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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 fiber is quite rapid; however, the rate and extent to which fiber 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 fibrolytic 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 lignified stem tissue. This allows the ruminal microbes greater access to the plant stem and the digestible portions of the plant. New developments in fiber 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: 819S–823S, 1997.

KEY WORDS: ruminants fiber digestion

Ruminants have adapted to a variety of ecological niches because of diverse ruminal microbial populations, which con- sist primarily of bacteria, protozoa and fungi. Ruminant ani- mals have the ability to convert low quality feeds into high quality protein and to utilize feeds from land not suitable to grow crops for human consumption. This is made possible by the ruminal microorganisms that synthesize and secrete the b 1-4 cellulase enzyme complex, thereby allowing hydrolysis of plant cell walls. However, the actual conversion of feeds, espe- cially fibrous forages, to meat and milk is not very efficient. Only 10–35% of energy intake is captured as net energy be- cause 20–70% of cellulose may not be digested by the animal.

If a greater percentage of the total dietary energy from forages was available to ruminants, lower cost diets could be formu-

lated

and environmental resources would be used more ef-

ciently. Four major factors regulate ruminant fiber digestion: 1 ) plant structure and composition, which regulate bacterial ac- cess to nutrients; 2 ) nature of the population densities of the

1 Presented as part of the 37th Annual Ruminant Nutrition Conference ‘‘New

Developments in Forage Science Contributing to Enhanced Fiber Utilization

by

Ruminants,’’ given at Experimental Biology 96, April 14, 1996, Washington, DC.

This conference was sponsored by the American Society for Nutritional Sciences

and was supported by grants from Agway, Inc., Carl S. Akey, Inc., A. O.

Smith

Harvestore Products, Inc., Cargill-Nutrena Feed Division, Consolidated Nutrition, L.C., Eli Lilly and Co., Farmland Industries, Inc., Hoffmann-La Roche, Inc., Land

O’Lakes, Mallinckrodt Veterinary, Merck Research Laboratories, Monsanto, Moorman Manufacturing Co., Pioneer Hi-Bred International, Inc., Prince Agri

Products, Inc., and Purina Mills, Inc. Guest editor for this symposium was

G. C.

Fahey, Jr., Department of Animal Sciences, University of Illinois, Urbana, IL. To whom correspondence should be addressed.

2

predominant fiber-digesting microorganisms; 3 ) microbial fac- tors that control adhesion and hydrolysis by complexes of hy- drolytic enzymes of the adherent microbial populations; and 4 ) animal factors that increase the availability of nutrients through mastication, salivation and digesta kinetics (Cheng et al. 1991). The effect of plant structure has been discussed by Buxton and Redfearn (1997) in this symposium. This re- view will focus on limitations of the fibrolytic microorganisms in converting fiber to usable end products and the effect of animal and dietary factors on digestion of fiber.

FIBROLYTIC MICROORGANISMS

Bacteria. The major fibrolytic bacteria include Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus (Cheng et al. 1991). Butyrivibrio fibrisolvens also pro- duces a cellulase, but it is probably more important in the hydrolysis of hemicellulose. Fibrolytic bacteria tend to degrade the more readily digestible structures such as the mesophyll cells, although F. succinogenes digests parenchyma bundle sheaths, epidermal cell walls and leaf sclerenchyma (Akin 1989). F. succinogenes interacts synergistically with non-cellu- lolytic bacteria during forage digestion. Examples of these syn- ergistic actions include a twofold increase in the utilization of orchard grass hemicellulose and pectin by co-culture with the hemicellulolytic bacterium Prevotella ruminocola over that by F. succinogenes alone (Osborne and Dehority 1989). Fungi. Fungi account for approximately 8% of the micro- bial biomass in the rumen (Orpin and Joblin 1988). The fungi seem to have an important role in fiber digestion because they

0022-3166/97 $3.00 1997 American Society for Nutritional Sciences.

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are able to penetrate both cuticle

and cell wall

of

lignified

tissues (Akin 1986). This suggests that fungi have cutinase activity (Kolattukudy 1984). In addition, fungi can degrade more recalcitrant cell wall materials, including the scleren- chyma and vascular tissue (Akin 1989). Fungi degraded 37– 50% of barley straw, whereas rumen bacteria digested only 14–25% (Joblin et al. 1989). The fibrolytic activity of the fungi, which includes both cellulase and hemicellulase activi- ties, is enhanced by hydrogen-utilizing methanogens (Joblin et al. 1989), which decrease the repressive effect of hydrogen

losomes. Of considerable interest is the fact that a large frac- tion (10–50%) of cells within a tested population for each strain of cellulolytic bacteria do not adhere to feed particles, even upon prolonged incubation (Weimer 1993). This may be due to differences in the ages of individual cells or to some subtle genotypic variations within an apparently homogenous microbial population. Information regarding the activity and composition of the cellulase complex of these nonadherent populations could be useful for genetic selection and transfer into other species.

(Orpin and Joblin 1988). The fact that fungi do not predomi- Hydrolytic enzymes of ruminal microorganisms. The ru-

nate in the rumen is primarily a function of their slower genera- tion time in comparison with bacteria (6–9 vs. 0.5–3.5 h, respectively). In addition, fungal growth is repressed by culture with some bacteria, such as Ruminococcus spp. Research to elucidate the interactions of the bacteria and the fungi may result in the development of methods to circumvent this

minal bacteria possess an array of hydrolytic enzymes, includ- ing cellulases and hemicellulases. A detailed discussion of the

microbial enzymes has been presented elsewhere (Forsberg et al. 1986, White et al. 1993). The anaerobic fungi also possess a broad range of fibrolytic enzymes, including cellulases and xylanases. Neocallimastix frontalis has the highest cellulolytic

growth repression. activity of any organism ever reported in the literature.

Protozoa. In vitro studies have suggested that 19–28% of total cellulase activity can be attributed to protozoa (Gijzen et al. 1988). However, digestion seems to be limited to very susceptible tissue such as mesophyll cells (Akin 1989). Studies have demonstrated that defaunation reduces fiber digestion

Borneman et al. (1990) demonstrated the presence of both ferulic and coumaric acid esterase activities in two monocen- tric and three polycentric fungi. The protozoa also possess cellulases, xylanases and a broad range of glycosidases, al- though none have been purified (Cheng et al. 1991).

(Bonhomme 1990, Yang and Varga 1993). However, in the Microbial interactions. Although cells of F. succinogenes absence of protozoa there is an increased requirement for non- alone are unable to clear cellulose agar, these organisms can protein nitrogen because of an increase in the bacterial popula- digest, in combination with Treponema and Butyrivibrio spe- tion. A shortage of nitrogen may therefore account, at least cies, large areas of agar (Kudo et al. 1986). Fondevila and Dehor- in part, for this reduction in fiber digestion (Ushida and Jouany ity (1994) demonstrated through sequential addition studies 1990). Because the difficulty of culturing protozoa free of bacte- that hemicellulose utilization resulted from initial solubilization ria, this microbial group has been ascribed a minor role in the of the hemicellulose from the forage by non-hemicellulose uti- digestion of fiber. lizers (F. succinogenes A3c and R. flavefaciens 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 fi-

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

nins in forages present additional layers of recalcitrant material for the microorganisms to penetrate (Harbers et al. 1981). Bacteria usually gain access to readily digestible inner tissues through stomata, lenticels or damaged areas, and digestion essentially proceeds from the inside out. Ruminal fungi also

degrade the more vulnerable areas of plant tissue, but in addi- tion have the ability to penetrate the plant cuticle. This aids in reducing the tensile strength of the tissue and provides additional sites for bacteria to access and attach (Akin 1986). Ho et al. (1988) described the production of appresorium-like structures where rumen fungal rhizoids come into contact with undamaged rigid cell walls. The appresorium can produce a fine penetration peg at the site of cell wall contact, which penetrates and grows inside the plant cell, thereby forming

new strains of bacteria for improved fiber digestibility (Forsberg et al. 1986, Teather 1985) remains largely unrealized. Strate- gies proposed have included the following: 1 ) increasing the competitiveness of cellulolytic organisms (F. succinogenes, Ruminococcus) by conferring the ability to utilize xylose and pectins, thereby allowing earlier colonization of particulate matter; 2 ) inserting the cellulase gene into numerically pre- dominant species (B. ruminicola); 3 ) increasing the competi- tiveness of cellulolytic species present in the rumen in low numbers (C. polysaccharolyticum) by according the ability to

adhere to feed particles; 4 ) inserting an acid-tolerant cellulase gene into acid-tolerant bacteria (Lactobacillus) to allow fiber fermentation at a ruminal pH less than 6; 5 ) developing a cutinase activity in predominant bacteria; and 6 ) allowing

normal rhizoids. predominant species to degrade arabinose side chains, thereby

The digestion process begins with the association of the microbe with a particular feed. Attachment to substrate occurs through the process of adhesion via protein complexes called

overcoming the rate-limiting effect of lignin. There have been at least 50 scientific reports on the cloning of genes coding for fiber-degrading enzymes (Wallace 1994).

adhesins. Adhesion is followed by successive microbial coloni- To date, only genes encoding antibiotic resistance have

zation within the adherent population until active digestive consortia are formed and nutrients are released from substrate digestion (Cheng et al. 1991). Several researchers have dem- onstrated that attachment of ruminal microorganisms to their substrate is a prerequisite for the digestion of forage particles in the rumen. The degree of colonization and mode of attack are specific to each microbial species (Kudo et al. 1990). Cellu- lolytic bacteria adhere to substrates by means of an extracellu- lar glycocalyx coat and possibly by protuberances called cellu-

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been transferred and expressed in ruminal bacteria. Gene transfer and expression can be complicated by several factors:

1 ) maintenance of the plasmid within the bacteria; 2 ) meta- bolic burden of the plasmid on the host bacterium; 3 ) integra- tion of the inserted genes into the chromosomes of the bacte- ria; 4 ) a possible requirement for post-transcriptional modifi- cation; and 5 ) a requirement for DNA sequences to encode for the export of proteins synthesized by the new genes. Per- haps the most significant hurdle will be the successful establish-

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LIMITATIONS TO RUMINAL FIBER DIGESTION

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ment of these modified bacteria in the rumen, given the various interactions and cross-feeding that occur among ruminal spe- cies. Results from previous introductions of non-genetically modified bacteria have been mixed, indicating a complex set

intensively investigated. In vitro dry matter digestibility was increased 30% and 13% for rice straw leaf and stem, respec- tively (Karunanandaa et al. 1995). Fungal treatment enhanced digestion of the mesophyll tissue and improved access for rumi-

of factors governing the survival of introduced species. nal microorganisms by collapsing the vascular bundles. Effective fiber. Recent use of the term effective fiber (eNDF) acknowledges the different functionality of dietary

ANIMAL FACTORS

AFFECTING MICROBIAL

FIBER DIGESTION

Animal and feeding systems can have a significant effect on the digestion of fiber. Notably, intake, dietary interactions, feeding strategies and feed additives will, to some degree, in- fluence microbial growth and subsequent fiber digestion. Intake. The extent of fiber digestion is the result of compe- tition between the rates of digestion and passage and, as such, is not a static value. Rumen liquid and particulate turnover rates are positively correlated with intake. Thus, as intake increases, the digesta flowing from the rumen will contain feed particles at earlier stages of digestion, and this will result in a lower dry matter digestibility (Russell et al. 1992). Because the rate of degradation of structural carbohydrate is of the same

order as passage rate, at high levels of intake the depression in digestibility of structural carbohydrate can be two to three times greater than that of the faster degrading, nonstructural carbohydrate. Although a high level of intake may depress ruminal fiber digestion, compensation occurs through increases in gross energy intake and hindgut digestion (Bourquin et al.

1990).

Composition of dietary fiber. Rumen available energy nor-

mally limits growth of bacteria, and any additional

organic

matter fermented in the rumen as a result of changing the forage:concentrate ratio will probably increase microbial pro- tein synthesis by providing more energy. Sniffen and Robinson (1987) suggested that the yield of bacteria was maximized with a forage content of 70% in the diet dry matter. Because

structural carbohydrate–fermenting microbes are usually lim- ited by a ruminal pH less than 6 (Hoover 1986), the depression in fiber digestibility at higher inclusion rates of concentrate can most likely be explained by the rapid degradation of non- structural carbohydrate. It is likely that fiber digestion will not be maximized at a single forage:concentrate ratio; rather, it will depend on the various rates of digestion of structural and nonstructural carbohydrate supplied by the forage and the con- centrate. This may be shown indirectly by the studies of Tam- minga (1981), who reported no relationship between for- age:concentrate and bacterial yield. This study used by-product ingredients with a high fiber content, in contrast to the tradi- tional cereal grains. Particle size and chemical and biological treatments. Al-

though physical processing of forages by grinding and

pelleting

does provide a greater surface area for attack by enzymes, utili- zation of structural carbohydrate is not increased; rather, im- provements in animal performance arise primarily from an increased digestible energy intake (Bourquin et al. 1990). In fact, fiber digestibility is reduced by 3.3% as a result of reduced residency time in the rumen. Chemical treatments such as sodium hydroxide, potassium hydroxide and ammonia will par- tially solubilize hemicellulose and lignin, as well as hydrolyze acetic, phenolic and uronic acid esters. Oxidative treatment of forage with sulfur dioxide or peroxide results in the degrada- tion of lignin and extensive solubilization of structural carbo- hydrate (Fahey et al. 1993). Potential improvements in ber digestion could result from the use of alkaline hydrogen perox- ide, which is a combined hydrolytic and oxidative process. The use of white-rot fungi for converting lignocellulosic mate-

rials to more digestible feedstuffs for ruminants has also been

fiber. Milk fat, chewing rate, and particle size have all been used as an index of effective fiber. Currently, the Cornell Net Carbohydrate and Protein System (CNCPS) uses eNDF to adjust ruminal pH and passage rate (Sniffen et al. 1992). Fac- tors other than particle size that influence eNDF include the degree of lignification of the fiber, degree of hydration, and bulk density. The importance of eNDF can be seen in the reduced growth rate of structural carbohydrate–fermenting mi- croorganisms and the reduction in total microbial yield when ruminal pH is lower than 6.2 (this being related to a dietary eNDF of 20%). Research to further quantify the value of eNDF for a range of feeds is much needed. Feeding strategies. Robinson (1989) indicated that fiber digestion may be limited by the order and frequency of sub- strate presentation to the rumen. A total mixed diet provides an optimal balance of nutrients to the microorganisms, thereby stabilizing fermentation. The potential to modify the ruminal environment is perhaps greater when separate, twice-daily feeding of forage and concentrate is practiced. Feeding diets, especially those that are highly fermentable, more frequently than twice a day is generally thought to stabilize the ruminal environment. This reduction in diurnal variation of fermenta- tion end products, in conjunction with improved coupling of protein and energy release in the rumen, can increase the rate of fiber digestion (Robinson 1989). Additives. The addition of buffers and alkalinizing prod- ucts (sodium bicarbonate, sodium sesquicarbonate, magnesium oxide, sodium bentonite) to the diet of lactating dairy cows can improve fiber digestion by reducing the period of time during the day that ruminal pH is less than 6. A buffer may overcome limitations to fiber digestion in diets that have a high proportion of low pH silages, fermented feeds with a moisture content greater than 50%, an acid detergent fiber õ19%, finely chopped haylage, a high proportion of concen- trate, irregular feeding of high levels of concentrate, or finely ground concentrate (Hutjens 1992). The ionophore monensin can improve cellulose digestion of diets high in readily available carbohydrate by inhibiting the growth of lactate-producing bacteria, thereby decreasing lactate concentrations and increasing ruminal pH (Russell and Strobel 1989). Ruminal fill and rate of passage are also influ- enced. Other ionophores reported to produce similar results include lasalocid, salinomycin, lysocellin, narasin and tetro- nasin (Wallace 1994). Further research is required to deter- mine if these function as an antibacterial or an antifungal agent in the rumen and if the efficiency responses in the animal are a result of ruminal or post-ruminal effects. Yeast culture and their extracts, particularly Aspergillus ory- zae and Saccharomyces cerevisiae, have a highly variable effect on animal performance and efficiency. Directly fed microbials, or probiotics, are organisms with the ability to a maintain a bacterial balance in the host animal’s digestive tract during stressful or disease situations. It is currently thought that these additives remove oxygen from the ruminal environment, thereby increasing bacteria viability, and result in pH stability and increased rate (but not extent) of cellulolysis (Wallace 1994). The further development of strains of probiotics to stimulate the growth of specific types of ruminal bacteria may result in diet-specific additives. Recently, the use of extracellu-

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TABLE 1

Research areas that could lead to the enhancement of fiber digestion

Microbial

Animal

Attachment to substrate

Ruminal pH (5.9–6.5) Increase interaction with fungi Increase substrate accessibility Identify optimal particle size Increase substrate availability Evaluate microbial growth phase Identify microbial growth factors Increase rate of hydration of substrate Decrease antiquality factors (tannin, silica) Evaluate importance of spirochetes Determine serotypes of binding proteins

Penetration of substrate

Increase interaction with fungi Determine enzyme induction mechanism Determine surface area exposure necessary Evaluate plant structure and composition Evaluate importance of protozoa

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 nonfiber components Evaluate transfer of fungal enzymes into bacteria Select for bacteria that attack refractory portions of the cell wall

Feeding strategies

Increase frequency of feeding Evaluate timing of feeding Evaluate order of substrate presentation Evaluate direct use of enzymes Increase residence time of substrate Determine optimal carbohydrate availability in the rumen Increase mastication and insalivation

For poor and good quality forages

Evaluate interaction with nonfiber components Evaluate use of biological and chemical treatments Determine interaction with protein Determine hydration potential Determine cationic exchange capacity Determine optimal particle size

lar enzymes that have been protected from the digestive pro- cess has been proposed as a method to improve fiber digestion

involving the lignin-carbohydrate bonds could be isolated and examined for their specific enzymatic and adhering capabili-

(Wallace 1994). ties. The prospects for improved use of fibrous residues rely on enhancing the rate of fermentation of the more readily fermented cell wall constituents. More emphasis could be

ENHANCEMENT OF FIBER DIGESTION:

OPPORTUNITIES

The enhancement of fiber digestion in the rumen is depen- dent upon advances in a number of related areas. The charac- terization of enzymes involved in feed digestion is lagging be- hind the rate at which genes are being cloned. What are the rate-limiting enzymes involved in plant cell wall hydrolysis? The overall constraints to microbial digestion of feeds impede attempts to improve feed utilization via genetic engineering

placed on selection of these microorganisms and their enzyme proles. Feeding management strategies should include evaluation of the order of substrate presentation on cellulolysis as well as particle sizes of feeds to optimize mastication and microbial attachment. Evaluation of physical, chemical and biological treatments of fiber and direct incorporation of selected enzymes into the diet of ruminant animals should continue.

of ruminal microorganisms. A summary of some

opportunities

LITERATURE CITED

for enhancement of fiber digestion is presented in Table 1. Attachment, adhesion, penetration and consortia formation are all essential processes in the digestion of feed by ruminal microorganisms. Information is needed on the microbial fer- mentation rate vs. growth rate as affected by nutrient require-

ments of ruminal microorganisms. Success in improving feed utilization by ruminants, whether through processing of feeds by mechanical, biological or chemical methods or through genetic engineering of ruminal microflora, depends on under-

standing these processes in greater detail. The elucidation

of

the synergy and competition among different species of micro-

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