.9d 0.1 **
NS 0.1
Ligninb,c 5.3e 6.6d 0.1 5.6e 6.3d 0.1 6.0d 6.0d 0.1 6.0d 6.0d 0.1 *
NS 0.1
a
Nutrient composition is expressed on DM basis.
b
Analyses are on ash-free basis.
c
Lignin is permanganate lignin.
d,e
Means with no common superscripts within a row within Silage, Maturity, Enzyme or Microbe differ (p < 0.05).
f
Level of significance for interaction is denoted by: ** for (p < 0.01), * for (p < 0.05) and NS for not significant.
 P. Mandebvu et al. / Animal Feed Science and Technology 77 (1999) 317±329 323

Although the recoveries of DM, OM, CP, ADF, cellulose and ADL were high (95±110%),
the recoveries of NDF and hemicellulose were lower (69±90%) and probably resulted
from partial degradation of hemicellulose during ensiling. Correction for volatile losses
increased overall DM recovery by 6% but did not change treatment responses. This is
consistent with what was reported by Williams et al. (1995). Microbial inoculant
decreased (p < 0.05) the recoveries of OM (98.7% vs. 96.7%), NDF (88.4% vs. 86.5%)
and hemicellulose (76.1% vs. 72.7%). The decrease in OM recovery may have been a
result of increased microbial respiration and fermentation of the fiber fraction. This is
consistent with the decline in NDF and hemicellulose concentrations (Table 2). Fibrolytic
enzymes had no effect on the nutrient recovery.
Ferulic acid cross-links arabinoxylans and lignin polymers via ester and ether linkages
(Jung and Allen, 1995). While rumen bacteria and fungi possess phenolic acid esterases
which ultimately break down the ferulate ester linkages, anaerobic cleavage of ether linkages
is not known to occur (Jung and Allen, 1995). The majority of p-coumaric acid is esterified to
lignin and is, therefore, unlikely to directly affect polysaccharide digestion (Jung and Allen,
1995). The increase in the cellulose content (Table 1) of the cell wall with same or less lignin
would be expected to lead to greater T85 digestibility, since cellulose is not covalently linked
to lignin or any other cell wall polymer (Jung and Ralph, 1990). Although magnitudes of
change were small, other workers (Stokes, 1992; Chen et al., 1994; Harrison et al., 1994) also
observed a decline in silage fiber concentration with microbial inoculant treatment.
Differences in plant species treated with enzymes or microbial inoculants also determine the
extent of reduction in the fiber concentration.

3.2. Silage fermentation end products

Among silages, T85 had a higher concentration of butyrate (p < 0.01), but lower
concentrations of ammonia (p < 0.05) than CBG (Table 4). Bermudagrass harvested at 3-
week maturity had a higher (p < 0.01) pH, and higher (p < 0.01) concentrations of
ammonia, total VFA, acetate and butyrate, and lower (p < 0.01) concentration of lactate
than bermudagrass harvested at 6-week maturity. Enzyme treatment of bermudagrass
forages increased (p < 0.05) the concentrations of butyrate in silages. Treatment of
bermudagrass forages with microbial inoculant increased (p < 0.05) the concentrations of
ammonia, total VFA and acetate, and decreased (p < 0.05) those of butyrate and lactate,
and the lactate : acetate and lactate : total VFA ratios in silages. These results suggest
secondary fermentation of lactate to acetate. The subsequent increase in ammonia
concentration supports this suggestion. There were no interactions between enzymes and
microbial inoculant. Reports by other researchers on effects of forage treatment by
fibrolytic enzymes, microbial inoculant, or a combination of the two on silage
fermentation end products have been variable (Stokes, 1992; Chen et al., 1994; Harrison
et al., 1994; Williams et al., 1995; Sheperd and Kung, 1996).

3.3. In vitro and in situ digestion of forages before ensiling

In vitro DM and NDF disappearances of forages prior to ensiling decreased in the order
of 3-week T85, 6-week T85, 3-week CBG and 6-week CBG. Tifton 85 bermudagrass had
 324
P. Mandebvu et al. / Animal Feed Science and Technology 77 (1999) 317±329
Table 4
pH and concentration of fermentation end products of T85 and CBG silages (Silage) made from forages harvested at different maturities (Maturity) and treated with an
enzyme mixture (Enzyme) or microbial inoculant (Microbe) and ensiled for 6 weeks

Item Silage Maturity Enzyme Microbe Interactiond

T85 Coastal SE 3 weeks 6 weeks SE No Yes SE No Yes SE Silage  Enzyme  SE

maturity microbe

pH 4.4b 4.4b 0.01 4.5b 4.3c 0.02 4.4b 4.4b 0.02 4.4b 4.4b 0.02 NS NS 0.02
(mg/g DM)
Ammonia 0.41c 0.48b 0.01 0.53b 0.35c 0.01 0.44b 0.44b 0.01 0.38c 0.50b 0.01 **
NS 0.02
Lactate (L) 72.6b 72.8b 2.9 66.3c 79.1b 2.9 70.8b 74.6b 2.9 77.6b 67.8c 2.9 **
NS 4.1
Acetate (A) 32.8b 32.6c 1.7 38.0b 27.4c 1.7 33.3b 32.1b 1.7 28.2c 37.2b 1.7 NS NS 2.4
Propionate 0.12b 0.15b 0.01 0.15b 0.12b 0.01 0.12b 0.15b 0.01 0.13b 0.14b 0.01 NS NS 0.02
Butyrate 0.51b 0.16c 0.04 0.50b 0.17c 0.04 0.25c 0.41b 0.04 0.42b 0.24c 0.04 **
NS 0.06
a
Total VFA (T) 35.3b 32.9b 2.2 40.5b 27.7c 2.2 33.7b 34.6b 2.2 30.6c 37.6b 2.2 * **
3.1
L:A 2.4b 2.4b 0.2 1.8c 3.1b 0.2 2.3b 2.6b 0.2 3.0b 1.9c 0.2 *
NS 0.3
L:T 2.4b 2.4b 0.2 1.8c 3.1b 0.2 2.3b 2.5b 0.2 2.9b 1.9c 0.2 *
NS 0.2
WSC 8.7b 9.6b 0.5 8.6b 9.6b 0.5 9.4b 8.8b 0.5 8.5b 9.7b 0.5 NS NS 0.7
a
Total volatile fatty acids.
b,c
Means with no common superscripts within a row within Silage, Maturity, Enzyme or Microbe differ (p < 0.05).
d
Level of significance for interaction is denoted by: ** for (p < 0.01), * for (p < 0.05) and NS for not significant.
 P. Mandebvu et al. / Animal Feed Science and Technology 77 (1999) 317±329 325

Table 5
In vitro and in situ DM and NDF disappearance (%) and digestion constants for T85 and CBG forages harvested
at different maturities

Item T85 Coastal

3-week 6-week 3-week 6-week SE

In vitro digestion
IVDMDa 61.7g 56.9h 51.4i 50.8i 1.1
IVNDFDb 60.6g 55.6h 42.6i 41.0i 1.6

In situ DM digestion
Disappearance
48 h 54.9g 52.5g 46.2h 43.2h 1.5
72 h 65.9g 56.3h 51.4i 51.3i 1.0
Digestion constantsc
Potentially digestible fraction, % 75.2g 61.1hi 54.3ij 53.1j 2.8
Readily digested fraction, % 14.1g 11.2g 13.2g 12.5g 1.1
Slowly digested fraction, % 61.1g 49.2h 41.1i 40.6i 2.6
Rate of digestiond, % hÿ1 3.5g 3.4g 3.5g 3.8g 0.5
Indigestible fraction, % 24.8j 38.9ij 45.7hi 46.9g 2.8
(R2)e 0.97 0.98 0.98 0.95

In situ NDF digestion

Disappearance
48 h 52.5g 49.5g 42.7h 39.8h 1.6
72 h 63.8g 53.1h 48.9i 43.7j 1.2
Digestion constantsc
Potentially digestible fraction, % 72.2g 58.3h 51.6h 48.5h 4.5
Rate of digestiond, % hÿ1 2.9h 3.8gh 2.7h 4.7g 0.6
Indigestible fraction, % 27.8h 41.7g 48.4g 51.5g 4.5
(R2)e 0.97 0.98 0.86 0.94
Discrete lagf, h 1.8 2.9 1.8 2.5
a
In vitro DM disappearance at 48 h of incubation with subsequent acid pepsin digestion for an additional 48 h.
b
In vitro NDF disappearance at 48 h of incubation.
c
In situ digestion constants were calculated using the nonlinear model p ˆ a ‡ b…1 ÿ eÿct † of érskov et al.
(1980).
d
Rate of digestion of the slowly digested fraction of DM (or potentially digestible fraction of NDF).
e
Coefficient of determination.
f
Lag time before NDF digestion.
g,h,i,j
Means with no common superscripts within a row differ (p < 0.05).

higher (p < 0.05) IVDMD and in vitro NDF disappearance at 48 h of incubation than
CBG (Table 5). Tifton 85 bermudagrass harvested at 3-week maturity had higher
(p < 0.05) IVDMD than T85 harvested at 6-week maturity. Maturity had no effect on
IVDMD for CBG or in vitro NDF disappearance at 48 h of incubation for T85 and CBG.
In situ DM and NDF disappearances of forages prior to ensiling decreased in the order of
3-week T85, 6-week T85, 3-week CBG and 6-week CBG (Table 5). Tifton 85
bermudagrass had higher (p < 0.05) in situ DM and NDF disappearances at 48 and
72 h of incubation and higher (p < 0.05) potentially digestible fractions of DM and NDF
than CBG.
 326
Table 6
In vitro and in situ DM and NDF disappearances (%) and digestion constants for T85 and CBG silages (Silage) made from forages harvested at different maturities
(Maturity) and treated with an enzyme mixture (Enzyme) or microbial inoculant (Microbe) and ensiled for 6 weeks
Item Silage Maturity Enzyme Microbe Interactionm

T85 Coastal SE 3 weeks 6 weeks SE No Yes SE No Yes SE Forage  Enzyme  SE

P. Mandebvu et al. / Animal Feed Science and Technology 77 (1999) 317±329

microbe microbe

In vitro DM and NDF digestion

IVDMDa 55.6k 48.9l 0.3 52.4k 52.1k 0.3 51.9k 52.6k 0.3 52.6k 51.9k 0.3 **
NS 0.5
IVNDFDb 51.0k 36.1l 0.3 44.2k 42.8l 0.3 43.6k 43.4k 0.3 44.4k 43.6k 0.3 **
NS 0.5
In situ DM digestion
Disappearance
48 h 52.2k 43.9l 0.7 48.5k 47.6k 0.7 47.6k 48.5k 0.7 48.3k 47.8k 0.7 NS NS 1.0
72 h 53.6k 46.7l 1.0 50.1k 50.1k 1.0 49.4k 50.8k 1.0 48.8k 51.4k 1.0 NS NS 1.4
Digestion constantsc
PDFd, % 62.5k 51.3l 1.6 57.7k 58.3k 1.8 55.6k 58.2k 1.8 54.5l 59.7k 1.9 NS NS 1.9
ae, % 19.2k 18.3k 0.5 18.9k 18.6k 0.6 18.6k 18.9k 0.6 18.1k 19.4k 0.6 NS NS 0.6
bf, % 43.3k 33.0l 1.5 36.8k 39.7k 1.7 37.0k 39.3k 1.7 36.4k 40.3k 1.8 NS NS 1.8
cg, %hÿ1 2.5k 2.9k 0.2 2.9k 2.4k 0.3 2.8k 2.5k 0.3 3.0k 2.3k 0.3 NS NS 0.3
Ifh, % 37.5l 48.7k 1.6 44.3k 41.7k 1.8 44.4k 41.8k 1.8 54.5k 40.3l 1.9 NS NS 1.9
(R2)i 0.91 0.93 0.87 0.91 0.88 0.89 0.88 0.90
In situ NDF digestion
Disappearance
48 h 40.6k 34.1l 1.0 38.1k 36.6k 1.0 36.8k 37.9k 1.0 38.5k 36.2k 1.0 NS NS 1.4
72 h 43.5k 36.8l 0.9 40.3k 40.0k 0.9 39.4k 41.0k 0.9 38.4l 42.0k 0.9 NS NS 1.3
Digestion constantsc
PDFd, % 53.2k 42.2l 3.3 46.7k 49.1k 3.7 46.7k 48.9k 3.7 44.4k 52.1k 3.9 NS NS 3.8
cg, % 3.0k 3.4k 0.4 3.5k 2.8k 2.8 3.2k 3.1k 0.4 3.7k 2.7k 2.6 NS NS 1.9
Ifh, % 46.8l 57.8k 3.3 53.3k 50.9k 3.7 53.3k 51.1k 3.7 55.6k 47.9k 3.9 NS NS 3.8
(R2)i 0.86 0.85 0.82 0.85 0.83 0.84 0.81 0.87
Discrete lagj, h 4.3 3.9 4.2 4.1 4.1 4.1 4.0 4.3
a
In vitro DM disappearance at 48 h of incubation with subsequent acid pepsin digestion for an additional 48 h.
b
In vitro NDF disappearance at 48 h of incubation.
c
In situ digestion constants were calculated using the nonlinear model p ˆ a ‡ b…1 ÿ eÿct † of érskov et al. (1980).
d
Total potentially digestible fraction.
e
Readily digested fraction.
f
Slowly digested fraction.
g
Rate of digestion of the slowly digested fraction of DM (or potentially digestible fraction of NDF).
h
Indigestible fraction.
i
Coefficient of determination.
j
Lag time before NDF digestion.
k,l
Means with no common superscripts within a row within Silage, Maturity, Enzyme or Microbe differ (p < 0.05).
m
Level of significance for interaction is denoted by: ** for (p < 0.01) and NS for not significant.
 P. Mandebvu et al. / Animal Feed Science and Technology 77 (1999) 317±329 327

The significant difference in the AIL between CBG and T85, the greater concentration
of ferulate ether linkages in CBG when compared with T85, and the higher amount of
glucose in the cell walls of T85 when compared with CBG may explain why the cell wall
fraction of T85 is more digestible than that of CBG at similar stages of maturity. Lignin is
the most commonly recognized limitation to the potential extent of cell wall digestion,
but its concentration is not well correlated with the rate of cell wall digestion (Van Soest,
1985).

3.4. In vitro and in situ digestion of silages made from forages treated with enzymes or
microbial inoculant

Following treatment and ensiling T85 had higher (p < 0.05) IVDMD and in vitro NDF
disappearance at 48 h of incubation than CBG (Table 6). Bermudagrass maturity had no
effect on IVDMD. Bermudagrass harvested at 3-week maturity had higher (p < 0.05) in
vitro NDF disappearance at 48 h of incubation than bermudagrass harvested at 6-week
maturity. The interaction (p < 0.05) between silage and maturity for IVDMD was because
T85 harvested at 3-week had a higher (p < 0.01) IVDMD than T85 harvested at 6-week,
while no differences were observed between the two maturities for CBG. Treatment of
bermudagrass forage with fibrolytic enzymes or microbial inoculant had no effect on
IVDMD or in vitro NDF disappearance at 48 h of incubation of silages. Other workers
reported that treatment of grass, legume, or mixture of the two forages with fibrolytic
enzymes, microbial inoculant or a combination of the two produces inconsistent effects
on DM or fiber digestion (McHan, 1986; Umana et al., 1991; Stokes, 1992; Sheperd and
Kung, 1996). Harrison et al. (1994) also reported that treatment of a variety of legumes
and grasses with enzymes and/or inoculants increased DM digestibility.
Following treatment and ensiling T85 had higher (p < 0.05) in situ DM and NDF
disappearance and higher (p < 0.05) potentially digestible fractions of DM and NDF than
CBG (Table 6). Bermudagrass maturity had no effect on in situ DM or NDF
disappearance or digestion constants of silages. Treatment of bermudagrass forages with
fibrolytic enzymes had no effect on in situ DM or NDF disappearance or digestion
constants of silages. Treatment of bermudagrass forage with microbial inoculant
increased in situ NDF (p < 0.05) disappearance at 72 h of incubation and increased the
potentially digestible fraction of DM (p < 0.05) of silages. There were no interactions
between silage and maturity, or enzymes and microbial inoculant on in situ DM or NDF
disappearances or digestion constants. Lewis et al. (1996) and Chen et al. (1994) reported
that treatment of forages with fibrolytic enzymes or microbial inoculants improved in situ
DM and NDF digestion, while other workers observed no effect on in situ DM or NDF
disappearance (Chen et al., 1994).

4. Conclusions

Tifton 85 had higher content of cell wall polysacharides, lower total lignin and ether-
linked ferulic acid concentration and higher DM and NDF in vitro and in situ
digestibilities than CBG. Treatment of bermudagrass at ensiling with fibrolytic enzymes
328 P. Mandebvu et al. / Animal Feed Science and Technology 77 (1999) 317±329

had no effect on cell wall composition, end products of silage fermentation or digestion.
Treatment of bermudagrass at ensiling with an inoculant containing LAB slightly
decreased fiber concentration and tended to enhance in situ digestion. However,
investigation of the effects of adding energy sources such as molasses to assist microbial
fermentation of ensiled grasses is recommended.

Acknowledgements

Authors would like to thank L.B.J. Pakke, New York for partial finacial support and
supply of the enzymes and microbial inoculant, and Mr. B.G. Mullinix for statistical
assistance.

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