In vitro determination of ruminal dry matter and cell wall degradation, and production of fermentation end-products of various by-products
In vitro determination of ruminal dry matter and cell wall degradation, and production of fermentation end-products of various by-products
M. Dolor 0
s MEGÍAS 0
0 Department of Animal Production, University of Murcia , Campus de Espinardo 30071, Murcia , Spain
- The samples used in the experiment were nine types of by-products: giant pumpkin, red pepper, melon, broccoli, brewer's grains, fresh and boiled artichoke, lemon peel and orange peel. The dry matter degradability of each by-product was determined by in vitro fermentation with ruminal fluid of the goat. The materials were incubated at 39 ºC for 12, 24, 48 and 72 h. At each time, pH, dry matter disappearance, neutral-detergent fibre disappearance, volatile fatty acids, lactic acid and ammonia productions were measured. Approximately 50% of the total dry matter loss of broccoli, melon and peels occurred at 12 h incubation. In addition, the total dry matter loss of the giant pumpkin, fresh and boiled artichoke, and brewer's grains was low (< 50% of dry matter loss) at 12 h. The molar proportion of acetate and propionate was influenced by the type of the feedstuffs. Thus, the acetate proportion was > 70% for all by-products, except for brewer's grains (63.8%), and the propionate proportion was < 20% for all by-products, except for the brewer's grains (21.3%). In conclusion, the ruminal dry matter and cell wall degradations, and production of fermentation end-products by in vitro determination offers a convenient method to compare the microbial digestion of the different byproducts.
citrouille, de l’artichaut et de la drèche de brasserie a été faible (< 50 % des pertes de matière sèche)
12 heures après. La proportion molaire d’acétate et de propionate a été influencée par le type
d’aliment utilisé. Ainsi, la proportion d’acétate a été supérieure à 70 % pour tous les sous-produits, sauf
pour la drèche de brasserie (63,8 %), et la proportion de propionate a été inférieure à 20 % pour tous
les sous-produits, sauf pour la drèche de brasserie. Ce travail permet de conclure que la détermination
de la dégradation ruminale in vitro, de la matière sèche et du NDF, et la mesure des produits terminaux
de fermentation de divers sous-produits offre une méthode appropriée qui permet de comparer la
digestion microbienne de divers sous-produits.
fermentation in vitro / dégradation de la matière sèche / dégradation du NDF / sous-produits
The effective use of by-products from
agricultural industries as feedstuffs is
dependent on several factors. These include
the production, nutrient composition in
relation to the nutrient requirements of the
available animals, processing cost, the
uniformity of supply of the by-product and the
marked availability of competitively priced
feedstuffs. Large quantities of by-products
are used in the ruminant’s diet in
agro-industrial areas [
]. However, little is known
about their fermentation pattern in the
rumen and a better understanding of their
digestion and products of fermentation is
necessary in order to properly balance their
introduction into the diets [
addition, the new systems to predict animal
performance and milk production and
composition include a nutritional model
that requires digestion rates of the fibre and
soluble carbohydrate fractions [
The prediction of the extent of
degradability using an in situ method has
advantages and is now widely used and
]. This technique proposed by
Ørskov et al.  can be used for the
prediction of dry matter intake, digestible dry
matter intake and animal performance.
However, this technique has several
disadvantages: it does not allow individual
adaptation of micro-organisms to the solid
substrates and fermentation end-products
cannot be monitored.
Other methods have been developed to
predict the extent of degradability. Thus,
some studies conducted by Luchini et al.
], Feng et al.  and Wilman et al. [
propose in vitro methods for determining
ruminal feed degradation because of a
greater speed and lower expense than in
vivo and in situ methods.
The by-products used in this work have
been studied by Megías et al. [
determine the chemical composition and in vitro
gas production. The objective of these
experiments was to evaluate and compare the
extent of digestion of DM and NDF and the
production of fermentation end-products
of by-products in standardised conditions
2. MATERIALS AND METHODS
The samples studied in the experiment
were the nine by-product types mainly used
for the feeding of dairy cows feeding in the
Murcia Region (Spain) and reported by
Martínez-Teruel et al. [
]: giant pumpkin
(Curcubita ficifolia, Bouché), red pepper
(Capsicum annuum, var. annuum), whole
melon (Cucumis melo), stem broccoli
(Brassica olareacea, var. italica), brewer’s
grains, inflorescences of fresh artichoke
and inflorescences of boiled artichoke
(Cynara scolymus), lemon peel (Citrus limon)
and orange peel (Citrus aurantium). The
samples were oven-dried and ground through a
1 mm screen. Table I shows the chemical
composition of the raw materials: DM
DM: dry matter; CP: crude protein; NDF: neutral-detergent fibre; ADF: acid-detergent fibre.
a Values except DM are expressed on a DM basis.
b Permanganate lignin.
by drying at 60 ºC for 48 h, OM by ashing
at 550 ºC for 3.5 h, CP by Kjeldahl × 6.25,
fibre and lignin by Van Soest et al. [
2.2. Collection of ruminal fluid
The dry matter degradability of each by
product was determined by in vitro
fermentation with ruminal fluid. Ruminal fluid
was collected approximately 4 h after
feeding from three mature Murciano-Granadina
goats consuming alfalfa hay ad libitum.
Ruminal fluid was transported to the
laboratory in a sealed thermos and was
immediately squeezed through four layers of
cheesecloth. The resulting ruminal fluid
was purged with deoxygenated CO2 before
use as the inoculum.
2.3. In vitro procedure
In vitro incubations were conducted as
described by the first stage of the Tilley and
Terry procedure [
]. Thus, 10 mL of
ruminal inoculum and 40 mL of buffer
solution were added to 0.5 g of the sample of
each by-product, in bottles. The buffer
] was used by Tilley and Terry
]. The bottles were flushed with CO2
and sealed. Duplicate bottles were
incubated in a 39 ºC shaking water bath for each
of two replicates for 12, 24, 48 and 72 h. At
each time, pH, dry matter (DM)
disappearance, neutral-detergent fibre (NDF)
disappearance, volatile fatty acids (VFA), lactic
acid and ammonia productions were
measured. After each respective incubation
interval, the fermentation medium was
decanted into plastic tubes, and then the
tubes were centrifuged at 3000 × g for
20 min. Supernatants were decanted into
glass bottles, 1 mL of 50% sulphuric acid
was added and they were frozen at –20 ºC
until analysis. The residues of each sample
after incubation were filtered through
Whatman® 541 paper and were washed
sequentially with water. The samples were
dried and weighed to determine the in vitro
DM disappearance. In vitro dry matter
disappearance was calculated as follows:
(1 – (( DM residue - blankDM) / DM
original)) × 100, where DM residue is the DM
recovered after 12, 24, 48 and 72 h of
fermentation, blankDM is the DM recovered in
the corresponding blank after the same
fermentation time, and DM original is the DM
of the substrate placed in the tube. Also,
NDF residues were determined on the DM
residue by in vitro NDF disappearance
using a modified method (neutral-detergent
digestion with heat-stable α-amylase)
described by Van Soest et al. [
The supernatants were analysed for
VFA concentration by capillary gas
chromatography described by Madrid et al.
], using 4-methyl-n-valeric acid as the
internal standard, for lactic acid and
ammonia concentrations by spectrophotometric
methods described by Madrid et al. [
and Chaney and Marbach [
2.4. Mathematical model and statistical analysis
DM disappearance, NDF disappear
ance, pH, VFA, lactic acid and ammonia
values were analysed using ANOVA for
two-way comparison with interactions
]. The model used was:
Yij = µ + Ai + Bj + ABij + ε
where A, B and AB are the effects of the
incubation time, the by-product type and the
incubation time × by-product type
3. RESULTS AND DISCUSSION
3.1. Dry matter degradation
The in vitro DM apparent disappearance
of by-products is given in Table II.
Approximately 50% of the total DM loss of
broccoli, melon and peels occurred at 12 h
incubation. The total DM loss of the giant
pumpkin, fresh and boiled artichoke, and
brewer’s grains was, however, low (< 50%
of DM loss) at 12 h.
The incubation time significantly (P <
0.001) influenced DM disappearance. The
major part of the DM loss occurred at 48 h.
Herbert and Thomson [
] used 48 h losses
of DM to account for the disappearance of
fractions of four barley straw genotypes
using the nylon bag procedure. The
by-products with a major percentage of DM loss at
48 h were orange and lemon peels (89.8 and
86.0%, respectively) and broccoli (86.1%).
It is reasonable to expect a good efficiency
in ruminant degradation when the citrus
byproducts are studied because these feeds are
highly fermentable [
]. Also melon,
boiled and fresh artichoke had high values
(77.5, 73.0 and 65.0%, respectively). These
results were expected because Gasa et al.
] reported high values of organic matter
digestibility in sheep for by-products of the
processing industry of the artichoke (75%).
In addition, in our work the red pepper had
intermediary degradability (60.9%) at 48 h
incubation, and Gasa et al. [
indicated an intermediary digestibility value of
61.5% for pepper by-products.
The by-products with less DM loss at
48 h were the brewer’s grains (48.1%) and
giant pumpkin (40.0%). The low values of
DM disappearance were similar to those
observed by Kabatange and Shayo [
maize stover (42.2%) after 48 h incubation
using an in situ method.
The variations in dry matter loss may be
related to the differences in chemical
] or to variations in physical
structure, such as the distribution within the
tissues of lignified cells [
]. Thus, the
byproducts with a high loss of dry matter
(peels, broccoli and melon) had high levels
of cell content (ranging from 70.9 to
75.7%) and low contents of lignin (ranging
from 2.3 to 4.2%). Van Soest [
that the cell content is almost completely
digestible and lignin is the main factor
limiting the digestibility in forages.
In addition, the incubation time × feed
stuff interaction was significant (P < 0.001)
because an increased level of DM loss was
the marker for the peels, broccoli and
melon, followed by artichoke by-products.
These results agreed with those of studies
which showed that DM degradability is
a,b,c,d Means in the same row with different following letters are significantly different.
* P < 0.05; ** P < 0.01; ***P < 0.001.
generally influenced by incubation time
and the type of incubated feed [
9, 11, 25
3.2. Neutral-detergent fibre degradation
The fermentation characteristics of the
NDF of by-products are given in Table II.
There were significant differences (P < 0.001)
in NDF disappearance between the
feedstuffs. Also, incubation time significantly
(P < 0.001) influenced the NDF
disappearance. The major part of the NDF loss of
byproducts occurred after 12 h incubation and
there were no high differences in NDF
disappearance between 48 and 72 h of
incubation. The highest NDF loss at 48 h was
observed in orange and lemon peels (79.2%
and 70.1%, respectively) and broccoli
(70.9%), and the lowest NDF loss at 48 h
was observed for the red pepper (37.0%),
giant pumpkin (34.3%) and brewer’s grains
These results showed that citrus peels
and broccoli by-products are digestible
fibre sources. These characteristics and the
high extent of their fibre digestion in the
rumen support that they can be characterised
as an energy feed, and have more in
common with spring forage, sugar beet pulp or
root crops than with cereal grains. In
addition, Duran et al.  indicated that cell-wall
constituents of citrus and beet pulp are
more extensively degraded than the other
by-products, such as wheat bran or straw.
The melon, and boiled and fresh
artichoke had a medium-loss of NDF at 48 h
(53.5, 59.0 and 46.6%, respectively).
However, melon had a high percentage of DM
loss at 48 h (> 75%) and it could also be
considered as an energy feed with a very
different carbohydrate profile than the
usual raw materials used by stock feed
manufacturers. On the contrary, boiled
artichoke had higher NDF and DM losses
(59.0 and 73.0%, respectively) than the
fresh artichoke (46.6 and 65.0%,
respectively). These results were probably due to
the effect of boiled industrial processes on
the chemical and physical structure of the
In addition, the incubation time × by
product type interaction was significant
(P < 0.001) because an increased level of
NDF loss was a marker for peels and
broccoli followed by melon and artichoke
byproducts more so than in the red pepper,
giant pumpkin and brewer’s grains.
3.3. Volatile fatty acids, lactic acid and ammonia production
The average concentration of volatile
fatty acids and the molar proportions of
each individual acid in the rumen liquor at
72 h of incubation are presented in
Table III. The total concentrations of VFA at
72 h did not significantly (P > 0.05) differ
for the by-products. However, the
by-product type significantly (P < 0.001)
influenced the VFA concentration in bifactorial
analysis. The values of VFA at 72 h
incubation in the red pepper (62.5 mM), brewer’s
grains (57.1 mM) and giant pumpkin
(52.9 mM) were lower than those of the
other by-products (ranging from 85.4 to
96.8 mM), reflecting the lower DM and
NDF degradabilities. Pitt et al. [
confirmed the importance of these fermenting
structural carbohydrates and fermenting
non-structural carbohydrates on ruminal
VFA. Also, Khandaker et al. [
that a higher concentration of VFA suggests
an increased rumen microbial activity due
to more quantities of organic matter being
fermented in the rumen. Durand et al. 
used the rumen simulation technique to
compare various by-products with a wide
range of fermentability which could be
divided into 4 groups with decreasing values
according to organic matter digestibility
and total VFA production: pulp
by-products (80%, 93 mM), cereal by-products
(68%, 69 mM), NaOH-treated straw and
hay (55%, 60 mM) and untreated and
NH3treated straw (34%, 40 mM).
The molar proportion of acetate and
propionate were influenced by the type of
feedstuffs. Thus, the acetate proportion was
> 70% for all by-products, except for the
brewer’s grains (63.8%), and the
propionate proportion was < 20% for all
by-products, except for the brewer’s grains
(21.3%). Durand et al.  reported that in
comparison with other groups (citrus pulp,
beet pulp, maize gluten meal and
roughages), cereal by-products showed a
particular VFA pattern with a high molar
proportion of propionate and low
proportion of acetate, probably because of their
residual starch content. It is agreed with
respect to the molar proportion of VFA that
high levels of acetate usually occur in
animals fed rations containing large amounts
of roughage, whereas lower levels are
associated with concentrates. Even though, the
citrus peel is an energetic concentrate feed
for ruminants, the effect of incubation
citrus peel in the rumen liquor did not reduce
the acetic acid as expected. Schaibly and
] reported that rations with citrus
pulp increased the molar percent acetic
acid. Ben-Ghedalia et al. [
barley with citrus pulp in diets for sheep and
observed greater acetate: propionate ratios
in the ruminal fluid of the sheep fed the
citrus pulp. This effect may be explained by
the higher pectin content of citrus
by-products. These structural carbohydrates are
rapidly fermentable and are related to acetic
In addition, the isovalerate proportion
was influenced by the type of by-products.
The highest molar percentage of isovalerate
was observed in the brewer’s grains (3.4%).
This is supported by the high level of crude
protein of this by-product. Thus, Yan et al.
] observed that supplements of
soyabean meal and fish meal significantly
increase the average molar proportions of
isobutyrate and isovalerate in the rumen.
This effect is probably derived from the
increased deamination of branched-chain
amino acids in the rumen. Thus, brewer’s
barley grains had the highest levels of
ammonia concentration at 12 h incubation
(5.3 mg·100 mL–1) (Tab. IV). Barber and
] however, reported that this
byproduct is scarcely degradable and its
protein fraction is relatively insoluble in the
rumen, because the grains are heated during
malting and mashing.
Several in vitro studies have shown that
maximum microbial growth occurs when
the ammonia-N concentration ranges from
5 to 8 mg·100 mL–1 [
]. The major part of
the by-products studied in our experiment
had lower ammonia-N levels at 12 h
incubation than the minimum concentration for
the maximum efficiency of microbial
Incubation time significantly (P < 0.001)
influenced the ammonia concentration, but
tiiaupkpnnGm reeepppdR leonM litrcceoboSm ir’rrseaegnBw tir-rsaehF eckoh ltiir-aedoB eckho leeepnoLm lreeeapgnO tilirfacaoB ilssaayn iitacccadL ianoAmm pH ,,,cadbitseannhM.;050***<<PP
this effect was similar to that of Luchini
et al. [
] and may have resulted from the
catabolism of lysed cells because some
nutrients become exhausted, some toxic waste
products accumulate, balanced growth can
no longer continue, and the bacteria enter a
stationary phase during incubation in vitro.
Lactic acid is another product of
carbohydrate fermentation [
]. In our case, the
feedstuffs significantly (P < 0.001)
influenced the lactic acid concentration
(Tab. IV). The carbohydrates that can lead
to the accumulation of lactic acid are starch,
maltose, sucrose, lactose, cellobiose,
fructose and glucose. The formation of lactic
acid during fermentation is correlated with
the fermentation rate, and the soluble
sugars, sucrose, glucose and raffinose produce
the highest lactic acid concentrations .
Thus, the by-products with the highest
solubility (melon, broccoli and orange peel)
had the highest levels of lactic acid at 12 h
In addition, the incubation time × feedstuff
interaction was significant (P < 0.001)
because the ranges of the levels of lactic acid
at different times from in vitro
fermentations were variable for each by-product.
Thus, the lactic acid concentration of the
giant pumpkin, orange peel and brewer’s
grain fermentations tended (P < 0.05) to
decrease when the incubation time was
increased. This result was similar to that
of Piwonka and Firkins [
], which indicated
that lactic acid could be partially fermented;
they reported that the in vitro digestion trials
contain bacteria that use lactic acid, as
expected in a mixed culture from the rumen,
and that lactic acid is fermented to
propionate (30 to 35%) and butyrate (65 to 70%).
Even so, the lactic acid of the fermentations
of other by-products, such as red pepper,
melon, broccoli, fresh artichoke and lemon
peel were not affected (P > 0.05) by
incubation time, except boiled artichoke.
In vitro pH decreased when the incuba
tion time increased (Tab. IV). This effect
could be caused by an accumulation of
VFA and lactic acid in the in vitro medium.
] reported that low ruminal pH
lowers microbial growth efficiency and
inhibits fibre fermentation. However, in vitro
pH was not less than 6.0 in any case in our
In conclusion, the in vitro determination
of ruminal dry matter and cell wall
degradations, and production of fermentation
endproducts offers a convenient method to
compare microbial digestion of various
byproducts. Thus, the extent of by-product
digestion and their characteristics of ruminal
fermentation support that broccoli, citrus
peels and melon can be characterised as
high-degradable feeds for ruminants
followed by artichoke by-products more than
the red pepper, giant pumpkin and brewer’s
The technical assistance of Antonio Pelegrín is gratefully acknowledged. This work has been support by the Autonomy Government of Murcia Region (Spain) (PCT95/100).
 Åman P. , Nordkvist E. , Chemical composition and in vivo degradability of botanical fractions of cereal straw , Swed. J. Agric. Res . 13 ( 1983 ) 61 - 67 .
 Barber W.P. , Lonsdale C.R. , By-products from cereal, sugar beet and potato processing , in: Ørskov E.R. (Ed.), By-products and Wastes in Animal Feeding, Occasional Publication No. 3, British Society of Animal Production (BSAP) , UK, 1980 , pp. 61 - 69 .
 Ben-Ghedalia D. , Yosef E. , Miron J. , Est Y. , The effects of starch- and pectin-rich diets on quantitative aspects of digestion in sheep , Anim. Feed Sci. Technol . 24 ( 1989 ) 289 - 298 .
 Chaney A.L. , Marbach E.P. , Modified reagents for determination of urea and ammonia , Clin. Chem . 8 ( 1962 ) 130 - 132 .
Cullen A.J. , Harmon D.L. , Nagaraja T.G. , In vitro fermentation of sugar, grains and by-product feeds in relation to initiation of ruminal lactate production , J. Dairy Sci . 69 ( 1986 ) 2616 - 2621 .
Doane P.H. , Schofield P. , Pell A.N. , Neutral detergent fiber disappearance and gas and volatile fatty acid production during the in vitro fermentation of six forages , J. Anim. Sci . 75 ( 1997 ) 3342 - 3352 .
Feng P. , Hunt C.W. , Pritchard G.T. , Julien W.E. , Effect of enzyme preparations on in situ and in vitro degradation and in vivo digestive characteristics of mature cool-season grass forage in beef steers , J. Anim. Sci . 74 ( 1996 ) 1349 - 1357 .
Res. 9 ( 1993 ) 321 - 330 .
 Gasa J. , Castrillo C. , Baucells M.D. , Guada J.A. , By-products from the canning industry as feedstuff for ruminants: Digestibility and its prediction from chemical composition and laboratory bioassays , Anim. Feed Sci. Technol . 25 ( 1989 ) 67 - 77 .
 Grigsby K.N. , Kerley M.S. , Paterson J.A. , Weigel J.C. , Site and extent of nutrient digestion by steers fed a low-quality bromegrass hay diet with incremental levels of soybean hull substitution , J. Anim. Sci . 70 ( 1992 ) 1941 - 1949 .
 Herbert F. , Thomson E.F. , Chemical composition, intake, apparent digestibility and nylon-bag disappearance of leaf and stem fractions from straw of four barley genotypes , Anim. Prod . 55 ( 1992 ) 407 - 412 .
 Hoover W.H. , Chemical factors involved in ruminal fiber digestion , J. Dairy Sci . 69 ( 1986 ) 2755 - 2766 .
 Kabatange M.A. , Shayo C.M. , Rumen degradation of maize stover as influenced by Leucaena hay supplementation , Livest. Res. Rural Dev . 3 ( 1991 ) 19 - 22 .
 Khandaker Z.H. , Steingass H. , Drochner W. , Supplementation of wheat straw with sesbania on voluntary intake, digestibility and ruminal fermentation in sheep , Small Rumin. Res . 28 ( 1998 ) 23 - 29 .
 Khazaal K. , Dentinho M.T. , Ribeiro J.M. , Ørskov E.R. , A comparison of gas production during incubation with rumen contents in vitro and nylon bag degradability as predictors of apparent digestibility in vivo and the voluntary intake of hays, Anim . Prod. 57 ( 1993 ) 105 - 112 .
 Lanza M. , Priolo A. , Biondi L. , Bella M. , Ben Salem H. , Replacement of cereals grain by orange pulp and carob pulp in faba bean-based diets fed to lambs: effects on growth performance and meat quality , Anim. Res . 50 ( 2001 ) 21 - 30 .
 Luchini N.D. , Broderick G.A. , Combs D.K. , In vitro determination of ruminal protein degradation using freeze-stored ruminal microorganisms , J. Anim. Sci . 74 ( 1996 ) 2488 - 2499 .
 Madrid J. , Matínez-Teruel A. , Hernández F. , Megías M.D. , A comparative study on the determination of lactic acid in silage juice by colorimetric, high-performance liquid chromatography and enzymatic methods , J. Sci. Food Agric . 79 ( 1999 ) 1722 - 1726 .
 Madrid J. , Megías M.D. , Hernández F. , Determination of short chain volatile fatty acids in silages from artichoke and orange by-products by capillary gas chromatography , J. Sci. Food Agric . 79 ( 1999 ) 580 - 584 .
 Marten G.C. , Barnes R.F. , Prediction of energy digestibility of forages with in vitro rumen fementation and fungal enzyme systems , in: Pigden W.J., Balch C.G. , Graham M . (Eds.), Standardization of Analytical Methodology for Feeds , Proceedings of workshop held in Ottawa, Ottawa, Canada, 1980 , pp. 61 - 71 .
 Martínez-Teruel A. , Madrid J., Megías M.D. , Gallego J.A. , Rouco A. , Hernández F. , “Uso de forrajes y subproductos en las explotaciones de vacuno de leche de la Región de Murcia” (“Using of forages and by-products in dairy cows farms of Murcia Region”), Arch . Zootec. 44 ( 1998 ) 33 - 42 .
 McDougall E.I. , Studies on ruminant saliva. I. The composition and output of sheep´s saliva, Biochem . J. 43 ( 1948 ) 99 - 109 .
 Megías M.D. , Hernández F. , Madrid J., Martínez A. , Feeding value, in vitro digestibility and in vitro gas production of different by-products for ruminant nutrition , J. Sci. Food Agric . 82 ( 2002 ) 567 - 572 .
 Mustafa A.F. , Christensen D.A. , McKinnon J.J., In vitro and in situ evaluation of fenugreek (Trigonella foenum-graecum) hay and straw , Can. J. Anim. Sci . 76 ( 1996 ) 625 - 628 .
 Ørskov E.R. , Hovell F.D.DeB ., Mould F. , The use of the nylon bag technique for the evaluation of feedstuffs, Trop . Anim. Prod. 5 ( 1980 ) 195 - 213 .
 Pitt R.E. , Van Kessel J.S. , Fox D.G. , Pell A.N. , Barry M.C. , Van Soest P.J. , Prediction of ruminal volatile fatty acids and pH within the net carbohydrate and protein system , J. Anim. Sci . 74 ( 1996 ) 226 - 244 .
 Piwonka E.J. , Firkins J.L. , Effect of glucose fermentation on fiber digestion by ruminal microorganism in vitro , J. Dairy Sci . 79 ( 1996 ) 2196 - 2206 .
 Ramanzin M. , Bailoni L. , Beni G. , Varietal differences in rumen degradation of barley, wheat and hard wheat straws , Anim. Prod . 53 ( 1991 ) 143 - 150 .
 Satter L.D. , Slyter L.L. , Effect of ammonia concentrations on rumen microbial protein production in vitro , Brit. J. Nutr . 32 ( 1974 ) 199 - 208 .
 Schaibly G.E. , Wing J.M. , Effect of roughage concentrate ratio on digestibility and rumen fermentation of corn silage-citrus pulp rations , J. Anim. Sci . 38 ( 1974 ) 697 - 701 .
 Steel R.G.D. , Torrie J.H. , Principles and Procedures of Statistics: A Biometrical Approach , 2nd ed., McGraw-Hill Book Co., New York, 1980 .
 Sutton J.D. , Rumen fermentation and gastro-intestinal absorption: carbohydrates , in: NeimannSorensen A. (Ed.), New Developments and Future Perspectives in Research on Rumen Function, Proceedings of seminar CEE , Forsogsanloeg Foulum , Denmark, 1986 , pp. 21 - 38 .
 Taniguchi K. , Zhao Y. , Uchikawa H. , Obitsu T. , Digestion site and extent of carbohydrate fractions in steers offered by-product diets, as determined by detergent and enzymatic methods , Anim. Sci . 68 ( 1999 ) 173 - 182 .
 Tilley J.M.A. , Terry R.A. , A two-stage technique for the in vitro digestion of forage crops , J. Brit. Grassl. Soc . 18 ( 1963 ) 104 - 111 .
 Van Soest P.J. , Nutritional Ecology of the Ruminant , 2nd ed., Cornell University Press, Ithaca, New York, NY, 1994 .
 Van Soest P.J. , Robertson J.B. , Lewis B.A. , Methods for dietary fiber neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition , J. Dairy Sci . 74 ( 1991 ) 3583 - 3597 .
 Vanzant E.S. , Cochran R.C. , Titgemeyer E.C. , Stafford S.D. , Olson K.C. , Johnson D.E. , Jean G.St. , In vivo and in situ measurement of forage protein degradation in beef cattle , J. Anim. Sci . 74 ( 1996 ) 2773 - 2784 .
 Wilman D. , Foulkes G.R. , Givens D.I. , A comparison of four methods of estimating the rate and extent of cell wall degradation in grass silages , Anim. Feed Sci. Technol . 63 ( 1996 ) 99 - 109 .
 Yan T. , Offer N.W. , Roberts D.J., The effects of dietary nitrogen sources and levels on rumen fermentation, nutrient degradation and digestion and rumen microbial activity by wether sheep given a high level of molasses, Anim . Sci. 63 ( 1996 ) 123 - 131 .