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Effect of maturity on rumen degradation of tropical and temperate forage cell wall polysaccharides from… Mbugua, David M. 1993

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EFFECT OF MATURITY ON RUMEN DEGRADATION OF TROPICAL ANDTEMPERATE FORAGE CELL WALL POLYSACCHARIDES FROM LEAVES ANDSTEMSbyDavid Mwaura MbuguaB.Sc.(Agric.), University of Nairobi, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDepartment of Animal ScienceWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril, 1993© David Mwaura Mbugua, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of  A--Ai ,^cThe University of British ColumbiaVancouver, CanadaDate DE-6  (2/88)ABSTRACTTwo main experiments were conducted in this study with theaim of better understanding the factors that influence thedegradability of forages by ruminants. In the first part ofthe study, the degradability of leaf and stem fractions of twomature tropical forages, bana grass (Pennisetum purpureum)and silverleaf desmodium (Desmodium uncinatum) were determinedby means of the In situ nylon bag technique.The effective degradabilities of both bana grass stems andleaves DM (50h)^were low (48.2% and 45.3% respectively) anddid not differ (P > 0.05) for the two fractions. Desmodiumstems were less degradable (P < 0.05) than the leaves (40.8%and 59.7% respectively). Neither grass leaves nor stemsdiffered (P > 0.05) in effective degradability of NDF (49.0%vs 51.6% respectively) or its constituent polysaccharides.Bana leaves were higher (P < 0.05) in the potentiallydegradable DM, than the stems but did not differ (P > 0.05) inthe rate at which this fraction was degraded. On the otherhand, desmodium leaves were higher in the potentiallydegradable fraction and the rate at which it was degraded thanthe respective stems. NDF, cellulose and hemicellulose showeda similar trend.The second part of this study was aimed at determiningwhether the degree of plant maturity has any effect on thedistribution of cellulose and linear and branched fractionsof hemicelluloses. Two temperate forages, orchard grassii(Dactylis qlomerata) and tall fescue (Festuca arundinacea)harvested at different stages of growth and separated intoleaves and stems were used. The cell wall content (NDF) andhemicellulose A (linear xylan) showed an increasing trend withmaturity in orchard grass but not in tall fescue.Hemicellulose B (branched xylan) in the sample used in thisstudy did not seem to change with increasing maturity.Stems (DM) of medium and late cut orchard grass were lesseffectively degradable (P < 0.05) than leaves (58.1% vs 62.0%and 52.6% vs 59.2% respectively). The potentially degradablefraction did not differ (P > 0.05) with maturation for the twofractions. The rate of degradation of this fraction was higherfor leaves (P < 0.1). Tall fescue fractions did not differ (P> 0.05) in effective degradability and rate of degradation ofthe potentially degradable fraction.Cellulose and hemicellulose A and B showed a decliningtrend in their effective degradabilities (50h). Among thesepolysaccharides hemicellulose B showed a relatively higherdegradability than the other polymers. Hemicellulose A seemedthe least degradable. It appeared that the polysaccharides ofleaves were relatively more degradable than those of stems.Tall fescue polysaccharides seemed to differ in effectivedegradability in both leaves and stems at the two cuttingdates other than for hemicellulose A. Hemicellulose B inorchard grass was associated with relatively high rates ofdegradation compared to the other polymers.Table of ContentsAbstract ^Table of Contents^ ivList of Tables viiList of Figures ^ ixList of Appendix Tables^Acknowledgement ^ xi1 GENERAL INTRODUCTION^ 12 LITERATURE REVIEW 52.1 PLANT FACTORS THAT INFLUENCE FORAGE QUALITY^ 52.1.1 Introduction^ 52.1.2 Plant maturity 52.1.3 Plant anatomy^ 82.1.4 Plant biochemistry 102.1.5 Chemical composition of forages^ 112.1.6 Summary statement^ 222.2 CELL WALL CHEMISTRY, ITS RELATION TO DIGESTIBILITY^ 232.2.1 Introduction^ 232.2.2 Cellulose 242.2.3 Hemicellulose ^ 262.2.4 Phenolic-Hemicellulose complexes^ 282.3 DIGESTION OF PLANT CELL WALLS BY RUMEN MICROBES^ 312.3.1 Introduction^ 312.3.2 Mechanism of cellulose degradation^ 332.3.3 Mechanism of hemicellulose degradation^ 362.3.4 Summary statement^ 392.4 METHODS OF EVALUATING THE NUTRITIVE VALUE OF FORAGES ^ 41iv2.4.1 Introduction^ 412.4.2 Chemical procedures^ 412.4.3 Biological procedures 432.4.3.1 Enzymatic methods^ 432.4.3.2 In vitro methods 452.4.3.3 In situ techniques^ 472.4.5. Summary statement 502.5 OVERALL SUMMARY^ 523 EXPERIMENTAL: DEGRADATION OF TROPICAL FORAGES ^ 543.1 Introduction^ 543.2 MATERIALS AND METHODS^ 553.2.1 Forages^ 553.2.2 In situ incubations^ 563.2.3 Experimental design 573.2.4 Chemical analyses^ 583.3 RESULTS AND DISCUSSION 593.3.1 Chemical composition^ 593.3.2 Degradation of DM, the cell wall andconstituents^ 613.4 SUMMARY AND CONCLUSIONS^ 734 EXPERIMENTAL: DEGRADATION OF TEMPERATE FORAGES ^ 764.1 Introduction^ 764.2 Objectives 774.3 MATERIALS AND METHODS^ 784.3.1 Forages^ 784.3.2 In situ incubations^ 794.3.3 Experimental design 804.3.4 Chemical procedures^ 824.4 RESULTS AND DISCUSSION^ 874.4.1 Chemical composition 874.4.2 Rates and extents of DM and cell walldegradation^ 924.4.2.1 DM degradation^ 924.4.2.2 Degradation of cell wall polymers^ 994.5 SUMMARY AND CONCLUSIONS^ 120BIBLIOGRAPHY^ 122APPENDIX 135viList of Tables Table 3.1 Chemical composition of bana grass and desmodiumleaves and stems^ 60Table 3.2 Extent of degradation of DM in bana grass andsilverleaf desmodium 62Table 3.3 Extent of degradation of NDF in bana grass andsilverleaf desmodium^ 64Table 3.4 Extent of degradation of hemicellulose in banagrass and silverleaf desmodium^ 65Table 3.5 Extent of degradation of cellulose in banagrass and silverleaf desmodium 66Table 3.6 Bana and desmodium DM degradation parameters^ 68Table 3.7 Bana and desmodium NDF degradation parameters^ 68Table 3.8 Bana and desmodium Cellulose degradationparameters^ 69Table 3.9 Bana and desmodium Hemicellulose degradationparameters 69Table 3.10 Effective degradabilities of DM at differentrumen passage rates^ 71Table 3.11 Effective degradabilities of NDF at differentrumen passage rates 71Table 3.12 Effective degradabilities of cellulose atdifferent rumen passage rates^ 72Table 3.13 Effective degradabilities of hemicellulose atdifferent rumen passage rates 72Table 4.1 Chemical composition of orchard grass.leaf andstem fractions^ 91Table 4.2 Chemical composition of tall fescue.leaf andstem fractions 92Table 4.3 Extent of DM degradation for orchard grassleaf and stem fractions^ 95Table 4.4 Extent of DM degradation for tall fescue.leafand stem fractions 95Table 4.5 Orchard grass DM degradation constants^ 97viiTable 4.6 Effective DM degradation values for orchardgrass^ 98Table 4.7 Tall fescue DM degradation constants^ 98Table 4.8 Effective DM degradation values for tall fescue.. ^ 99Table 4.9 Extent of degradation of cellulose in orchardgrass^ 101Table 4.10 Orchard grass cellulose degradation constants^ 102Table 4.11 Extent of degradation of cellulose in tallfescue grass^ 102Table 4.12 Tall fescue cellulose degradation constants^ 102Table 4.13 Extent of degradation of hemicellulose A inorchard grass^ 106Table 4.14 Orchard grass hemicellulose A degradationconstants 106Table 4.15 Extent of degradation of hemicellulose A intall fescue grass^ 107Table 4.16 Tall fescue hemicellulose A degradationconstants^ 107Table 4.17 Extent of degradation of hemicellulose B inorchard grass 111Table 4.18 Orchard grass hemicellulose B degradationconstants^ 111Table 4.19 Extent of degradation of hemicellulose B intall fescue grass^ 112Table 4.20 Tall fescue hemicellulose B degradationconstants^ 112vii iList of Figures Figure 3.1 DM degradability for desmodium leaves and stems ^ 63Figure 4.1 Schematic presentation of the carbohydratefractionation procedure^ 84Figure 4.2 Orchard grass DM degradability curve^ 94Figure 4.3 Orchard grass hemicellulose B degradabilitycurve^ 110Figure 4.4 Cellulose degradability in orchard grass^ 116Figure 4.5 Linear xylan degradability in orchard grass^ 116Figure 4.6 Branched xylan degradability in orchard grass... ^ 117Figure 4.7 Cellulose degradability in tall fescue grass^ 117Figure 4.8 Linear xylan degradability in tall fescue grass. ^ 118Figure 4.9 Branched xylan degradability in tall fescuegrass^ 118ixList of Appendix Tables Appendix Table 1 ANOVA for tropical forages DM (48h) ^ 137Appendix Table 2 ANOVA for orchard grass DM (48h) 135Appendix Table 3 ANOVA for tall fescue grass DM (48h) ^ 135Appendix Table 4 ANOVA for tropical forages DM (96h) ^ 135Appendix Table 5 ANOVA for orchard grass DM (72h) 136Appendix Table 6 ANOVA for tall fescue grass DM (72h) ^ 136Appendix Table 7 Degradation values for orchard grasscellulose^ 136Appendix Table 8 Degradation values for orchard grasshemicellulose A^ 137Appendix Table 9 Degradation values for orchard grasshemicellulose B 138Appendix Table 10 Degradation values for tall fescue grasscellulose^ 139Appendix Table 11 Degradation values for tall fescue grasshemicellulose.A^ 140Appendix Table 12 Degradation values for tall fescue grasshemicellulose B 141Appendix Table 13 Effective degradability for orchard grasscellulose^ 141Appendix Table 14 Effective degradability for orchard grasshemicellulose A^ 142Appendix Table 15 Effective degradability for orchard grasshemicellulose B 142Appendix Table 16 Effective degradability for tall fescuegrass cellulose^ 142Appendix Table 17 Effective degradability for tall fescuegrass hemicellulose.A^ 143Appendix Table 18 Effective degradability for tall fescuegrass hemicellulose B 143xACKNOWLEDGEMENTI wish to express my most sincere gratitude to my researchsupervisor Dr. R.M. Tait, Associate Professor, Department ofAnimal Science, for providing me with his support andinvaluable guidance throughout the entire duration of thisstudy.I am also most grateful to the other members of mygraduate committee Dr. J.A. Shelford, Dr. L.J. Fisher and Dr.M. Pitt for their helpful comments and advice during thecourse of this research.Sincere thanks also go to the Kenya Government and theCanadian International Development Agency (C.I.D.A.) forawarding me the scholarship that made this study possible. Mygratitude also goes to J.N. Mwangi of Simon Fraser Universityfor his help in data analyses.Finally, I would like to dedicate this thesis to my mum, areally wonderful 'mami'. Thanks to God who made it happen.x iCHAPTER ONEGENERAL INTRODUCTIONForty per cent or more of the earth's landmass is composedof rangeland which is more suited for grazing than forcultivation (Church, 1988). Products useful to humans fromsuch areas would be greatly reduced if grazing animals werenot available to utilize the vegetation to some degree.Forages provide the majority of feed for ruminant animals(Minson, 1990). Ruminants have played a major role in farmingproduction, providing mankind with meat, milk, clothing anddraft power. The tremendous diversity in these animals makesit possible for them to adapt to a wide range of climates;they are endowed with a capacity to feed on a wide range oftemperate and tropical vegetation (Hobson, 1988). The uniquepresence of the rumen, a complex anaerobic microbial ecosystemin the foregut of these animals enables them to fermentforages of diverse nature and origin.The grazing animal exists in a highly dymamic situation inwhich its performance , in terms of growth , milk or woolproduction is determined by the quantity and quality offorage available. The principal nutritional constraint onanimal productivity on a world-wide basis is the intake ofdigestible nutrients, particularly available energy (Reid andJung, 1982). The primary sources of energy found in foragesare the structural polysaccharides, cellulose, hemicelluloseand pectins ( Dehority, 1991).Within a sward , forage quality varies not only with1species of plant, but also with the stage of growth, plantparts, and with climatic conditions ( Norton, 1982). Foragequality can be evaluated in terms of digestibility orfermentation of plant constituents and in terms of quantityof feed that ruminants consume (Akin, 1989). Variation in thedigestibility of organic matter in forages is related tovariation in the content of indigestible entities and thevariable digestibilities of their potentially digestiblestructural carbohydrates (Ellis et al., 1988). The extendedincubation of forage and cereal straws in the rumen leaves aresidual fraction of cell wall polysaccharides highlyresistant to further microbial degradation (Gordon et al.,1983). Variation in the digestibility of structuralcarbohydrates is due to both their intrinsic properties and tostructural and chemical properties of the plant tissues formedby these structural carbohydrates and other chemical entitiesof cell wall contents.Factors limiting microbial degradation of plant cell wallshave previously been attributed to features such as physicalencrustation with lignin and cellulose structure ( Raymond,1969). However Morrison (1973) suggested that lignin is moreclosely associated with hemicelluloses than with cellulose.Harkin (1973) suggested a possible existence of covalentlinkages between lignin and the cell wall polysaccharides.Brice and Morrison (1982) reported that hemicellulose finestructure, particularly its xylose to arabinose ratio, wassignificant in influencing how this polysaccharide was2degraded in the rumen. This ratio is known to increase withplant maturation.Hemicelluloses of pasture plants consist of a mixture ofthe following three major polysaccharide types:^(a)linear A (hemicellulose A),^a^water-insolubleheteroxylan containing uronic acid but only smallamounts of arabinose;(b) a more soluble hetero-xylan containing much morearabinose and less uronic acid than linear A;(c) a water soluble, highly branched polymer which inaddition to pentoses, is rich in galactose and uronicacid(b+c) = hemicellulose B (Gaillard et al., 1965).In their pure forms these polysaccharides are highlydegraded by mixed cultures of rumen bacteria (Bailey andGaillard, 1965).Factors that limit the utilization by ruminants ofcellulose and hemicellulose need to be better understood.Factors such as cellulose crystallinity and linearity andbranching of hemicelluloses have been suggested as potentiallimitations to cell wall digestibility by ruminants (Morrison,1979). These factors might reflect on voluntary intake of theruminant animal and hence productivity.In the work presented here, two mature tropical forages,bana grass (Pennisetum purpureum) and the legume silverleafdesmodium (Desmodium uncinatum) were separated into leaf andstem fractions and their cell wall degradation3characteristics studied. Their cellulose and hemicellulosecomponents were also studied for these characteristics. Theresults were interpreted in relation to concentrations ofthese polysaccharides and lignin and suggestions were made totry to overcome some of the problems related to theirindigestibility.To further understand the factors that may lead to lowdegradability of these polysaccharides in the rumen, afractionation scheme for cell wall components was adoptedwhich facilitated the separation of linear and more branchedfractions of hemicellulose. This work was done using twotemperate grasses; orchard grass (Dactylis qlomerata) and tallfescue (Festuca arundinacea) at different stages of maturity.They were also separated into leaf and stem fractions.Digestion kinetic models were developed for the various cellwall polymers. An effort has also been made to relate theobserved results to possible effects on parameters that affectproduction, such as voluntary intake. The rapid decline in thedigestibility of orchard grass within short periods of growth(Shelford and Fisher, 1988) warranted a closer study of thepossible factors leading to this.4CHAPTER TWOLITERATURE REVIEW2.1^PLANT FACTORS THAT INFLUENCE FORAGE QUALITY2.1.1 IntroductionForages are the major source of feed for ruminants in manyparts of the world. The quality of a forage has its ultimateexpression in terms of animal performance when it is fed asthe sole source of digestible nutrients. Forages, however, arediverse in their characteristics, and this diversity resultsin variations in quality as an animal feed. Pasture plantsdiffer in species, stage of maturity, morphology, anatomy,biochemistry, and in chemical components in cell contents andcell walls (Norton, 1982). These factors influence voluntaryintake, digestibility and efficiency of utilization ofabsorbed nutrients. In the following sections a briefdiscussion is given covering differences in stage of maturity,anatomy, and biochemistry in relation to their effects onvoluntary intake and digestibility. The role of chemicalcomposition in this respect will be given a more thoroughdiscussion.2.1.2 Plant maturityThe factors which influence forage growth are dynamic, andchange significantly with time. Consequently the chemical andphysical characteristics of forages are influenced by thestage and the rate of growth of the crop, as well as by the5previous management of the sward. Mowat et al. (1965) studiedthe effect of maturation on crude protein (CP) content and invitro dry matter digestibility (IVDMD) of leaves and stems ofvarious temperate forages. They reported that the CP contentof both leaf and stem portions decreased with increasingmaturity. Norton (1982) made a similar observation for bothtemperate and tropical forages.In the early stages of growth, the cell contents (cellnucleus and the cytoplasm) may account for at least two-thirdsof forage dry matter, with protein being the major contributor(Gill et al., 1989). As the plant matures, the proportion ofthe cell walls and its constituent fractions increases and thecell content decreases (Van Soest, 1982; Jung and Vogel,1992). This situation leads to a decline in digestibility offorages. With maturation the protein content of legumesdeclines only slowly compared to grasses (Norton, 1982).Older leaves show the greatest decrease. The higher proteincontent in legumes than grasses and its maintenance withmaturity may be associated with the continuous supply ofnitrogen available from rhizobial fixation. Variations betweenlegume species in protein content probably reflects on theeffectiveness of rhizobial nitrogen fixation under differentenvironmental conditions (Norton, 1982).Maturation has been reported to lead to decreased IVDMD ofboth leaf and stem fractions in grasses (Mowat et al., 1965).These workers observed that at early growth stages thedigestibility of stems of grasses was higher than that of6leaves. In orchard grass, even at head emergence, stems wereslightly more degradable than leaves. Shelford and Fisher,(1988) have also reported on the effect of advance in maturityin grasses on DM digestibility. These researchers reported adecline of 11 percentage points in the digestibility oforchard grass harvested within a space of 10 days. Gill et al.(1989) reported that cellulose, hemicellulose and lignin allincrease with advancing maturity but indicated that it is theincreased lignin, in particular its spatial distribution,which has the most significant negative effect on the rate andextent of digestion of a forage.Leaves of tropical grass species tend to be lessdigestible than those of temperate grasses (Hacker and Minson,1981). These workers reported that in maize and tropicalgrasses, high temperatures during growth result in decreasedleaf and stem digestibility. Increasing day temperature wasreported to decrease digestibility in leaves of Cenchrus ciliaris and Pennisetum clandestinum; however high nighttemperatures increased the digestibility of leaves of P.clandestinum (Hacker and Minson, 1981). Both tropical andtemperate grasses decline in dry matter digestibility at amean rate of about 0.6% units per degree centigrade (Minsonand Wilson, 1980).72.1.3 Plant anatomy2.1.3.1 Leaf bladesThe amount and arrangement of tissues in forages caninfluence their quality. Tissues in the leaf blades of allgrasses include vascular tissue (divided into phloem and xylemcells), parenchyma bundle sheath(s) surrounding the vasculartissue, sclerenchyma patches connecting the vascular bundlesto the epidermises, single-layered abaxial and adaxialepidermal cells covered by a protective cuticle, and mesophyllcells between the vascular bundles and epidermal layers(Metcalfe, 1960).The first stable products of photosynthesis in tropicalgrasses are four-carbon compounds, while those of temperategrasses and dicotyledons are three-carbon compounds. Hencethese plants are referred to as C4 and C3 respectively.Generally, grasses possessing the C3 pathway forphotosynthesis have a higher ratio of mesophyll to vasculartissue than grasses that possess the C4 photosyntheticpathyway (characterized by a specialized leaf anatomy - theKranz anatomy)(Akin, 1986). The mesophyll, devoid of lignin,is the tissue most easily degraded by microorganisms in therumen. In tropical grasses intercellular air spaces representonly 3-12% of leaf volume compared with 10-35% in temperatespecies (Norton 1982). The lower surface area to weight ratiofor both mesophyll and bundle sheath tissues restrictsaccessability of plant cells to microbial digestion in the8rumen (Hanna, et al 1973), thereby decreasing the rate ofdigestion of the bundle sheath and enclosed vascular tissue(Akin and Burdick, 1975).In C3 plants, the parenchyma bundle sheath is a distinctstructure but its cell wall appears to degrade as rapidly asthe wall of the mesophyll (Akin, 1986). This structure in C4plants appears to be a major factor that often occupies aprominent part in the undegradable residue of C4 plants. StemsForage stems contain a large proportion of lignifiedtissues which resist microbial degradation. Stem anatomy of C4and C3 plants seems to be similar, with the epidermis,sclerenchyma ring and vascular xylem occupying 28-34% of thecross-sectional area (Akin, 1989). These tissues are highlylignified and are totally indigestible. Parenchyma tissuesvary in lignin histology and digestibility depending on ageand species. In general, grasses tend to show the followingtrend in tissue degradability:mesophyl, phloem > epidermis, parenchyma bundle sheath >sclerenchyma > lignified tissues (Akin, 1982).Legume stems can be highly digestible in young plants, butas they mature they become lower in digestibility andcontribute primarily to the indigestibility of the plant. Inlegumes it appears that parenchyma tissues do not lignify withplant maturity as in grasses (Akin, 1989). As such, thistissue is totally degraded in legumes.^Differential9lignification of legume tissues relative to the grasses is afactor responsible for differential breakdown, which mayaccount for greater intake of legumes over grasses for a givenstage of maturity (Demarquilly and Jarrige, 1974).2.1.4 Plant biochemistryThe main factors regulating the intake of pasture byanimals are the density of the sward, the level of fiber andits physical composition, provided there is adequate proteinminerals and vitamins. Feed intake is severely depressed whencrude protein content of pasture falls below 6-8% (Minson,1982). Tissue nitrogen content of C4 plants is usually lowerthan that of C3, mainly because of the higher efficiency ofnitrogen use associated with growth of these tropical plants(Norton, 1982). Brown (1978) indicated that the higher growthrate, nitrogen use efficiency and lower tissue nitrogencontent of C4 plants than C3 plants is related to differencesin the pathways of carbon fixation. The major difference inthe biochemical pathways for C3 and C4 plants that mightinfluence quality is that in C4 plants, the firstphotosynthetic products are oxaloacetic acid, malic acid andaspartic acid whereas in C3 plants phosphoglyceric acid isproduced (Norton, 1982).In C3 plants, fraction 1 protein (ribulose-1,5-biphosphate) represents about 50% of the soluble protein foundin the mesophyll cells. The low activity of this enzyme isreported to be the main factor limiting carbon fixation during10photosynthesis. On the other hand the concentration of thisenzyme is low (about 20%) in C4 plants and restricted to thebundle sheath cells (Bjorkmann et al., 1976).Phosphoenolpyruvic acid carboxylase is found in relatively lowconcentrations in C4 mesophyll cells but its higher activityresults in high rates of CO2 fixation per unit of cellularprotein when compared to C3 plants. Norton (1982) concludedthat the low protein content often found in tropical grasses,even when fertilized with nitrogen, is an inherentcharacteristic of C4 plants, which is closely related to theirsurvival under conditions of low soil fertility. Colman andLazenby (1970) have shown that at higher temperatures (23-35°C), the efficiency of nitrogen use by C4 plants is higherthan at low temperatures (13-24°C), but tissue nitrogencontent is also decreased by high temperatures.2.1.5 Chemical composition of foragesThe availability of nutrients in a feeddetermined by the chemical composition of therespect to the concentrations of availablecomponents and secondly, through organicis essentiallyfeed: first withand unavailablestructures andinhibitors that may limit the availability of the componentswith which they are associated.As pasture plants mature there is usually an increase inthe proportion of fiber and a reduction in the protein andnon-structural carbohydrates of the cell contents. In a studyof 82 samples of grass and legumes, Van Soest (1965) reported11significant correlations between intake and neutral detergentfiber (r = - 0.65), protein (r = 0.54), and acid detergentfiber (r = -0.53) but not with lignin (r = -0.13). Thevoluntary intake of forage has been positively related to thedigestibility of dry matter and energy (Minson, 1982). Thisrelationship is associated with the main factors that controlvoluntary intake, namely the proportion of indigestibleresidue in the feed, the transit time of this residue throughthe rumen and the size of the rumen. The chemical compositionof any given forage will vary depending on its species, stageof maturity, soil fertility, plant part and climaticconditions. The following sections focus on protein andcarbohydrate sources in forages and how they relate tovoluntary intake and digestibility. Herbage proteinsThe crude protein fraction, i.e. total nitrogen (N), offorages consists of proteins (true), amino acids, amides,ureides and nitrates (NPN). True protein constitutes about 60-80% of total nitrogen (Minson, 1990). The protein in freshforages may be classified into three main categories: fraction1 - a single protein, i.e., ribulose-1,5-biphosphate (RUDP)carboxylase; fraction 2 - a mixture of cytoplasmic and otherchloroplastic proteins; and fraction 3 - a mixture includingchloroplastic, nuclear, and mitochondrial membrane proteins(Mangan, 1982). About 50% of the cell protein is RUDPcarboxylase or fraction 1 protein. Fractions 1 and 2 are12soluble and are likely to be fermented in the rumen, althoughrates may vary for individual proteins (Nugent and Mangan,1981). The extent of dietary protein degradation in the rumenhas been correlated with protein solubility (Buttery andLewis, 1982). Forage protein solubility may be influenced bythe presence of tannins (Norton, 1982) which also reduce thesusceptibility of the protein to microbial fermentation.Temperate grasses generally contain more crude protein(Cl') than tropical grasses, with mean concentration of 12.9and 10.0% DM, respectively (Minson, 1990). Temperate andtropical legumes have the same leaf anatomy, C3 pathway ofphotosynthesis,^and similar levels of crude protein;concentration of Cl' were 17.5 and 16.6% DM,^respectively(Minson, 1990). The low protein content of tropical grassesposes a major limit to intensive forms of animal production,and the inclusion of tropical legumes in these pasturesimproves animal production by increasing protein availabilityfor grazing animals (Norton, 1982). Immature forages containhigh levels of crude protein of which more than 80% is trueprotein, found mainly in the cytoplasm. As immature foragescontain low levels of cell wall constituents and are readilyfermentable, they form a rich source of both protein andenergy for rumen microbes (Hogan, 1982). As the plant maturesthere is a rapid decline in protein content, especially ingrasses as the proportion of leaf decreases, which poses amajor criterion in the management of forage quality forgrazing animals. Protein content decreases in both stems and13leaves as the plant ages, but the decline is slower withleaves than with stems.Minson (1990)^reported that there is a rapid decline involuntary intake when crude protein in a feed material fallsbelow about 7.0% DM. When low quality roughages are notlimited in quantity, protein is the most beneficial supplement(DelCurto, 1991). However, the exact mechanisms involved withthe stimulation of feed intake are not clearly defined, andin all likelihood are a combination of several factors. Thedepressing effect on intake of a deficiency of protein appearsto be caused by factors other than rumen distension, sincegrinding and pelleting protein deficient herbage has littleeffect on intake (Minson, 1967). The depression of intakeappears to be caused by a deficiency of circulating aminoacids,^since intake of a protein deficient diet can beincreased if casein (but not urea)^is infused into theduodenum (Egan, 1965). Herbage carbohydratesCarbohydrates are the main repository of photosyntheticenergy in plants and comprise roughly 50-80% of the dry matterof forages and cereals. The nutritive characteristics of thesecarbohydrates for animal feeding are variable, depending uponsugar components and linkages (Van Soest, 1982). Nutrientavailability depends on the capability of microbial enzymes tocleave glycosidic bonds in plant carbohydrates and between thecarbohydrates and other substances. Most polysaccharides14entering the rumen can be considered as belonging to one oftwo general types: plant non-structural carbohydrates such assugars, starch and fructosans, and the structuralpolysaccharides which compose the greater part of all plantcell walls and which are considered to form the fibrouscomponent of animal feedstuffs.Non-structural carbohydrates are readily available sourcesof energy to ruminant animals (Smith, 1973). The structuralpolysaccharides have a skeletal function in the living plantand are, by their very nature and organisation within the cellwall, far more resistant to microbial attack. However , it isthe ability to utilise such materials as an energy source thatprovides ruminants with their particular ecological niche, andthe ability of rumen micro-organisms to degrade plantpolysaccharides efficiently is of paramount importance to thesurvival of the mature animal. Non-structural carbohydratesIn this section a brief discussion on the majornonstructural polysaccharides in relation to theirdistribution and degradability will be given. Suffice tomention that the monosaccharides, glucose and fructose, andthe disaccharide sucrose, are the predominant sugars found inplant tissues and are mainly found in the cell contents. Theyare rapidly fermented in the rumen to yield volatile fattyacids (VFA), the major energy sources for the ruminant animal. Herbage storage polysaccharidesStarches and fructosans are the most abundant non-structural polysaccharides. Starches are glucose polymerscomposed of D-glucopyranose units joined principally throughal-4 glycosidic links (Smith, 1973). Starch is a composite ofstructurally distinct polymers: amylose and amylopectin(Chesson and Forsberg, 1988). Amylose consists of anessentially linear chain of al-4 linked sugar residues.Amyloses are degraded by randomly acting a-amylases whichbring about a rapid reduction in viscosity and the release oflow molecular weight oligosaccharides (maltodextrins) and by8-amylases which remove successive maltose units from the non-reducing end of the chain (Chesson and Forsberg, 1988; VanSoest, 1982). On the other hand amylopectin is a highlybranched molecule with al-6 links at the branch points and al-4 links within the chains of each branch. About 50% ofamylopectin can be degraded to maltose by the action of B-amylases, leaving a residue known as 'B-limit dextrin' whichis protected from further attack by the 6-linked branchpoints. Complete hydrolysis of amylopectin requires the actionof enzymes capable of hydrolysing the al-6 linkages, notablythe glucoamylases and 'debranching enzymes'.Polymers of fructose occur in two forms, the inulins whichserve as reserve carbohydrates in tubers in which thefructofuranosyl units are linked B2-1 and the levans in whichthe units are linked B2-6 (Smith, 1973). The latter arecommonly found in temperate grasses, particularly in stems,16where they can reach levels of 1-5% of total dry matter(Smith,1973). Fructosans are rapidly and completely degradedin the rumen by organisms which include the holotrich(Williams, 1986) and entodiniomorph protozoa (Coleman, 1980).Many bacteria are able to utilise inulins (Hungate, 1966).The relationship between cellular components and feedintake depends on the contribution made by a specificconstituent to the structure or volume of the plant. The rateof organic matter removal from the rumen plays a major role inlimiting feed consumption. Organic matter can be removed fromthe rumen by digestion and by passage to the lower gut (Blacket al., 1982). Soluble carbohydrates can be degraded by rumenmicrobes 30 times faster than storage carbohydrates which, inturn, are degraded 5 times faster than structuralcarbohydrates (Maeng and Baldwin, 1976). Thus, largedifferences in the proportions of these broad groups of plantconstituents can markedly affect the rate of organic matterremoval from the rumen and hence voluntary intake. Ulyatt(1981) suggested that the lower ratio of structural to readilyfermentable carbohydrates in clover than in ryegrass was themajor reason for the reduced apparent mean retention time oforganic matter in the rumen and hence the greater voluntaryfeed consumption of sheep eating clover. Since structuralcarbohydrates are degraded at the slowest rate, theycontribute most to dietary organic matter accumulation in therumen (Black et al., 1982). This aspect is the subject of thenext topic. Herbage structural polysaccharidesCell walls form 30-80% of plant dry matter and vary as asource of energy. The cell wall of plants is a complex matrixcomposed of polysaccharides, proteins, phenolics (includinglignin and tannins), water and minerals (Hatfield, 1989).Bailey (1973) has given a comprehensive review of thechemistry and biosynthesis of these polysaccharides.Structural carbohydrates in higher plants are represented bythe polysaccharides cellulose, hemicelluloses and the pecticsubstances. The pectic substances include those substancesthat are more or less readily extracted from plant tissueswith hot water, cold dilute acid, hot ammonium oxalate or hotEDTA solution (Bailey, 1973). The hemicellulosic fraction isdefined as the group of polysaccharides extracted under alkaliconditions after pectic material and lignin are removed fromthe matrix. The remaining material after the sequentialextraction of pectins, lignin and hemicelluloses is defined ascellulose, or more precisely as a-cellulose, a pure celluloseB-glucan (Bailey, 1973).The nutritional value of a forage given to an animal is afunction of the amount of forage eaten and the nutrients madeavailable during its digestion. Provided the energy demand ofthe animal is not satisfied, differences in intake of plantmaterial may result from either differences in the rate ofremoval of organic matter from the rumen and (or) differencesin the amount of digesta which can accumulate in the rumen(Black et al., 1982). The importance of plant cell wall as the18primary restrictive determinant of intake has beendemonstrated by Mertens (1973) as cited by Van Soest (1984).Using 187 forages, he showed that neutral detergent fiber(NDF), the fraction that is most representative of the totalplant cell wall, was negatively correlated (r= -0.75) withvoluntary intake.Functional fiber is needed to maximize chewing, salivaproduction and rumen function and to minimize the incidence ofdigestive disorders. On the other hand, productivity of cattlefed high forage diets is limited by the amount of forage thatcan be consumed (Beauchemin, 1991). In this case, forageintake and utilization are maximized by enhancing breakdown,digestion and throughput of fiber alleviating gut fill.Several concepts have been advanced regarding the action ofthe fill mechanism. Mertens (1973) point to the limitingeffect of rumen fill. Dermaquilly et al. (1965) have suggestedthat the volume of indigestible matter is limiting throughflow in the lower gut. Campling and Balch (1961) showed thatremoval of swallowed hay from the rumen had an immediateeffect on the time the cow stopped eating. Cows could beencouraged to eat for very much longer than normal by removingthe swallowed hay. Further Campling et al. (1961) as cited byCampling (1969) observed that cows fed hay or dried grassstopped eating when the reticulorumen contained similaramounts of dry matter and that the quantity of each roughageeaten was directly related to its rate of disapperance fromthe reticulorumen. Van Soest (1982) observed that the capacity19of the intestines to transport digesta was not a limitation tointake because daily fecal output increases with increasingvoluntary roughage intake.Since the amount of cell wall is more highly associatedwith intake than any of its components or rate of fermentationparameters (Van Soest , 1975), strong support is given to astructural volume theory relating voluntary intake and foragequality. This involves two parts: the structural volume theory(relates to cell wall content) and rate of passage. The intakeof a forage is usually increased when it is ground andpelleted (Minson, 1982), a result of decreased effectivevolume (Van Soest, 1975). However, the process of digestiondoes not decrease volume unless a reduction in particle sizeoccurs. Van Soest (1975) considered the aspect of volume asrelating to cell wall content which therefore influences therumen fill effect. To alleviate the fill effect two mechanismscome into play, namely, rumination with reduction in particlesize and passage rate.When feed enters the rumen, undigested feed residues mustbe reduced in size before they can leave the reticulorumen(Campling, 1969). Particle size reduction of feeds is mainlythe result of chewing during eating and rumination. Microbialdegradation and abrasion during movement within the rumencontribute to a lesser extent. Chewing of feeds during eatingand rumination is a response to the physical property offiber. Large feed particles are chewed more and reduced to agreater extent than smaller ones during eating (Beauchemin,201991). For forages, between 15 and 55% of the long particles(by weight) are broken down during eating to a size that canbe passed from the rumen. Rumination is usually the principalmeans by which particle size of feed is reduced, but notalways. This is because the net contribution of rumination tobreakdown of ingested feed particles depends on the efficiencyof breakdown during feeding (Beauchemin, 1991). Once the largeparticles are swallowed after chewing, rumination veryeffectively reduces them. Fine particles pass from the rumenmore rapidly than large ones (Campling, 1969). Thus, thehigher intake of ground and pelleted forages could beinterpreted as having saved the ruminant the trouble ofruminating forage particles to a size that will pass. If thisis the case, the cell wall properties that determine the rateof breakdown and the amount of work required will affectintake and digestibility (Van Soest, 1975).212.1.6 SUMMARY STATEMENTThe foregoing discussion has stressed the role ofindigestible components of forages on voluntary intake. Thefraction of the fiber that is indigestible has been estimatedto constitute up to 1/3 of the total fiber in grasses andabout 1/2 in legumes (Fahey and Garleb, 1991). Variableintakes have been reported for diets with comparable amountsof cell walls (NDF) and this has been explained to be as aresult of possible differences in rate and extent of cell walldigestion (Varga and Hoover, 1983).Since the overall rate and extent of cell wall digestionwill depend on the rates and extents of digestion ofindividual polymers constituting the cell wall, a study of thelatter parameters would cast some light on the understandingof overall digestion of the cell walls. This could alsopossibly explain why diets containing comparable levels ofcell walls may lead to differences in voluntary intake. Thepresent study was proposed as an effort to understand howdifferent cell wall polymers are degraded within the rumen andto try and relate the results to variations in intake.222.2 CELL WALL CHEMISTRY, AND ITS RELATION TO DIGESTIBILITY2.2.1 IntroductionThe degradability of plant cell walls is likely to belimited not only by the complex interrelationships betweenpolysaccharide structures of the wall, but also by non-carbohydrate materials such as lignin (Ford and Elliot, 1987).Low molecular weight phenolic compounds associated with thecell wall have also been implicated as inhibitors of foragedigestion in the rumen (Akin and Chesson, 1989). In thissection the interrelationships amongst the wall compounds andhow they relate to forage digestion in the ruminant animal arediscussed. The pectins, whose degradability is known to behigh, will not be discussed.The plant cell wall is the principal structural componentof the plant cell and surrounds the protoplast and exterior ofthe cell membrane, the plasmalemma. Depending on cell type andthe stage of maturity, the plant cell wall can be composed ofup to three layers, the middle lamella, the primary cell wailand the secondary cell wall (Selvendran and O'Neill, 1987).The first cell wall structure to be synthesized by the cell isthe middle lamella which is composed of pectic substances,primarily the Ca++ salts of galacturonate andrhamnogalacturonate. The next cell wall layer to be formed isthe primary cell wall, which is usually found in youngundifferentiated cells that are still growing (Selvendran andO'Neill, 1987). This layer consists mainly of polysaccharides,23primarily cellulose, hemicellulose and pectins. Besides thesepolysaccharides, this layer also contains approximately 10%protein, primarily extensin, with 30% of the amino acidresidues being hydroxyproline (Fahey and Garleb, 1991). Thesecondary cell wall layer is formed once the cell has reachedmaturity. It is a rigid and thickened layer that has adistinct shape and property. The thickening is as a result ofthe laying down of the polyphenolic compound lignin, whichbegins in the primary cell wall region and extends onward tothe middle lamella (Fahey and Garleb, 1991).2.2.2 CelluloseCellulose is both a component of the primary and secondarywall material. It is composed of B-linked glucose residueswhich form chains of up to 14,000 residues in length (McNeilet al., 1984). The unique properties of cellulose areconferred by its secondary, rather than its primary,structure. The linear chains of B-1,4-linked glucose unitsaggregate through intra- and extramolecular hydrogen bondingto form microscopically visible fibrils (Delmer, 1987). Thedegree of order found within and between fibrils varies fromregions in which the glucan chains are held firmly inparallel, and where X-ray diffraction studies indicate a highdegree of crystallinity, to regions in which this order issomewhat reduced (amorphous regions)(Chesson and Forsberg,1988). Other polysaccharides and lignin are closely associatedwith cellulose, but there is no evidence to suggest that24cellulose has covalent bonds with them (Hatfield, 1989).Cellulose is present in all plants and is probably thestructural polysaccharide which contributes most to therigidity and strength of plant structures (Bailey, 1973).The degradation of cellulose has been extensively studiedin a number of microorganisms. One of the restraints todegradation either by enzymatic or chemical systems is theextensive hydrogen bonding that occurs in microfibrils to givecellulose a crystalline structure. Other factors postulated aspotentially affecting cellulose digestion include encrustationby lignin and hemicellulose and a limited enzyme accessiblespace for cellulose hydrolysis (Fahey and Garleb, 1991).Kerley et al. (1988) defined enzyme accessible space by thesize, shape and surface properties of microscopic and sub-microscopic capillaries within fiber in relation to the size,shape diffusibility of microbial cellulase enzymes. Cellulosecrystallinity is perceived to be a factor that regulates therate as opposed to the extent of cellulose digestion. VanSoest (1982) reported that alfalfa cellulose prepared bychlorite delignification and alkaline extraction ofhemicellulose had a slower digestion and greater overalldigestibility than cellulose in intact alfalfa cell wall. Heargued that the removal of lignin and hemicellulose may haveallowed cellulose chains to become more closely aligned andthus resulted in greater crystallinity. The same worker(1975) indicated that lignin is more highly correlated to thedegree of cellulose digestion than it is to rate of digestion.252.2.2 HemicelluloseThe constituents of the cell wall fall into three groups:the fibrillar polysacharides (mainly celluloses discussed inthe previous section), the matrix polysaccharides (Bailey,1973) and the encrusting substances (Selvendran and DuPont,1984). The matrix polysaccharides are made up of linearlyoriented polymers which are present at all stages of thedevelopment of the wall and also of highly branchedpolysaccharides that are deposited at particular stages ofgrowth (Selvendran and DuPont, 1984). There are two majorfractions in the matrix polysaccharides: (1) the pecticsubstances; (2) the hemicelluloses, which are represented bythe polysaccharide soluble in alkali.Hemicellulose is the most complex of the plantpolysaccharides. Hatfield (1989) has categorisedhemicelluloses into four distinct components namely: thexylans, 8-glucans, xyloglucans and the mannans. The xyl'anfraction, assembled from 8-1,4-linked xylose residues, is themain hemicellulosic component found in grasses. The xylan issubstituted with acetyl, arabinosyl and glucuronyl residues(Cheng et al., 1991; Hatfield, 1989). In ryegrass, wheat andbarley straw, and white clover, 50% or more of the xyloseresidues may be substituted in the 0-2 and 0-3 positions, with50 to 70% of these sustituents being acetyl groups (Cheng etal., 1991). The extent of substitution on the xylan backboneby arabinose varies from one in every two to eight xylose26residues (Hatfield, 1989). A proportion of the arabinoseresidues are further esterified with feruloyl and coumaroylresidues. In barley straw for example, 1 in every 31 arabinoseresidues was esterified with p-coumaric acid, and 1 in every15 with ferulic acid (Mueller-Harvey et al., 1986).The other component of hemicelluloses, the B-glucansrepresent a unique molecule that seems to be restricted toimmature tissues of grasses (Morrison, 1979) and endospermcell walls of cereals (Hatfield, 1989). About 70% of themolecule is made up of glucose residues linked B-(1,4) and theremaining 30% linked 8-(1,3). Xyloglucans on the other handare composed of a backbone of B-(1,4) linked glucopyranoseunits. This backbone is substituted with xylopyranose units byan a-(1,6) linkage. Xyloglucans are most abundant in primarycell walls of dicotyledons and may possess additional sugarsmainly galactose and fucose (Hatfield, 1989). The mannans arefrequently found as components of many seeds (galactomannans),but are also found as cell-wall components of many plants. Theglucomannans and galactoglucomannans are basically linearmolecules composed of B-(1,4) linked glucopyranose andmannopyranose residues.A series of hemicellulases exist in the rumen which can beisolated from cell free rumen fluid (Brice and Morrison,1982). These workers studied the degradability of lignifiedand delignified hemicelluloses isolated from perennialryegrass harvested at different maturity stages. They foundthat as the ratio of xylose:arabinose increased with plant27maturity (Morrison, 1980), there was an increase inhemicellulose degradability. The reason for this increase wasthat isolated hemicellulases cleave between two unsubstitutedxylose residues. As a plant matures, however, lignin contentincreases and overrides the favorable effects of the increasedxylose:arabinose ratio. However, it is also suggested thatlinear xylan may have a high affinity for the cellulosefibrils (McNeil et al. 1975). An increase in hydrogen bondingbetween xylan and cellulose microfibrils could lower thefermentability of both fiber fractions (Fahey and Garleb,1991). It has been concluded that with young grass, theproportion of side chains is the major controlling factor ofhemicellulose digestion while in older tissue the lignincontent is the dominant factor (Morrison 1979).2.2.4 Phenolic-hemicellulose complexesLignin is generally considered to be the overiding factorin the limitation of cell wall degradation by microorganismsand their cell-free enzymes (Wallace et al., 1991). In theintact cell wall, lignin has been shown to be covalently boundto the hemicellulose (Morrison, 1979) and to exist, in part atleast, as a lignin-carbohydrate complex (LCC), (Scalbert etal., 1985).In an attempt to understand the nature of LCCs, Morrison(1973) isolated an LCC from ryegrass (Lolium multiflorum).Further to this he investigated the structure of thismolecule, isolated from (Lolium perenne), and reported that28there were at least three types of bonds between lignin andcarbohydrate (Morrison, 1974). One type of bond could becleaved by borohydride reduction, another cleaved by alkaliand a third type was resistant to alkali. Saponification ofplant fibers with alkali has shown that p-coumaric and ferulicacids and dimeric phenolic acids are bound to graminaceouscell walls (Hartley and Ford 1989). Treatment of bermudagrass(Cynodon dactylon) leaf blades for 24 hours with 0.1M sodiumhydroxide resulted in a release of 86% of the ferulic acid,65% of the dimers and 50% of the p-coumaric acid (Akin et al.,1992). Digestibility was increased from 6.5% in the untreatedcontrol to 56.6%. Lignin level was reduced after the alkalitreatment. A higher level of alkali (14 NaOH) resulted in anincrease in digestibility to 72%, and increased biodegradationof mesophyll and phloem tissues (Akin et al., 1992).Lignin-carbohydrate complexes have been isolated from therumen (Gaillard and Richards, 1975; Neilson and Richards,1982; Conchie et al., 1988). These workers postulated thatthese complexes are likely to represent phenolic richfragments of the wall from which most of the sugar has beenremoved by the action of rumen microflora. Further theythought that any remaining sugar was likely to provideevidence of the nature of the LCCs in the intact wall.Gaillard and Richards (1975), have reported that the LCC fromthe rumen are soluble at pH 7 or higher but precipitate at pH3. Neilson and Richards (1978) found that the soluble LCCproduced in the rumen were not significantly affected by29further digestion in the rumen after passing into solution.Since this complex precipitated in the low pH of the abomasumand eventually appeared in the feces, they concluded that theamount of the complex digested was unlikely to be significantafter it passed from the rumen.The complexes isolated from both the ovine and bovinerumen, tended to have either a high (> 100kDa) or low (< 4kDa)molecular weight (MW), the higher MW containing a highercarbohydrate concentration (Wallace et al., 1991). Reducingsugars (principally xylose and arabinose) were found in thelow MW fraction, thus suggesting ether linkages to thephenolics (Wallace et al., 1991).302.3 DIGESTION OF PLANT CELL WALLS BY RUMEN MICROBES2.3.1 IntroductionThe fundamentally important process of plant cell walldigestion by ruminants is dependent on colonization anddigestion within the very complex microbial ecosystem of therumen. Bacteria, fungi and protozoa colonize practically allplant materials that enter the rumen, with the exception ofintact, outer plant surfaces which are reportedly notcolonized by bacteria (Chesson and Forsberg, 1988). The rumencontains microbial species adapted to adhere to the specificincoming plant components in the physical condition theyattain through ruminant feeding behavior. Adhesion is followedby successive microbial colonization within the adherentpopulation, until effective digestive consortia are formed andthe substrate is digested to release nutrients at maximal rate(Cheng et al., 1991). Adhesion is a prerequisite for theinitiation of digestion of plant materials because thisprocess strategically positions the microbial cell in contactwith the substrate (Cheng et al., 1991).The major route of invasion of plant tissues by microbesappears to be via broken epidermal lesions (Chesson andForsberg, 1988). Colonization by entry through stomata iscomparatively unimportant with stem fragments of legumes andgrasses, but it can be of greater importance for colonizationof leaves (Cheng et al., 1983/84). The major species ofbacteria that attach are the cellulolytic bacteria31Ruminococcus albus, R. flavefaciens and Fibrobactersuccinogens (formerly Bacteroides succinogens Montgomery etal., 1988) which are commonly found on cut edges of cell wallsand damaged areas of cell surfaces (Akin, 1986). TheRuminococcus species appear to be loosely associated with cellwalls, while F. succinogens exhibits a tight adhesion,frequently conforming to the surface of the material beingdigested (Chesson and Forsberg, 1988). The mechanism ofattachment of different rumen microorganisms to cellulose andindeed other complex fibrous materials may involve specificbinding by: cell-surface associated enzymes; adhesins(molecules on the microbial cell surface that bind toreceptors on the plant material; or, possibly, non-specificionic interaction (Forsberg, 1986).Rumen fungi and protozoa colonize plant fragments anddegrade them to differing degrees (Akin, 1986). Thecellulolytic fungus Neocallismatix frontalis has been shown toproduce an active cellulolytic complex that carries out cell-free cellulose digestion (Wood et al., 1986). This is the onlycell-free cellulolytic complex characterised from a rumenorganism to date (Cheng, et al., 1991). Even though the modeby which these microorganisms attach themselves to plantmaterials is poorly understood, it has been found thatanaerobic fungal zoospores have a preference for stomata andbroken areas of plant particles from which soluble sugarsdiffuse and to which zoospores demonstrate a chemotaxicresponse (Chesson and Forsberg, 1988). Among the studied rumen32ciliate protozoa, EDidinium ecaudatum has been found toproduce cellulase of high activity (Chesson and Forsberg, 1988).2.3.2 Mechanism of cellulose degradationThe rate of microbial digestion of feeds depends on threegeneral factors: (1) plant structures that regulate bacterialaccess to nutrients; (2) microbial factors that controladhesion and the development of microbial consortia; and (3)complexes of oriented hydrolytic enzymes of the adherentmicroorganisms (Cheng et al., 1991). Isolated plant structuralcarbohydrates are readily digested by rumen microorganisms;however, their availability in the intact plant can be limitedand varies with both plant species and maturity (Dehority,1991).Rumen microbes responsible for cellulose digestion,produce cellulase enzymes in an active form. The cellulasesystem usually consists of three major types of enzymes whichfunction synergistically in the hydrolysis of crystallinecellulose (Chesson and Forsberg, 1988). These are: endo-1,4-B-glucanase, cellobiohydrolase and B-glucosidase. Theendoglucanase attacks cellulose in a random fashion thatresults in a rapid decrease in chain length and gives rise tocello-oligosaccharides. The cellobiohydrolase degradescellulose by splitting off cellobiose units from the non-reducing end of the chain. The B-glucosidases hydrolysecellobiose and soluble cello-oligosaccharides with a lowdegree of polymerisation, to glucose but cellulose is not33degraded.In a study of cellulose digestion from eleven foragesusing pure and mixed cultures of rumen bacteria as well asthrough in vivo digestion trials, Dehority (1991), reported ahigher extent of cellulose digestion with F. succinogens A3ccompared with R. flavefaciens B34b and in vivo cellulosedigestion. Similar findings have been reported by Cheng et al(1983/84). succinogens is one of the rumen bacteria mostactive in growth related degradation of recalcitrant forms ofcellulose such as cotton fibers and cellulose powder (Chessonand Forsberg, 1988). These gram negative, rod shaped bacteriaare reported to produce high levels of endoglucanase and B-glucosidase (Groleau and Forsberg, 1981). Cells grown oncellulose produced 7-8 times more endoglucanase than eithercellobiose-grown or glucose grown cells (Groleau and Forsberg,1981) and it was suggested that endoglucanase is subject to atype of catabolite repression.The bacteria F. succinocrens is reported to form intimateadhesion to cellulose substrate (Cheng et al., 1983/84). Theyfurther reported that these bacterial cells were alsoassociated with pit formation in the cellulosic cell walls ofthe grass leaves used in their study and that in many casesthey were associated with complete digestion of these cellwalls. Besides F. succinogens, other gram-positive coccusbacteria have been noted to be associated with pit formationand are reported (Cheng et al., 1983/84) to resemble those ofpure cultures of Ruminococcus flavefaciens used by Latham et34al (1978a). Pettipher and Latham (1979) have studied thecharacteristics of the cellulase enzymes produced by R.flavefaciens. They reported that the pH optimum was between6.4 and 6.6 and the temperature optimum between 39 and 450C.Cellulase^activity was^found^to^be mainly cell^associatedduring^exponential^growth,^but cell-free enzyme accumulatedduring the stationary phase.^Another important cellulolyticbacteria is^Ruminococcus^albus.^Leatherwood (1965) hasreported that^this^bacterium produces^about 20%^of theendoglucanase in rumen fluid, with some 58% of the cellulasebeing produced from the cells. Stack and Hungate (1984)discovered that 3-phenylpropionic acid in rumen fluid was theactive compound which, upon inclusion in the growth medium inthe place of rumen fluid, caused albus to grow faster andto synthesise a very active, cell-associated, high molecularweight cellulase. Cheng et al. (1983/84) concluded that thismay indicate that selected endoglucanases are secreted duringgrowth.Since rumen microorganisms and indeed those of thedigestive tract have complex nutritional requirements and canonly utilise one or two of the major polysaccharides,synergism among microorganisms can be important for theefficient use of forages by the ruminant animal. Synergism hasbeen observed in the way of crossfeeding of hydrolysisproducts, utilization of end-products or production of anessential nutrient (Dehority, 1991). Russell (1985) reported agood example of crossfeeding of hydrolysis products when he35showed that non-cellulolytic bacteria could utilise cello-detrins released by cellulolytic bacteria. The combination ofthe cellulolytic species F. succinogens, R. flavefaciens or R.albus with non-cellulolytic Treponema or Butyrivibrio speciessharply accelerates the rate of cellulose digestion, and whilecells of F. succinogens alone are unable to clear celluloseagar, these microorganisms can clear large areas of the agarif they are combined with Treponema or Butyrivibrio species(Kudo et al., 1986). Ruminant cellulose digestion is veryefficient because cellulolytic organisms are closelyassociated with symbiotic species that remove the products oftheir digestive enzymes, and thereby accelerate the digestiveprocess (Cheng et al., 1991).2.3.3 Mechanism of hemicellulose degradationDigestion of hemicelluloses has been recognised for a longtime. Dehority et al. (1962) traced this activity to the rumenmicrobial population and studied it in vitro with mixedcultures. They reported that, similar to cellulose digestionfrom forages, rate and extent of hemicellulose digestiondecreased markedly with plant maturity.Xylanases are the main hemicellulose hydrolysing enzymes.They are more widely distributed than are cellulases amongrumen bacteria (Chesson and Forsberg, 1988). Microorganismsoften produce more than one xylanase, and F. succinogens, forexample produces two (Chesson and Forsberg, 1988). B-xylosidase, B -glycosidase and a-L-arabinofuranosidase are36essential for the complete degradation of oligomeric fragmentsarising from the hydrolysis effected by the polysaccharide-degrading enzymes widely distributed among rumen bacteria(Williams et al., 1984).The major hemicellulolytic bacteria in the rumen are:Butyrivibrio fibrisolvens, Bacteroids ruminicola andRuminicoccus species (Yokoyama and Johnson, 1988). Most of thepredominant cellulolytic ruminicoccus species will degrade andutilise hemicellulose. Recent findings indicate that speciesof B. fibrisolvens, the most frequently isolatedhemicellulose-digesting ruminal microorganism, representseveral strains but almost all strains are hemicellulolytic(Hespell, 1988). Unlike the cellulolytic species, strains ofthis bacteria use hemicellulose degradation products forgrowth. On the other hand not all strains of B. ruminicola arehemicellulolytic. Hespell (1988) attributes this to severalfactors, among them, the type of substrate used to grow theorganism. He showed that the digestibility of alfalfahemicellulose decreases with the age of the plant irrespectiveof the bacterial species that carries out the degradation.Coen and Dehority (1970) observed that even though B.ruminicola strain D31d digested alfalfa hemicellulose, thisstrain was almost incapable of digesting bromegrasshemicellulose. This could be due to the chemical andstructural differences between the two hemicelluloses sincethe bromegrass has no rhamnose or uronic acids andconsiderably more glucose than alfalfa (Hespell, 1988).37The biochemistry and enzymology of hemicellulosedegradation by ruminal bacteria is not well understood. InRuminococcus albus, the cellulase and other enzymes appear tobe organized into highly structured, high molecular weight,extracellular complexes of proteins and polysaccharides (Stackand Hungate, 1984). With F. succinogens, cellulase andxylanase activities are associated with large membrane-associated complexes (Groleau and Forsberg, 1981). 11,fibrisolvens produces extracellular polysaccharides having acomplex sugar composition. Hespell (1988) speculated thatthese extracellular complexes could aid the cells of variousspecies to associate with the insoluble plant materials andaid in hindering diffusion of the enzymatically generatedproducts away from the cell.The glycanases and glycosidases with roles inhemicellulose digestion are subject to carbon source-dependentregulation (Greve et al., 1984). The xylanase from R. albus isproduced when cells are grown on filter paper but not whenthey are grown on cellobiose (Greve et al., 1984), andxylanase from R. flavefaciens is produced when cells grow oneither filter paper or cellobiose (Pettipher and Lantham,1979). Forsberg et al. (1981) reported that xylanase from F.succinogens could only be produced by cells grown on filterpaper and on Avicel but not on glucose. Chesson and Forsberg(1988) suggested that such findings could imply that xylanasesfrom R. albus and F. succinogens are subject to repression.The distribution of glycanases and glycosidases among38rumen ciliate protozoa is widespread (Chesson and Forsberg,1988). The highest activities have been found in Epidiniumecaudatum caudatum, Eremoplastron bovis Ostracodinium obtusumbilobum and Eudiplodinium maggi. Hemicellulase activity wasfirst detected in the rumen ciliate protozoon Epidiniumcaudatum (Bailey et al., 1962). A wide range of glycosidehydrolases are synthesized by rumen protozoa (Williams et al.,1984). The glycoside hydrolases involved in the degradation ofplant cell wall structural polysaccharides, including a-L-arabinofuranosidase, B-D-galacturonidase, B-D-glucosidase andB-D-cellobiosidase, were higher in the cellulolyticentodiniomorphid ciliates. On the other hand anaerobic rumenfungi have also been reported to secrete a wide range ofglycanases and glycosidases (Mountfort and Asher, 1985).Neocallismatix frontalis has been reported to produce a highlyactive, extracellular cellulase, several-fold higher inactivity than that of the aerobic fungus Trichoderma reese(Wood et al., 1986).2.3.4 SUMMARY STATEMENTThe foregoing discussion has dealt with the main microbesinvolved in cell wall degradation and on the necessity of awhole complement of them in cell wall degradation. Thesignificance of synergism has been stressed. In most studiesdealing with degradation of cell wall polymers, isolated pureforms have been used. However, in this situation anyinteractions between the wall polymers and their effect on39degradation cannot be detected.In the present study, the plant materials studied wereincubated in their intact forms in the rumen with the hopethat isolated polymers from the residues would give an idea ofrecalcitrant polysaccharides in the intact plant. Thisapproach was preferable to incubating the isolated polymersand assessing their disappearance.402.4^METHODS OF EVALUATING THE NUTRITIVE VALUE OF FORAGES2.4.1 IntroductionSeveral techniques have been developed that attempt toestimate the contribution of feed protein or carbohydrate tothe rumen. These methods are either animal or laboratorybased. The following discussion will focus on chemical methodsof assessing forage nutritive value as well as in vitro and insitu methods.2.4.2 Chemical proceduresThe conventional system of feed evaluation that has beenin use for the longest time is the so-called proximate systemof analysis. This system partitions dry matter into etherextract, crude fiber, nitrogen, nitrogen-free extract and ash(Van Soest, 1982). It is the basis on which TDN is calculatedusing the following assumptions: 1. ether extract recoverslipids and fats which contain 2.25 times the energy ofcarbohydrate. 2. All nitrogen is in protein which contains 16%nitrogen. 3. Crude fiber recovers the least digestible fibrousand structural matter of the feed. 4. The NFE representshighly digestible carbohydrates. Van Soest (1982) who hasgiven an extensive review of this system argues that none ofthese assumptions are entirely true.The NFE contains the cumulative errors of all the otherdeterminations. However, the greatest and most fundamental41error of the proximate system of analysis is the division ofcarbohydrates into crude fiber and NFE (Van Soest, 1982).Crude fiber has in some instances been found to be moredigestible than NFE especially in grasses that contain morehemicellulose and soluble lignin.Other systems of feed evaluation based on comparativereplacement values of feeds are used in Europe. They includethe Scandinavian feed unit system and the starch equivalehtsystem (Van Soest, 1982). In the case of the Scandinavian feedunit system, the value of barley is taken as 100 and therelative quantity of other feeds required to replace theproductive value of a unit of barley is taken as its feed unitequivalent. The starch equivalent is expressed as the netenergy (NE) value of feed in units relative to the NE of lkgof starch.A significant improvement in forage quality evaluationmethodology was the description of plant composition in termsof cell content and cell walls (Van Soest, 1967). According tothis system, the cell contents comprise components that aredigestible by enzymes secreted in the digestive tracts of allanimals (Barnes, 1973), while the cell wall is comprised ofthose components that are only partially digestible and thenonly by rumen and cecal microorganisms. The cellular contentsare soluble in neutral detergent and include minerals, lipids,sugars, organic acids, pectin, starch, soluble proteins andnon-protein nitrogen. Van Soest and Moore (1966) estimated thecellular contents were 98% digestible and not affected by42lignification. The cell wall constituents (CWC) or neutraldetergent fiber (NDF) are insoluble in neutral detergent andare only partially available. These constituents includemainly cellulose, hemicellulose, lignin, cutin and ash.Acid detergent fiber (ADF) on the other hand ,quantitatively recovers cellulose, lignin, silica andunavailable nitrogen. The importance of ADF is that itrepresents a fractionation of the plant cell wall wherebyhemicellulose is dissolved and partitioned from the celluloseand lignin which remain in the residue (Van Soest, 1984). ADFis highly negatively correlated (r = -0.75) to digestibilitywhile NDF which reflects more on the volume of a forageconsumed is highly negatively correlated (r = -0.76) tovoluntary intake (Van Soest, 1984).2.4.3 Biological proceduresIn this section the use of enzymatic, in vitro and in situtechniques of evaluating the nutritive value of forages willbe discussed. Enzymatic methodsEnzymatic techniques have been employed to study thedigestibility of the nutritional components of by foragesvarious workers (Jones and Hayward, 1973; 1975; Hungate etal., 1983). Jones and Hayward (1973), observed that there areseveral advantages of using enzymatic methods overfermentation methods. They cited the variations observed43between animals and those due to different diets as exampleswhich can be overcome by use of enzymes. The enzymes are eitheracquired from commercial sources or extracted from the rumenfluid. The most common sources of cellulase are the fungiTrichoderma viride and Trichoderma reesei. Proteases are usedto study protein degradability.Even though there are advantages of using commercialenzyme preparations (low cost, time reduction and lesscontamination of feed residue) over live microbial culturesthere are concerns, particularly with commercial proteases,about their specificity in relation to ruminal proteolyticactivity (Nocek, 1988). Mahadevan et al. (1987) concluded thatthe use of non-rumen proteases in an in vitro system forprediction of feed protein degradation may be of limited valueor misleading, since protease other than of ruminal origin maynot have the same action on feed proteins.Enzymes derived from different fungal sources have beenreported to differ in their ability to solubilize herbage andcellulose (Jones and Hayward, 1975). Among four differentfungi, Trichoderma viride was found to be the most active,solubilising 70% of cellulose paper in 24 hours. Nocek andHall (1984) have further reported that use of enzymeprocedures generally results in less solubilization of drymatter than does use of ruminal microbes. They observed lowercell wall digestion with enzyme combinations than with in situvalues. They concluded that this may be associated withlimited enzyme, change in incubation environment with time, or44inadequate complements of enzyme types to simulate ruminaldigestion. The possibility of lack of physical association ofthe commercial enzyme preparation with fiber particles couldnot be ruled out.Nocek (1988) has concluded that enzymatic digestiontechniques may be more suitable for measuring relativedifferences between feedstuffs than providing absolutedigestibility values. Accuracy of prediction appears to dependgreatly on the forage or feedstuff in question, and therelative complement of enzymes used in the incubation. In vitro methodsThe most commonly used in vitro system is the batchfermentation method. Under this we have the single-stage andthe two-stage systems. Even though there have been manymodifications to the original method used by Tilley and Terry(1963) the basic elements remain the same.The first stage of a batch in vitro procedure simulatesrumen digestion of structural carbohydrates. The two-stagefermentation technique attempts to measure not only thedigestible fibrous fraction but also the digestible solublefraction. The second stage involves the extraction of theresidue from the first stage with acid-pepsin (Tilley andTerry, 1963) or neutral detergent solution (Van Soest et al.,1966). The acid-pepsin digestion stage simulates the in vivobreakdown of feed and microbial protein by the digestiveenzymes of the abomasum in the ruminant. Dry matter45disappearance is greater following neutral detergentextraction due to the solubilization of the cell wall. Thus,in vitro procedures with neutral detergent have been equatedto true digestibility and procedures with acid pepsin equatedto apparent digestibility (Van Soest et al., 1966).High correlations between one-stage in vitro and in vivocellulose digestion or dry matter disappearance have beenreported but interlaboratory comparisons reveal highvariations. Barnes (1967) reported mean in vitro cellulose(24h) values ranged from 40.0 to 63.9% among 14 laboratories,and in vitro dry matter digestibility (24h) ranged from 38.7to 53.5% among five laboratories. The addition of the second-stage acid-pepsin digestion gave higher correlationcoefficients and lower errors compared to results from theone-stage technique.The greatest source of uncontrolled variation in any invitro system is the inoculum (Barnes, 1973). Use of washedcell suspensions or rumen fluid strained through many layersof cheesecloth have been used successfully to reducevariability between analysis (Rode and Satter, 1984). Howeverthis method may not be recommendable since the removal offorage particles is likely to remove the most activecellulolytic microorganisms. Other sources of random errorinclude donor diet, buffer medium, sample size, and sample(Barnes, 1973). Besides random error, in vitro techniques areassociated with predictive errors. These are errors emanatingfrom the use of mathematical models to predict in vivo46measurements from in vitro values (Barnes, 1973).Inability of the batch culture to adequately account forthe dynamic situation within the rumen led to the developmentof semi-continuous and continuous systems that more closelyapproximate ruminal conditions (Abe and Kumeno, 1973;Czerkawski and Breckenridge, 1979). Slyter and Putnam (1967)indicated protozoal numbers were lower and viable bacterianumbers slightly higher when compared with ruminal environmentof a steer fed the same diet. Czerkawski and Breckenridge(1977) on the other hand, observed that protozoa numbers weresimilar between in yivo data and in vitro simulations.Mathematical descriptions of continuous culture kinetics havebeen expanded to account for the more complex behavior ofsubstrates and microbes in activated sludge and otherheterogenous ecosystems (Hashimoto et al., 1982) but have notbeen used to describe the rumen ecosystem to date. In situ techniquesThe in situ technique, also variously referred to as thedacron bag, nylon bag, in sacco technique has been usedextensively to study rumen degradability of nitrogen (N), drymatter (DM), organic matter (OM) and fiber fractions (Susmelet al., 1990). This method involves suspension of feedmaterial in the rumen and allows intimate contact of the testfeed with the ruminal environment. The method allows thedescription of a degradability kinetic (Orskov et al., 1980),which generally follows a first-order model, and the quantity47of nutrient effectively degraded in the rumen can becalculated from kinetic coefficients of degradability and theoutflow rate of feed from the rumen.The nylon bag technique is subject to considerablevariability. Sources of variation include: size and type ofbags; cloth mesh size; sample size and fineness of grind;number of samples per trial; diet of the host animal; methodof suspension in the rumen; location and time in the rumen;and method of cleaning and rinsing bags after removal from therumen (Barnes, 1973). An extensive review by Nocek (1988) ofthe in situ technique has addressed these problems andproduced some recommendations. On the question of sample andbag size, he suggests the optimum sample size to be that whichwill provide adequate residue at the end of extended rumenincubation for chemical analysis without overfilling the bagso as to delay bacterial attachment, increase lag time andunderestimate digestion rate. He recommends a range in samplesize to surface area ratio of 10 to 20 mg/cm2 for both forageand concentrate diets.Grinding of forages increases surface area per unit weightof sample and the surface area accessible for microbialattachment. Fineness of grind is important as it determineshow much material is passively lost through the pores of thebags and also the digestion rate. Generally, longer andcoarser materials are associated with slower rates ofdigestion and greater variation (Nocek, 1988). Grinds of lessthan lmm have little effect on digestion rate. Van Karen and48Heinemann (1962) demonstrated no difference in DMdisappearance for forages ground through screens of 0.28, 0.42and 0.84 mm. However, decreasing particle size to < 0.6mm mayalso cause clumping of the sample, thus decreasing digestionrate (Figroid et al., 1972). Nocek (1988) suggests a grindof 2mm for protein supplements and by-product type ingredientsand 5mm for forage materials.Utilization of forages involves complex interactions amongplant components, microorganisms in the rumen and the animal.These factors interact to influence both digestibility andvoluntary intake. Disappearance of feed from the digestivetract can be described by two major processes, digestion andrate of passage (Mertens and Ely, 1982). Waldo et al. (1972)proposed that fiber exists in two definable components --potentially digestible and indigestible. While theindigestible fraction disappears from the rumen by passageonly, the potentially digestible fraction disappears bypassage, digestion and absorption.The digestive process can be divided into rates ofdigestion, digestion lag and potentially digestible fractions(Mertens and Ely, 1982). Initial work on digestion kineticsdid not consider a lag phase and only assumed the existence ofthree pools (Orskov et al., 1980): pool A that was consideredto be rapidly degraded in the rumen; pool B that wasconsidered to be potentially degradable with time and pool Crepresenting a fraction that appeared undegradable in therumen irrespective of time. For protein degradation, DM and49other components, Orskov and McDonald (1979) had shown that ifthe percentage protein disappearance (p) from samplesincubated for time t is described by the equation p = a + b(1 _ e-ct) and if k is the fractional rate of passage from therumen, then the effective degradability can be calculated as P= a + bc/(c + k) wherep = the actual degradation after time 't';a = the component of protein degraded rapidly and formsthe intercept of the degradation curve at time zero,b = the potential degradability of the component ofprotein which will, in time, be degraded.c = the rate constant for the degradation of 'b' (%/hr).P = the effective degradability of protein.With fibrous feeds there is often a lag phase in thedegradation of DM due to the time taken for adherence ofcellulolytic organisms to the substrate (Orskov, 1991). Assuch McDonald (1981) has improved on the above equation byincorporating a lag time factor associated with thedegradation of the b component.2.4.4 SUMMARY STATEMENTChemical and biological (in vitro and in situ) methods ofassessing the nutritive value of feed materials for ruminantswere discussed. The weak and the strong points of each methodwere also mentioned.The in situ method was chosen for this study. This waspartly because it eliminates variations (in in vitro systems)50emanating from use of inoculum which has been filtered throughcheesecloth and may therefore be associated with lowcellulolytic and hemicellulolytic activity. It was also feltthat more substrate material could be used with this methodthan with an in vitro one.512.5 OVERALL SUMMARYThe foregoing literature review has dealt with aspectsthat influence or have a potential influence on thedegradability of forages by ruminants. Such plant factors asthe species, maturity and anatomy of morphological parts werediscussed. The role of chemical composition, with an emphasison the cell walls as the primary physical factor regulatingvoluntary intake in ruminants was stressed.Having established the role played by the cell walls inforage utilization, a discussion of the main factors thatinfluence the breakdown of its constituent polysaccharides wasgiven. Such factors as cellulose crystallinity, linearity andbranching of hemicelluloses, formation of covalent linkageswith other simple or complex phenolics of the wall werediscussed. Microbial degradation of cell walls was covered,with an emphasis on the significance of synergism in wallbreakdown. Finally the various methods used in the laboratoryto study digestibility were discussed.The present study was initiated with the aim of firststudying the factors that limit utilization of two popularKenyan forages harvested during the dry season. While it iswell accepted that the concentration of the cell walls andlignin are the dominant factors regulating forage utilization,a study of the contribution of the constituent polysaccharidesto this aspect have not. This is particularly so with thesetwo forages.Past research has shown that the degree of linearization52or branching of hemicelluloses may contribute significantly tothe overall degradation of plant cell walls. However, factorsleading to differential degradations of these wallconstituents have not been studied and are mainlyspeculations. In this study it was postulated that suchpolymers would be associated with different rates and extentsof degradation in the rumen. Such differences were thought tolead to differences in overall cell wall degradation. In thesecond part of this work, the effect of maturity on thedegradability of these polymers in leaves and stems werestudied.^Their rates and extents of degradation weredetermined^with the hope of identifying recalcitrantpolysaccharides and the factors leading to this.53CHAPTER THREEEXPERIMENTAL: DEGRADATION OF TROPICAL FORAGES3.1 IntroductionThere is an enormous amount of literature on utilizationof both tropical and temperate forages by ruminants (Minson,1990; Mowat et al., 1965). One of the major limiting factorsto animal production in Kenya is related to the seasonality offorage production which is the uneven distribution ofrainfall. During the wet season there is abundant forage andmilk production during this period is usually high. However,during the dry season there is a marked decline in milk yielddue mainly to a scarcity of adequate forage of high quality tomeet the energy demands of the lactating cow.In a country like Kenya where forage plays the major roleof meeting both the energy and protein demands of the cow, anunderstanding of the factors that limit forage utilization isvery important. It is generally believed that theconcentration of the cell wall in any forage is the dominantfactor regulating energy intake. During the dry season, theavailable forage for cattle feed in Kenya is usually of amature nature and therefore can be a major limitation toenergy and protein intake. It is well documented that proteinconcentration declines with forage maturation while the cellwall concentration increases (Minson, 1990). These effects aredetrimental to animal production.54The broad objective of this study was to determine thefactors that may limit forage utilization of two widely usedtropical forages, bana grass (Pennisetum purpureum) and thelegume silverleaf desmodium (Desmodium uncinatum) harvestedduring the dry season in Kenya. Proper selection of rationingredients is important in avoiding possible depressions indigestibility and hence available supplies of energy and aminoacids. Therefore the present study was undertaken with thefollowing specific objectives in mind:(a) To determine the rate and extent of degradation of thecell wall and its constituents in leaf and stemfrations of bana grass and silverleaf desmodium.(b) To compare the degradation characteristics of thegrass versus that of the legume.(c) Provide some possible suggestions of alleviating theproblem of having inadequate feed of a high qualityduring the dry season.3.2 MATERIALS AND METHODS3.2.1 ForagesTwo tropical forages were used in this study. Bana grassand silverleaf desmodium were harvested in Kenya during thedry period of September to November of 1991. Bana grass washarvested on Oct. 24th and was a four month regrowth.Silverleaf desmodium was harvested on Oct. 17, 18 and 19th.Like bana grass, desmodium was at the flowering stage during55harvesting. Only one sample of each forage was used because itwas felt that replications could be generated by using severalcows. Also the forages were initially intended to be used fora different experiment (involving a lot of chemical work)rather than the one reported here.After each harvest the legume and the grass were manuallyseparated into leaves and stems as soon as could be possibleand then put into polythene bags which were placed in a deepfreezer at -8°C. After completion of the harvesting the foragefractions were dried in a forced-draught oven maintained at600C until sufficiently dry for grinding. The weights of thedried materials were taken and recorded. The stem to leafratios determined on the dried materials were 1.75 and 1.35for bana grass and desmodium respectively. The foragefractions were then ground in a laboratory hammer mill(Christy and Norris, UK) to pass a 2mm sieve screen.3.2.2 In situ incubationsTo determine the degradability characteristics of theseforage fractions, they were incubated in three Holsteinheifers adapted (three weeks) and maintained on a purely grasshay diet. The heifers were fitted with permanent rumencannulae. About 7g of the ground substrate was weighed intonylon bags measuring 10cm x 20cm with a mean pore size of 53gm(Bar Diamond, Inc. Idaho, U.S.A.). The mouth of each bag wastied and secured with a nylon string before the bags were put56in a weighted mesh bag and placed in the ventral sac of therumen. The substrate to bag surface area ratio was maintainedat 17.5mg/cm2.To model the degradability of DM, cell wall (NDF),cellulose and hemicellulose, duplicate substrate samples wereincubated in each cow for 12, 18, 24, 48 and 72h. In thisfirst run degradabilities did not reach an asymptote andtherefore this experiment was repeated for the followingincubation times 12, 18, 24, 48, 72, 96 and 120 hours.Incubations were conducted in reverse time order such that allthe bags were removed at the same time at the end of 120h.Zero hour disappearance of dry matter was estimated by placingsubstrate samples in static water at room temperature for 30minutes.All the bags were washed under gently flowing tap water(Orskov et al., 1980) before drying in a forced-draught ovenat 60°C for 48h. The dried bag plus residue were weighed todetermine DM disappearance.3.2.3 Experimental designDegradability data for the two forages and their leaf andstem fractions were analysed in an 8 (incubation time) x 2(forage) x 2 (plant part) factorial arrangement in arandomised complete block design. The three cows served as theblocks. The interaction between forage and plant part was alsodetermined.57Degradability parameters, 'a' the soluble fraction, 'b'the insoluble but fermentable fraction and 'c' the rate Ofdegradation of the insoluble fraction 'b' (Orskov and McDonald,1979) were estimated using the Eureka program (1987). Toestimate the effective degradabilities of the DM, the cellwall and its constituents assumed values for the outflow rate(k) were chosen (0.02 and 0.04/h).All statistical analyses and comparisons were conductedusing the General Linear Model of SAS 1985).3.2.4 Chemical analysesDM content of the forage fractions was determined bydrying samples to constant weight at 1050C in a forced-draughtoven. Ash was determined by igniting the samples at 500°C in amuffle furnace. Crude protein was determined by the method ofParkinson and Allen (1975). NDF and ADF were determined by themethod of Waldern (1971). Lignin (permanganate) and cellulosewere determined by the method of Goering and Van Soest (1970).Hemicellulose was calculated as the difference between NDF andADF.3.3 RESULTS AND DISCUSSION3.3.1 Chemical compositionTable 3.1 presents the chemical composition values for thetwo forages and their leaf and stem fractions. As statedearlier the forages were harvested at the flowering stage. Thelegume fractions contained a higher CP content than the grassfractions. In both cases, leaves showed a trend of higher CPcontent than stems. Bana stems had the highest cell wallcontent (78.29%) indicating very low cell contents. Desmodiumleaves had the lowest cell wall content. Leaves in both caseswere lower in cell wall than stems. Lignocellulose (ADF)content was highest in desmodium stems (62.61%) than in theother fractions. Hemicellulose concentration was lower in bothlegume fractions relative to the grass fractions. Grass leavesalso contained a higher hemicellulose content than grassstems. The grass leaves and stems had a higher concentrationof cellulose than the legume. In both cases stems were higherin cellulose than the leaves.Lignin concentration was observed to be greater in stemsthan in leaves in both the grass and the legume. Legume stemshad a very high concentration of lignin (15.43%) relative tothe other forage fractions. Ash content was higher in grassleaves than in grass stems but the situation was reversed inthe case of the legume.59Table 3.1 Chemical composition of bana grass and desmodiumleaves and stems (%DM)ItemTreatmentsBana Desmodiumstems leaves stems leavesCP 4.4 6.8 9.0 25.5NDF 78.29 73.26 71.34 49.08ADF 51.62 45.77 62.61 41.35Hemi. 26.67 27.49 8.73 7.73Cellulose 36.08 33.40 25.67 19.31Lignin 10.75 6.37 15.43 9.24Ash 9.93 12.39 10.67 10.17The results reported here are consistent with previousreports on differences between leaves and stems and betweenlegumes and grasses for both temperate and tropical forages.Norton (1982) for example, indicated that the cell wallcontent of leaves is usually lower than that of stem, withdifferences between leaf and stem being greater in legumesthan in grasses. In this study, stems were higher than leavesin cell wall content by 5.03 percentage points while inlegumes the difference was 22.26 percentage points. The lowconcentration of hemicelluloses in legumes is well documented.Bailey (1973) reported a lower concentration of totalhemicellulose and cellulose in legume leaves compared withgrass leaves but no marked differences were observed betweenlegume and grass stems. Lignin and cellulose concentrationswere higher in stems than in leaves of the two forages whichindicates on the former organ's rigid structure required to60hold the plant erect. Ash levels in both forages were high andit was speculated that this may have been the result ofcontamination of soil minerals effected by the dusty and windyconditions that prevailed during the period prior toharvesting of these forages.3.3.2 Degradation of DX, the cell wall and its constituents3.3.2.1 Extent of degradation of DX and the cell wall.Table 3.2 shows the in situ degradation values of DM forthe two forage fractions. Only the 48h incubation statisticalcomparisons are shown in this table, but data on all the otherincubation times are also included. Appendix Table 1 shows theANOVA for DM at 48h for the two forages. 48h comparisons wereshown as it was felt that these values could reflect on theextent of degradation at normal rumen retention time (about50h Van Soest, 1975). At 48h, bana stems DM was moredegradable (P < 0.05) than that of bana leaves. This wasunexpected but it should be noted that the extent ofdegradation at 48h depended on the rate at which degradationwas changing for both leaf and the stem fractions at thatparticular time. Desmodium leaves DM was significantly moredegraded (P < 0.05) than stems. This is consistent with otherfindings (Akin, 1989) which indicate that the lbwdigestibility of mature legume plants is primarily caused bythe low digestibility of the stems. This has been attributedto the presence of heavily lignified xylem, phloem cap and61interbundular cells which form a formidable barrier todegradation (Akin, 1989). A significant (P < 0.05) interactionbetween forage and plant components was observed. This waspossibly because the plant parts were obtained from the sameoriginal material.Table 3.2 Extent of degradation of DM in bana grass andsilverleaf desmodium.Treatment INCUBATION TIME (h)0 12 18 24 48 72 96 120B. Stems 18.5 30.8 38.6 44.3 59.8a 63.0 65.8 67.6B. Leaves 16.0 24.2 34.2 41.3 56.2b 61.6 65.7 67.7D. Stems 17.5 24.9 33.8 37.6 49.7c 52.9 55.3 56.8D. Leaves 19.6 39.8 57.5 62.2 73.0d 74.7 76.2 78.2S.E.M. 0.15 1.41 1.30 1.49 0.59 0.52 0.43 0.36a,b,c,d Means followed by a different letter within the 48 andcolumn differ (P < 0.05).DM degradabilityDesmodium stems and leavesFigure 3.1Table 3.3 Extent of degradation of NDF in bana grass andsilverleaf desmodium.Treatment INCUBATION TIME (h)0 12 18 24 48 72 96 120B. Stems 18.5 30.1 37.5 44.2 60.2a 63.8 66.6a 68.6B. Leaves 19.9 28.2 40.5 48.0 63.2b 68.0 71.0b 73.2D. Stems 19.8 23.2 32.6 37.7 49.4c 53.2 54.8c 57.2D. Leaves 37.9 49.8 65.1 70.5 77.9d 79.5 80.4d 82.3S.E.M. 0.21 1.17 1.22 1.37 0.62 0.48 0.42 0.31a,b,c,d Means followed by a different letter within the 48 and96h columns differ (P < 0.01).Degradation of bana grass cell walls (NDF, Table 3.3) washigher (P < 0.01) for leaves than for stems at 48h and 96h. Asimilar situation was observed with the legume. Grass stemcell walls were more degradable (P < 0.01) than those oflegume stems, but legume leaf cell walls were more degradable(P < 0.01) than those of grass leaves. It is well establishedthat legumes have higher lignin concentrations than grassesand that the concentration is greater in stems than in leaves(Jung, 1989). Smith et al. (1972) reported that legume cellwalls had a lower extent of digestion than grass cell wallsand were also associated with higher lignin concentration's.This could explain the observation in this study where grassstems were more degradable than those of legumes. In the caseof leaves, Van Soest (1982) notes that grass leaves have botha metabolic and a structural role through the midrib which may64result in lower degradability of this tissue compared withlegume leaves which mainly have a metabolic role.Grass leaf cellulose was degraded to the same extent at48h. as stem cellulose (Table 3.5). Similarly no significantdifference was observed between grass leaf and stem degradedat 96h. In the case of the legume significantly more (P <0.05) leaf cellulose was degraded compared with that of stern.Degradation of the hemicellulose in grass fractions did notdiffer (P > 0.05) at 48h but that of leaves was moredegradable (P < 0.05) than that of stems at 96h (Table 3.4).On the other hand, the legume fractions differed (P < 0.05) intheir hemicellulose degradability at the two incubation times.Desmodium stems had the lowest extent of hemicellulosedigestion at 48h and 96h.Table 3.4 Extent of degradation of hemicellulose inbana grass and silverleaf desmodium.Treatment INCUBATION TIME (h)0 12 18 24 48 72 96 120B. Stems 18.5 34.3 37.4 45.4 61.6a 64.4 66.2a 68.1B. Leaves 15.9 24.4 39.2 45.6 64.0a 68.3 70.1b 73.0D. Stems 17.4 21.3 21.9 27.7 37.2b 42.8 41.0c 44.6D. Leaves 22.2 23.4 56.3 65.2 77.7c 80.6 81.0d 83.0S.E.M. 1.21 2.89 1.63 2.56 2.15 1.01 1.17 1.13a,b,c,d Means followed by a different letter within the 48 and96h columns differ (P < 0.05).65Table 3.5 Extent of degradation of cellulose in banagrass and silverleaf desmodium.Treatment INCUBATION TIME (h)0 12 18 24 48 72 96 120B. Stems 18.5 35.3 38.8 44.5 63.9a 67.6 71.4a 72.7B. Leaves 16.0 27.0 38.3 45.7 63.5a 69.1 72.7a 74.2D. Stems 17.4 26.0 37.6 39.3 56.9b 59.3 60.7c 63.2D. Leaves 14.1 31.4 48.2 48.3 63.5a 63.8 65.2d 68.6S.E.M. 1.02 2.61 1.47 1.95 0.81 1.11 0.51 0.50a,b,c,d Means followed by a different letter within the 48 and96h columns differ (P < 0.05). DEGRADATION CHARACTERISTICS OF DM, THE CELL WALL ANDITS CONSTITUENTSTables 3.6 to 3.8 are a presentation of the degradationkinetic parameters, as defined by Orskov and MacDonald (1979),for DM, the cell wall and its constituents. Leaves ofdesmodium had a higher (P < 0.05) insoluble but potentiallydegradable (fraction ,b,) DM compared with desmodium stems.However, grass leaves did not differ (P > 0.05) from grassstems in this fraction. Grass leaf and stem fractions did notdiffer (P > 0.05) in their cell wall (NDF) fraction 'b'content. This could be compared with the results of Fritz etal. (1990) who observed a higher concentration of cell wallsin leaves than in stems of sorghum x sudangrass genotypes. Therates of degradation associated with this fraction in the twomorphological parts did not differ (P > 0.05). This wasunexpected more so because leaves are known to contain highlydegradable and unlignified cells (for example the mesophylls).It is however noted that lignin is not related to rate of66degradation of the cell wall but rather determines the extentof degradation.The potentially degradable hemicellulose was higher (P <0.05) in grass leaves than in stems. However, this fractiondid not differ (P > 0.05) for cellulose in both leaves andstems of the grass. A similar situation existed with thelegume. No difference (P > 0.05) was observed in the rates ofdegradation of these polymers in both grass and leaffractions. The rate of degradation of legume cellulose andhemicellulose were 1.3 and 1.5 higher respectively in leavesthan in stems. The amounts of these polymers potentiallydegradable in the rumen were higher (P < 0.05) in leaves thanin the stems. Generally the grass fractions were higher thanthe legume fractions in potentially degradable cellulose andhemicellulose. This could be related to the lower levels ofthese polymers in the original legume plant materials. Amongstthe grass and the legume plant parts, legume leaves hadsignificantly (P < 0.05) higher rates of degradation of thecell wall constituents. The grass and the legume did notdiffer in lag phase possibly because of the large error. Smithet al. (1972) observed that legume cell walls were digested ata faster rate than grass cell walls, even though theygenerally had a higher lignin content and lower extents ofdigestion than grasses. The higher rate of degradation oflegume leaves in this study, could lead to higher rate ofdigestion of the whole plant.Table 3.6 Bana and desmodium DM degradation parametersTreatment DEGRADATION PARAMETERSa b lag (h) cBana^Stems 19.0 54.7 ac 3.8 0.03Bana Leaves 16.0 58.2 c 7.0 0.04Desmodium Stems 17.4 45.1 b 7.1 0.05Desmodium Leaves 14.1 52.7 a 5.3 0.08S.E.M. 1.42 1.46 1.4 0.01atbfc Means followed by different letter(s) within a columndiffer (P < 0.05).a= soluble material (%)b= potentially degradable material (%)c= rate of degradation of 'b' (%)Table 3.7 Bana and desmodium EDF degradation parametersTreatment DEGRADATION PARAMETERSa b lag (h) cBana^Stems 18.5 a 50.1 a 5.6 0.04 aBana Leaves 19.9 b 52.3 a 8.2 0.05 ab,Desmodium Stems 20.1 b 35.9 b 10.1 0.05 abDesmodium Leaves 37.9 c 42.5 c 8.6 0.1 bS.E.M. 0.17 0.64 1.1 0.01a(b/c Means followed by different letter(s) within a columndiffer (P < 0.05).Table 3.8 Bana and desmodium cellulose degradation parametersTreatment DEGRADATION PARAMETERSa b lag (h) cBana^Stems 18.9 54.7 a 3.8 0.03 aBana Leaves 16.0 58.2 a 7.0 0.04 aDesmodium Stems 17.4 45.1 b 7.1 0.05 aDesmodium Leaves 14.1 52.7 a 5.3 0.08 bS.E.M. 1.42 1.46 1.4 0.01a t b Means followed by different letter(s) within a columndiffer (P < 0.05).Table 3.9 Bana and desmodium hemicellulose degradationparametersTreatment DEGRADATION PARAMETERSa b lag (h) cBana^Stems 18.5 50.0 a 3.5 a 0.04 aBana Leaves 15.9 56.0 b 8.7 b 0.06 aDesmodium Stems 14.8 29.3 c 13.0 c 0.06 cDesmodium Leaves 20.8 60.0 b 10.9 bc 0.1 bS.E.M. 1.69 1.62 0.8 0.01a ( b i c Means followed by different letter(s) within a columndiffer (P < 0.05). EFFECTIVE DEGRADABILITY OF DM, THE CELL WALL ANDITS CONSTITUENTSResults for the effective degradabilities (based on boththe rate of digestion and the assumed rates of passage throughthe rumen) are presented in Tables 3.9 to 3.12. The effectivedegradability of DM was higher (P < 0.05) for grass leavesthan for the stems. Effective degradability of cellulose andhemicellulose did not differ (P > 0.05) between bana leavesand stems. However, cell walls were more degradadable (P <0.05) in leaves than in stems. Reid et al. (1973) made thesame observations with the same grass in Uganda where theyreported no significant differences in DM digestibilitybetween leaf and stem until the regrowth was 16 weeks. In thisstudy the grass was harvested as a 4 months regrowth. In theirstudy Reid et al. (1973) observed that the digestibility ofthe whole plant at 16 weeks was 48.9%. In this study at 50hthe degradability of stems was 48.2% and that of leaves 45.3%(Table 3.9).In the case of the legume leaf and stem fractions, cellwall components were more effectively degraded in leaves thanin stems (P < 0.05). DM was significantly (P < 0.05) moreeffectively degraded in bana stems compared with desmodiumstems. But desmodium leaf DM was more degradable (P < 0.05)compared to that of bana leaves. In Australia, Graham (1967)described a mature sample of uncinatum as highly lignifiedmaterial of low digestibility (about 40%), but found that70sheep in metabolism cages ate relatively large amounts of it,so that levels of energy retention were unusually high forsuch a coarse feed. The low degradability of desmodium stemscould be related to the observed low and slow rate ofdegradation. The low digestibility of the whole plant may beas a result of the low degradability of stems as observed inthe present study. Stems were also associated with the highestconcentration of lignin (15.43%).Table 3.10 Effective degradabilities of DX at differentrumen passage rates.Treatment MEAN FLOW RATESk = 0.02 k = 0.04Bana Stems 48.2a 38.8aBana Leaves 45.3b 34.9bDesmodium Stems 40.7c 33.0bDesmodium Leaves 59.6d 49.1cS.E.M. 0.31 0.67a ( b I c t d Means followed by different letter(s) within a columndiffer (P < 0.05).Table 3.11 Effective degradabilities of EDF at differentrumen passage rates.Treatment MEAN FLOW RATESk = 0.02 k = 0.04Bana Stems 48.2 a 30.4aBana Leaves 51.0 b 40.4bDesmodium Stems 40.9 c 33.3cDesmodium Leaves 67.6 d 59.4dS.E.M. 0.42 0.46acb,c,d Means followed by different letter(s) within a columndiffer (P < 0.05). 71Table 3.12 Effective degradabilities of cellulose atdifferent rumen passage rates.Treatment MEAN FLOW RATESk = 0.02 k = 0.04Bana Stems 51.2 a 40.9 aBana Leaves 50.2 a 38.5 abDesmodium Stems 44.8 b 35.8 bDesmodium Leaves 50.0 a 40.4 aS.E.M. 0.88 1.15a/b, Means followed by different letter(s) within a columndiffer (P < 0.05).Table 3.13 Effective degradabilities of hemicellulose atdifferent rumen passage rates.Treatment MEAN FLOW RATESk = 0.02 k = 0.04Bana Stems 49.2 c 39.9 cBana Leaves 49.7 c 38.2 cDesmodium Stems 30.6 a 24.3 aDesmodium Leaves 61.8 b 49.5 bS.E.M. 1.00 1.45a f b f c Means followed by different letter(s) within a columndiffer (P < 0.05).3.4 SUMMARY AND CONCLUSIONSUnderstanding the factors that limit forage utilization,particularly in those countries that depend on these materialsas the sources of energy and protein for their livestock, isvery important. In this study two locally popular tropicalforages, bana grass and silverleaf desmodium (legume) wereharvested during the dry season in Kenya and studied for theirrates and extents of degradation in the rumen. Stems of bothforages were found to be high in cell wall content, ADF andlignin. Desmodium leaves were low in both the cell wall andADF. The legume fractions were low in hemicellulose relativeto the grass. However, their lignin content were highparticularly in the stems. Cellulose and hemicellulose werehigh in the grass fractions.Results from this study indicated that the two forageswere poorly degraded in the rumen. Desmodium stems wereparticularly so, a finding that seems to agree with the reportof Akin (1989) that the low digestibility of the mature wholeplant may be because of the low digestibility of the stems.Desmodium leaves were however moderately degradable. Thiscould be related to a high content of rapidly degradable celltypes like the mesophyll cells (Minson and Wilson, 1980).There was no difference in the rate of degradation of DM inthe grass fractions but this was higher in desmodium leavescompared to the stems. Grass leaves were higher in content ofpotentially degradable cellulose and hemicellulose than the73stems. The rates of disappearance of these polymers in bothleaves and stems was not different.In terms of the effective degradabilities of DM and thecell wall components the results indicated that the grassfractions were not different. Legume fractions were howeverdifferent in this respect, with the leaves having highereffective degradabilities than stems. The effect of higherpassage rates especially with forages of poor quality is toreduce their degradability in the rumen and this was evidentin this study by comparing two passage rates, 0.02 and 0.04/h.The low degradability of the two tropical forages in thisstudy may be related to their high cell wall and lignincontents. It was clear with the desmodium stems that lignincould have played a major role in limiting the degradation ofthe cell wall and its constituents. Because of the highdegradability of the legume leaves and its low cell wallcontent and high crude protein content it is suggested thatthis forage could be combined with the grass during the dryseason to improve intake. It is however noted that desmodiumis associated with high levels of tannins, but since itsintake seems to be relatively high in sheep (Graham, 1967) itis possible that with cattle the intakes may even be better.'It is suggested that during the wet season when there isabundant forage of high quality (high protein content and lowcell wall concentration) some form of conservation,particularly ensiling, can be adopted. Fisher and Shelford74(1988) have enumerated the possible ways through which forageslose their nutritive value during the ensiling process.Delaying cutting should be avoided as it is one of the waysnutritive loss occurs.CHAPTER FOUREXPERIMENTAL: DEGRADATION OF TEMPERATE GRASSES4.1 IntroductionThe role of the cell walls in regulating voluntary intakeand digestibility is well documented. Since the cell wallshave a major role contributing to the fill effect inruminants, factors which influence their rate and extent ofdegradation will reflect on voluntary intake. Such factors ascellulose crystallinity, linearity and branching Ofhemicelluloses as previously discussed, have profound effectson cell wall degradation parameters (rate and extent ofdegradation). These parameters will also differ depending onthe type of cells being degraded. Mesophyll cells, whichcomprise most of the leaf tissue, are practically unlignifiedand therefore highly degradable. Most experiments on celluloseand hemicellulose degradation have been conducted on theisolated polymers which reportedly are rapidly degraded. Thisscenario may be very different from that of the intact plant.In proposing this study, it was hypothesized that theoverall rate and extent of cell wall degradation will bedetermined by the rates and extents of degradation ofindividual polymers. It was speculated that linear andbranched hemicellulosic polymers will differ in theseparameters and that maturity could have a significant effectin this respect. The main objective of this study was to76determine the degradability of cellulose,^linear xylan(Hemicellulose A) and a mixture of linear and branched short-chain heteroxylans (Hemicellulose B) by incubating the intactplant parts in the rumen and then extracting those polymersthat survive rumen incubation.4.2 ObjectivesThe specific objectives of these experiment were:1. to determine the effect of maturity and species on thelevels of cellulose, linear xylan and branched heteroxylanin both leaf and stem fractions of orchard grass and tallfescue;2. to investigate whether there are any differences in thedegradability of each of these polymers between leaves andstems;3. to investigate whether there are differences between thedegradability of linear xylan (hemicellulose A) from mixedlinear and branched heteroxylans (hemicellulose B) withinthe same plant part;4. to relate the degradabilities of the various polymers tolignin and its concentration on the respective plantparts.5. to interprete the observed results in terms of solvingpractical animal production problems.774.3 MATERIALS AND METHODS4.3.1 ForagesTwo forages, orchard grass (Dactylis alomerata) cv. Mobiteand tall fescue (Festuca arundinacea) cv. Courtney, were usedin this experiment. They were harvested in the spring of 1992from plots at Agriculture Canada Research Station - AgassizB.C. Orchard grass was harvested on three different dates; May11th., May 20th., and May 29th. This particular grass showsrapid decline in digestibility within short growth periods(Shelford and Fisher, 1988). Tall fescue was harvested on twodates; May 20th. and May 29th. A third harvest, June 5th. wasdiscarded after it was found that it was not a first cut asoriginally intended.These grasses were manually separated into leaves andstems. Leaves were defined as those parts that form the leafblade while stems included the leaf sheaths. Dead leaves werediscarded. The separate fractions were then dried in a forceddraught oven maintained between 55 - 60°C until they weresufficiently dry for grinding. The weights of the dried leafand stem fractions of the two grasses for each harvest datewere taken and recorded. Wet weights of the separated leaf andstem fractions were not taken. After drying the materials werethen ground to pass through a 2mm sieve in a laboratory hammbrmill (Orskov et al., 1980).784.3.2 In situ incubationsThree Holstein heifers fitted with permanent rumencannulae were used in this study. They were adapted (twoweeks) and maintained on a purely grass hay diet fed twicedaily (0800 and 1400hrs). About 7g of the ground substratewas weighed into nylon bags ( size 10cm x 20cm with mean poresize of 53Am) and the mouth of each bag tied and secured witha nylon string. The substrate weight to bag area ratio was17.5mg/cm2 which is well within the recommended range of 10 to20mg/cm2 (Nocek, 1988). Only one run was possible because of ashortage of cannulated animals at the time, inadequacy oforiginal forage material and a tight schedule for theavailable animals.To model the degradability of DM, cellulose, linear xylanand branched heteroxylan components of the forage, duplicatesubstrate samples were incubated in each cow for 12, 18, 24,48 and 72 hours. The bags were secured in a weighted mesh bagwhich was held about 50cm from the mouth of the cannula. Allthe bags were placed in the ventral sac of the rumen. Samplesto be exposed for 72 hours were placed into the rumen fir'stabout one hour before feeding time. Introduction of theremaining samples was conducted in reverse order over time,such that, the last samples (12h incubation) were introduced60h after introduction of the 72h samples. Thus, all bags wereremoved at the same time, 72h after entry of the first setwith the hope of minimizing variation in washing. Zero hour79disappearance of DM was determined by placing substrate samplebags in static water in a basin for 30 minutes.All the bags were washed under gently flowing tap wateruntil rinsings were clear. This took approximately 2 minutesper bag. After gentle squeezing (Orskov, 1980) to removeexcess water the bags were dried in a forced draught oven at55°C for 48h. The bags were then weighed to determine DMdisappearance.4.3.3 Experimental designData on orchard grass and tall fescue were analysedseparately since the available information (physiologicalmaturity) could not justify their combined analysis andcomparison. Data on orchard grass DM degradability wereanalysed as a 6 (incubation time) x 3 (stage of maturity) x 2(plant part) factorial in a completely randomised blockdesign. Data on tall fescue DM degradability were analysed asa 6 x 2 x 2 factorial also in a randomised complete blockdesign, where cows (3) served as the different blocks. A totalof 216 observations for orchard and 144 for tall fescue weremade.Due to a severe limitation in the amount of DM residueleft after incubation, particularly for the earlier stages ofgrowth, residues from all the cows were pooled which renderedthe polymer results statistically not analysable. Incubationof about 7g resulted in less than lg of residue which after80treatment with neutral detergent solution would have beenreduced by half. While there was adequate amount of leavesfrom the earlier stages of growth for triplicate incubations,stems were severely inadequate. Other options were to conductthe experiment in several runs but the demand for the fewcannulated cows made this impossible. Polymer degradabilityresults are therefore reported as trends rather thanstatistically defined differences.The results were fitted to the following equation usingEureka program which performs Least Square Fit to find thefunction (of the required form) that best matches the pointsx, f(x) where x is the % degradation value and f(x) is theincubation time intervals:p = a + b(1 _ et)where 'p' is the percentage disappearance at time t, 'a'the soluble degradable fraction or physically lost material,'b' the insoluble but degradable fraction, and 'c' thedegradation rate constant. When t = 0, p was fixed as thedisappearance due to washing. All these constants were laterused to calculate effective DM, cellulose, linear xylan orbranched heteroxylan degradation (P) using the formula:bcP= a + ^c + kwhere k is the assumed outflow rates ( 0.02, 0.04,^0.06and 0.08).All the statistical analyses and comparisons wereconducted using the General Linear Model (least square means)81of the SAS package (1985).4.3.4 Chemical proceduresWith the exception of the degradation measurements, DMconcentration was determined by drying samples to constantweight in a forced-draught oven at 100°C. Ash was measured byigniting samples in a muffle furnace at 500 to 505°C. Nitrogenwas determined by the sulfuric acid oxidation method(Parkinson and Allen, 1975) and then CP calculated as N x6.25. Neutral detergent fiber (NDF), acid detergent fiber(ADF), cellulose and permanganate lignin analyses were carriedout according to the method of Goering and Van Soest (1970).Hemicellulose was calculated as the difference between NDF andADF. Determination of cellulose,^linear zylan andbranched heterozylanThe residues were ground to pass through a lmm sieveopening in a Wiley mill before further analysis. All analysesfor each polysaccharide were conducted in triplicate frompooled samples (across cows).To facilitate the extraction of the various polymers anycell contents and lignin remaining in the residues were firstremoved by treating the materials with neutral detergentsolution and then delignifying them using sodium chlorite(Gaillard and Bailey, 1968). Ten grams (10g) of the ground82residue was boiled with 300m1 of neutral detergent solutionfor one hour over a heating block. The neutral detergentresidue (NDR) was then dried at 400C for 48h. A 6g sample ofthe NDR was heated with 170m1. of water containing 1.9g ofsodium chlorite and 0.35m1. of glacial acetic acid, in awater bath under a hood for lh. at 70 - 80°C (Wise et al.,1946; Morrison, 1975). The reaction flask was sealed byinverting a 25ml. erlenmeyer flask onto its neck (Wise et al.1946). At intervals of 15 minutes equal amounts of acetic acidand sodium chlorite were added and the mixture gently stirred.After lh the delignified residue was washed free of chloritewith large amounts of water and then air-dried after washingwith acetone. At this stage the material (holocellulose) wasready for polymer extraction.Figure 4.1. Schematic presentation of the carbohydratefractionation procedureRumen sample residue (ground lmm)[Boil in neutral detergent solution lh.]Neutral detergent residue[Na chlorite, 80°C, lh.]Holocellulose[10% KOH, 16h, room temp.]Hemicellulose^ a-cellulose(soluble) (residue)[pH=4.5-5, 4°C, 16h]Centrifuge; 17,300g, 30min.Linear Xylan^ Supernatant(sediment)[4 vol. 95% ethanol]^ [Centrifuge; 17,300g, 30min]Mixed linear^Supernatantand branched heteroxylans^(discarded)(sediment) a-celluloseTriplicate samples (1g) of the delignified residues wereextracted by shaking with potassium hydroxide (10% w/v, 20m1.)under nitrogen at room temperature for 16h in 50m1 centrifugebottles (Gordon and Gaillard, 1976). The extract was filteredand then centrifuged at 300 x g to remove any particulatematter. The sediment was combined with the extracted residueand then washed free of alkali with water followed by acetoneand dried to constant weight. This residue formed the firstpolymer, a-cellulose. After drying its weight was taken andrecorded. Linear zylan - ALinear xylan was determined on the supernatants from theprevious step. The supernatant was acidified with 50% aceticacid to pH 4.5-5 to precipitate the linear xylan (Gordon andGaillard, 1976). The solution was placed in a refrigeratorovernight to facilitate this process. The precipitate,creamish in color, was centrifuged off at 17,300 x g for 30minutes (Jung et al, 1991). After washing this precipitate, itwas dried in a freeze drier overnight. The weight was thentaken and recorded. Since this precipitate contained a lot ofsalt from the alkali extraction step it was found necessary toash it to determine the absolute weight of the linear xylan.Ashing was done at 6000C since at 5000C it was found to leavea residue of uncombusted carbon. Absolute linear xylan was85calculated as the difference between the previously recordedvalue and the ash value. Mixed linear and branched heteroxylansBranched heteroxylan was determined on the supernatantsfrom the previous step by decanting it into 4 volumes of 95%ethanol (Jung et al., 1991; Gordon and Gaillard, 1976). Theprecipitate, which formed immediately, was collected bycentrifugation at 17,300 x g and then dried in a freeze-drierovernight. The weight was recorded. After ashing as in thecase of linear xylan, the weight of the absolute branchedheteroxylan was calculated as the difference in weight betweenthe original unashed material and the weight of the ash.4.4^ RESULTS AND DISCUSSION4.4.1 Chemical compositionThe chemical composition of both orchard grass and tallfescue leaf and stem fractions are presented in Tables 4.1 and4.2 respectively. The leaf to stem ratios of early, medium andlate cut orchard grass were 1.71, 1.20 and 1.16 respectively.Those of early and medium cut tall fescue were 2.42 and 3.10respectively. The quality of orchard grass declined from theearly cut to the late cut as indicated by the decrease in CPand the increase in NDF and ADF in both leaves and stems.Leaves showed a trend of higher CP than stems at the threestages of growth. Neutral detergent fiber (NDF), a measure ofthe cell walls (Van Soest, 1982), rose by 9.35 percentagepoints within two weeks for stems and by 7.41 percentagepoints within the same period of time for leaves. Aciddetergent fiber (ADF), a measure of lignocellulose andindigestibility rose by 8.33 percentage points between earlyand late cut stems. With leaves ADF rose by 5.77 percentagepoints between the two cuts.The results with tall fescue were unexpected. Crudeprotein values were very low as shown in Table 4.2. While thelow CP values would probably indicate that this grass was pastheading visual observation and the leaf to stem ratioindicated otherwise. The crude protein content of forages isinfluenced by the stage of growth and the level of nitrogen87fertilizer applied during growth (Jones and Wilson, 1987).Young vegetative growth is high in protein but the proteincontent rapidly falls as the proportion of leaf decreases.Protein content decreases in both leaves and stems as theplant ages but the decline is more rapid with stems than withleaves (Norton, 1982). Our results for orchard grass are inagreement with these reports. However, those of tall fescuewere inconsistent. Crude protein content was determined atU.B.C. and verified at Agriculture Canada Research Station -Agassiz.Cellulose: This important structural component of plantsrose from 28.32% for early maturity stems to 33.79% for latematurity stems in orchard grass. Leaves had a tendency to belower cellulose than stems at all stages of maturity. Theincrease in cellulose content with maturity was smaller withleaves compared with stems. This reflects on the role ofcellulose, particularly in stems, of providing strength andrigidity to the whole plant. Tall fescue showed a similartrend to orchard grass but the increase in cellulose wassmaller. Both orchard grass and tall fescue leaves showed atrend of lower cellulose than stems. These results are inagreement with past findings regarding changes in cellulosewith growth. For example, Bailey (1973) indicated thatcellulose content in leaves may rise rapidly from the levels(8 - 10%) of the early stages of growth to much higher levels(21 - 22%) as was the case with ryegrass.88Hemicellulose A: Linear^xylan,^referred to^ashemicellulose-A in Tables 4.1 and 4.2, was first defined byGaillard (1962) as a water insoluble heteroxylan containinguronic acid and a small proportion of arabinose. The levels ofthis polymer were observed to increase with maturity for bothleaves and stems of orchard grass. For stems, there was anincrease in this polymer by 3.75 percentage points from earlyto late maturity. Apart from the early maturity stage, leavesand stems had a similar trend in linear xylan content. On theother hand, tall fescue showed the same trend. Few studieshave been conducted in which the quantitative distribution ofthis polymer in leaves and stems has been determined. However,past research has shown that as a plant matures the ratio ofxylose to arabinose and glucose residues increases, indicatingan increase in linearity of the hemicellulose (Reid andWilkie, 1969; Morrison, 1980).Hemicellulose B: Branched heteroxylans or hemicellulose Bhave been defined as a mixture of small molecular weightxylans which are richer in side chains together with morecomplex molecules containing galactose, glucose and rhamno .se(Bailey, 1973). The distribution of these polymers did notseem to differ in both leaves and stems of orchard grass withmaturity. Tall fescue showed the same trend as orchard grass.Chemical fractionation of hemicellulose B has shown that itcontains both a linear and a highly branched polymer(Gaillard, 1965). Morrison (1980), looked at the change in89proportion between the linear and the branched polymers withmaturity in both leaves and stems of S26 cocksfoot (Dactylisqlomerata) and S170 tall fescue (F. arundinacea). In bothcases, and for both leaves and stems, ageing resulted in anincrease in the linear molecule at the expense of the branchedone. The overall effect of this change on hemicellulose B isto make this molecule more linear at later stages of maturity,even though no change may be observed in total extractablehemicellulose B. Data presented here show trends that suggestno changes in this cell wall component, but it is possiblethat the linear component associated with it may haveincreased at the expense of the branched polymer withmaturity.Lignin content in both leaves and stems of orchard grassand tall fescue were low as shown in Tables 4.1 and 4.2. Otherthan in the early stage of maturity of orchard grass steinsshowed a higher content than leaves. Tall fescue showed thesame trend.Table 4.1. Chemical composition of orchard grass leaf andstem fractions (DM).Item(%)TreatmentsEarlyStems EarlyLeaves MediumStems MediumLeaves LateStems LateLeavesCP 8.1 16.2 6.0 14.3 5.2 13.2NDF 58.5 54.8 61.3 56.3 67.8 62.2ADF 34.2 30.2 38.0 31.2 42.5 36.0a-Cell. 28.3 25.7 30.6 26.4 33.8 28.8Hemi-A 8.6 10.4 10.7 9.7 12.4 11.4Hemi-B 11.9 10.3 10.8 10.1 11.6 11.2TotalHemi A+B 20.6 20.7 21.5 19.8 24.0 22.5Lignin 3.0 2.9 4.0 3.4 5.8 4.6^.Ash 10.3 8.8 8.28 9.7 9.9 8.8Table 4.2 Chemical composition of tall fescue leaf and stemfractions (% DM).Item TreatmentsEarlyStem EarlyLeaves MediumStems MediumLeavesCP 4.9 9.9 4.5 9.6NDF (%) 54.6 52.8 55.1 53.1ADF (%) 33.2 31.7 34.3 31.8a-Cell. 27.1 26.2 27.9 26.7Hemi-A 9.8 9.8 9.8 10.4Hemi-B 9.6 10.9 11.5 10.7TotalA + B 19.4 20.7 21.3 21.1Lignin 3.0 3.0 4.0 3.6Ash 5.6 6.0 7.5 RUMEN RATES, EXTENTS AND EFFECTIVE DEGRADABILITIES OFDM AND CELL WALL POLYMERS4.4.2.1 Degradation of forage DMResults on the extent of DM degradation in the rumen fororchard grass and tall fescue are presented in Tables 4.3 and4.4 respectively.^Appendix Tables 2, 3, 5, and 6 are apresentation of the ANOVAs of DM for the two forages.^.Theeffect of maturity on DM disappearance is well documented(Norton, 1982; Van Soest, 1982). In this study, thedisappearance of DM was significantly higher (P < 0.05) with92leaves than with stems. Comparisons were made for all theincubation times but statistical comparisons are shown onlyfor 48 and 72h. Significant differences (P < 0.05) wereobserved between leaves at the early stage and the latterstages of growth in 72h DM disappearance. However medium andlate harvested leaves did not differ (P > 0.05) in DMdisappearance. A drop of 6.45 percentage points in DMdisappearance between early and late cut leaves was observed.Stems were significantly different (P < 0.05) in DM loss at72h at all the three stages of growth. A drop in DMdisappearance at 72h of 11.3 percentage points occurred forstems. Figure 4.2 shows the degradation curve of orchard grassDM at early and late maturity for both leaves and stems.Disappearance of tall fescue leaf and stem fractions did notestablish a clear or obvious trend. Leaves were significantlyhigher (P < 0.05) in DM disappearance (72h) compared to stemsat the early maturity stage but not at the second stage.These results are in agreement with previous reportsregarding digestibility of leaves and stems. Leaves areusually more digestible than stems (Akin, 1986) but can beequal or only slightly less digestible at earlier stages ofgrowth (Buxton et al., 1985). This is related to differencesin cell-wall concentration and chemical composition betweenleaves and stems (Hornstein et al., 1989). The fact that nosignificant differences were observed between either leaves orstems at the two harvest stages of tall fescue may indicate93that this particular grass does not change in quality withinshort growth periods.DM degradabilityOrchard grassFigure 4.295Table 4.3 Extent of DM degradation for orchard grass leafand stem fractions.Treatments TIME OF INCUBATION (h)0 12 18 24 48 72Early Stems 26.62 40.70 51.87 62.84 75.17a 78.83aEarly Leaves 24.82 43.16 56.83 68.83 77.04a 82.67dMedium Stems 23.14 38.83 44.41 53.55 66.88b 74.58b.Medium Leaves 20.28 36.33 52.31 67.41 73.73c 78.04aLate Stems 17.77 34.25 42.15 49.61 63.99d 67.53cLate Leaves 18.53 40.06 48.97 59.93 68.70b 76.22abs.e.m. 0.368 1.17 1.91 1.00 0.80 0.86a,btc,d Means within the same column (48 and 72h) followedby different letter(s) differ (P < 0.05).Table 4.4 Extent of DX degradation for tall fescue leafand stem fractions.Treatments TIME OF INCUBATION (h)0 12 18 24 48 72Early Stems 35.36 52.23 57.40 59.95 70.87b 77.21bEarly Leaves 31.27 52.22 61.24 62.19 77.18a 83.70aMedium Stems 33.57 49.46 56.13 62.39 74.43a 80.69a -Medium Leaves 31.46 50.63 59.58 66.92 75.15a 82.40as.e.m.^11^0.39 1.25 1.17 1.54 1.51 0.94a,b Means within the same column (48 and 72h) followedby different letter differ (P < 0.05).A noticable difference between early and late harvests wasin their fraction 'a', the immediately soluble material. Latecut forage fractions were associated with significantly lower(P < 0.05) 'a' values than early-cut fractions. This couldpossibly indicate the lower concentration of soluble matter as96reflected by an increase in NDF content. It could also probablyreflect on the greater resistance to breakage associated witholder more lignified tissues which may have led to lowerpassive loss of dry matter from the bags. The second fractiondenoted by constant 'b'^is insoluble but potentiallydegradable (Orskov, 1991). No significant differences (P >0.05) were observed between leaves and stems in this fractionat the three stages of growth of orchard grass. In tall fescue(Table 4.7) leaves had significantly higher (P < 0.05) DM 'b'fraction than stems and also the older material wassignificantly lower (P < 0.05) than younger material in thisfraction. No significant differences (P > 0.05) were observedin the rates of disapperance, denoted by the letter 'c',the 'b' fraction between leaves and stems and amongstdifferent maturities for both orchard grass and tall fescue(Tables 4.5 and 4.7 respectively).The extent of degradation occurring in the rumen isdetermined by both the rate of digesta passage within therumen and its rate of degradation. Tables 4.6 and 4.8 show theeffective DM degradation values of orchard grass and tallfescue respectively, calculated for various rates of passage.Other than for the early maturity stage, leaves and stems oforchard grass differed (P < 0.05) in effective DM degradation.Late cut stems were significantly lower (P < 0.05) ineffective degradability than stems of early cut but not themedium one. Early and medium cut leaves did not show any97differences (P > 0.05) in effective degradability. However,late cut leaves were significantly lower (P < 0.05) thanearlier cuts in effective degradation. Tall fescue leaves andstems did not have any significant differences (P > 0.05) ineffective degradability either at the earlier or lattermaturity stage. This can be explained by the fact that thecharacteristics of the two cuts were not sufficientlydifferent in maturity.Table 4.5 Orchard grass DM degradation constantsTreatments DM DEGRADATION CONSTANTSa^(%) b^(%) cEarly Stems 24.97c 60.13de 0.035cEarly Leaves 23.30f 63.30de 0.043hMedium Stems 21.87f 62.33de 0.028cMedium Leaves 18.40k 64.27d 0.044hLate Stems 16.97k 56.73e 0.034cLate Leaves 17.90k 62.33de 0.043hs.e.m. 0.49 2.30 0.005a= % solubility;b= potentially degradable fraction [%] andc= rate of degradation of the 'b' fraction (/h].ccd,e,f,k Means within the same column followed bydifferent letter(s) differ (P < 0.05).c,h Means within the last column followed by differentletter differ (P < 0.1)Table 4.6 Effective DM degradation values (%) for orchardgrassTreatments Mean outflow rate variablesk=0.02 k=0.04 k=0.06 k=0.08Early^Stems 62.97db 52.83ab 46.90bc 43.10abEarly Leaves 66.07b 55.73a 49.43b 45.20aMedium Stems 58.13ad 47.47bc 41.63cd 37.97bdMedium Leaves 62.03db 51.53ab 45.17bc 40.80abLate Stems 52.60a 42.93c 37.43d 33.83dLate Leaves 59.16cd 49.00bc 42.93cd 38.83bdI^s.e.m. 1.84 1.98 1.94 1.86^Ik= rumen digesta flow rate Uh].acb,c,d Means within the same column followed bydifferent letter(s) differ (P < 0.05).Table 4.7 Tall fescue DM degradation constantsTreatments DM Degradation Constantsa^(%) b^(%) c^(%)Early Stems 35.80a 53.73a 0.036a^*Early Leaves 30.97b 56.50a 0.044aMedium Stems 33.33c 44.03b 0.032aMedium Leaves 31.00b 52.77ab 0.047as.e.m. 0.71 2.79 0.008a ( b f c Means within the same column followed bydifferent letter(s) differ (P < 0.05).Table 4.8 Effective DM degradation values (%)^for tallfescue grass.Treatments Mean outflow rate variablesk= 0.02* k= 0.04 * k= 0.06^* k= 0.08*Early Stems 63.97 56.53 52.23 49.40Early Leaves 68.16 59.10 53.67 50.00Medium Stems 65.87 56.67 51.67 48.37Medium Leaves 67.03 58.56 53.33 49.80 .I^s.e.m. 1.57 2.02 2.08 2.04*means within these columns not different (P > 0.05) Degradation of forage cell wall polymers4. CelluloseDegradation of cellulose was determined by isolating thefraction that survived rumen incubation. Table 4.9 shows theextent of cellulose disappearance from the rumen at varioustimes. Degradation of this polymer was high in orchard grassat all stages of growth at 72h ranging from 71.5% in late cutstems to 84.7% in early stems. Based on the actual degradationvalues and the standard deviations (72h), it would seem thatboth leaves and stems at the early stage of harvest of orchardgrass were not different. Similarly, it would appear that morecellulose disappeared from leaves than from stems at the latematurity stage of growth. Akin (1986) observed that planttissues differ in rates of disappearance, suggesting thattissue morphology also may affect accessibility and rate ofdigestion. The surface area that is accessible to enzymes waspositively related to rate of digestion when surface area was10 0increased by swelling of cotton linters (Stone et al., 1969).There was a general trend of cellulose degradabilitydeclining in both leaves and stems as these morphologicalparts matured. Results of rate of degradation presented inTable 4.10 show that cellulose had a trend of being morerapidly degraded in leaves than in stems at all stages ofgrowth other than in the early one in orchard grass. However,maturity did not seem to result in much change in rate ofcellulose degradation in stems. Younger leaves seemed morerapidly degraded relative to older, late harvested leaves. Thepotentially degradable fraction, 'b', declined from 73.37% atearly maturity to 65.10% at late maturity for stems. Leavesshowed a similar trend declining from 70.63% to 62.70%. Basedon the standard deviations it would appear that both leavesand stems of early and medium harvests possibly were notdifferent in their potentially degradable fraction.Table 4.11 shows the results for the extent of degradationof tall fescue cellulose. No clear trend was observed in thedegradation of this polymer in both leaves and stems. However,based on the 72h degradation values and their standarddeviations it would appear that earlier cut stems were lessdegradable than the corresponding leaves (79.5± 0.04 vs 84.4±0.04). Rate of cellulose degradation did not seem to differ instems at the two growth stages (Table 4.12). However, leafcellulose seemed to be degraded faster in the medium stage ofgrowth than in the early one.101Digestion of cellulose is incomplete but this depends onthe type of tissues and cells being digested (Chesson andForsberg, 1988). Darcy and Belyea (1980) reported thatcellulose of late cut orchard grass was less digested thanthat of early cut. Mowat et al. (1965), also observed with invitro studies that leaves of earlier cut orchard grass wereless digested than stems. The results reported here showed atrend consistent with their findings.Table 4.9 Extent of degradation of cellulose in orchard grass.(Mean ±SD; n=3)Treatments TIME OF INCUBATION (h)0 12 18 24 48 72Early Stems 26.7 37.1 50.1 62.9 77.6 84.7+ 0.11 ± 0.18 ± 0.16 ± 0.18 ± 0.18 ± 0.10Early Leaves 24.9 52.8 54.5 67.8 76.9 84.3± 0.14 ± 0.56 ± 1.44 ± 0.42 ± 0.31 ±^0.38 .Medium Stems 22.5 37.9 43.3 53.6 73.4 77.8± 0.47 ± 0.62 ± 2.23 ± 0.77 ± 0.45 ± 0.06Medium Leaves 20.3 33.8 43.1 66.2 74.3 78.9± 0.02 ± 0.50 ± 0.32 ± 0.09 ± 0.25 ± 0.17Late Stems 17.8 31.7 41.3 51.2 70.4 71.5± 0.41 ± 0.86 ± 0.26 ± 0.03 ± 3.76 ± 0.31Late Leaves 18.5 40.9 48.8 60.4 67.7 79.1± 0.09 ± 0.46 ± 0.36 ± 0.40 ± 0.24 ± 0.28Table 4.10 Orchard grass cellulose degradation constants(Mean ±SD; n=3)Treatments CELLULOSE DEGRADATION CONSTANTSa (%) b (%) cEarly Stems 24.10± 0.100 73.37± 1.041 0.025± 0.0006Early Leaves 25.17± 0.058 70.63± 0.651 0.044± 0.0021Medium Stems 21.37± 0.252 71.03± 1.950 0.024± 0.0010Medium Leaves 17.43± 0.116 69.97± 0.611 0.035± 0.0032Late Stems 16.23± 0.451 65.10± 0.854 0.029± 0.0006Late Leaves 18.43± 0.058 62.70± 0.854 0.039± 0.0010Table 4.11 Extent of degradation of cellulose in tall fescuegrass (Mean ±SD; n=3).Treatments TIME OF INCUBATION (h)0 12 18 24 48 72Early Stems 35.2 52.4 57.3 61.3 73.0 79.5+ 0.21 ± 0.17 ± 0.79 ± 0.28 ± 0.14 ± 0.04Early Leaves 31.5 48.2 59.1 59.9 76.6 84.4± 0.44 ± 0.22 ± 0.68 ± 0.19 ± 0.75 ± 0.04Medium Stems 33.6 52.0 58.4 66.2 78.1 84.8± 0.10 ± 0.14 ± 0.11 ± 0.13 ± 0.71 ± 0.08Medium Leaves 31.5 48.8 57.0 64.9 75.3 83.6± 0.06 ± 0.40 ± 1.10 ± 0.42 ± 0.07 ± 0.12Table 4.12 Tall fescue cellulose degradation constants(Mean ±SD; n=3)Treatments CELLULOSE DEGRADATION CONSTANTSa (%) b (%) c^.Early Stems 35.77± 0.058 56.00± 0.600 0.033± 0.0012Early Leaves 31.37± 0.058 60.00± 0.458 0.030± 0.0006Medium Stems 33.43± 0.153 47.60± 0.265 0.034± 0.0000Medium Leaves 31.20± 0.100 56.77± 0.404 0.034± 0.0015103Cellulose degradability is thought to be influenced byseveral factors. On the one hand extensive hydrogen bonding incellulose microfibrils is thought to influence the rate atwhich this polymer is degraded while on the other hand ligninshielding is thought to impact on the extent of itsdegradation (Hatfield,1989). Morrison (1979) indicated thatsince the structure of cellulose does not change significantlyduring the growing period, then cellulose of a young plantought to be as digestible as that of an older plant. Ligninwas found to increase with maturity in the present study andmore so with stems than with leaves. The presence of a highproportion of unlignified mesophyll cells in leaves (Akin,1986) would probably explain the higher degradability of leafcellulose than that of stems. The fact that tall fescue didnot seem to show any differences in cellulose digestibility atthe two stages of growth would indicate that this grass washarvested at a fairly young stage and the cutting interval tooshort for any significant changes to be detected. Hemicellulose ATable 4.13 shows the results of the extent of linear xylan(hemicellulose A) degradation at various incubation times.These results suggest that there was a rapid decline indegradability of this polymer, dropping from 75.94 to 57.02%in two weeks for orchard grass stems. In leaves however, nosuch drastic changes were observed. Degradability of this104polymer remained high (k$ 70%). Based on the standarddeviations these results suggest that hemicellulose A may nothave been differently degraded in both leaves and stems oforchard grass at the earlier stage of growth. The rate ofdegradation of this polymer tended to increase for stems withmaturation (Table 4.14). The same situation was observed withleaves. While this seems to contradict the observation on theoverall extent of degradation, it has been reported that bothrate and extent of degradation of a cell wall polymer areinfluenced by different factors (Hatfield, 1989). Lignininfluences the extent of degradation while factors likeaccessibility of the cell wall by hydrolytic enzymes,intermolecular bonding will affect the rate but notnecessarily the extent of polymer degradation.These results would tend to support the hypothesis thatlignin does not influence the rate of hydrolysis of cell wallpolymers but rather sets an upper limit to the extent to whichthese polymers can be degraded. There is good evidence that,in grasses, at least part and maybe all of the lignin iscovalently bound to hemicellulose (Morrison, 1974). Gordon andGaillard (1976) showed that in wheatstraw about 78% of thelignin in this material was associated with linear xylan(hemicellulose A) while only 8% was associated with the morebranched fraction (hemicellulose B). Increase in linearity ofhemicellulose is believed to have positive effects onhemicellulose degradability (Brice and Morrison, 1982). This105is because of an increase in the proportion of contiguousunsubstituted xylose residues. Hydrolysis of hemicelluloses byrumen hemicellulases is reported to take place at the bondbetween two unsubstituted xylose residues. An increase inlignin content however counteracts the positive effects ofhemicellulose linearization that accompany maturation(Morrison, 1979).Tall fescue showed the same trend as orchard grass in the72h extent of linear xylan degradation (Table 4.12). Likecellulose in earlier harvested stems, hemicellulose A tendedto be less degradable than the corresponding polymer in leavesat the same stage of growth. The rate of degradation of thepotentially degradable fraction, 'b', showed an increasingtrend in stems with maturation but not in leaves (Table 4.7).The potentially degradable fraction, b, dropped from 60.30 to55.07% in stems from early to medium maturity.Table 4.13 Extent of degradation of hemicellulose A in orchardgrass (Mean ±SD; n=3).Treatments TIME OF INCUBATION (h)0 12 18 24 48 72Early Stems 25.1 34.2 42.5 50.9 64.9 75.9^.± 0.11 ± 1.86 ± 1.10 ± 1.79 ± 0.47 ± 0.57Early Leaves 23.7 37.1 42.5 55.2 66.1 75.7± 0.24 ± 0.70 ± 4.03 ± 0.87 ± 0.91 ± 1.42Medium Stems 21.5 31.7 32.7 37.1 56.9 63.5± 0.07 ± 1.41 ± 0.58 ± 0.01 ± 1.87 ± 0.61Medium Leaves 15.9 23.8 30.4 53.1 65.6 68.5± 0.11 ± 1.34 ± 0.71 ± 0.50 ± 3.08 ± 0.73Late Stems 16.4 23.4 32.4 38.4 53.1 57.0± 0.39 ± 5.80 ± 1.68 ± 1.05 ± 1.52 ± 0.51Late Leaves 18.5 39.8 48.1 55.1 63.1 72.4± 0.16 ± 3.12 ± 1.72 ± 1.59 ±0.73 ± 0.63Table 4.14 Orchard grass hemicellulose A degradation constants(Mean ±SD; n=3)Treatments HEMICELLULOSE A DEGRADATION CONSTANTSa (%) b (%) cEarly Stems 24.10± 0.656 72.60± 1.212 0.014± 0.0006Early Leaves 23.83± 0.322 66.00± 1.253 0.022± 0.0023Medium Stems 21.47± 0.231 62.87± 1.563 0.018± 0.0012Medium Leaves 15.80± 0.458 71.87± 2.702 0.021± 0.0015Late Stems 16.53± 0.058 54.07± 4.055 0.021± 0.0025Late Leaves 18.63± 0.666 54.67± 2.401 0.043± 0.0031Table 4.15 Extent of degradation of hemicellulose A in tallfescue grass (Mean ±SD; n=3).Treatments TIME OF INCUBATION (h)0 12 18 24 48 72Early Stems 31.1 43.9 47.4 48.4 62.6 71.0± 0.15 ± 0.91 ± 2.03 ± 1.90 ± 0.18 ± 026Early Leaves 33.5 36.1 47.4 45.0 65.3 78.2± 0.07 ± 2.28 ± 0.43 ± 0.56 ± 1.25 ± 0.75Medium Stems 31.5 48.7 51.4 56.6 69.6 78.3± 0.57 ± 1.54 ± 0.60 ± 1.08 ± 0.94 ± 0.94Medium Leaves 31.6 42.0 47.3 57.1 66.1 79.0± 0.04 ± 0.67 ± 1.65 ± 1.43 ± 0.68 ± 0.54Table 4.16 Tall fescue hemicellulose A degradation constants(Mean ±SD; n=3)Treatments HEMICELLULOSE A DEGRADATION CONSTANTSa (%) b (%) cEarly Stems 35.10± 0.781 60.30± 1.229 0.012± 0.0006Early Leaves 31.80± 0.872 65.73± 0.551 0.015± 0.0006Medium Stems 34.03± 0.231 55.06± 0.987 0.022± 0.0000Medium Leaves 31.17± 0.416 63.23± 1.050 0.016± 0.00234. Homicellulose BThe degradability of the more branched hemicellulose(hemicellulose B) at various incubation times is presented inTables 4.17 and 4.19 for orchard grass and tall fescuerespectively. These values suggest that for orchard grass,hemicellulose B was likely to be more degradable (72h) instems than in leaves at the earlier stage of growth (88.3±0.05 vs 86.1± 0.67) but this scenario changed at late maturity108when more of this polymer tended to be more degraded in leavesthan in stems (81.0± 0.67 vs 73.8± 1.71). Irrespective of themorphological part, degradability of this polymer declinedwith maturity. It has been shown that as grass matures, thelinear component of hemicellulose B increases (Morrison,1980). Since this component is more degradable than thebranched one it would be expected that hemicellulose B wouldbecome more degradable as grass matures. However, it has beenshown that the linear component is the one closely associatedwith lignin. An increase in this cell wall component withmaturity would therefore lead to a decline in thedegradability of hemicellulose B. Based on the standarddeviations leaf hemicellulose B in tall fescue tended to bemore degradable than that of stems at the earlier growthstage, but did not at the later stage of maturity. This couldbe related to an increase in the more degradable linearcomponent of hemicellulose B and only a limited increase inlignin.Figure 4.3 shows the rumen degradation curve forhemicellulose B for both the leaf and stem fractions oforchard grass. The rate of degradation of the potentiallydegradable fraction of this polymer did not seem to differwith maturation for stems but showed a tendency to increase inleaves (Tables 4.18 and 4.20). Based on the standarddeviations it would appear that fraction 'b' was higher inearly than in late cut stems in orchard grass but not in109leaves. Similarly leaves appear not to have differed fromstems in each of the growth stages. Other than for the earlycut leaves, the degradation rate of this polymer seemed thesame for all the plant parts of tall fescue.Hemicellulose B degradabilityOrchard grassFigure 4.3111Table 4.17 Extent of degradation of hemicellulose B in orchardgrass (Mean ±8D; n=3).Treatments TIME OF INCUBATION (h)0 12 18 24 48 72Early Stems 25.8 50.0 59.4 71.4 83.3 88.3+ 0.28 ± 1.71 ± 2.81 ± 1.92 ± 0.55 ± 0.05Early Leaves 23.4 44.4 58.3 71.1 80.0 86.1+ 0.40 ± 1.20 ± 1.01 ± 1.22 ± 0.59 ± 0.67 .Medium Stems 22.2 40.8 45.9 55.5 72.9 78.0± 0.14 ± 0.18 ± 0.95 ± 1.71 ± 0.86 ± 0.56Medium Leaves 17.9 36.9 44.8 67.0 75.5 78.6± 0.06 ± 2.59 ± 2.18 ± 2.75 ± 0.19 ± 0.79Late Stems 16.8 39.0 53.3 58.8 72.8 73.8± 0.07 ± 2.47 ± 1.34 ± 0.49 ± 2.39 ± 1.71Late Leaves 17.8 44.4 54.9 63.9 72.5 81.0± 0.07 ± 0.95 ± 1.27 ± 0.54 ± 0.12 ± 0.67Table 4.18 Orchard grass hemicellulose B degradation constants(Mean ±SD; n=3)Treatments HEMICELLULOSE B DEGRADATION CONSTANTSa (%) b (%) cEarly Stems 25.87± 0.404 66.27± 1.002 0.042± 0.0020Early Leaves 23.37± 0.404 66.50± 0.265 0.042± 0.0015Medium Stems 22.10± 0.200 65.97± 1.002 0.028± 0.0017Medium Leaves 17.90± 0.700 67.73± 1.716 0.037± 0.0012Late Stems 16.80± 0.200 60.73± 1.550 0.047± 0.0031Late Leaves^_17.75± 0.570 63.55± 1.345 0.048± 0.0021Table 4.19 Extent of degradation of hamicellulose B in tallfescue grass (Mean ±SD; n=3).Treatments TIME OF INCUBATION (h)0 12 18 24 48 72Early Stems 25.5 53.0 61.7 62.3 73.7 80.1± 0.58 ± 3.04 ± 3.72 ± 1.24 ± 0.37 ± 0.84Early Leaves 23.1 56.0 59.5 67.8 76.8 85.5± 0.54 ± 0.89 ± 1.92 ± 1.39 ± 0.53 ± 0.14Medium Stems 22.1 57.5 61.5 66.0 78.1 84.7± 0.12 ± 1.22 ± 0.33 ± 1.05 ± 0.45 ± 0.53Medium Leaves 17.6 55.2 61.7 68.2 78.1 86.1± 0.53 ± 1.55 ± 1.39 ± 0.79 ± 0.79 ± 1.08Table 4.20 Tall fescue hemicellulose B degradation constants(Mean±SD; n=3)Treatments HEMICELLULOSE B DEGRADATION CONSTANTSa (%) b (%) cEarly Stems 35.77± 0.058 56.00± 0.600 0.033± 0.0012Early Leaves 31.27± 0.153 59.03± 2.043 0.030± 0.0000Medium Stems 33.43± 0.153 47.60± 0.265 0.034± 0.0006Medium Leaves 31.20± 0.100 56.77± 0.404 0.034± 0.0015As indicated earlier hemicellulose B is composed of alinear and a branched component both of which determine thetotal degradability of hemicellulose B. Bailey and Gaillard(1965) isolated both linear hemicellulose A xylan andhemicellulose B and studied the action of rumen microbialenzymes on these polymers. They reported that the branched Bpolymers ( isolated from hemicellulose B by complexing withiodine), from both grass and legume were more resistant tohydrolysis than the linear polymers. Of the linear polymers113(including linear A), the B fraction was hydrolysed mostrapidly which may explain why hemicellulose B was moredigestible than hemicellulose A in red clover (Bailey andMacrae, 1970). The resistance to hydrolysis of the branched Bpolymers by the rumen microorganisms was suggested to be due,most probably , to the high content of uronic acid in thispolymer. In this study hemicellulose B had a trend of higherdegradability after 72 hours of rumen incubation thancellulose or hemicellulose A. It appeared that hemicellulose Awas the least degradable of the polymers studied. Theobservations of Gordon and Gaillard (1976) that only a smallproportion of the total lignin is associated withhemicellulose B may explain the high extent of degradation ofhemicellulose B observed in these experiments.Arabinofuranosidase activity has been found in cell freeextracts of the rumen (Williams and Strachan, 1984) and iscapable of removing the arabinose residues from the xylans.The activity of such glycosidases renders highly substitutedarabinoxylans more susceptible to xylanase degradation.However, the three dimensional organization of the matrix mayplay a role in the extent and/or rate of such side chainremoval (Hatfield, 1989). Greve et al. (1984) observed that anarabinofuranosidase from Ruminococcus albus extensivelyremoved arabinose residues from isolated hemicellulosicmaterials (47% removal) but had a more limited activityagainst isolated cell walls (1.2%). The arabinofuranosidase114has a molecular weight of approximately 300,000. Such a largeenzyme may have difficulty penetrating the matrix to removearabinose side chains. This limitation may in turn limit theactivity of xylanases that can penetrate to the xylansubstrates. It is possible then that the high degradability ofhemicellulose B could be related to a lower proportion of thehighly branched fraction relative to the linear one andtherefore a higher activity of the xylanases. Indeed, Morrison(1980) showed the ratio of the linear to the branchedcomponent to be about 65:35 in leaves and 83:17 for stems.This ratio was dependent on maturity, being higher with oldermaterial. The decline in degradability of hemicellulose B withage could be related to the formation of hydrogen bondsbetween the linear xylans and cellulose (Hatfield, 1989) orlignin (Buxton and Brasche, 1991).The rate of digestion of fiber components is a constrainton total fiber digestion and factors altering this rate affectfiber digestibility (Allen and Mertens, 1988). Similarly,digestibility of fiber decreases as the rate of rumen turnoverincreases. Figures 4.4 to 4.9 present the results for theeffective degradabilities of the various cell wall polymers ata rumen passage rate of 0.02/hr. Data for these bar charts arepresented in appendix Tables 10 to 15. In first-order systems,retention times are reciprocals of fractional rates ofpassage. In this study relative comparisons were made only fora 0.02/hr. passage rate which corresponds to a rumen mean115retention time of 50h. The results obtained indicate that theeffective degradability of the polymers studied was far fromcomplete ranging from 44.10% (hemicellulose A) to 70%(hemicellulose B). Further the results indicate thatirrespective of the forage material, stage of growth or plantmorphological part, hemicellulose B was the most effectivelydegradable cell wall polymer. This is likely to be associatedwith the high rate of digestion of this polymer relative tothe others. Bailey et al. (1976) reported in their in vivowork using grass hemicelluloses, that all grass hemicellulosefractions were present in the feaces with a slightly lowerproportion of hemicellulose B relative to other hemicellulose.Compared to feed fractions arabinose was lower in faecalhemicellulose B but not in the other fractions. Their resultswere comparable to those of Ford (1973) who observedpreferential removal of hemicellulose arabinose, glucose andgalactose relative to xylose with a proportional increase inhemicellulose A xylan in the faeces compared to the feed.Maturity tended to have a lowering effect on theeffective degradability of all the polymers studied in orchardgrass fractions. No clear pattern was observed with the tallfescue. As a plant matures and the hemicellulose componentincreases in linearity there is a tendency for the linearxylans to form hydrogen bonds with cellulose (Morrison, 1979;Hatfield, 1989). The extent of this interaction might preventthe binding of cellulolytic microorganisms or impede the116hydrolytic activity of a microorganism's cellulolytic complex(Hatfield, 1989), thus lowering the extent of degradation ofthese polymers.Fig. 4.4^Cellulose degradabilityOrchard grassFig. 4.5^Linear xylan degradabilityOrchard grass117Fig. 4.6^Branched xylan degradabilityOrchard grass1 1E".::1 stems20^29^11cutting date (May/92)Leaves20 29Fig.^4.7 Cellulose degradabilityTall fescue grass^20^24^20^29Cutting date (May/92)^=] stems^MN Leaves118Fig. 4.8^Linear xylan degradabilityTall fescue grassFig. 4.9 Branched xylan degradabilityTall fescue grass1194.4.3 SUMMARY AND CONCLUSIONSStructural carbohydrates of forages are not completelydigestible by ruminants. The nature of these limitations arepoorly understood. In this study, an attempt was made tounderstand the effect of maturity on the major cell wallcarbohydrates of leaf and stem fractions of two temperategrasses, orchard grass and tall fescue. Further to this, anattempt was made to understand the factors that may lead totheir reduced degradability. The polymers studied werecellulose, a linear molecule of hemicellulose commonlyreferred to as hemicellulose A and another fraction that isassociated with high levels of arabinose and therefore highlybranched, simply referred to as hemicellulose B.This study showed that orchard grass declines rapidly inquality with maturity within short growth intervals. Tallfescue however did not show this trend. Cellulose in bothleaves and stems increased with maturation. Similarobservations were made with Hemicellulose A, which was alsoobserved to be equally distributed between leaves and stem's.Hemicellulose B was equally distributed betweem leaves andstems, but maturation did not seem to influence its level.Hemicellulose B was the most effectively degradablepolymer among those studied. This was related to its high rateof degradation and possibly to the presence of a highlydegradable linear xylan associated with it. Maturity had asignificant lowering effect on the degradability of all the120polymers. Hemicellulose A was the least effectively degradedof the polymers studied. Leaf polysaccharides were moredegradable than those of stems other than in the case of theearliest stage of growth when it was observed that some stempolysaccharides were more degradable. Tall fescue did not showa good trend in polymer breakdown. However, irrespective ofthe stage of growth, or plant part, hemicellulose B was themore degradable fraction compared with the others.From this study it is concluded that the rapid decline inquality of orchard grass could be related to the presence ofrecalcitrant cell wall polymers. Cellulose and hemicellulose Awere observed to increase with maturity and were bothincompletely degraded. Hemicellulose B was highly degradableat the earlier growth stages but declined with maturity. Thepossibility that linear xylans could have interacted throughhydrogen bonding with cellulose and thus rendered therespective hydrolases ineffective on these polymers cannot beruled out. In the present study lignin may not have been themajor player in limiting utilization of these polymers sinceits level was fairly low. However, it is now well establishedthat it is the chemical composition of lignin rather than itsquantity that is important in inhibiting digestibility.This study tends to support the hypothesis that the amountof linear xylan, relative to the branched xylan, may be animportant factor influencing both the rate and extent of cellwall digestion. Presumably, linear xylans have a greater121propensity to form physical and/or chemical associations withother cell wall polymers, especially lignin, that woulddecrease cell wall digestibility. This study showed thatpolymers associated with leaves were more degradable thanthose of stems.It is concluded from this study that a breeding programaimed at reducing some of the recalcitrant polymers, oralternatively reducing the lignin level may be beneficial toanimal production. Similarly a breeding program aimed atincreasing the leafiness of the forages could enhance theirutilization by livestock. 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In: The ruminant animal, digestivephysiology and nutrition, Church, D.C.), Prentice Hall,Engelwood Cliffs, New Jersey, pp. 125-144.135APPENDIXAppendix table 1 ANOVA for tropical forages (48h)Source^df^SS^MS^F^P > FTreatment^3forages(F)^1^66.6^66.6^31.6^0.0001components(C) 1^577.7^577.7 274.3^0.0001F x C^1 1089.6^1089.6 517.3^0.0001Block (cow) 2^39.9 20.0^9.5^0.0015Error^18 37.9^2.1Corrected total 23^1811.8Appendix table 2 ANOVA for orchard grass DX (48h)Source df^SS MS F P > FTreatment 5component(C) 1^98.5 98.5 25.8 0.0001maturity(M) 2 576.4 288.2 75.4 0.0001CXM 2^12.1 6.0 1.6 0.2235Block (cow) 2^343.5 171.7 44.9 0.0001Error 28^107.0 3.8Corrrected total 35^1137.4Appendix table 3 ANOVA for tall fescue grass DX (48h)Source df^SS MS F P > FTreatment 3component(C) 1 74.0 74.0 5.44 0.0315maturity(M) 1 3.5 3.5 0.26 0.6181C x M 1 47.0 47.0 3.45 0.0796Block (cow) 2 48.7 24.4 1.79 0.1955Error 18 244.9 13.6Corrrected total 23 418.1136Appendix tableSource4^ANOVA for tropical forages (96h)df^SS^MS^F P > FTreatment^3forages(F) 1^0.03 0.03 0.02 0.8797components(C) 1^646.0 646.0 582.0 0.0001F x C 1^661.6 661.6 596.1 0.4697Block (cow) 2 1.7 0.9 0.8 0.0001Error 18 20.0 1.1Corrected total 23 1329.3Appendix table 5SourceANOVA for orchard grass DMdf^SS^MS^F(72h)Pr>FTreatment 5component(C) 1 248.0 248.0 55.7 0.0001maturity(M) 2 446.2 223.1 50.1 0.0001C x M 2 49.9 25.0 5.6 0.0092Block (cow) 2 229.6 114.8 25.8 0.0001Error 27 120.2 4.5Corrrected total 34 1140.9Appendix table 6SourceANOVA for tall fescue grass DMdf^SS^MS(72h)P > FTreatment 3component(C) 1 74.0 74.0 5.44 0.0315maturity(M) 1 3.5 3.5 0.26 0.6181C x M 1 47.0 47.0 3.45 0.0796Block (cow) 2 48.7 24.4 1.79 0.1955Error 18 244.9 13.6Corrrected total 23 418.1137Appendix table 7 Degradation values for orchard grasscelluloseTreatment Incubation time (h)0 12 18 24 48 72EARLYSTEMS26.62 37.32 50.27 62.91 77.4626.83 36.96 50.06 62.70 77.7926.67 37.10 49.96 63.05 77.50LEAVES25.03 52.16 53.02 67.55 76.5424.83 52.85 55.90 67.59 77.1024.76 53.27 54.64 68.30 77.06MEDIUMSTEMS22.77 38.52 42.71 53.92 73.92 77.7322.73 37.28 41.43 54.22 73.36 77.8422.93 37.81 45.86 52.76 73.02 77.78MEDIUMLEAVES20.25 33.28 43.23 66.25 74.60 79.0520.27 34.27 43.33 66.15 74.10 78.7120.29 33.89 42.73 66.08 74.29 78.91LATE18.27 32.40 41.03 51.17 74.68 71.81STEMS17.72 30.72 41.49 51.17 68.46 71.2117.47 31.87 41.48 51.12 67.92 71.39LATE18.53 41.05 49.17 60.90 67.52 78.83LEAVES18.44 41.21 48.61 60.20 40.34 48.49 60.23 67.99 79.39138Appendix table 8 Degradation values for orchard grasshemicellulose ATreatment Incubation time (h)0 12 18 24 48 72EARLYSTEMS24.99 32.49 41.18 51.14 65.08 75.63'25.10 36.19 43.16 49.00 65.32 75.5925.21 34.01 43.01 52.54 64.42 76.59EARLYLEAVES23.40 36.30 40.05 54.27 65.00 74.1823.83 37.19 40.26 56.00 66.65 75.9123.83 37.67 40.14 55.33 66.50 75.00MEDIUMSTEMS21.40 30.29 32.91 37.12 57.42 62.8421.47 31.79 33.11 58.43 63.8321.54 31.11 32.02 37.11 57.80 63.95MEDIUMLEAVES15.80 23.31 29.98 53.67 65.48 68.4816.00 22.90 31.24 52.69 65.56 67.7716.01 23.10 30.05 53.00 66.64 69.21LATE15.93 25.99 32.62 37.25 53.82 57.36STEMS16.53 25.18 31.97 39.32 52.07 56.4316.67 24.89 31.58 38.60 52.33 57.27LATE18.32 32.52 49.89 53.55 62.61 72.17LEAVES18.46 34.00 48.05 55.08 62.71 71.8518.63 33.52 48.42 55.23 63.93 73.06139Appendix table 9 Degradation values for orchard grasshemicelluloseTreatment Incubation time (h)0 12 18 24 48 72EARLYSTEMS25.87 49.92 61.70 72.02 83.59 88.3625.47 51.74 60.64 69.23 83.56 88.3726.00 48.33 61.37 72.92 82.63 88.28EARLYLEAVES22.97 43.10 58.74 72.48 80.61 85.3023.37 44.65 57.14 70.18 79.45 86.4223.76 44.48 59.02 70.58 80.24 86.49MEDIUMSTEMS22.10 40.59 46.19 54.79 73.29 78.3922.34 40.94 46.61 55.70 73.49 78.31'22.10 40.85 45.80 54.18 71.91 77.39MEDIUMLEAVES17.90 35.88 45.44 65.49 75.40 78.7617.96 35.72 46.71 65.23 75.37 79.3417.84 35.14 45.30 65.24 75.72 77.77LATE16.73 40.50 52.33 58.96 71.49 74.80STEMS16.80 39.05 52.80 58.24 73.95 74.8116.87 39.57 54.85 59.17 70.10 71.84LATE17.77 43.57 54.15 64.39 72.37 81.72LEAVES17.81 45.45 55.34 63.39 73.43 80.9417.67 44.23 54.12 64.09 72.60 80.38140Appendix table 10 Degradation values for tall fescue grasscelluloseTreatment Incubation time (h)0 12 18 24 48 72EARLYSTEMS35.35 52.40 56.43 60.96 73.11 52.50 57.79 61.50 72.84 79.4134.97 52.16 57.82 61.33 72.96 79.58EARLYLEAVES31.23 48.21 59.20 59.74 76.44 84.3732.02 48.48 58.31 60.11 76.72 84.3931.27 48.05 59.65 59.82 76.66 84.44MEDIUMSTEMS33.66 52.17 58.53 66.07 77.27 84.8133.56 51.95 58.47 66.32 78.47 84.8833.47 51.91 58.32 66.27 78.48 84.72MEDIUMLEAVES31.45 48.37 57.64 65.38 75.23 83.7331.55 49.19 57.54 64.73 75.20 83.6631.46 48.92 55.68 64.60 75.34 83.50141Appendix table 11 Degradation values for tall fescue grasshemicellulose ATreatment' Incubation time (h)I^0 12 18 24 48 72EARLYSTEMS31.11 44.98 49.63 48.29 62.43 70.7630.97 43.47 46.94 49.56 62.66 70.9031.27 43.34 45.66 50.36 62.78 71.26EARLYLEAVES33.56 47.61 45.53 66.56 79.0833.59 34.45 47.65 45.06 64.10 77.7033.46_31.4734.9849.7246.8851.5844.4256.1464.9270.0077.9078.30MEDIUMSTEMS 32.10 49.45 50.71 57.88 70.30 79.3130.97 46.93 51.85 55.91 68.54 77.43.MEDIUMLEAVES31.58 42.62 45.44 57.71 65.63 79.3431.51 41.97 48.66 55.50 65.85 79.2931.58 41.28 47.69 58.17 66.90 78.38142Appendix table 12 Degradation values for tall fescue grasshemicellulose BTreatment' Incubation time (h)0 12 18 24 48 '^72^IEARLYSTEMS25.75 50.21 61.18 61.26 73.98 79.3824.81 52.48 60.31 61.97 73.46 79.8825.87 52.53 61.69 63.68 81.02EARLYLEAVES23.37 57.03 58.06 58.84 77.44 85.3023.37 55.36 61.69 58.32 76.59 85.5723.43 55.68 58.81 56.22 76.47 85.51MEDIUMSTEMS22.22 61.92 38.88 66.73 78.19 81.0221.98 61.37 36.77 66.57 78.41 84.2422.10 61.33 36.78 64.83 77.55 84.24'MEDIUMLEAVES17.90 56.35 63.18 67.42 78.86 87.0116.99 55.42 60.43 68.99 78.05 86.4917.90 55.75 61.48 68.08 77.28 84.93Appendix Table 13 Orchard grass cellulose effective DegradabilityTreatments Mean outflow rate variablesk=0.02 k=0.04 k=0.06 k=0.08Early Stems 65.34± 0.207 52.78 46.09 41.93Early Leaves 67.03± 0.321 57.14 51.03 46.88Medium Stems 60.00± 0.200 47.90 41.57 37.68Medium Leaves 61.91± 1.448 50.05 43.18 38.71Late Stems 54.57± 0.304 43.40 37.27 33.39Late Leaves 59.84± 0.053 49.33 43.08 38.93143Appendix Table 14 Orchard grass hemicellulose A effectiveDegradabilityTreatments Mean outflow rate variablesk=0.02 k=0.04 k=0.06 k=0.0.8Early Stems 55.40± 0.608 44.23 39.10 36.13Early Leaves 58.50± 1.510 47.37 41.63 38.17Medium Stems 51.53± 0.757 41.20 36.17 33.17Medium Leaves 52.07± 0.611 40.10 34.07 30.40Late Stems 44.10± 0.400 35.03 30.43 27.67Late Leaves 55.70± 0.361 46.73 41.20 37.50Appendix Table 15 Orchard grass hemicellulose B effectiveDegradabilityTreatments Mean outflow rate variablesk=0.02 k=0.04 k=0.06 k=0.08Early Stems 70.70± 0.100 59.73 53.10 48.63Early Leaves 68.40± 0.300 57.40 50.73 46.27.Medium Stems 60.57± 0.586 49.27 43.10 39.20Medium Leaves 61.77± 0.493 50.33 43.60 39.20Late Stems 59.37± 0.404 49.57 43.47 39.27Late Leaves 62.60± 0.608 52.30 43.83 41.37Appendix Table 16 Tall fescue cellulose effective degradabilityTreatments Mean outflow rate variablesk= 0.02 k= 0.04 k= 0.06 k= 0.08Early Stems 65.44± 0.071 57.32 52.69 49.70Early Leaves 67.11± 0.068 56.82 51.13 47.52Medium Stems 68.76± 0.189 59.23 53.75 50.19Medium Leaves 66.82± 0.232 57.17 51.63 48.04144Appendix Table 17 Tall fescue hemicellulose A effectivedegradabilityTreatments Mean outflow rate variablesk= 0.02 k= 0.04 k= 0.06 k= 0.08Early Stems 57.87± 0.208 49.17 45.27 43.07Early Leaves 59.60± 1.054 49.43 44.70 41.97Medium Stems 62.93± 0.569 53.63 48.87 45.97Medium Leaves 59.63± 1.823 49.53 44.77 41.97Appendix Table 18 Tall fescue hemicellulose 13 effectivedegradabilityTreatments Mean outflow rate variablesk= 0.02 k= 0.04 k= 0.06 k= 0.08 _.Early Stems 65.43± 0.058 57.33 52.73 49.73Early Leaves 66.43± 1.155 56.30 50.70 47.17Medium Stems 68.73± 0.208 59.27 53.73 50.20Medium Leaves 66.83± 0.208 57.20 51.63 48.03145


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