Conference: New Developments in Forage Science

Contributing to Enhanced Fiber Utilization by Ruminants

Microbial and Animal Limitations to Fiber Digestion and Utilization
1
Gabriella A. Varga
2
and Eric S. Kolver
Department of Dairy and Animal Science, Pennsylvania State University, University Park, PA 16802
ABSTRACT The ruminal microbial populations attack, degrade and ferment structural carbohydrates in forage
cell walls and thereby provide volatile fatty acids and protein to the host animal. Microbial colonization of ber is
quite rapid; however, the rate and extent to which ber is degraded is determined to a considerable degree by
factors such as microbial accessibility to substrate, physical and chemical nature of the forage and kinetics of
ruminal digestion. The physical and chemical nature of forages can present a barrier to their complete digestion
in the rumen, especially the association of lignin with polysaccharide constituents. Adhesin proteins allow bacteria
with cell-bound enzymes to come into intimate contact with their substrates, ensuring that the degradation
products are preferentially available. Research on various brolytic enzymes and cellulose binding domains may
allow for the transfer of novel genetic material to bacteria for enhancing the hydrolysis of plant cell walls. Fungi
may also play an important synergistic role in the ruminal digestion of forages by physically disrupting the lignied
stem tissue. This allows the ruminal microbes greater access to the plant stem and the digestible portions of the
plant. New developments in ber utilization by ruminants are currently under investigation and include genetic
manipulation of ruminal bacteria, chemical and biological treatments of forages, and manipulation of dietary inputs
and feeding management. J. Nutr. 127: 819S823S, 1997.
KEY WORDS: ruminants ber digestion
Ruminants have adapted to a variety of ecological niches predominant ber-digesting microorganisms; 3) microbial fac-
because of diverse ruminal microbial populations, which con- tors that control adhesion and hydrolysis by complexes of hy-
sist primarily of bacteria, protozoa and fungi. Ruminant ani- drolytic enzymes of the adherent microbial populations; and
mals have the ability to convert low quality feeds into high 4) animal factors that increase the availability of nutrients
quality protein and to utilize feeds from land not suitable to through mastication, salivation and digesta kinetics (Cheng
grow crops for human consumption. This is made possible by et al. 1991). The effect of plant structure has been discussed
the ruminal microorganisms that synthesize and secrete the b by Buxton and Redfearn (1997) in this symposium. This re-
1-4 cellulase enzyme complex, thereby allowing hydrolysis of view will focus on limitations of the brolytic microorganisms
plant cell walls. However, the actual conversion of feeds, espe- in converting ber to usable end products and the effect of
cially brous forages, to meat and milk is not very efcient. animal and dietary factors on digestion of ber.
Only 1035% of energy intake is captured as net energy be-
cause 2070% of cellulose may not be digested by the animal.
FIBROLYTIC MICROORGANISMS
If a greater percentage of the total dietary energy from forages
was available to ruminants, lower cost diets could be formu-
Bacteria. The major brolytic bacteria include Fibrobacter
lated and environmental resources would be used more ef-
succinogenes, Ruminococcus avefaciens and Ruminococcus
ciently.
albus (Cheng et al. 1991). Butyrivibrio brisolvens also pro-
Four major factors regulate ruminant ber digestion: 1)
duces a cellulase, but it is probably more important in the
plant structure and composition, which regulate bacterial ac-
hydrolysis of hemicellulose. Fibrolytic bacteria tend to degrade
cess to nutrients; 2) nature of the population densities of the
the more readily digestible structures such as the mesophyll
cells, although F. succinogenes digests parenchyma bundle
sheaths, epidermal cell walls and leaf sclerenchyma (Akin
1
Presented as part of the 37th Annual Ruminant Nutrition Conference New
1989). F. succinogenes interacts synergistically with non-cellu-
Developments in Forage Science Contributing to Enhanced Fiber Utilization by
lolytic bacteria during forage digestion. Examples of these syn-
Ruminants, given at Experimental Biology 96, April 14, 1996, Washington, DC.
This conference was sponsored by the American Society for Nutritional Sciences ergistic actions include a twofold increase in the utilization of
and was supported by grants from Agway, Inc., Carl S. Akey, Inc., A. O. Smith
orchard grass hemicellulose and pectin by co-culture with the
Harvestore Products, Inc., Cargill-Nutrena Feed Division, Consolidated Nutrition,
hemicellulolytic bacterium Prevotella ruminocola over that by
L.C., Eli Lilly and Co., Farmland Industries, Inc., Hoffmann-La Roche, Inc., Land
OLakes, Mallinckrodt Veterinary, Merck Research Laboratories, Monsanto, F. succinogenes alone (Osborne and Dehority 1989).
Moorman Manufacturing Co., Pioneer Hi-Bred International, Inc., Prince Agri
Fungi. Fungi account for approximately 8% of the micro-
Products, Inc., and Purina Mills, Inc. Guest editor for this symposium was G. C.
bial biomass in the rumen (Orpin and Joblin 1988). The fungi
Fahey, Jr., Department of Animal Sciences, University of Illinois, Urbana, IL.
2
To whom correspondence should be addressed. seem to have an important role in ber digestion because they
0022-3166/97 $3.00 1997 American Society for Nutritional Sciences.
819S
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820S SUPPLEMENT
are able to penetrate both cuticle and cell wall of lignied losomes. Of considerable interest is the fact that a large frac-
tion (1050%) of cells within a tested population for each tissues (Akin 1986). This suggests that fungi have cutinase
activity (Kolattukudy 1984). In addition, fungi can degrade strain of cellulolytic bacteria do not adhere to feed particles,
even upon prolonged incubation (Weimer 1993). This may more recalcitrant cell wall materials, including the scleren-
chyma and vascular tissue (Akin 1989). Fungi degraded 37 be due to differences in the ages of individual cells or to some
subtle genotypic variations within an apparently homogenous 50% of barley straw, whereas rumen bacteria digested only
1425% (Joblin et al. 1989). The brolytic activity of the microbial population. Information regarding the activity and
composition of the cellulase complex of these nonadherent fungi, which includes both cellulase and hemicellulase activi-
ties, is enhanced by hydrogen-utilizing methanogens (Joblin populations could be useful for genetic selection and transfer
into other species. et al. 1989), which decrease the repressive effect of hydrogen
(Orpin and Joblin 1988). The fact that fungi do not predomi- Hydrolytic enzymes of ruminal microorganisms. The ru-
minal bacteria possess an array of hydrolytic enzymes, includ- nate in the rumen is primarily a function of their slower genera-
tion time in comparison with bacteria (69 vs. 0.53.5 h, ing cellulases and hemicellulases. A detailed discussion of the
microbial enzymes has been presented elsewhere (Forsberg et respectively). In addition, fungal growth is repressed by culture
with some bacteria, such as Ruminococcus spp. Research to al. 1986, White et al. 1993). The anaerobic fungi also possess
a broad range of brolytic enzymes, including cellulases and elucidate the interactions of the bacteria and the fungi may
result in the development of methods to circumvent this xylanases. Neocallimastix frontalis has the highest cellulolytic
activity of any organism ever reported in the literature. growth repression.
Protozoa. In vitro studies have suggested that 1928% of Borneman et al. (1990) demonstrated the presence of both
ferulic and coumaric acid esterase activities in two monocen- total cellulase activity can be attributed to protozoa (Gijzen
et al. 1988). However, digestion seems to be limited to very tric and three polycentric fungi. The protozoa also possess
cellulases, xylanases and a broad range of glycosidases, al- susceptible tissue such as mesophyll cells (Akin 1989). Studies
have demonstrated that defaunation reduces ber digestion though none have been puried (Cheng et al. 1991).
Microbial interactions. Although cells of F. succinogenes (Bonhomme 1990, Yang and Varga 1993). However, in the
absence of protozoa there is an increased requirement for non- alone are unable to clear cellulose agar, these organisms can
digest, in combination with Treponema and Butyrivibrio spe- protein nitrogen because of an increase in the bacterial popula-
tion. A shortage of nitrogen may therefore account, at least cies, large areas of agar (Kudo et al. 1986). Fondevila and Dehor-
ity (1994) demonstrated through sequential addition studies in part, for this reduction in ber digestion (Ushida and Jouany
1990). Because the difculty of culturing protozoa free of bacte- that hemicellulose utilization resulted from initial solubilization
of the hemicellulose from the forage by non-hemicellulose uti- ria, this microbial group has been ascribed a minor role in the
digestion of ber. lizers (F. succinogenes A3c and R. avefaciens B34b) and subse-
quent fermentation of the soluble polysaccharide by the utiliz-
ing, but non-degrading organism (P. ruminocola H2b).
MICROBIAL ADHESION AND HYDROLYSIS
Association and attachment of microorganisms to -
GENETIC MANIPULATION OF
ber. For digestion to proceed, microorganisms often must pen-
MICROBIAL SPECIES
etrate resistant barriers such as epicuticular waxes and the
cuticle layer that can restrict enzymatic attack. Silica and tan- The potential of recombinant DNA technology to develop
new strains of bacteria for improved ber digestibility (Forsberg nins in forages present additional layers of recalcitrant material
for the microorganisms to penetrate (Harbers et al. 1981). et al. 1986, Teather 1985) remains largely unrealized. Strate-
gies proposed have included the following: 1) increasing the Bacteria usually gain access to readily digestible inner tissues
through stomata, lenticels or damaged areas, and digestion competitiveness of cellulolytic organisms (F. succinogenes,
Ruminococcus) by conferring the ability to utilize xylose and essentially proceeds from the inside out. Ruminal fungi also
degrade the more vulnerable areas of plant tissue, but in addi- pectins, thereby allowing earlier colonization of particulate
matter; 2) inserting the cellulase gene into numerically pre- tion have the ability to penetrate the plant cuticle. This aids
in reducing the tensile strength of the tissue and provides dominant species (B. ruminicola); 3) increasing the competi-
tiveness of cellulolytic species present in the rumen in low additional sites for bacteria to access and attach (Akin 1986).
Ho et al. (1988) described the production of appresorium-like numbers (C. polysaccharolyticum) by according the ability to
adhere to feed particles; 4) inserting an acid-tolerant cellulase structures where rumen fungal rhizoids come into contact with
undamaged rigid cell walls. The appresorium can produce a gene into acid-tolerant bacteria (Lactobacillus) to allow ber
fermentation at a ruminal pH less than 6; 5) developing a ne penetration peg at the site of cell wall contact, which
penetrates and grows inside the plant cell, thereby forming cutinase activity in predominant bacteria; and 6) allowing
predominant species to degrade arabinose side chains, thereby normal rhizoids.
The digestion process begins with the association of the overcoming the rate-limiting effect of lignin. There have been
at least 50 scientic reports on the cloning of genes coding microbe with a particular feed. Attachment to substrate occurs
through the process of adhesion via protein complexes called for ber-degrading enzymes (Wallace 1994).
To date, only genes encoding antibiotic resistance have adhesins. Adhesion is followed by successive microbial coloni-
zation within the adherent population until active digestive been transferred and expressed in ruminal bacteria. Gene
transfer and expression can be complicated by several factors: consortia are formed and nutrients are released from substrate
digestion (Cheng et al. 1991). Several researchers have dem- 1) maintenance of the plasmid within the bacteria; 2) meta-
bolic burden of the plasmid on the host bacterium; 3) integra- onstrated that attachment of ruminal microorganisms to their
substrate is a prerequisite for the digestion of forage particles tion of the inserted genes into the chromosomes of the bacte-
ria; 4) a possible requirement for post-transcriptional modi- in the rumen. The degree of colonization and mode of attack
are specic to each microbial species (Kudo et al. 1990). Cellu- cation; and 5) a requirement for DNA sequences to encode
for the export of proteins synthesized by the new genes. Per- lolytic bacteria adhere to substrates by means of an extracellu-
lar glycocalyx coat and possibly by protuberances called cellu- haps the most signicant hurdle will be the successful establish-
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821S LIMITATIONS TO RUMINAL FIBER DIGESTION
ment of these modied bacteria in the rumen, given the various intensively investigated. In vitro dry matter digestibility was
increased 30% and 13% for rice straw leaf and stem, respec- interactions and cross-feeding that occur among ruminal spe-
cies. Results from previous introductions of non-genetically tively (Karunanandaa et al. 1995). Fungal treatment enhanced
digestion of the mesophyll tissue and improved access for rumi- modied bacteria have been mixed, indicating a complex set
of factors governing the survival of introduced species. nal microorganisms by collapsing the vascular bundles.
Effective ber. Recent use of the term effective ber
(eNDF) acknowledges the different functionality of dietary
ANIMAL FACTORS AFFECTING MICROBIAL
ber. Milk fat, chewing rate, and particle size have all been
FIBER DIGESTION
used as an index of effective ber. Currently, the Cornell Net
Carbohydrate and Protein System (CNCPS) uses eNDF to
Animal and feeding systems can have a signicant effect
adjust ruminal pH and passage rate (Sniffen et al. 1992). Fac-
on the digestion of ber. Notably, intake, dietary interactions,
tors other than particle size that inuence eNDF include the
feeding strategies and feed additives will, to some degree, in-
degree of lignication of the ber, degree of hydration, and
uence microbial growth and subsequent ber digestion.
bulk density. The importance of eNDF can be seen in the
Intake. The extent of ber digestion is the result of compe-
reduced growth rate of structural carbohydratefermenting mi-
tition between the rates of digestion and passage and, as such,
croorganisms and the reduction in total microbial yield when
is not a static value. Rumen liquid and particulate turnover
ruminal pH is lower than 6.2 (this being related to a dietary
rates are positively correlated with intake. Thus, as intake
eNDF of 20%). Research to further quantify the value of eNDF
increases, the digesta owing from the rumen will contain feed
for a range of feeds is much needed.
particles at earlier stages of digestion, and this will result in a
Feeding strategies. Robinson (1989) indicated that ber
lower dry matter digestibility (Russell et al. 1992). Because
digestion may be limited by the order and frequency of sub-
the rate of degradation of structural carbohydrate is of the same
strate presentation to the rumen. A total mixed diet provides
order as passage rate, at high levels of intake the depression in
an optimal balance of nutrients to the microorganisms, thereby
digestibility of structural carbohydrate can be two to three
stabilizing fermentation. The potential to modify the ruminal
times greater than that of the faster degrading, nonstructural
environment is perhaps greater when separate, twice-daily
carbohydrate. Although a high level of intake may depress
feeding of forage and concentrate is practiced. Feeding diets,
ruminal ber digestion, compensation occurs through increases
especially those that are highly fermentable, more frequently
in gross energy intake and hindgut digestion (Bourquin et al.
than twice a day is generally thought to stabilize the ruminal
1990).
environment. This reduction in diurnal variation of fermenta-
Composition of dietary ber. Rumen available energy nor-
tion end products, in conjunction with improved coupling of
mally limits growth of bacteria, and any additional organic
protein and energy release in the rumen, can increase the rate
matter fermented in the rumen as a result of changing the
of ber digestion (Robinson 1989).
forage:concentrate ratio will probably increase microbial pro-
Additives. The addition of buffers and alkalinizing prod-
tein synthesis by providing more energy. Sniffen and Robinson
ucts (sodium bicarbonate, sodium sesquicarbonate, magnesium
(1987) suggested that the yield of bacteria was maximized
oxide, sodium bentonite) to the diet of lactating dairy cows
with a forage content of 70% in the diet dry matter. Because
can improve ber digestion by reducing the period of time
structural carbohydratefermenting microbes are usually lim-
during the day that ruminal pH is less than 6. A buffer may
ited by a ruminal pH less than 6 (Hoover 1986), the depression
overcome limitations to ber digestion in diets that have a
in ber digestibility at higher inclusion rates of concentrate
high proportion of low pH silages, fermented feeds with a
can most likely be explained by the rapid degradation of non-
moisture content greater than 50%, an acid detergent ber
structural carbohydrate. It is likely that ber digestion will not
19%, nely chopped haylage, a high proportion of concen-
be maximized at a single forage:concentrate ratio; rather, it
trate, irregular feeding of high levels of concentrate, or nely
will depend on the various rates of digestion of structural and
ground concentrate (Hutjens 1992).
nonstructural carbohydrate supplied by the forage and the con-
The ionophore monensin can improve cellulose digestion
centrate. This may be shown indirectly by the studies of Tam-
of diets high in readily available carbohydrate by inhibiting
minga (1981), who reported no relationship between for-
the growth of lactate-producing bacteria, thereby decreasing
age:concentrate and bacterial yield. This study used by-product
lactate concentrations and increasing ruminal pH (Russell and
ingredients with a high ber content, in contrast to the tradi-
Strobel 1989). Ruminal ll and rate of passage are also inu-
tional cereal grains.
enced. Other ionophores reported to produce similar results
Particle size and chemical and biological treatments. Al-
include lasalocid, salinomycin, lysocellin, narasin and tetro-
though physical processing of forages by grinding and pelleting
nasin (Wallace 1994). Further research is required to deter-
does provide a greater surface area for attack by enzymes, utili-
mine if these function as an antibacterial or an antifungal
zation of structural carbohydrate is not increased; rather, im-
agent in the rumen and if the efciency responses in the animal
provements in animal performance arise primarily from an
are a result of ruminal or post-ruminal effects.
increased digestible energy intake (Bourquin et al. 1990). In
Yeast culture and their extracts, particularly Aspergillus ory-
fact, ber digestibility is reduced by 3.3% as a result of reduced
zae and Saccharomyces cerevisiae, have a highly variable effect residency time in the rumen. Chemical treatments such as
on animal performance and efciency. Directly fed microbials, sodium hydroxide, potassium hydroxide and ammonia will par-
or probiotics, are organisms with the ability to a maintain a tially solubilize hemicellulose and lignin, as well as hydrolyze
bacterial balance in the host animals digestive tract during acetic, phenolic and uronic acid esters. Oxidative treatment
stressful or disease situations. It is currently thought that these of forage with sulfur dioxide or peroxide results in the degrada-
additives remove oxygen from the ruminal environment, tion of lignin and extensive solubilization of structural carbo-
thereby increasing bacteria viability, and result in pH stability hydrate (Fahey et al. 1993). Potential improvements in ber
and increased rate (but not extent) of cellulolysis (Wallace digestion could result from the use of alkaline hydrogen perox-
1994). The further development of strains of probiotics to ide, which is a combined hydrolytic and oxidative process.
stimulate the growth of specic types of ruminal bacteria may The use of white-rot fungi for converting lignocellulosic mate-
rials to more digestible feedstuffs for ruminants has also been result in diet-specic additives. Recently, the use of extracellu-
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822S SUPPLEMENT
TABLE 1
Research areas that could lead to the enhancement of ber digestion
Microbial Animal
Attachment to substrate Feeding strategies
Ruminal pH (5.96.5) Increase frequency of feeding
Increase interaction with fungi Evaluate timing of feeding
Increase substrate accessibility Evaluate order of substrate presentation
Identify optimal particle size Evaluate direct use of enzymes
Increase substrate availability Increase residence time of substrate
Evaluate microbial growth phase Determine optimal carbohydrate availability in the rumen
Identify microbial growth factors Increase mastication and insalivation
Increase rate of hydration of substrate
Decrease antiquality factors (tannin, silica)
Evaluate importance of spirochetes
Determine serotypes of binding proteins
Penetration of substrate For poor and good quality forages
Increase interaction with fungi Evaluate interaction with nonber components
Determine enzyme induction mechanism Evaluate use of biological and chemical treatments
Determine surface area exposure necessary Determine interaction with protein
Evaluate plant structure and composition Determine hydration potential
Evaluate importance of protozoa Determine cationic exchange capacity
Determine optimal particle size
Rate of digestion
Elucidate enzymatic activity of consortium
Identify microbial synergistic and/or competitive interactions
Decrease phenolic concentration of plants
Decrease generation time of organisms
Determine importance of catabolic and/or regulatory enzyme expression
Determine effect of nonber components
Evaluate transfer of fungal enzymes into bacteria
Select for bacteria that attack refractory portions of the cell wall
lar enzymes that have been protected from the digestive pro- involving the lignin-carbohydrate bonds could be isolated and
examined for their specic enzymatic and adhering capabili- cess has been proposed as a method to improve ber digestion
(Wallace 1994). ties. The prospects for improved use of brous residues rely
on enhancing the rate of fermentation of the more readily
fermented cell wall constituents. More emphasis could be
ENHANCEMENT OF FIBER DIGESTION:
placed on selection of these microorganisms and their enzyme
OPPORTUNITIES
proles.
Feeding management strategies should include evaluation
The enhancement of ber digestion in the rumen is depen-
of the order of substrate presentation on cellulolysis as well as
dent upon advances in a number of related areas. The charac-
particle sizes of feeds to optimize mastication and microbial
terization of enzymes involved in feed digestion is lagging be-
attachment. Evaluation of physical, chemical and biological
hind the rate at which genes are being cloned. What are the
treatments of ber and direct incorporation of selected enzymes
rate-limiting enzymes involved in plant cell wall hydrolysis?
into the diet of ruminant animals should continue.
The overall constraints to microbial digestion of feeds impede
attempts to improve feed utilization via genetic engineering
of ruminal microorganisms. A summary of some opportunities
LITERATURE CITED
for enhancement of ber digestion is presented in Table 1.
Attachment, adhesion, penetration and consortia formation Akin, D. E. (1986) Chemical and biological structure in plants as related to
microbial degradation of forage cell walls. In: Control of Digestion and Metab-
are all essential processes in the digestion of feed by ruminal
olism in Ruminants (Milligan, L. P., Grovum, W. L. & Dobson, A., eds.), pp.
microorganisms. Information is needed on the microbial fer-
139157. Prentice-Hall, Englewood Cliffs, NJ.
mentation rate vs. growth rate as affected by nutrient require- Akin, D. E. (1989) Histological and physical factors affecting digestibility of
forages. Agric. J. 81: 1725.
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Bonhomme, A. (1990) Rumen ciliates: their metabolism and relationships with
utilization by ruminants, whether through processing of feeds
bacteria and their hosts. Anim. Feed Sci. Technol. 30: 203266.
by mechanical, biological or chemical methods or through
Borneman, W. S., Hartley, R. D., Morrison, W. H., Akin, D. E. & Ljungdahl, G.
(1990) Feruloyl and p-coumaroyl esterase from anaerobic fungi in relation genetic engineering of ruminal microora, depends on under-
to plant cell wall degradation. Appl. Microbiol. Biotechnol. 33: 345352.
standing these processes in greater detail. The elucidation of
Bourquin, L. D., Garleb, K. A., Merchen, N. R. & Fahey, G. C., Jr. (1990) Effects
the synergy and competition among different species of micro-
of intake and forage level on site and extent of digestion of plant cell wall
monomeric components by sheep. J. Anim. Sci. 68: 24792495. organisms is necessary to further our knowledge in this area.
Buxton, D. R. & Redfearn, D. D. (1997) Plant limitations to ber digestion and
Opportunities exist to improve overall utilization of ligno-
utilization. J. Nutr. 127: 814S818S.
cellulosic materials as ruminant feeds. Organisms with the ca-
Cheng, K. J., Forsberg, C. W., Minato, H. & Costerton, J. W. (1991) Microbial
ecology and physiology of feed degradation within the rumen. In: Physiologi- pacity to continue their attack on the most refractory sources
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823S LIMITATIONS TO RUMINAL FIBER DIGESTION
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