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Effect of fibrolytic enzymes on the nutritive value of tropical grasses and dairy cattle performance


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EFFECT OF FIBROLYTIC ENZYME S ON THE NUTRITIVE VALUE OF TROPICAL GRASSES AND DAIRY CATTLE PERFORMANCE By DERVIN BARTOLO DEAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Dervin B. Dean

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To my lovely wife Domenicch ella, and my dear kids, Sher yll, Homer, and Stephanie

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iv ACKNOWLEDGMENTS I would like to give thanks to my supervisory committee chair (Dr. Adegbola Adesogan) for his valuable guidance during my Ph.D. program and to the rest of my committee (Dr. Charles Staples, Dr. Lynn Sollenberger, Dr. Ramon Littell and Dr. Ann Wilkie) for their time and dedication to my research activities. I thank my sponsor (the University of Zu lia) for covering the expenses required to complete my program, the Department of Animal Sciences of the University of Florida, for giving me the opportunity to improve my knowledge and the crew of the Dairy Research Unit for their help during my in vivo trial. I would also like to thank to all of my lab supervis ors and partners (Nathan Krueger, Sam-Churl Kim, Kathy Arriola, Susan Chikagwa-Malunga, John Funk, Jamie Foster, Nancy Wilkinson, Pam Miles, Max Huisden, Alvin Boning, Bruno Amaral, Ashley Hughes, Tolu Ososanya, Mustapha Sa lawu and Sergei Sennikov) for their help during my field and lab activities. I thank Dr. Dario Colombatto for helping me to determine the enzyme activities. Finally I thank my friends (German Por tillo, Maria Padua, Tomas Belloso, Andres Kowalski, Carlos Lucena, Lucia Holsthausen, Carlos Rodriguez, and Carlos Vargas) for their support during the last five years.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv TABLE OF CONTENTS.....................................................................................................v LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................xi LIST OF ABREVIATIONS.............................................................................................xii ABSTRACT.....................................................................................................................xi ii CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................4 Cell Wall Differences between Tropical and Temperate Forages................................4 Methods for Improving Forage Nutritive Value...........................................................6 Ammoniation................................................................................................................7 Ammonia Treatment Methods.............................................................................10 Animal Response to Feeding Ammoniated Forages...........................................11 Feed Enzymes.............................................................................................................13 Definition, Types, and Classification..................................................................13 Commercial Exogenous Fibrolytic Enzymes......................................................17 Mode of Enzyme Action.....................................................................................18 Factors Affecting Enzyme Action.......................................................................19 Enzyme Stability in the Digestive Tract..............................................................21 Methods of Determining Enzyme Activity.........................................................24 Effect of Enzyme Treatment on Chewing Behavior...........................................26 Effect of Enzyme Treatment on th e Ruminal Microbial Population...................27 Effect of Enzyme Treatment on Ruminal Fibrolytic Capacity............................29 Effect of Enzyme Treatment on Fibe r Concentration Before Ingestion..............31 Effect of Enzyme Treatment on DM and Fiber Digestibility Post Ingestion......32 Effect of Enzyme Treatment on Silage Fermentation.........................................34

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vi Effect of Enzyme Treatment on Hay Nutritive Value.........................................35 Effect of Enzyme Treatment on Animal Performance........................................36 Effect of Enzyme Treatme nt on Blood Metabolites............................................42 Effects of Combining Enzyme and Chemical Treatments..................................42 3 EFFECT OF TREATMENT WITH A MMONIA OR FIBROLYTIC ENZYMES ON THE NUTRITIVE VALUE OF HAYS PRODUCED FROM TROPICAL GRASSES...................................................................................................................46 Introduction.................................................................................................................46 Materials and Methods...............................................................................................47 Enzyme Application............................................................................................47 Laboratory Analysis............................................................................................48 Statistical Analysis..............................................................................................50 Results and Discussion...............................................................................................51 Chemical Composition of Tropical Hays............................................................51 Effect of Promote and Ammoniation on Chemical Composition in Experiment 1....................................................................................................52 Effect of Promote and Ammonia Application on in vitro DM, NDF, and ADF Digestibility in Experiment 1...........................................................................53 Effect of Fibrolytic Enzyme a nd Ammonia Application on Chemical Concentration of C4 Forages in Experiment 2.................................................57 Effect of Enzyme Treat ment and Ammoniation on in vitro DM, NDF, and ADF Digestibility in Experiment 2..................................................................61 Effect of Enzyme Treatments and Ammoniation on in situ DM Degradation....66 Conclusions.................................................................................................................69 4 EFFECT OF FIBROLYTIC ENZYMES ON THE FERMENTATION CHARACTERISTICS, AEROBIC STABILITY, AND DIGESTIBILITY OF BERMUDAGRASS SILAGE....................................................................................71 Introduction.................................................................................................................71 Materials and Methods...............................................................................................72 Enzyme Application............................................................................................72 Laboratory Analysis............................................................................................74 Statistical Analysis..............................................................................................76 Results and Discussion...............................................................................................77 Chemical Composition of Freshly-treat ed Bermudagrass before Ensiling.........77 Chemical Composition, Microbial C ounts and Aerobic Stability of Bermudagrass Silages......................................................................................77 Organic Acid Concentration of Bermudagrass Silages.......................................84 In vitro DM and Fiber Digestibility of Bermudagrass Silages............................86 Conclusions.................................................................................................................88

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vii 5 EFFECT OF METHOD OF DIETA RY ADDITION OF A FIBROLYTIC ENZYME ON THE PERFORMANCE OF LACTATING DAIRY COWS.............90 Introduction.................................................................................................................90 Material and Methods.................................................................................................91 Diets.....................................................................................................................92 Sample Collection and Analysis..........................................................................94 Statistical Analysis..............................................................................................97 Results and Discussion...............................................................................................98 Chemical Composition of th e Dietary Ingredients..............................................98 Voluntary Intake..................................................................................................99 Effect of Promote on Milk Production and Composition..................................100 Body Weight Gain and Body Condition Score.................................................104 Blood Glucose, Urea-N and -Hydroxybutyrate...............................................105 Ruminal pH and Concentration of VFA and NH3-N.........................................106 In situ DM disappearance..................................................................................116 Conclusions...............................................................................................................120 6 GENERAL SUMMARY, CONCLU SIONS AND RE COMMENDATIONS.........121 APPENDIX A ABSTRACT FOR CHAPTER 3..............................................................................130 B ABSTRACT FOR CHAPTER 4..............................................................................133 C ABSTRACT FOR CHAPTER 5..............................................................................135 LIST OF REFERENCES.................................................................................................137 BIOGRAPHICAL SKETCH...........................................................................................149

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viii LIST OF TABLES Table page 2.1 Some fibrolytic enzyme-producing microorganisms and the enzymes they produce.....................................................................................................................15 2.2 Effect of spraying enzymes onto feeds prior to feeding on milk production in recent studies............................................................................................................38 3.1 Actual enzyme application rates used......................................................................49 3.2 Manufacturer-stipulated enzyme activities..............................................................49 3.3 Chemical composition of the untreated hays...........................................................53 3.4 Effect of Promote or ammonia trea tment on chemical composition (% DM) of tropical grass hays....................................................................................................54 3.5 Effect of Promote or ammonia applica tion on the IVDMD of tropical grass hays..55 3.6 Effect of Promote or ammonia appl ication on the IVNDFD of tropical grass hays........................................................................................................................... 56 3.7 Effect of Promote or ammonia appl ication on the IVADFD of tropical grass hays........................................................................................................................... 57 3.8 Effect of fibrolytic enzyme or ammonia application on the NDF, ADF and hemicellulose concentrations (%) of tropical hays..................................................58 3.9 Effect of fibrolytic enzyme or ammonia application on the WSC and CP concentrations (%) of tropical hays..........................................................................59 3.10 Effect of fibrolytic enzyme or ammonia application on the IVDMD (%) of tropical hays.............................................................................................................63 3.11 Effect of fibrolytic enzyme or ammonia application on the IVNDFD (%) of tropical hays.............................................................................................................64 3.12 Effect of fibrolytic enzyme or a mmonia application on the IVADFD (% of DM) of tropical hays.........................................................................................................65

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ix 3.13 Effect of X-20 or ammonia applicat ion on the in situ kinetics of DM disappearance of bermudagrass and bahiagrass.......................................................67 3.14 Effect of A-20 or ammonia applicat ion on the in situ kinetics of DM disappearance of bermudagrass and bahiagrass.......................................................68 4.1 Chemical composition of bermudagrass forages before ensiling (g/kg DM)..........78 4.2 Effect of fibrolytic en zymes on pH, concentrations of DM (g/kg) and ammoniaN (g/kg total N), DM losses (%), micr obial counts (log cfu /g) and aerobic stability (h) of bermudagrass silage.........................................................................79 4.3 Effect of fibrolytic enzymes on th e chemical composition of bermudagrass silage (g/kg DM)......................................................................................................80 4.4 Effect of fibrolytic enzymes on the organic acid concentration (g/kg DM)of bermudagrass silage.................................................................................................85 4.5 Effect of fibrolytic enzymes on in v itro digestibility of DM (g/kg), NDF, ADF and hemicellulose (Hem) in bermudagrass silage after 6 or 48-h of digestion (g/kg DM).................................................................................................................87 5.1 Ingredient and chemical compositi on of the basal untreated diet............................93 5.2 Chemical composition of the enzyme -treated and untreated forages and concentrate (% DM) (n= 4 replicates per mean)....................................................101 5.3 Effect of method of enzyme addition on diet digestibility and voluntary intake...102 5.4 Effect of method of enzyme additi on on milk production and composition..........103 5.5 Effect of method of enzyme addition on body weight and condition score, and blood metabolites...................................................................................................106 5.6 Effect of method of enzyme addition on ruminal fluid pH....................................108 5.7 Effect of method of enzyme addition on ruminal NH3-N concentration...............109 5.8 Effect of method of enzyme addition on ruminal acetic acid molar percentage....111 5.9 Effect of method of enzyme a ddition on ruminal propionic acid molar percentage...............................................................................................................112 5.10 Effect of method of enzyme addition on ruminal butyric acid molar percentage..113 5.11 Effect of method of enzyme addition on ruminal acetic:propionic acid ratio........114 5.12 Effect of method of enzyme a ddition on total VFA concentration........................115

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x 5.13 Effect of method of enzyme add ition on ruminal isobutyric acid molar proportion...............................................................................................................117 5.14 Effect of method of enzyme add ition on ruminal isovaleric acid molar percentage...............................................................................................................118 5.15 Effect of method of enzyme additi on on kinetics of in situ feed DM disappearance in lactating Holstein Cows..............................................................119

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xi LIST OF FIGURES Figure page 5.1 Effect of method of enzyme addition on ruminal fluid pH....................................108 5.2 Effect of method of enzyme add ition on ruminal NH3-N concentration...............109 5.3 Effect of method of enzyme addition on ruminal acetic acid molar percentage....111 5.4 Effect of method of enzyme a ddition on ruminal propionic acid molar percentage...............................................................................................................112 5.5 Effect of method of enzyme addition on ruminal butyric acid molar percentage..113 5.6 Effect of method of enzyme addition on ruminal acetic:propionic acid ratio........114 5.7 Effect of method of enzyme a ddition on total VFA concentration........................115 5.8 Effect of method of enzyme add ition on ruminal isobutyric acid molar proportion...............................................................................................................117 5.9 Effect of method of enzyme add ition on ruminal isovaleric acid molar percentage...............................................................................................................118

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xii LIST OF ABREVIATIONS A-20 Biocellulase A-20 ADF acid detergent fiber ADFD acid detergent fiber digestibility BA bahiagrass BCS body condition score BE bermudagrass HBA beta hydroxybutyrate BUN blood urea nitrogen BW body weight BWG body-weight gain CA Cattle-Ase CP crude protein CPP crude protein production CPD crude protein digestibility DM dry matter DMD dry matter digestibility DMI dry matter intake FCM fat-corrected milk FP fat production Glc glucose IVADFD in vitro acid detergent fiber digestibility IVDMD in vitro dry matter digestibility IVNDFD in vitro neutral detergent fiber digestibility MCF milk crude fat MCP milk crude protein NDF neutral detergent fiber NDFD neutral detergent fiber digestibility NFC non-fiber carbohydrates NH3 ammonia NH3-N ammonia nitrogen Pr Promote RIN relative intake SCC somatic cell counts TDN total digestible nutrients TMR total mixed ration VFA volatile fatty acids WSC water soluble carbohydrates WSN water soluble nitrogen X-20 Biocellulase X-20

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xiii Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF FIBROLYTIC ENZYME S ON THE NUTRITIVE VALUE OF TROPICAL GRASSES AND DAIRY CATTLE PERFORMANCE By Dervin Bartolo Dean December 2005 Chair: Adegbola T. Adesogan Major Department: Animal Sciences Five experiments were conducted to determ ine whether the nutritive value of hay or silage made from tropical grasses and anim al performance can be improved by addition of fibrolytic enzymes. Experiments 1 and 2 determined the effect on the digestibility of Coastal bermudagrass and Pensacola bahiagrass hays of applying NH3 or four fibrolytic enzymes: Promote (Pr), Biocellulase X-20 (X -20), Biocellulase A-20 (A-20), and CattleAse (CA) at 0, 0.5, 1, and 2x the rates reco mmended by the respective manufacturers. Biocellulase X-20 and A-20 improved the 6-h and 48-h digestion of the forages, but ammoniation was more effectiv e. In Experiment 3, Tifton 85 bermudagrass was ensiled without treatment (Control), or after treatment with the enzymes used in Experiments 1 and 2. Compared to Control silages, Prom ote-treated silages had lower pH and dry matter (DM) losses, and lower concentrati ons of ammonia-N, neutral (NDF) and acid detergent fiber (ADF), and gr eater concentrations of resi dual water soluble carbohydrates (WSC), in vitro DM, NDF, and ADF digestibility. The other enzymes also increased

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xiv fiber hydrolysis, but did not im prove indices of fermentation quality. Therefore, Promote was the most promising enzyme for improving the fermentation and nutritive value of silages. Experiments 4 and 5 tested the eff ect of Promote on the performance of thirty Holstein lactating dairy cows fed a ration c onsisting of bermudagrass silage, corn silage, and concentrate ad libitum for two 28-d periods. Treatment s were the following: Control, enzyme applied at ensiling to the bermudagrass (TS), or at feeding to the concentrate (EC), the total mixed ration (ETMR) or the be rmudagrass silage (EF). Voluntary intake, apparent digestibility, milk production, and bl ood glucose concentration were unaffected by treatment. Cows fed ETMR tended to ha ve lower beta hydroxybutyrate and blood urea-N concentrations and greater milk fat and protein concen trations than cows fed the control diet. In Experiment 5, five ruminally fistulated lactating co ws were fed the same diets as in Experiment 4 for three, 15-d pe riods. Ruminal pH wa s decreased by feeding EC, whereas acetate:propionate ratios were reduced by feeding ETMR. In situ DM disappearance was unaffected by enzyme treat ment. These experiments suggest that ETMR was the most promising treatment.

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1 CHAPTER 1 INTRODUCTION Forages represent the most important, cost effective feed resource in ruminant nutrition (Jung and Allen, 1995). However, th e relatively low quality of tropical forages militates against their use as the sole feed for actively growing or high-performing ruminants. Several attempts have been made to improve forage quality genetically or by chemical or biological treatments. One of the most important goals in this regard is to improve the fiber digestibility of the forages. Some chemical and biological treatments have been effective at ac hieving this objective. Ammoniation is one of the most studied chemical treatments for enhancing fiber digestibility and several reports have desc ribed its effectiveness for improving both forage quality and animal performance. Ammoniation increases forage crude protein (CP) concentration and substant ially reduces the concentratio n of neutral detergent fiber (NDF) in forages. Most of the loss of NDF is due to hydrolysis of hemicellulose, though the disruption of chemical linkages between lignin and hemicellulose also occurs (Weiss and Underwood, 1995; Barrios-Urdaneta and Vent ura, 2002). Additional benefits of ammoniation include reduced y east and mold growth, and le ss aerobic deterioration of high moisture hay and silage (Woolford and Tetlow, 1984; Bates et al., 1989b). Consequently, feeding ammoniated forage ofte n results in increased daily gain and dry matter (DM) intake in be ef cattle (Vagnoni et al., 1995; Brown and Pate, 1997). However, the use of ammonia for improving forage quality has been limited because of the corrosive nature of the alkali which can be hazardous to operators and their

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2 equipment (Lalman et al., 2005). Ammoniati on also contributes to N importation to farms and therefore represents a small but impor tant threat to air qua lity because of its contribution to surface water eutrophication and nitrate contamination of ground water (Ishler, 2005). In developing countries, the labor required for ammoniation and problems of delivering anhydrous ammonia have limited adoption of the technique. Fibrolytic enzyme application is one of the most studied biological treatments for improving forage quality and animal performance. Such enzymes have been effective at improving the utilization of a wide range of diets containing roughages (Rode and Beauchemin, 1998) due to improved fiber hydr olysis (Colombatto et al., 2003b) which often results in increased digestibility (Christensen, 1997; Rode et al., 1999) and voluntary intake (Pinos-Rodriguez et al., 2002 ). Nevertheless, another study has shown that exogenous enzymes did not consistently improve fora ge utilization by ruminants (Lewis et al., 1999). This inconsistency is attributable to several factors such as differences in enzyme type and activity, treatment duration, application method, diet composition and level of animal performance. Additional factors that may be impli cated include suboptimal prevailing temperature and pH for enzyme action, presence of inhibitors or abse nce of cofactors and inadequate enzyme to substrate ratios. Neve rtheless, feed enzymes have been used to improve the utilization of a wide range of diets containing legumes, grasses, haylage, straw and other feedstuffs (Beauchemin et al., 2003). The mode of action of these enzymes in ruminants is not fully understood. In addition to incr easing fiber hydrolysis, they can enhance feed colonization by incr easing the numbers of ruminal fibrolytic microbes (Morgavi et al., 2000; Nsereko et al., 2000a) and ther eby increase the rate of

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3 degradation of feeds in the rumen (Yang et al., 1999). Additionally, modes of action include improved palatability (Adesogan, 2005), changes in gut viscosity (Officer, 2000), and changes in the site of digestion (Rode and Beauchemin, 1998; Hristov et al., 2000). Most of the studies of fibrolytic enzyme treatment of ruminant feeds have been done using feedstuffs grown under temperat e conditions. Little is known about their effectiveness on tropical or subtropical forages which tend to be less digestible. Yet there is greater scope for improving th e quality of tropical forages than there is for temperate forages due to the greater nutritive value of the latter. The aim of this series of experiments was to evaluate the effect of ammonia and proprietary fibrolytic enzyme application on the nutritive value of tropical forages and animal performance.

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4 CHAPTER 2 LITERATURE REVIEW Cell Wall Differences between Tropical and Temperate Forages Even under the intensive concentrate f eeding systems of ruminant animal production in developed countries, forages continue to represent the single most important feed resource; however, cell-wall concentration and digestibility limit the intake potential and energy availability from forage crops in beef and dairy production systems (Jung and Allen, 1995). Depending on the stage of maturity of the plant, cell walls represent between 30 and 80% of plant dry matter (DM) in grasses so that under some circumstances (high forage diet s) the bulk of carbohydrate fermented to metabolizable volatile fatty acids (VFA) by ru men micro-organisms may be derived from cell wall polysaccharides and in senescent gr asses, almost all fermentable carbohydrate arises from wall polysacchar ides (Stone, 1994). Cellulose is the predominant wall polysaccharide. The cellulosic microfibrils are embedded in a matrix composed of noncellulosic polysaccharides and some proteins The major matrix polysaccharides in grasses are glucuronoarabinoxylan s, together with smaller amounts of heteroglucans (xyloglucans) and glucans (Stone, 1994). The main reason why the digestibility of tropical grasses is less than that of temperate perennial grasses is because of di fferences in cell wall composition. There are differences in polysaccharide composition in cell walls of different types and also considerable quantitative differences in co mponents, e.g., mesophyll cells which are more abundant in temperate forages are relativel y cellulose-rich (Gor don et al., 1977, cited by

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5 Stone, 1994) and readily degraded by rumen micro-organisms; but tropical grasses are rich in lignified and secondarily thickene d cells of xylem (Akin and Burdick, 1975). Differences in leaf anatomical structure between Panicoid trop ical and Festucoid temperate grasses associated with the C4 and C3 photosynthetic pathways have been known to botanists for many years (Wilson and Hacke, 1987). These researchers determined that leaves of C4 (tropical) grasses consistently had less mesophyll and more of the less-digestible epidermis, bundle shea th, sclerenchyma and vascular tissues than leaves of C3 temperate and legumes grasses. Wils on and Hattersley (1989) also found that anatomical differences between the l eaf structural groups were consistently expressed, with C3 species having higher proportions of mesophyll (53-67 vs. 28-47%) and lower proportions of bundle sheath (5-20 vs 12-33%) and vascular tissue (3-9 vs. 612%) than the C4 species. According to Wilson and H acke (1987) the anatomy associated with either C4 tropical or C3 temperate grass genera clearly contribut es to difference in DM digestibility between leaves. Comparisons of C4 or C3 leaf anatomy in a wide range of summergrowing Panicum species grown under the same environmental conditions determined that the C4 anatomy of tropical grass genera causes their leaves to have lower digestibility and higher cell wall concentr ation than grasses with C3 anatomy (Akin et al., 1983). Quantitative analysis of leaf anatomy of a number of grasses indicated that leaves of the tropical species had 25 percentage units more of the slowly di gested cell tissues than had the temperate grasses (Akin and Burdick, 1975). According to Buxton and Redfearn (1997), energy availability from forages is limited by fiber concentration because fiber is slowly and incompletely digested, whereas

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6 cell soluble are almost completely digested. Thus, the proportion of fiber to cell soluble is a major determinant of energy availability in forages. Ruminants digest about 40 to 70% of grass fiber, and some fiber fragment s cannot be digested no matter how long they remain in the rumen. Lignin interferes with microbial degradation of fiber polysaccharides by acting as a physical barrier and by being cross-linked to polysaccharides by ferulate bridges (Moore and Jung, 2001). Lignin and ferulate cross linkages are more abundant in C4 than C3 grasses. This is the chemical basis for the lower digestibility of C4 grasses (Ramalho, 1991). Voluntary intake of forages is a critical determinant of animal performance and cell wall concentration is negatively related to intake of ruminants consuming high-forage diets. Cell walls affect intake by contributi ng to ruminal fill. Cell wall concentration and rate of passage are the most critical para meters determining ruminal fill (Jung and Allen, 1995). Ruminal fill is typically greater in ruminants consuming C4 grasses than in those consuming C3 grasses because of the poorer digestibility of C4 grasses. Methods for Improving Forage Nutritive Value Different chemical and biological treatmen ts have been used for enhancing the nutritive value of low quality forages and roughage s. The main effect of such treatments is due to modifications of cell wall component s. The changes that take place when low quality roughages are treate d with alkali (e.g., ammonium hydroxide, NaOH) are of a physical as well as of a chemical nature (Ramalho, 1991). It is well known that the roughages are normally softer after chemical tr eatment and this may be one of the reasons for the higher intake found for treated fo rage (Ramalho, 1991). Another important change that takes place during alkali treatment is a swelling of the plant cell wall. This is probably most pronounced for forage treated with NaOH solution (Harbers et al., 1982).

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7 There are a number of chemical reactions taki ng place during alkali treatment of forages. Saponification of ester linkages between acetic acid and phenolic acids, and polysaccharides and or lignin as well as such linkages between uronic acid residues of xylans in hemicelluloses and lignin occur during the alkali treatment of straw (Harbers et al., 1982). If the temperature is high enough in the presence of al kali, lignin undergoes cleavage of other linkages between phenyl propane units and free phenolic groups are formed. As a result of the accompanying decrease in the molecular weight and cleavage of linkages to hemicellulose, an increased solubility of lignin in the alkaline solution will occur (Theander and Aman, 1984; cited by Ra malho, 1991). Sundstizil (1998) reported that the OM digestibility of alkali-treated rye straw incr eased from about 46 to 71%. Disruptions of ferulate bridge s by ammoniation have been also associated with improving fiber digestion (Brown and Adjei, 1995, Ba rrios-Urdaneta and Ventura, 2002), voluntary intake (Glenn, 1990; Vagnoni et al., 1995; Lines and Weiss, 1996) and animal performance (Rasby and Ward, 1989; Br own, 1993; Brown and Pate, 1997). Ammoniation Ammoniation is one of the most studied chemical treatments for improving forage digestion in the past few years (Chaudhry, 1998). Chemical treatments of low quality forages, such as ammoniation, have been s hown to economically extend the use of such forages into more nutritionally challenging peri ods of the production cycle, such as late gestation and early lactation (Wiedmeier et al., 2003). Lo w quality forages are treated with ammonia for the two following reasons: 1) ammonia is an effective preservative for hay containing up to 30% moisture and 2) tr eatment of mature grass hays and poorer quality crop residues is a cost-effective way for improving their feeding value (Weiss and Underwood, 1995). Ammoniation in creases crude protein (CP) in the treated forages by

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8 adding about 50 to 80% of the N in NH3 to the forage. Some of the retained nitrogen is converted by microbes present on the forage into microbial protei n and another fraction of the retained nitrogen is bound in an unknow n manner to the forage fiber components (Weiss and Underwood, 1995). Barrios-Urdaneta and Ventura (2002) observed that their dry ammoniation method improved the CP of koroniviagrass ( Brachiaria humidicola ) from 3.2 to 8.3% Brown (1993) observed that ammoni ation (4% of DM) of stargrass ( Cynodon nlemfuensis ) hay increased (P < 0.01) total N concentration (from 1.0 to 1.4%). A similar increase (0.9% N, P < 0.01) was obtaine d by Lines et al. (1996) in alfalfa hay. Ammoniation also improves forage digestib ility. This is due to hydrolytic action on linkages between lignin and structural pol ysaccharides, thus increasing organic matter (OM) potentially available for utilizati on by the ruminal microorganisms (BarriosUrdaneta and Ventura, 2002). Ammoni a treatment substantially reduces the concentration of neutral deterg ent fiber (NDF) in forages and most of the loss of NDF is due to hydrolysis of hemicellulose and disr uption of chemical linkages between lignin and hemicellulose, making the hemicellulose more digestible (Weis and Underwood, 1995). Cellulose digestibility also increas es since lignified hemicellulose encases cellulose (Chaudhry, 1998). Ammoniation partially breaks down the structure of cellulose by disrupting hydrogen bonds. This reaction causes a swelling of the fiber and allows cellulase better acce ss to the fiber for digestion (Church, 1988). Lines et al. (1996) reported lower NDF (58.8 vs. 56.2%, P < 0.01) and hemicellulose (13.2 vs. 9.4%, P < 0.01) concentrations in ammoniated alfalf a hay compared to the untreated hay. This agrees with results obtained by Brown and Adjei (1995) who found lower NDF (-5%, P <

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9 0.05) and hemicellulose (-17%, P < 0.01) c oncentrations in urea-treated (6% DM) guineagrass ( Panicum maximum ) hay compared to the untreated hay Ammonia treatment also changes the physic al characteristics of forages making them more pliable and increases their hydration. Hydration rate has an important role in digestion rate; the faster a fora ge particle is hydrated, the fa ster it is digested (Weiss and Underwood, 1995). Barrios-Urdaneta and Ve ntura (2002) showed that ammoniation increased the in vitro NDF digestibility (by 10.9%) of koroniviagrass. Brown (1993) observed that ammoniation increased in vitro OM, NDF, and ADF digestibility and decreased (P < 0.01) NDF concentration in st argrass hay. Vagnoni et al. (1995) showed that ammoniation of mature bermudagrass increased both the in situ rate (P < 0.05) and the potential extent (P < 0.01) of forage DM and NDF disappearance in lactating cows. Zorrila-Rios et al. (1991) found that the in vitro DM digestibility (IVDMD) of wheat straw was increased by 54% due to ammoniation. In that study, ammoniation also almost doubled the CP concentration of the stra w compared to untreated straws. Woolford and Tetlow (1984) observed that ammoniation of high-moisture hay reduced the growth of yeasts and molds, and decreased the rate of aerobic deterioration. Bates et al. (1989b) found a substantial reduction of external molding when ammonia was metered into the sealed plastic container of round bale sila ge; however, they observed that ammoniation was associated w ith undesirable fermentation characteristics, especially when direct-cut, low DM tropical forages were ensiled. Dry matter recovery and intake of ammoniated, direct-cut, berm udagrass round bale silage was very poor. Although application of ammonia to bermudagr ass wilted to 40 to 50% DM improved the quality of round bale silage, th ese authors did not recommend this practice because of the

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10 high level of management required for success, and because treatment of silage and hay with ammonia has, on occasi on, been toxic to cattle. Ammonia Treatment Methods Gaseous anhydrous ammonia has been used for forage treatment in developed countries while, in developing countries, sp raying ammonia soluti ons and dipping hay in urea solutions are pref erred (Chenost and Kayouli, 1997). According to these authors, using anhydrous ammonia is more effective, but its high cost and re quirement for special delivery and storage facilities have hindered it s utilization by farmers. The use of a urea solution is a simple, low cost technique; however, it has not become widely accepted. The labor involved in handling the materi al and the appearance of molds as a consequence of the water added has limited adoption by commercial producers (BarriosUrdaneta and Ventura, 2002). The latter re searchers recently deve loped a method that they called dry ammoniation by adding water and urea into plastic co ntainers (19-l) and suspending 1 kg hay bales 5-8 cm over the perf orated cover of the container. Thereafter the hay and container were hermetically sealed with a plastic tarp and stored for 14 or 21 days. The method was found to increase CP concentration and in vitro NDF digestibility by 190% and 37%, respectively. Barrios-Urdaneta and Ventura (2002) evaluated the effect s of storage time (14 and 21 days), water volume (200 and 400 ml/kg DM of hay) and urea quantity (20 and 40 g/kg DM of hay) on the CP concentration and in vitro digestibility of NDF of koroniviagrass of 1 kg hay bales. The best increase in nutritive value was obtained when the hay was stored for 21 days and treated with 200 ml of water and 40 g of urea/kg. According to Dolberg (1992), ammoniation treatment time may vary from one to five weeks. However, temperature and treatment time are inversely related; and

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11 therefore, more time is required in the winter or cold weather. Simple tests of successful treatment of straw are a browni ng in the color of the forage, a strong smell of ammonia, and absence of rotten and molded straw (Lal man et al., 2005). The amount of anhydrous ammonia necessary to improve digestibilit y is between 2 and 4% of DM (Weiss and Underwood, 1995). The reaction between ammonia and fiber is dependent on temperature, so if forages are treated w ith ammonia during cold weather a five-week treatment period is recommended (Dolberg, 19 92). After this treatment period, forage can remain covered for extended periods without problems. It is recommended that the forage be left uncovered for at least 3 to 5 da ys prior to feeding to allow free ammonia to escape (Lalman et al., 2005). This may not be necessary, but sometimes animal acceptance may be poor initially if ammoniated bales are not aired out prior to feeding (Weiss and Underwood, 1995). Brown and Adjei (1995) applied a urea solu tion (0, 4, 6, or 8% of the forage DM) to guineagrass hay harvested at different moisture concentrations (25 or 40%) and observed that CP concentration and in vitro OM digestibility (IVOMD) increased linearly (P < 0.01), whereas concentrations of hemi cellulose (P < 0.01) and ADL (P < 0.05) decreased linearly with increasing amount of urea applied. The same researchers treated guineagrass hays with urea at 0, 4, or 6% of the forage DM. The urea solution was sprayed onto the flat sides of the bales, or a pplied by low pressure (10 psi) injection. The greatest improvements in CP and NDF concen tration and IVOMD we re obtained at the 25% forage moisture concentration usi ng the low pressure injection method. Animal Response to Feeding Ammoniated Forages Several reports show that ammoniation of low quality forages can improve animal performance. Weiss and Underwood (1995) stat ed that ammonia treatment increases the

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12 DMI and DMD of low to medium quality gr ass hay by 5 to 10 per centage units. The increase in both variables results in a subs tantial increase in cons umption of digestible energy by animals fed ammoniated forages as compared to those fed untreated forage. Consequently, ammoniated stra w can provide adequate energy and protein to maintain lactating beef cows and ewes under most conditions while untreated straw can not. Vagnoni et al. (1995) fed crossbred beef steers anhydrous ammoniated (3% of hay DM) mature bermudagrass hay or supplemented their diets with urea and observed that ammoniation, unlike urea supplementation, increased ADG and DMI (P < 0.05), which suggests that ammoniation resulted in greater growth of ruminal microorganisms. In two digestion and growth trials, round bales of hay were sprayed with solutions of 0, 4, or 6% urea and fed to beef steers. Hay intake in creased in a quadratic (P < 0.05) manner with increasing urea concentration. Apparent NDF a nd ADF digestibility increased linearly (P < 0.05) with increasing urea concentration and linear improvements in ADG (P < 0.05) and gain/feed (P < 0.07) were obser ved (Brown and Adjei, 1995). According to Rasby and Ward (1989), when animal requirements for protein are high, as during lactation, the N needed by rumen bacteria ca n be supplied using ammoniated forages and by supplementing wi th a source of ruminally undegraded protein (RUP) but is digested in the small intestine. The latter meets the remaining protein need and may enhance animal performance because of the improved amino acid profile reaching the small intestine from diet ary RUP. Because the energy requirements of lactating dairy cows are quite high, th e amount of ammoniated low quality forages included in the diet should be limited (Weiss and Underwood, 1995).

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13 The only dried forages that should be cons idered for 3% ammoniation are straws, mature grass hays and corn stover. No more than 1% of NH3 should be applied to high quality forages such as alfalfa ( Medicago sativa ), immature orchardgrass ( Dactylis glomerata ), fescue ( Festuca arundinacea ), sudangrass ( Sorghum sudanense ), cereal grain hays or any moderate to early harvested gr ass hay (including both cool and warm season species) because the resulting product is of ten toxic to livestock (Weiss and Underwood, 1995). Ammoniation of high quality roughages can lead to toxicity problems known as crazy cow syndrome or bovine bonkers. Symptoms include hyperexcitability, circling, convulsions, and even death. Toxic ity is caused when cattle consume sufficient quantities of the toxic compound, 4-methylim idazole, which is formed when soluble sugars in the roughage react with ammonia. This compound passes through the milk to affect nursing calves, which seem to be more susceptible to the toxicity than mature animals. Mature roughages have low sol uble sugar content a nd present little NH3 toxicity risk (Lalman et al., 2005). The foregoing indicates that ammonia trea tment is a viable method of increasing the nutritive value of low quality forage s and improving the animal performance of ruminant livestock fed such grasses. However, the use of NH3 is limited due to the high investment in infrastructure required for delivering NH3, treating the forages, and storing the treated forage and concerns about th e hazardous nature of the alkali. Feed Enzymes Definition, Types, and Classification Enzymes are naturally occurring globular protein molecules that catalyze specific chemical reactions in biologi cal systems. Two mechanisms have been propounded to explain enzyme action. The first, the lock and key mechanism, was proposed by Emil

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14 Fischer in the late 1800s, which postulates th at enzymes accommodate substrates with specific shape that complement the enzyme active site (Scrutton, 1999). However, in 1958 Daniel Koshland proposed the induced fit th eory, which postulates that the substrate can induce conformational changes in an enzy me structure to bri ng the catalytic groups of the enzyme into the proper alignment for binding the substrate (Koshland, 1994). Both theories are now accepted mech anisms for enzyme action. Enzymes are involved in the digestion of complex feed molecules into their chemical constituents (e.g., glucose, amino aci ds) in both bacteria and the host animal. Digestive enzymes are essential to animal s because complex feeds are not readily absorbed from the digestive tract unless th ey are degraded to simpler molecules (Kung, 2001) Enzymes are classified broadly by the s ubstrate on which they act and by their specificity. Commercial en zyme products are fermentati on extracts of bacterial ( Bacillus spp.) or fungal ( Trichoderma and Aspergillus spp.) origin (Beauch emin et al., 2004a), and contain a unique array of enzymatic activities (Table 2.1). Enzyme activity can be assayed using in vitro methods by measuring end products of hydrolysis (i.e., reducing sugars, amino acids or peptides) per unit time using a specified substrate under defined conditions. These substrates are often purifie d or modified to simplify measurements of activity (Kung, 2001). Cellulose is hydrolyzed thr ough a complex process involving cellulases. Numerous specific enzymes contribute to cellulase activity, including endocellulase, exocellulase, and -glucosidase. In general, endog lucanases hydrolyze cellulose chains at random to

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15 Table 2.1 Some fibrolytic enzyme-produci ng microorganisms and the enzymes they produce Microorganism Enzymes Aspergillus niger 1, 5 -amylase, endoxylanase, -xylosidase, acetylxylan esterase, -L-arabinofuranosidase Aspergillus ficuum 3 -glucanase Aspergillus candidus 3 Cellulase Aspergillus sydowi 4 Phytase, -D-fructofuranosidase Microorganism Enzymes Aspergillus oryzae 1, 2 -amylase, protease Bacillus licheniformis 3 -amylase Bacillus subtilis 3 Phytase, -amylase Trichoderma viridae 3 Xylanase, -glucanase, protease, cellulase Saccharomyces cerevisae 1, 3 -galactosidase Humicola insolens 6 -glucanase 1 Beauchemin et al., 2004a 2 Carlsen et al., 1996 3 Hutcheson, 2001 4 Muramatsu and Nakakuki, 1995 5 Noel et al., 1998 6 Schulein, 1997 produce cellulose oligomers of varying degrees of polymerization; exoglucanases hydrolyze the cellulose chain from the non-reducing end, producing cellobiose, and glucosidases hydrolyze short-chain cellulose oligomers and cellobiose to glucose (Beauchemin et al., 2003). The main enzymes involved in de grading the xylan core polymer to soluble sugars are xylanases and -1,4 xylosidase (Bhat and Hazlewood, 2001). The xylanases include endoxylanases, which yield xyloo ligomers and -1,4-xylosidases, which in turn yield xylose. Other hemicellulase enzymes involved primarily in the digestion of side chains include -mannosidase, -L-arabinofuranosidase, -D-glucuronidase, -Dgalactosidase, acetyl xylan esterases, and fe rulic acid esterase (W hite et al., 1993, cited by Beauchemin et al., 2003; Bhat and Hazlewood, 2001).

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16 According to Fanutti et al. (1995), endo-1,4-xylanase hydrolyzes the -1,4linked polysaccharide backbones of xylans, which form the major hemicellulose component of forages. Some studies on the structure of xylanases have revealed that some enzymes are comprised of single catal ytic domains while other xylanases are modular, consisting of single or multiple cata lytic domains fused via linker sequences to noncatalytic sequences, some of which const itute cellulose binding domains (Fanutti et al., 1995). Hemicellulases derived from aerobic microorganisms do not appear to associate into multi-enzyme complexes, while anaerobic organisms often synthesize multi enzyme cellulase-hemicellulase complexe s (Fanutti et al., 1995). These researchers have focused their studies on plant cell wall -degrading enzymes of anaerobic fungi that are particularly active against the more recalcitrant plant structural polysaccharides and have observed that these organisms produce ce llulases and hemicellulases that associate into large molecular weight, multi-enzyme complexes and bind tightly to cellulose, exerting their cellulolytic effect. Murashima et al. (2003) noted that plant cell walls are comprise d of cellulose and hemicellulose and other polymers that are intertwined, and this complex structure presents a barrier to degradation by pur e cellulases or hemicellulases. They determined the synergistic effects on corn ( Zea mays ) cell wall degradation by the action of xylanases and cellulases from Clostridium cellulovorans Xylanase and cellulase were found to degrade corn cell walls synergistically but not purified substrates such as xylan and crystalline cellulose. The mixture of xylanases and cellulases at a molar ratio of 1: 2 gave the highest synergistic effect on corn cell wall degradation. The amounts both of

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17 xylooligosaccharides and cellooligosaccharides liberated from corn cell walls were increased by the synergistic action of xylanases and cellulases. Pectin, a minor component of grass cell wa lls, is digested in the rumen either by strictly pectinolytic species or by specie s possessing a combination of pectinases (e.g., pectin lyase, polygalacturonase, pectin met hyl esterase) and xylanases (Cheng et al., 1996). Commercial Exogenous Fibrolytic Enzymes Commercial enzyme products are relativel y concentrated and purified, and they contain specific enzyme ac tivities (Beauchemin et al ., 2004a). Use of exogenous enzymes can be beneficial when the enzyme preparation and the feed are complimentary. The use of fibrolytic enzymes as additives for ruminant diets has been the focus of considerable research recently following pos itive responses to en zyme supplementation in feeding trials (Beauchemin et al., 1995; Kung et al., 2000). However, in contrast to the case in non-ruminants (Bedford and Schulze, 1998), the mode of action of these enzyme additives in ruminants is not fully unde rstood. As an alternative to costly in vivo trials, several in vitro studies have been conducted to ex amine the effects of enzymes on the degradation of feedstuffs, but the complexity of these feeds makes it difficult to identify which feed fractions are most influenced by enzymatic action. The use of purified xylans and cellulose can minimize this complexity and provide a more informative method of evaluating the mode of action of enzymes. However, the results of such studies may not always correlate to the en zyme effects on feedstuffs. Several feed enzyme products that contain a blend of en zymes have been shown to be effective at enhancing the utilization of ruminant diets (Rode and Beauchemin, 1998). Nevertheless, the enzyme levels and activit ies that will effectively improve dietary

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18 nutrients will vary with the diet being consid ered and the nature of the enzyme. Types of cellulases and hemicellulases in commercial enzyme products differ substantially, and differences in the relative proportions and activities of thes e individual enzymes determine the efficacy of cell wall degradat ion by these products (Beauchemin et al., 2003). In addition to fiber-degrading enzymes, these products also have secondary enzyme activities, including amylases, proteases, and pectinases, which contribute to their hydrolytic capacity. Various factors such as enzyme type and met hod of preparation and application, amount of enzyme applied and fr action of the diet targeted, and animal differences have lead to inconsistencies in re sults of trials in wh ich enzymes have been added to ruminant feed s (Bowman et al., 2002). Rode and Beauchemin (1998) evaluated commercial enzyme preparations in vitro using alfalfa hay or barley ( Hordeum vulgare ) silage as a substrate. Effectiveness of enzyme products differed for the two substrat es, indicating that an enzyme product that elicits a positive response in one diet may not be effective if evaluated using a different diet. According to Newbold (1995, cited by McAllister et al., 2001) destruction of the multi-enzyme complexes during the extraction process may explain why enzymes from mixed ruminal microorganisms failed to release much soluble sugar from hay and straws. Mode of Enzyme Action The mode of action of exogenous enzymes is generally to hydrolyze some plant components that impede digestion, thereby in creasing the nutritive value of the feed. A number of different mechanisms of enzyme action have been postulated, including direct hydrolysis (Sheperd and Kung, 1996b; Colomb atto et al., 2003b), stimulation of microbial numbers and attachment to substrat e (Morgavi et al., 2000a), improvements in palatability (Adesogan, 2005), changes in gut viscosity (Officer, 2000), and changes in

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19 the site of digestion (Rode and Beauchemin, 1998; Hristov et al., 2000). Some of these factors increase the hydrolytic capacity of th e rumen, which indirectly reduces gut fill, and hence enhances feed intake (Adesogan, 2005) Morgavi et al. ( 2000a) suggested that synergy between ruminal fibrolytic enzymes and added enzymes may also be responsible for improvements in animal production when ruminants are fed enzyme-supplemented feeds. Lack of information about enzyme produc ts used and method of providing the product to animals makes it difficult to comp are the results from early studies to more recent studies. Inconsistent results seem to be caused by a number of factors including diet composition, type of enzyme preparati on used, complement of enzyme activities, level of enzyme provided, enzyme stabil ity and method of application (Rode and Beauchemin, 1998). Factors Affecting Enzyme Action It is essential to determine the conditions necessary for optimizing effects of supplemental fibrolytic enzymes on animal performance. When viewed across a variety of enzyme products and e xperimental conditions, the response to feed enzymes by ruminants has been variable. This variation can be attributed to differences in the lactation stage of cows (Lewis et al., 1999; Rode et al., 1999), enzyme type, activity and characteristics (Dawson and Tricar ico, 1999), under or over-supplementation with enzymes (Beauchemin et al., 1995; Yang et al., 1999; Beauchemin et al., 2000; Kung et al., 2000), and inappropriate method of supplying the enzyme product to the animal (Bowman et al., 2002; Sutton et al., 2003). A ccording to Beauchemin et al. (2003) animal responses to fibrolytic enzymes are al so greater at times wh en fiber digestion is compromised and when energy is the first-limiting nutrient in the diet.

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20 Different experiments have examined the impact of delivery method on the effectiveness of exogenous enzymes. Bowman et al. (2002) compared a Control diet to diets in which a fibrolytic enzyme product (Promote) was applied to the concentrate (45% of TMR), or to a pelleted portion of th e supplement (4% of TMR), or to a premix (0.2% of TMR). All diets that were suppl emented with the enzyme product delivered about 1.0 g per cow per day. Digestibility of OM, NDF and ADF in the total tract was increased in comparison to the Control wh en enzymes were added to the entire concentrate. Enzyme application to smalle r portions of the diet produced only numerical increases in digestibility over the Control. However, there was an increase in microbial N synthesis in cows fed the enzyme-supplemented premix. Enzyme supplementation did not affect milk production and compos ition, but cows receiving the enzymesupplemented concentrate had numerically higher fat-corrected milk (FCM) production compared to the Control cows. These result s indicate that the propor tion of the diet to which the enzyme is applied must be maximized to ensure a beneficial response. Lewis et al. (1996) examined the effect of a solution containing cellulases and xylanases on the digestion of a forage-based diet. Ruminally cannulat ed beef steers were assigned randomly to a Control diet (70:30 grass hay: barley ratio DM ba sis) or diets in which an enzyme was added to the forage 24 h before feeding (F-24), to the forage 0 h before feeding (F-0), to the barley 0 h before feeding (B-0), or infu sed ruminally 2 h after feeding (RI). Dry matter and NDF intakes were not different across treatments. In situ rate of NDF disappearance of the enzyme-treated barley or forage wa s greater (P < 0.05) than that of the untreated diet. Ruminal in fusion of enzymes compared with F-24 and F-0 produced lower disappearance of DM and NDF at 96-h (P < 0.05). In situ rate of DM

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21 disappearance of enzyme-treated grass tended to be greater (P < 0.10) in steers fed B-0 and Control than in those fed F-24 and F-0. Total tract digestibilities of DM, NDF, and ADF were greater (P < 0.10) in cows fed F24 and F-0 than those fed the Control diet. Forage transit time was shorter (P < 0.10) fo r B-0 than for F-24 and F-0; however, all other contrasts for particulate passage did not differ (P > 0.10). Results from this study indicate that direct applica tion of enzymes to forage is capable of improving forage digestion. Enzyme Stability in the Digestive Tract Several digestive enzymes have been su ccessfully used to enhance poultry and swine performance, but they have not been used traditionally in diets fed to ruminants. The primary reason for this practice was due to the fact that enzymes are proteins and thus would be subject to degradation by microbial proteases in the rumen and/or inactivated by proteases in the small intes tine (Kung, 2001). Stability in the rumen is critical for enzyme effectiveness. Cons iderable variation ex ists among fibrolytic enzymes in their ability to maintain activity in the ruminal environment. Some enzymes lose their activity rapidly when incubated in ruminal fluid due to proteolysis or adverse pH and temperature conditions that limit en zyme activity, whereas other enzymes show little or no loss in activity ev en after 12 hours of ruminal in cubation. This is partially because enzymes also have pH and temperature optima at which they are most effective. Kopecny et al., 1994 (cited by Kung, 2001) repor ted that a cellulase enzyme complex was rapidly degraded by ruminal bacterial pr oteases and its addition to ruminal fluid had no effect on in vitro fiber digestion. According to Morgavi et al. (2001) the cellulase enzyme complex from Trichoderma spp. has a pH and temperature optima of 4.5 and 50C, respectively.

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22 Colombatto et al. (2004a) found that the xylanase activity of two fibrolytic enzymes (Depol 40 and Liquicell 2500) showed optimal activity at pH of 5.6 and both products retained at least 70% of their xylanase act ivity after 48-h incubation at 15 or 39C in ruminal fluid. Vicini et al. (2003) analyzed the in vitro activities of two commercial fibrolytic enzymes and observed that all major cell ulose and hemicellulose-degrading activities were present; however, the optimal pH range was more acidic, and the optimal temperature (approximately 50C) was greater than the norma l pH and temperature in the rumen. The authors concl uded that it appears that a considerable part of the potential activity of these enzyme preparations was lost due to conditions in the rumen. Kung et al. (2002) indicated that the activ ity of similar fibrolytic enzymes may be optimized under different conditions. They ev aluated two different xylanases (B and C) and reported that at 40C, the activity of xyl anase C was greatest at a pH of 6.5 but was substantially reduced as the pH decreased. In contrast, xylanase B showed greatest activity at pH 5 and activity of xylanase C wa s twice that of xylanase B at pH 5.5 and 6. Fontes et al. (1995) reported that several xylanases were resi stant to several proteases but only one cellulase from a mesophilic organism was resistant to proteolytic attack. Hristov et al. (2000) observed that increasing ruminal doses of exogenous polysaccharide-degrading enzymes in heifers increased ruminal fluid carboxymethylcellulase and xylanase activities linearly (P < 0.01) and that elevated levels of fibrolytic activities in the rumen re sulted in increased (quadratic, P < 0.01) carboxymethylcellulase, xylanase and -glucanase activities in duodenal digesta. Duodenal amylase activity a nd reducing sugar concentra tion also were increased (quadratic response, P < 0.01, and P < 0.05, re spectively) by polysaccharidase enzyme

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23 supplementation. Xylanase activity of fecal DM was increased linearly (P < 0.05) with increasing ruminal exogenous polysaccharidases enzyme levels. Xylanases have been shown to be much mo re stable in the rumen than cellulases (Hristov et al., 1996, cited by Rode and B eauchemin, 1998). This may due to the relatively large and more complex structure of cellulases compared to xylanases. Morgavi et al. (2001) observed that polysaccharidase activities of commercial preparations from T longibrachiatum incubated for up to 6-h within ruminal fluid were remarkably stable. Cellulase and cellulose 1, 4-beta-cellobiosidase activities were least stable, followed by xylanase, whereas beta-g lucanase activity was not affected. Feed enzyme supplements may exert their e ffect on feed digestib ility in the small intestine as well as in the rumen (Rode and Beauchemin, 1998). Ther efore, stability is very important if these enzymes are to remain active in the intestines as well as in the rumen. According to Fontes et al. (1995), th e stability of xylanases and cellulases in the rumen may be related to glycosylation, whic h may protect them from inactivation from temperature and proteases. Many xylanases and cellulases from bacteria and fungal sources are glycosylated. Gl ycosylation involves covalent bonding of monosaccharides to specific amino acid side chains in enzyme s and glycosylation has been shown to confer resistance to proteolysis in monogastrics and ruminal fluid (Font es et al., 1995). The survival of exogenous enzyme activ ities in the rumen may also depend upon the proteolytic environment of the host animal, which can be variable (Falconer and Wallace, 1998). For example, stability of exogenous enzymes varied depending upon the donor cow, and ruminal fluid obtained from cows before feeding inactivated polysaccharidases to a greater extent than ru minal fluid taken after feeding (Morgavi et

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24 al., 2001). This was probably due to the highe r ruminal feed conten t after feeding, which allowed the proteolytic microor ganisms to colonize the feed particles and exert their enzymatic effect, thereby decreasing the resi dual proteolytic activity in the rumen fluid. The activity of enzymes deri ved from mesophilic (e.g. Trichoderma and Aspergillus spp ) or thermophilic (e.g., Thermoascus aurantiacus ) sources will not be optimized when used as ruminant feed a dditives (Beauchemin et al., 2004a). These authors suggested that enzymes should be selected that work at lower temperatures. The organisms that produce these enzymes are ps ychrophilic (Beauchemi n et al., 2004a), and their potential to improve th e initial rate of OM degr adation of corn has been demonstrated by Colombatto et al. (2004b). Cummings and Black (1999) reported that a psychrophilic, gram-negative bacterium has b een isolated and has abundant xylanolytic activity. Crude enzyme activity was measured in the supernatant at temperatures ranging from -5 to 50C. The bacterium gave fast er growth at 15C, however optimal enzyme temperature was observed at 37C. The is olation of the enzymes secreted by these microorganisms is potentially promising for improving ruminal fiber degradation. Some researchers have sugge sted that feeding unprotec ted enzymes may be more useful in immature ruminants where ru minal microbial populations are not fully developed. For example, Baran and Km et (cited by Kung, 2001) reported that a pectinase-cellulase enzyme additive improved ruminal fermentation in newly weaned lambs but not in adult sheep (with established ruminal microflora). Methods of Determining Enzyme Activity According to Beauchemin et al. (20 03) fiber-degrading enzyme activities are generally determined by measuring the rate of release of reducing sugars from pure substrates, with enzyme units expressed as the quantity of reducing sugars released per

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25 unit of time per unit of enzyme. Reducing sugars, which include monosaccharides, disaccharides and some oligosaccharides, can be measured colorimetrically using the Nelson/Somogyi copper method (Somogyi, 1952) or the di nitrosalicyclic acid method (Miller, 1959). The most commonly used substrate for measuring cellulase activity (endo--1,4-glucanase activity) is carboxymethyl ce llulose (Wood and Bhat, 1988). Exoglucanase activity can be measured using crystalline cellu lose preparations, such as Avicel. -glucosidase activit y is determined by measuring the release of glucose from cellobiose, or the release of p -nitrophenol from p -nitrophenyl--D-glucoside (Bhat and Hazlewood, 2001). Xylanase activity is most co mmonly measured by determining the release of reducing sugars from prepared xylan, such as oat ( Avena sativa ) spelt or birchwood xylan. Xylanases are specific for the internal -1,4 linkages within the xylan backbone, and are generally considered endoxylanases (Bhat and Hazlewood, 2001). Endoxylanases can be considered to be debranching or non-debranching based on their ability to release arabinose in addition to hydrolyzing the main xylan chain. -xylosidase activity can be determined by using p -nitrophenyl derivatives (Bhat and Hazlewood, 2001). Enzyme activity measurements must be conducted under conditions closely defined with respect to temperature, pH, ionic strength, substrate concentration, and substrate type, since all of these factors affect the enzyme activit y. The optimal temperature and pH for most commercial fibrolytic enzymes is approximately 60C and optimal pH is between 4 and 5 (Coughlan, 1985). However, these optima are not representative of the conditions in the rumen, which are closer to a pH of 6.0 to 6.7 and 39C (Van Soest, 1994).

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26 Wallace and Hartnell (2001) evaluated enzymatic and tracer methods for detecting and measuring the quantity of fibrolytic en zyme preparations added to corn silage, ryegrass ( Lolium multiflorum ) silage and a total mixed ration and observed that the quantity of enzyme preparations added to th e feeds could not be detected using their enzymatic activities. Glycosidase activities of soluble washed from the feed were more than an order of magnitude greater than glycosidase in the added enzymes. Carboxymethylcellulase and xylanase activity de terminations which used reducing sugar release as the measurement, were subject to interference from reducing sugars present in the feed. A fluorescent tracer method, using fluor escein added at a rate of 1 g/L of feed enzymes, or 2 g/t of feed, was developed that enabled sensitiv e detection of liquid enzyme additions to feeds (Wallace and Hartnell, 2001). Effect of Enzyme Treatment on Chewing Behavior Alterations in mechanical processing (B eauchemin and Rode, 1997) and chemical properties (Beauchemin and Buchanan-Smit h, 1989; cited by Rode and Beauchemin, 1998) of feeds can significan tly alter chewing behavior and consequently saliva production. Therefore, the use of exogenous fi brolytic enzymes in dairy cow diets may alter feeding behavior and sa liva production. Increasing the rate of fermentation within the rumen leads to a decrease in ruminal pH, which can decrease fiber digestion (Russell and Wilson, 1996). Supplemental fibrolytic enzymes have b een shown to increase fiber digestion (Rode et al., 1999; Yang et al., 2000), and ru minal pH has been lowered in some cases (Lewis et al., 1996), but not others (Yang et al., 1999). Thus, applying fibrolytic enzymes to feed before feeding may decrease both chewing time and saliva output and increases the risk of acidosis (Bowman et al., 2003). Th e latter researcher s investigated

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27 the effects of enzyme supplementation on the chewing and feeding behavior, saliva secretion, and ruminal pH in lactating dairy cows fitted with ruminal cannulas. Enzyme supplementation did not alter daily time spen t eating or ruminating, but increased saliva production, with no difference among enzyme application treatments. These results indicate that applica tion of this fibrolytic enzyme product did not alter the physical structure of the feed measured by feeding a nd chewing variables. The increase in total saliva production observed in cows fed enzyme -supplemented diets may be attributed to a physiological response to compensate for the increase in fermentation products produced during digestion. Beauchemin et al. (2000) ev aluated two doses of a fibrol ytic enzyme fed to dairy cows in a diet containing 45% forage and 50% concentrate. They observed that the time spent eating each day was similar for cows re gardless of diet, even though cows fed the enzyme-treated diets ate more than the cows fed the Control diet. Thus, adding enzyme to the diet decreased the time spent eati ng per unit of DM, NDF, or ADF, with no difference between the low and high amount of enzyme supplementation. According to the authors, decreased time spent eating per unit of fiber suggests the enzyme mixture may have had a pre-ingestive effect on the feed that enhanced the ease of ingestive mastication, which contradicts the conclusion of Bowman et al. (2003). Beauchemin et al. (2000) also measured rumination activity and did not detect effect of an enzyme supplementation on this variable. Effect of Enzyme Treatment on the Ruminal Microbial Population The enzyme activities that exist in the ru men are diverse, and include those that degrade cellulases, xylanases, -glucanases, pectinases, am ylases, proteases, phytases and those that degrade specific plant toxins (e.g., tanninases) (Wang and McAllister,

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28 2002). The variety of enzymes present in th e rumen arises from the diversity of the microbial community and the multiplicity of fibrolytic enzymes pr oduced by individual microorganisms. Efficient digestion of co mplex substrates in the rumen requires the coordinated activities of these enzymes. Limitations to cell wall digestion in the rumen can result from insufficient quantities or t ypes of enzymes produced by ruminal microbes or from an inability of degradative enzyme(s) to interact with target substrates, or from an unconducive environment for optimal enzyme activity (e.g., low ruminal pH) (McAllister et al., 2001) According to Morgavi et al. (2000), feed enzyme additives used to improve digestion in ruminants inte ract not only with the f eed but also with ruminal microorganisms. These authors reported overall increases in the rumen microbial population due to the addition of an exogenous fi brolytic enzyme to different substrates. However, it is not clear whet her this effect was due dire ctly to microbial growth stimulation or indirectly by m odifying feed structure. Morg avi et al. (2000) studied the effect of an enzyme preparation from T. longibrachiatum (TE) on growth of F. succinogenes in a medium containing cellobiose, crysta lline cellulose or corn silage fiber. Fiber disappearance and fermentation products were evaluated. The growth rate of F. succinogenes on cellobiose was not affected by TE (P > 0.05), but growth on cellulose was increased by TE though substrate disappear ance and gas production were unaffected. When corn silage fiber was used, the addi tion of TE increased NDF disappearance (P < 0.05) at 24 and 48-h (33 and 52% in Controls versus 53% and 65% in TE treatments, respectively). These results suggest that the Trichoderma enzyme preparation did not supply nutrients or growth factors to F. succinogenes Fibrobacter succinogenes digests

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29 cellulose efficiently and addition of e xogenous cellulases did not further increase cellulose disappearance. However, TE increase d corn silage fiber degradation probably by providing an enzyme(s) that limited degradation, but was not secreted by F. succinogenes Enzyme additives have been shown to enhance colonization of feed by ruminal microorganisms and increase the rate of degradation in the rumen (Yang et al., 1999). Morgavi et al. (2000a) found th at an enzyme pr oduct derived from Trichoderma longibrachiatum worked in synergy with ruminal enzymes to release sugars from corn silage, xylan, and cellulose, thereby e nhancing ruminal hydrolytic activity. Nsereko et al. (2000a) supplemented two da iry cow diets with 0, 1, 2, 5 or 10 L of enzyme per ton of DM. Incremental levels of this enzyme stimulated numbers of total viable ruminal bacteria (P < 0.05) by 100, 330, 390 and 250% (quadratic effect, P < 0.05). Of the rumen bacteria, the most notable increases in numbers were for cellobioseutilizing (P < 0.01), xylanolyt ic (P < 0.05) and amylolytic (P < 0.05) subgroups. The numbers of cellulolytic bacteria were unaffected (P < 0.05). Increasing concentrations of the enzyme had a convex, quadratic effect on protozoal numbers (P < 0.05), and the lower protozoa numbers partially explain the increased number of bacteria. These data suggest that exogenous enzymes can enhance feed digestion at least, in part, by increasing numbers of rumen bacteria that utilize hemicellulose and secondary products of cellulose digestion. Effect of Enzyme Treatment on Ruminal Fibrolytic Capacity The inclusion rate of exogenous enzymes in ruminant diets is usually in the range of 0.01 to 1% of the diet, contributing about 10 to 100-times greater fibrolytic activity per gram of feed than when silage additive s are used (Christensen, 1997). Based on the estimated average fibrolytic activity normally present in the fluid fraction in the rumen, it

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30 has been estimated that supplemental enzy mes may contribute up to 15% of the total fibrolytic activity (Rode and Beauchemin, 1998). However, the activity of commercial enzymes is measured at pH and temperature ranges that generally differ from that of rumen fluid. Thus, once ingested, exogenous enzymes likely contribute considerably less fibrolytic activity than calculated. Furtherm ore, fibrolytic enzyme activity associated with particulate matter is notably higher than in the ruminal fluid (Wang and McAllister, 2002; Rode and Beauchemin, 1998). This is probably because the attachment of the microbes to the feed particle allows the enzy me to act directly on the substrate, thereby increasing the catalytic ac tion of the enzyme. Thus, the contribution of exogenous enzymes to ruminal fibrolytic activity is difficult to estimate and is probably less than that commonly indicated on commercial enzyme containers. Colombatto et al. (2003 a) observed that enzyme addition to rumen fluid in vitro increased (P < 0.05) the initial (up to 6-h) xylanase, endoglucanase, and -D-glucosidase activities in the liquid fraction by an aver age of 85%. Xylanase and endoglucanase activities in the solid fraction also were in creased (P < 0.05) indica ting an increase in fibrolytic activity by ruminal microbes. Fu rthermore, incremental addition of enzyme increased (P < 0.05) the rate of gas producti on of various substrates, suggesting that fermentation of cellulose and xylan was enzy me-limited. However, adding the enzyme at levels higher than 2.55 L/g of DM failed to further increase the ra te of gas production, indicating that the maximal level of stimul ation was already achieved at lower enzyme concentrations. Authors concluded that enzy mes enhanced the fermentation of cellulose and xylan by a combination of pre and postincubation effects (i.e., an increase in the release of reducing sugars duri ng the pretreatment phase and an increase in the hydrolytic

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31 activity of the liquid and solid fractions of th e ruminal fluid), which resulted in a higher rate of fermentation. Effect of Enzyme Treatment on Fiber Concentration Before Ingestion Previous findings indicate that applica tion of exogenous fibrolytic enzyme products to diets have pre-ingestive effects. The ad sorption of enzyme onto the substrate is an important prerequisite for hydrolysis (Pe ll and Schofield, 1993). Applying exogenous enzymes directly to feeds re leases reducing sugars (Hrist ov et al., 1998), and in some cases, partially solubilizes NDF and ADF (Kra use et al., 1998). Colombatto et al. (2003a) evaluated the effects of adding a commercial enzyme product on the hydrolysis and fermentation of cellulose, xylan, and a mixt ure of both substrates. They reported that addition of enzyme in the absence of rumina l fluid increased (P < 0.01) the release of reducing sugars from xylan and the mixture af ter 20 h of incubation at 20C. Hydrolysis of the fiber pre-feeding may i ndicate a modification of the plant cell wall structure, which could decrease the physical effectiveness of the fiber in the diet. When inadequate effective fiber is fed, chewing activity decr eases, which leads to less salivary buffer secretion, resulting in a more acidic ruminal pH, altered ruminal fermentation patterns and low ratios of acetate to propionate that ultimately result in modified animal metabolism and reduced milk fat synthesis (Mertens, 1997). Recent studies also have shown that enzy me preparations containing high ferulic acid esterase activity as well as xylanase a nd cellulase activity reduced the NDF and ADF concentrations and increased the digestion of hays made from 12-week regrowth of Tifton 85 bermudagrass, Coastal bermudagrass a nd Pensacola bahiagrass (Krueger et al., 2003). This enzyme also increased the rate and extent of in situ degradation of the forages and reduced the lag time before fora ge degradation commenced (Krueger et al.,

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32 2004). These studies suggest that enzyme tr eatment can improve the nutritive value of tropical grasses. Sheperd and Kung (1996b) observed that appl ying an enzyme additive containing cellulase and hemicellulase reduced NDF and ADF concentration of corn silage during the ensiling period. However, Mandevbu et al. (1999) observed that treatment of bermudagrass forages harvested after 3 or 6 wk regrowth periods with a mixture of cellulase, amylase and hemicellulase had no e ffect on silage fiber concentration or cell wall carbohydrate fraction. This discrepancy is probably due to differe nces in activity of the enzyme products used in both experiments and to differences in cell wall components of corn and bermudagrass silage. Effect of Enzyme Treatment on DM a nd Fiber Digestibility Post Ingestion There is increasing evidence that exoge nous fibrolytic enzymes improve fiber digestion within the rumen, thereby increasing feed utilization in ru minants (Lewis, 1999; Rode et al., 1999). According to Beauchemin et al. (2003) the focus of most enzymerelated research for ruminants has been on plant cell-wall degrad ing enzymes. Cellulose and hemicellulose, the major structural polysaccharides in plants (Van Soest, 1994), are converted to soluble sugars by enzymes collectively referred to as cellulases and hemicellulases. More than thirty years ago, various st udies showed significant improvements in average daily gain (ADG) and feed convers ion rate (FCR) of cat tle when fed diets supplemented with enzymes containing amyloly tic, proteolytic and cel lulolytic activities (Rode and Beauchemin, 1998). Improvements in animal performance were due to increased DM and fiber digestibility. Christ ensen (1997) found an increase (P < 0.05) in DM digestibility when fibrolytic enzymes were added to rations of steers at feeding time

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33 or 24 h prior to feeding. The NDF and ADF digestibilities of the rations increased numerically by approximately three percentage units. In the same study, a positive effect on rumen degradation of forage was observed, when a mixed cellulase, hemicellulase and pectinase enzyme additive was applied to alfalfa hay, grass hay, oat straw and barley silages at different rates. Christensen (1997) also observed that application of 600 IU/kg DM of xylanase had a positive effect on both in vitro and in situ degradation of both high-fiber and low-fiber forages. Applying fibrolytic enzymes prior to feedi ng can alter the struct ure of the cell wall, thereby making it more amenable to degrada tion (Beauchemin et al., 2004b). Nsereko et al. (2000b) applied an enzyme product to alfalfa hay that was then autoclaved to inactivate enzyme activities and washed to remove any product of the hydrolysis, eliminating the possibility of chemotactic e nhancement of digestion or synergy between microbial enzymes and exogenous enzymes. In vitro NDF digestibility was higher at 12 and 48-h for treated than for untreated hay a nd generally this effect was enhanced by longer pre-incubation with enzymes. Since th ese effects were observed in the absence of active ruminal enzymes and soluble hydr olysis products, the exogenous enzymes probably caused structural changes to th e forages that improved digestion. Rode et al. (1999) evaluate d the effect of exogenous fibrolytic enzyme (Promote) on DMI and digestibility in cows fed Contro l diets or diets in which an enzyme was added to the concentrate at a rate of 1.3 g/kg (DM basis). Enzyme addition did not affect DMI. However, total tract digestibilit y of nutrients as determined using Cr2O3, was increased by enzyme treatment (DM, 61.7 vs. 69.1%; NDF, 42.5 vs. 51.0%; ADF, 31.7 vs. 41.9%; CP, 61.7 vs. 69.8%). Neverthele ss, effects of supplemental enzymes on

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34 digestibility have been inconsistent. Enzy me products comprised mainly of xylanases and cellulases have increased digestibility (Rode et al., 1999; Yang et al., 2000), or not affected digestibility (Lewis et al., 1999) Other studies have shown that exogenous enzymes did not consistently improve animal performance, and the mechanism for improved growth was not always confir med by digestibility trials (Rode and Beauchemin, 1998). Mandevbu et al. (1999) observed that treatment of bermudagrass forages with fibrolytic enzymes had no effect on in vitro or in situ DM or NDF disappearance of silages. Hr istov et al. (2000) observed that the ruminally soluble fraction and effective degradability of feed DM in situ were increased (quadratic response, P < 0.01) by enzyme treatment in ru minally cannulated heifers, but apparent digestibility of DM, CP, and NDF were not affected. Effect of Enzyme Treatment on Silage Fermentation Applying cell-wall degrading enzymes duri ng the ensiling process can increase the release of fermentable sugars from the stru ctural polysaccharides thereby providing extra substrate for the microbial fermentation. Th is often increases th e production of lactic acid, which reduces the risk of clostridial fe rmentation (Van Vuuren et al., 1989). When used as silage additives, fibrolytic enzymes predigest plant cell walls and this can increase the extent and rate of degradati on of silage in the rumen, and consequently, improve digestibility and nutritive value (McHan, 1986). Rodrigues et al. (2001) reported that application of a mixture of cellulase and endoxylanase to ryegrass before ensiling reduced NDF, ADF and acetic acid (P < 0.01) concentration and increased lactic acid and sugar concentration (P < 0.01). Si milar results were obtained by Clavero and Razz (2002) with dwarf elephantgrass ( Pennisetum purpureum ) silage treated with a cellulase mixture. Selmerolse n (1993) showed that the fe rmentation of crops with low

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35 sugar concentration, such as tropical gra sses, was improved more by enzyme addition, while that of crops with high sugar concentr ation were improved more by lactic acid bacteria inoculation. In agreement, recent results also have shown that treatment of bermudagrass, which is low in sugars, with fi brolytic enzymes alone or with an enzymeinoculant blend (Adesogan et al., 2004) im proved the fermentati on, but contradictory results exist (Mandevbu et al., 1999). Clearly enzyme applic ation at ensiling to forage containing low sugar contents is logical because of potentia l sugar release from enzymeinduced fibrolysis, but the response depends on the enzyme activities and treatment conditions (Adesogan, 2005). Kung and Ranjit (2001) compared w hole-plant barley treated with L. buchneri and enzymes, or a mixture of L. plantarum, P. pentosaceus, P. freudenreichii and enzymes or a buffered propionic acid-based additive. They observed that silages treated with L. buchneri and enzymes had lower pH and higher c oncentrations of acetic and propionic acids and improved aerobi c stability when compared with untreated silage. These results indicate that enzymatic treatments can repr esent a viable strategy for improving the quality of silages, though they don t directly affect aerobic stability. Effect of Enzyme Treatment on Hay Nutritive Value Direct effects of fibrolytic enzyme tr eatment on chemical composition of treated hays before ingestion have not been studied previously. In stead, research has focused on the effect of such enzymes on ruminal ferm entation or voluntary intake of the treated hays. Pinos-Rodriguez et al. (2002) observed that applicatio n of an exogenous fibrolytic enzyme to alfalfa or ryegrass hays increased intake of DM (P < 0.01), OM and CP (P < 0.05) in lambs; however, NDF and ADF intake were not affected. The enzyme increased apparent digestibility of CP, hemicellulose (P < 0.05), and NDF (P < 0.10) in alfalfa. The

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36 enzyme also improved N balance (P < 0.05), and total VFA (P < 0.05) concentration in the rumen (after 3 and 6-h of incubation) for both hays. According to the authors, these results indicate that directly fed exoge nous fibrolytic enzymes may change ruminal fermentation, intake, and digestibility of fo rages. Dawson and Tricarico (1999) showed that when fescue hay was not treated or tr eated with preparations high in xylanase or cellulase activity, xylana se addition increased carbohydrate utilization and VFA production, cellulase addition altered VFA prop ortions, and addition of a mixture of the enzymes increased carbohydrate digestion and the acetate: propionate ratio. Novak et al. (2003) evaluated the eff ect of a fibrolytic enzyme containing carboxymethyl cellulase and xylanase on rumina l disappearance of DM, NDF and ADF, and intestinal DM digestibility of wheat stra w. Enzyme addition had no effect (P >0.05) on the effective degradability of DM, NDF and ADF, but increased DM, NDF and ADF disappearance after 4 and 6-hours of incubati on and decreased these measures after incubation for 12 and 24 hours. Differences in enzyme activity and stability in rumen fluid, application methods, and characteristics of rumen fluid due to donor animal diet partly explain the discrepancies between thes e studies. The conflicting results highlight the merits of further evaluation of the benefits of fibrolytic enzyme application to hays. Effect of Enzyme Treatment on Animal Performance Dry matter intake has been increased (B eauchemin et al., 2000) or unchanged (Beauchemin et al., 1999; Kung et al., 2000) by dietary supplementation with enzymes. Feed intake responses to enzyme supple mentation have generally been small and inconsistent (Yang et al., 1999; Rode et al ., 999; Schingoethe et al., 1999; Vicini et al., 2003) with only occasional significant (P < 0.05) increases (Lewis et al., 1999). Attempts to improve feed efficiency in dair y cows by the use of direct-fed fibrolytic

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37 enzymes applied at or a few hours before feeding have yielded variable production responses (Sutton et al., 2003). Milk production also has been increased in some studies in which dairy cow diets were supplemented with fibrolytic enzymes (Rode et al., 1999; Yang et al., 2000; Kung et al., 2002), but it has been unaffected in other studies (Sheperd and Kung, 1996b; Beauchemin et al., 2000; Vicini et al., 2003). Table 2.2 summarizes the results of supplementation with fibrolytic enzymes on m ilk production from several experiments. Milk yield responses have been generall y positive but often not significant, while changes in milk fat and protein concentration have been both positive and negative and are often not significant (Beauchemin et al., 1999; Lewis et al., 1999; Schingoethe et al., 1999; Yang et al., 1999; 2000; Kung et al., 2000; Phipps et al., 2000; Rode et al., 1999; Vicini et al., 2003). Rode et al (1999) used lactating Holste in cows in early lactation to investigate effects of exogenous fibrolytic enzyme (Promote) supplementation on DMI, milk production and digestibility. Enzyme addition did not affect DMI (P > 0.05) but tended (P < 0.1) to increase milk yield (35.9 vs. 39.5 kg/d) as a consequence of increased digestibility. Percentage of milk fat was lower (P < 0.05) and percentage of milk protein tended to be lower (P < 0.1) in cows fe d the enzyme-supplemented diet, such that component yields were similar (P > 0.05) for co ws fed either diet. Energy deficiency was numerically lower (P > 0.05) for cows fed the enzyme-supplemented diet than for cows fed the Control diet (-3.33 vs. -3.62 Mcal/d). Consequently, the au thors concluded that supplementing dairy cow diets with Promote ha s the potential to enhance milk yield and nutrient digestibility by cows in early lactation without changing feed intake.

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38 Table 2.2 Effect of spraying enzymes onto f eeds prior to feeding on milk production in recent studies Study Increase in milk production1, kg/d Dietary forage type P values Beauchemin et al., 1999 +0.3, +1.5 Barley silage + alfalfa haylage (45%) NS Lewis et al., 1999 +1.2, +6.3, +1.6 Alfalfa hay + alfalfa silage (41.6%) < 0.05 Rode et al., 1999 +3.6 Corn silage + alfalfa hay (38.5%) < 0.11 NS Schingoethe et al., 1999 Expt. 1: + 1.2, + 0.9, + 2.7 Expt. 2: + 1.3 Corn silage + alfalfa hay (55%) < 0.01 Yang et al., 1999 + 0.9, + 1.9, + 1.6 Barley silage + alfalfa cubes (52.8%) <0.05 Beauchemin et al., 2000 0.5, 0.5 Barley silage + alfalfa haylage (45%) NS < 0.10 Kung et al., 2000 Expt. 1: + 2.5, -0.8 Expt. 2: + 0.7, + 2.5 Corn silage + alfalfa hay (45%) < 0.10 Yang et al., 2000 + 0.1, + 2.1 Corn silage + alfalfa hay (38%) < 0.05 Zheng et al., 2000 + 2.0, + 4.1, + 1.5 Corn silage + alfalfa hay (50%) < 0.07 Bowman et al., 2002 + 0.6, 0.6, -1.5 Barley silage + alfalfa silage (55%) <0.10 Knowlton et al., 2002 + 1.8, 1.2 Corn silage + alfalfa silage (53%) NS 1. The increase in milk is relative to milk produc tion by Control cows, NS: no significant effect Studies have shown that application of low or high amounts of enzymes to forages or diets produced different responses. Yang et al. (1999) examined the effect of two doses of a cellulase-xylanase enzyme mixtur e applied to the forage or concentrate component of dairy cow diets. They obs erved that milk production increased in cows fed a high dosage of the enzyme compared with cows fed the Control diet, but effects on milk composition were minimal. The response to enzyme supplementation was affected more by amount of enzyme app lied than by the dietary component treated with the enzyme. The authors claimed that th e results demonstrated the benefits of using a fibrolytic enzyme to enhance feed di gestion and milk production by dairy cows.

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39 However, Beauchemin et al. (2000) found th at a high level of enzyme application was less effective than a low level at increasing total tract digestibility. Lewis et al. (1999) carried out two experi ments to evaluate the effectiveness of adding a mixture of cellulases and xylanases to dairy cow diets. In Experiment 1, cows were assigned to diets containing forages th at had or had not been treated with the enzyme between 8 and 24 h prior to feeding. They observed that cows consuming the enzyme-treated forage produced more milk (27.2 vs. 25.9 kg/d, P < 0.05) and digested more DM per day than did cows fed the Contro l forage. In Experiment 2, early lactation cows were assigned to one of four treatments for 16 wk: 1) no enzyme treatment, 2) a low (1.25 ml/kg of forage DM) enzyme treatment 3) a medium (2.5 ml/kg of forage DM) enzyme treatment, or 4) a high (5.0 ml/ kg of forage DM) enzyme treatment. Dry matter intake was similar across enzyme treatments and intake was greater than for cows fed the Control forage. Yield of milk, 3.5% fat-corr ected milk, and energy-corrected milk were greater by cows on Treatment 3 than by co ws on Treatment 1. Therefore, applying fibrolytic enzymes to the forage portion of the rations improved lactational performance of early and mid-lactation cows. Schingoethe et al. (1999) eval uated the response to a dir ect-fed (applied at feeding time) cellulase and xylanase enzyme mixture applied at 0, 0.7, 1.0 or 1.5 L/ton of DM to the forage portion (60% corn silage and 40% alfalfa hay) of a TMR for lactating cows just prior to feeding. Over the 12-wk trial period, milk production from cows assigned to the 1.5-L enzyme treatment increased by 10.8% relative to those in the Control (no enzyme addition) group, while fat and pr otein production increased by 20 and 13%, respectively. The lowest enzyme rate account ed for approximately on e-half of the milk

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40 production increases that occurred with th e highest enzyme application rate. The responses to enzyme-treated forages were in itially evident 2 to 4 wk after experiment started and they were maintain ed throughout the remainder of the experiment. Cows that started to receive enzyme-tr eated forage during the first 100 d postpartum produced 9 to 15% more milk and 16 to 23% more energy-cor rected milk than did cows fed the Control diet. However, milk production was not increas ed when cows were in mid-lactation (121 d postpartum) at the start of the ex periment (Schingoethe et al., 1999). The reason for the general poor response to low levels of enzyme application is obvious, but a lack of benefit for the high leve ls is less apparent. Such occurrences may be attributed partly to negativ e feedback inhibition which is one of the classical modes of regulation of enzyme action (Adesogan, 2005). This feedback mechanism occurs when enzyme action is inhibited by production of a critical concentration of a product of the enzyme-substrate interaction. For instance, fermentation of sugars produced by cell wall hydrolysis may reduce ruminal pH to levels that inhibit cell wall digestion (Adesogan, 2005) by the negative effect of low pH on ru minal fibrolytic microorganisms. An alternative hypothesis is that excessive enzyme application blocks the binding sites for enzymes or may prevent substrate co lonization (Beauchemin et al., 2003). Sutton et al. (2003) used multiparous cows fitted with rumen and proximal duodenal cannulas in early lactation to investigat e the effect of method of application of a fibrolytic enzyme product on digestive pro cesses and milk production. The enzyme was not applied (Control), sprayed on the TMR befo re the morning feed (TMR-E), or on the concentrate the day before feeding (Conc-E ), or infused into the rumen for 14 h/d (Rumen-E). There was no treatment effect on ei ther feed intake or milk yield but values

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41 were numerically higher in cows fed TMR-E than in the rest of the cows. Ruminal digestibility of DM, OM and starch were unaffected by the enzyme. Ruminal NDF digestibility was lowest in cows fed TMR-E, but these cows also had the greatest postruminal NDF digestibility. Total tract diges tibility of starch, DM and OM were highest in cows fed TMR-E. Ruminal retention time was reduced by all enzyme treatments but postruminal transit time was increased so the decline in total tract retention time with enzymes was not significant. It was suggest ed that the reduction in ruminal particle retention time would reduce time available for fi brolysis to occur; a nd therefore, partly explain the variability in the reported responses to enzyme treatment. Bowman et al. (2002) also investigated the effect on dairy cows (averaged 111 32 DIM) of a fibrolytic enzyme (Promote) added at 1.0 g/cow/d to the concentrate portion (45% of the dietary DM) of the TMR, to the pelleted supplement portion (4% of the dietary DM) of the TMR, or to a premix (0.2 % of the dietary DM). The effects of enzyme supplementation on milk production and composition were not significant (P >0.05), but cows receiving the enzyme-supplem ented concentrate had numerically higher FCM compared to the Control cows. Knowlton et al. (2002) evaluated the effect of a fibrolytic enzyme formulation on the intake, partitioning, and excr etion of N and P by dairy cows in early and late lactation. Cows fed diets containing the enzyme formulation gained more weight than those fed the enzyme-free diet, partic ularly in early lactation. Enzyme treatment did not affect apparent digestibility, excreti on of N and P, or retention of these nutrients in body tissues Interactions observed between the effects of stage of lactation and treatment indicated that the nature of the milk yield and manure excretion responses differed between early and late-lactation cows. Milk yield, fecal output and N

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42 excretion in cows fed enzyme-supplemented di ets were greater than those of Control cows in early lactation, but le sser in late-lactatio n. Energy requirements of early lactation cows are higher that those in late lactatio n; therefore, the enzyme supplementation in early lactation is potentially more promisi ng than in mid-lactation or late-lactation because it can improve energy balance (Jurkovich et al., 2002). Effect of Enzyme Treatment on Blood Metabolites Urea is the primary form of excretory N in mammals, and greater concentrations of blood urea N (BUN) have long been known to refl ect inefficient utilizat ion of dietary CP by ruminants (Broderick and Clayton, 1997). Few papers have reported the effect of fibrolytic enzymes on blood metabolites. Hris tov et al. (1998) found that blood glucose and urea concentration in lactating dairy cows were not affected by enzyme treatments. Hristov et al. (2000) observed that plasma beta hydroxybutyrate (BHBA) concentration was reduced (P < 0.01) in cows supplemented with fibrolytic enzymes. Jurkovich et al. (2002) also found a lower incidence of hype rketonaemia and lower acetoacetic acid and non-esterified fatty acid (NEFA) concentrati ons in the blood of cows supplemented with a mixture of fibrolytic enzymes, which indicates that enzyme supplementation can improve energy balance in lactating cows. Effects of Combining Enzyme and Chemical Treatments Though chemical treatments also have been successfully used to disrupt ferulate bridges and hydrolyze cell wa lls in tropical forages, little is known about the effectiveness of biological treat ment at achieving these objectiv es. Opportunities exist to improve overall utilization of lignocellulosic materials as ruminant feeds by using organisms or their secreted enzymes with the capacity to attack the most refractory fiber components that have lignin-carbohydrat e bonds (Varga and Kolver, 1997). The

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43 prospects for improved use of fibrous re sidues relies on enha ncing the rate of fermentation of the more readily fermente d cell wall constituents and increasing the extent of digestion of poor ly degraded constituents. Combinations of chemical and biological treatments have been applied to low quality forages, and the results show that they can act synergistic ally for improving the nutritive value of such roughages. Wang et al. (2004) carried out four experiments to study the effects of pre-treating wheat straw with alkali (5% of NaOH, wt/wt, or 3%, wt/wt of NH3) and then spraying it with an enzy me mixture (xylanase, -glucanase, carboxymethylcellulase, and amylase) on in vitro, in situ, and in vivo digestibility. In Experiment 1 enzymes increased (P < 0.01) gas produc tion and the incorporation of 15N into microbial N at 4 h from NaOH-treated wheat straw (P < 0.01 for gas; P < 0.05 for 15N) compared to untreated wheat straw. In Experiment 2, untreated and alkali-treated wheat straw were sprayed with enzymes at 0, 0.15, or 1.5 mg/g DM and incubated ruminally in nylon bags for up to 80 h to determine the in situ DM disappearance (ISDMD). Interactive effects (P < 0.05) of pretreatment and enzymes were observed on all ruminal degradation parameters. Alkali increased the rate (P < 0.01) and extent (P < 0.01) of ISDMD irrespective of enzymes. Enzyme application to untreated straw did not affect the extent of ISDM D, but increased (P < 0.01) that of alkali-treated straw. In Experiment 3, substrates from Experi ments 1 and 2 were incubated in acetate buffer for 24 h to measure the hydrolytic loss of DM and re lease of reducing sugars and phenolic compounds. Alkali pretreatment and enzymes each increased (P < 0.01) DM loss and the release of reducing sugars, and in combination, exerted additive effects (P < 0.01). Enzymes did not affect the release of phenolic compounds from the straw. In

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44 Experiment 4, wrapped straw bales were injected with NH3 four months before the study, and enzymes were applied immediately before feeding. Applying enzymes to ammoniated straw increased (P < 0.05) digestibility of DM, OM, and total N but did not affect the intake of DM or digestibility of ADF by crossbred beef cows in late gestation. According to the authors, pretreatment of straw with alkali enhanced the efficacy of exogenous enzymes, presumably by breaking esterified bonds and releasing phenolic compounds and/or by swelling the crystalline cellulose and enhancing enzyme penetration. Adogla-Bessa et al. (1999) also found that adding urea and fibrolytic enzymes to wheat silage was more effective than either treatment alone. However, using enzymes and chemicals for forage improveme nt is probably not economically viable. Including enzymes that mimic alkali hydrolysis (e.g., esterases) in commercial feed additives could improve substantia lly the effectiveness of enzyme products for ruminants. The conflicting results on the effectivene ss of fibrolytic enzymes for enhancing forage nutritive value and animal performa nce highlight the need for more concerted investigation of this subject. The fact that even less is known about the extent to which enzymes can improve the quality of tropica l forages and enhance animal performance from such forages, emphasizes the importan ce of future studies in this area. The aim of this series of experiments was to evaluate the effect of ammoniation and proprietary fibrolytic enzyme application on the nutritive va lue of tropical grasses and on animal performance. The specific objectives were: To evaluate the effect of applying ammonia or four commercial fibrolytic enzymes on the nutritive value of two C4 grass hays (Chapter 3).

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45 To evaluate the effect of a pplying different rates of four proprietary fibrolytic enzyme preparations at different rates, at ensiling, on the nutritive value of Tifton-85 bermudagrass silage (Chapter 4). To determine the effects of applying an en zyme to bermudagrass at ensiling, or to different components of the diet at feed ing 8 in feed intake, milk production and composition, blood metabolites and digestion ki netics of dairy cows (Chapter 5).

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46 CHAPTER 3 EFFECT OF TREATMENT WITH AMM ONIA OR FIBROLYTIC ENZYMES ON THE NUTRITIVE VALUE OF HAYS PRODUCED FROM TROPICAL GRASSES Introduction Feed enzymes have been shown to be eff ective in a wide range of diets containing roughages (Rode and Beauchemin, 1998). Their effectiveness is partly due to improved hydrolysis of the fiber fraction (Colombatto et al., 2003b) which increases digestibility (Christensen, 1997; Rode et al., 1999) and voluntary intake (Pinos-Rodriguez et al., 2002). Nevertheless, other studies have shown that exogenous enzymes do not consistently improve forage utilization. This inconsistency is attributable to factors such as differences in enzyme type and activit y, treatment duration, a pplication method, diet composition and level of animal performance. Fibrolytic enzymes seem to work by increa sing the rate, but not the extent of fiber digestion (Feng et al., 1996; Yang et al., 1999). This suggests that the fibrolytic enzyme products currently on the market for rumina nts may not be introducing novel enzyme activities into the rumen (Wang and McA llister, 2002). One of the few studies on fibrolytic enzyme treatment of tropical gr asses showed that enzyme treatment had no effect on silage fiber concentra tion, cell wall carbohydr ate fraction and in vitro or in situ DM or NDF disappearance of silages (Mandevbu et al., 1999). Yet due to the widespread use of C4 grasses which intrinsically have lo w nutritive values, it is important to determine if modifying treatment conditions will lead to enzyme-mediated enhancements in their quality.

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47 Ammoniation is one of the most studied chemical treatments for improving forage quality. Ammoniation improves forage diges tibility due to the hydrolytic action of the ammonia on linkages between lignin and struct ural polysaccharides, thus increasing the organic matter potentially available for utilization by the ruminal microorganisms (Barrios and Ventura, 2002). Ammoniati on also increases the crude protein (CP) concentration of the treated forages, and this improvement is through fixation of the applied nitrogen (Weiss and Underwood, 1995). The objective of this experiment was to evaluate the effect of applying ammonia or four commercial fibr olytic enzymes on the nutritive value of two C4 grass hays. Materials and Methods Enzyme Application In the first of two experiment s, the effects of applying NH3 or a fibrolytic enzyme complex (Promote, Pr) (Cargill, Minnet onka, MN) were measured on the DM and chemical composition and in vitro and in situ digestibility of two tr opical grass hays. The forages tested were 12-week regrow th of Coastal bermudagrass hay ( Cynodon dactylon ) (BE) and Pensacola bahiagrass hay ( Paspalum notatum ) (BA). The ammonia was applied at 40 g/kg DM and the enzymes were applied at 0 (Control), 0.5, 1 and 2 times the rates recommended by the respective ma nufacturers. This was done because the optimal application rate for C4 grass hays was unknown. The actual application rates are shown in Table 3.1. The enzymes were dissolve d in 500 ml of water and applied in a fine spray to 3 replicates of 2 kg of each hay. Treat ed hays were stored for 3 weeks in plastic bags (30 bags for Experiment 1 and 66 bags for Experiment 2) and then chemically characterized. The manufacturer-stipulated act ivities of the enzymes are shown in Table 3.2. Cellulase activity was also determined at 39oC and pH 5.5 using the filter paper

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48 method (Wood and Bhat, 1988) and the values obtained for Pr, X-20, CA and A-20 were 33.7, 22, 0 and 51.3 filter paper units/g, respecti vely, where one unit of activity is the amount of enzyme that releases exactly 2 mg of glucose from 50 g of filter paper in 60 min. Xylanase activity was determined at 39oC and pH 5.5 using the di-nitro salicylic acid procedure (Bailey et al., 1992) and the values obtai ned for Pr, X-20, CA and A-20 were 5190, 7025, 0 and 3530 mol of xylose released/min/ml, respectively. In the second experiment, the effects of applying NH3 or three fibrolytic enzymes were measured on the same variables as in the pr evious experiment. The enzymes studied in Experiment 2 were Biocellulase X-20 (X -20) (LodeStar, IL, USA), Cattle-Ase (CA) (Loveland Industries Inc, Greel ey, CO, USA) and Biocellula se A-20 (A-20) (LodeStar, IL, USA). Two separate experiments were conducted because the enzymes were not simultaneously available. Laboratory Analysis The NDF and ADF concentrations (Van Soest et al., 1991) of the samples and digested residues were determined wit hout amylase pretreat ment using an ANKOM200 Fiber Analyzer (ANKOM Technology, Macedo n, NY). Hemicellulose was calculated by difference from NDF and ADF concentrations Water soluble carbohydrates (WSC) were determined with the anthrone reaction assay (Ministry of Agriculture Fisheries and Food, 1986). Crude protein (CP) was determined by digesting 0.5 g of sample using a micro Kjeldahl apparatus (Labconco Corporation, Kansas City, MO) and the N concentration was determined (Noel and Hambleton, 1976) using a Technicon Auto Analyzer (Technicon, Tarrytown, NY, USA).

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49 Table 3.1 Actual enzyme application rates used Application rate Enzyme 0.5x 1x 2x Promote1 (mg/kg DM) 650 1300 2600 Biocellulase X-202 (mg/kg DM) 7.3 14.5 29 Biocellulase A-202 (mg/kg DM) 7.3 14.5 29 Cattle-Ase3 (mg/kg DM) 89 178 356 1 Cargill, Minnentoka, MN 2 LodeStar, Channahon, IL, USA 3 Loveland Industries Inc, Greeley, CO, USA Table 3.2 Manufacturer-stipulated enzyme activities. Enzymatic activity Enzyme Cellulase (Units/g) Xylanase (Units/g) -Glucanase (Units/g) Amylase (Units/g) Promote1 1,200 Biocellulase X-202 5,700 16,000 600 1,200 Biocellulase A-202 6,000 400 4,300 3,100 Cattle-Ase3 15,000 1 Cargill, Minnentoka, MN, USA 2 LodeStar, Channahon, IL, USA 3 Loveland Industries Inc, Greeley, CO, USA The in vitro digestibility of DM (IVDMD) NDF (IVNDFD) and ADF (IVADFD) were determined in duplicate runs after incubating forage samples in buffered rumen fluid for 6 or 48-h using two ANKOMII Daisy Incubators (ANKOM Technology, Macedon, NY). The buffer was prepared according to the ANKOM Technology procedure. The rumen fluid was obtaine d before feeding from two, non-lactating, fistulated cows, fed 9 kg of Coastal bermudagrass hay and 400 g of soybean ( Glycine max ) meal daily. In situ rumen degradability was measured only in hays treated with NH3, X-20 and A-20, because these treatments were found to be more effective at increasing in vitro digestibility than the others. Five g of ground (4 mm screen) hay samples were weighed into nylon bags (50 m pore size) in triplic ate and placed into th e two fistulated, non-

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50 lactating Holstein cows for 0, 3, 6, 9, 12, 24, 48, 72, 96, and 120 h. At each incubation time, bags were removed and rinsed with c ool water and frozen. At the end of each period, all bags were washed in a washing machine and dried for 48-h at 60 C. The cows used for this study were the same as those used as rumen fluid donors for the in vitro study. In order to avoid placing too many substrate-filled bags in the rumen, only bags for 3 treatments (Control, ammonia and X-20 or A-20) and one forage were simultaneously incubated (60 bags maxi mum incubated at the same time/cow). Statistical Analysis A completely randomized design with 3 re plicates per treatment was used to quantify the effects of enzyme or NH3 application on chemical components. The model used was: Yijk: + Ti + Ei Where: Yij: dependent variable : general mean Ti: treatment effect (enzyme*level) and NH3 Ei: experimental error A completely randomized design with 3 re plicates per treatment was used to quantify the effects of enzyme or NH3 application on digestibility after 6-h and 48-h. Data from 6-h and 48-h incubations were analyzed separately. The model used was: Yijk: + Ti + Rj + Eij Where: Yij: dependent variable

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51 : general mean Ti: treatment effect (enzyme*level) and NH3 Rj: run effect Eij: experimental error Data were analyzed using the GLM procedure of SAS (1995). Orthogonal contrasts were used to compare additive tr eatment means, and polynomial contrasts were used to determine the effect (linear, quad ratic and cubic) of in creasing the amount of enzyme application. Treatment significance wa s declared at the 5% level and tendencies were declared at the 15% level. The interaction treatment forage was not included in the previous models because the in vitro and in situ trial were done separately. The in situ ruminal degradation parameters were estimated using the model described by McDonald (1981): P= a + b (1-e-c (t-L)) where P = DM degraded at time t, a = wash frac tion, b = potentially de gradable fraction, a+b= total degradable fraction, c = the ra te at which b is degraded, t = time incubated in the rumen, and L = lag phase. Th e constants a, b, c, and L were estimated using the nonlinear regression (NLIN) proced ure of SAS (1995) a nd analyzed using the GLM procedure of SAS (1995). Results and Discussion Chemical Composition of Tropical Hays The chemical composition of untreated bermudagrass and bahiagrass hays is shown in Table 3.3. The low CP and high NDF, ADF, hemicellulose and lignin

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52 concentrations are typical of mature tropical grasses. Thes e values agree with Jung and Allen (1995) who concluded, that depending on th e stage of maturity, cell walls represent between 30 and 80% of plant DM in grasses so that under most circumstances, the bulk of carbohydrates in mature grasses are from cell wall polysaccharides. Effect of Promote and Ammoniation on Chemical Composition in Experiment 1 Enzyme treatment increased (P < 0.05) the NDF concentration of BE hay, suggesting that Pr also contained non-fibrolytic enzymes (T able 3.4). However, the NDF concentration of BA was decreased (P < 0.01) by both Pr and NH3 treatment, though NH3 treatment was more effective (P < 0.01) at hydrolyzing the NDF fraction than Pr. Bahiagrass hay had a lower (P < 0.01) concentration of ADF than BE. The ADF concentration of BE was decreased (P < 0.01) by NH3 and Pr (linear res ponse) treatments, but that of BA was not. Promote tended (P = 0.052) to be more ef fective at hydrolyzing the ADF of BE than NH3. The hemicellulose concentration of BE was increased (P < 0.01) by enzyme (quadratic response) and NH3 treatment, whereas that of BA was decreased (P < 0.01) by NH3 treatment. The WSC concentration of BA was increased (P < 0.01) by Pr and NH3 treatment (P < 0.01) and NH3 was more effective in this respect. Both treatments also numerically (P > 0.05) increased the WSC concentration of BE. The higher WSC concentration observed in the NH3-treated BA hay compared to other hays, shows that the chemical treatment was more effective at hydrolyzing th e fiber fraction of this forage. Enzyme treatment did not affect the CP concentr ation of either of the hays; however NH3 treatment produced greater (P < 0.01) values than those of Contro l and enzyme-treated hays.

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53 Table 3.3 Chemical composition of the untreated hays Forage Nutrient Bahiagrass Bermudagrass CP, g/kg DM 66 69 NDF, g/kg DM 792 821 ADF, g/kg DM 431 485 Lignin, g/kg DM 55 67 Hemicellulose, g/kg DM 336 354 Ash, g/kg DM 57 50 CP: crude protein, NDF: neutral detergent fiber, ADF: acid detergent fiber These results indicate that the treatments, particularly NH3, had different effects on cell wall components of the forage s and forage type affected the response. In BE, total cell wall concentration was not reduced by a dditive treatment but the NDF fraction was hydrolyzed by other additives, thus increas ing the ADF. Whereas in BA, the ADF fraction was unaffected by treatment, but NDF and hemicellulose fractions were hydrolyzed into sugars, which is in agreement with Colombatto et al. (2003), and Hristov et al. (1998). Promote treatme nt was less effective than NH3 treatment at cell wall hydrolysis. This result agrees with t hose of Brown (1993), who observed that ammoniation decreased (P < 0.01) the NDF concentr ation of stargrass ( Cynodon nlemfuensis ) hay. Effect of Promote and Ammonia Application on in vitro DM, NDF, and ADF Digestibility in Experiment 1 After 6-h of digestion, BA had greater (P < 0.01) IVDMD than BE, but this difference was no longer evident after 48-h of digestion. Promote-treatment increased (P < 0.01) the 6-h IVDMD of BE; however, ammoni ation was more effective (Table 3.5). Only NH3 treatment increased (P < 0.01) the 6h IVDMD of BA or the 48-h IVDMD of both forages. The effectiveness of NH3 at increasing the 6-h and 48-h digestibility of the hays concurs with the results of Zorrila-Rios et al. ( 1991), who observed that the

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54 Table 3.4 Effect of Promote or ammonia treatment on chemical composition (% DM) of tropical grass hays NDF ADF Hemicellulose WSC CP Additive Level BE BA BE BA BE BA BE BA BE BA Control 82.1 79.2 48.5 43.1 33.6 35.4 2.0 0.9 6.6 6.9 0.5x 85.2 78.1 46.7 43.8 38.4 35.0 2.6 4.1 6.7 6.7 1x 85.1 78.1 46.8 42.6 38.3 34.4 2.5 3.8 6.4 6.8 Promote 2x 86.2 78.5 44.5 44.3 41.7 34.2 2.6 3.2 6.4 7.1 Mean 85.5 78.2 46.0 43.6 39.5 34.5 2.6 3.7 6.5 6.9 Ammonia 83.6 75.4 46.7 43.7 37.0 31.7 2.7 4.9 17.1 12.8 s.e.m. 0.964 0.354 0.293 0.491 0.872 0.737 0.039 0.019 0.258 0.139 Contrasts P values Polynomial effects Promote level NS NS L** NS L* NS NS C* NS NS Promote vs. Control 0.015 <0.01 <0.01 0.452 <0.01 0.339 0.232 <0.01 0.477 0.285 Control vs. Ammonia 0.289 <0.01 <0.01 0.962 0.021 <0.01 0.234 <0.01 <0.01 <0.01 Promote vs. Ammonia 0.128 <0.01 0.052 0.485 0.593 0.098 0.785 <0.01 <0.01 <0.01 NDF: neutral detergent fiber, ADF: acid detergent fiber, WSC: water soluble carbohydrates, CP: crude protein, BE: bermudagrass, BA: bahiagrass, L: linear effect, Q: quadratic effect, C: cubi c effect, NS: no significant effect, *: P < 0.05, **: P < 0.01, 1Bahiagrass vs. bermudagrass

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55 Table 3.5 Effect of Promote or ammonia a pplication on the IVDMD of tropical grass hays DM digestibility, % After 6-h After 48-h Additive Level BE BA BE BA Control 10.1 14.2 49.8 47.9 0.5x 10.6 14.2 49.7 48.8 1x 11.4 14.9 49.0 49.3 Promote 2x 11.1 14.4 49.9 48.3 Mean 11.0 14.5 49.5 48.8 Ammonia 16.5 18.7 61.4 61.7 s.e.m. 0.237 0.308 0.463 1.582 Polynomial effects P values Promote level NS NS NS NS Contrasts Promote vs. Control <0.01 0.418 0.686 0.633 Control vs. Ammonia <0.01 <0.01 <0.01 <0.01 Promote vs. Ammonia <0.01 <0.01 <0.01 <0.01 DM: dry matter, BE: bermudagrass, BA: bahiagrass, NS: non significant effect IVDMD of the wheat ( Triticum aestivum ) straw was increased by approximately 54% by ammoniation. Dawson and Tricarico (1999) suggested th at the most active period for exogenous enzyme is the first 6 12 h of digestion, wh ich transpires prior to bacterial colonization of feed substrates or action of endogenous enzymes. This partly explains why Pr increased the 6-h IVDMD and not the 48-h IVDMD of BE. The 6 and 48-h IVNDFD of both hays were similar (Table 3.6), despite the lower NDF and lignin concentrations of BA. Treatment with NH3 increased (P < 0.01) the 6 and 48-h IVNDFD in both forages. Pr treatm ent tended (P < 0.15) to reduce the 6-h and 48-h IVNDFD of BE but did not affect the co rresponding values for BA. In contrast to the 6-h IVADFD, the 48-h IVADFD was higher (P < 0.01) in BE than in BA hay

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56 Table 3.6 Effect of Promote or ammonia a pplication on the IVNDFD of tropical grass hays NDF digestibility, % After 6-h After 48-h Additive Level BE BA BE BA Control 6.4 5.5 43.6 40.7 0.5x 5.1 4.9 44.5 42.9 1x 3.5 4.1 38.2 37.1 Promote 2x 6.3 5.8 40.2 38.9 Mean 5.0 4.9 41.0 39.6 Ammonia 8.1 7.9 58.4 59.4 s.e.m. 0.723 0.473 1.305 1.032 Polynomial effects P values Promote level Q* NS Q* Q* Contrasts Promote vs. Control 0.149 0.349 0.105 0.383 Control vs. Ammonia <0.01 <0.01 <0.01 <0.01 Promote vs. Ammonia <0.01 0.080 <0.01 <0.01 NDF: neutral detergent fiber, BE: bermudagr ass, BA: bahiagrass, Q: quadratic effect, NS: no significant effect, *: P < 0.05, **: P < 0.01, (Table 3.7). The 6-h and 48-h I VADFD were higher (P < 0.01) in NH3-treated hays than the other hays for both forage types. Pr tr eatment reduced (P < 0.15) most of the 6-h and 48-h IVADFD values of the hays. These results suggest that except for sli ghtly increasing the 6h IVDMD of BE, Pr treatment did not improve DM or cell wall diges tion in the forages. The fact that some of these measures were decreased by Pr tr eatment is surprising. In contrast, NH3 treatment increased 6-h and 48-h digestibility estimates, which reflect the rate and extent of digestion, respectively. Since intake is constrained by the rate at which the diet is digested (Romney and Gill, 2000), ammoniati on is more likely to increase intake and thereby increase animal performance than Pr treatment. Incremental addition of Pr did not have consistent beneficial effect s on the nutritive value of BA or BE.

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57 Table 3.7 Effect of Promote or ammonia a pplication on the IVADFD of tropical grass hays ADF digestibility, % After 6-h After 48-h Additive Level BE BA BE BA Control 3.7 5.4 48.0 41.9 0.5x 1.5 4.2 46.7 41.8 1x 3.1 3.1 45.0 37.2 Promote 2x 1.8 5.1 48.2 39.2 Mean 2.1 4.1 46.6 39.4 Ammonia 9.3 7.4 65.5 58.3 s.e.m. 0.855 0.511 1.345 0.880 Polynomial effects P values Promote level Q* NS Q* Q* Contrasts Promote vs. Control 0.148 0.052 0.413 0.032 Control vs. Ammonia <0.01 0.021 <0.01 <0.01 Promote vs. Ammonia <0.01 <0.01 <0.01 <0.01 ADF: acid detergent fiber, BE: bermudagrass, BA: bahiagrass, Q: quadratic effect, NS: no significant effect, *: P < 0.05, **: P < 0.01 Effect of Fibrolytic Enzyme and Ammoni a Application on Chemical Concentration of C4 Forages in Experiment 2 Unlike responses with BA, treating BE with NH3 (P < 0.01), X-20 (P < 0.05) or A20 (P < 0.01) decreased the NDF concentra tion and the CA enzyme gave the same tendency (P=0.073) (Table 3.8). Ammonia tr eatment was more effective than CA treatment. The ADF concentration of BE was decreased (P < 0.01) by NH3 treatment and increased (P < 0.05) by CA and A-20 treatment, but that of BA was only decreased by X20 treatment (P < 0.01). The hemicellulose concentration of BE was decreased (P < 0.01) by treatment with X-20 (cubic response), CA and A-20, but not NH3. Only X-20 treatment increased (P < 0.01) the hemicellulo se concentration of BA, other treatments had no effect.

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58 Table 3.8 Effect of fibrolytic enzyme or ammonia application on the NDF, ADF and hemicellulose concentrations (%) of tropical hays NDF ADF Hemicellulose Additive Level BE BA BE BA BE BA Control 87.1 78.7 46.5 47.9 40.6 30.9 NH3 84.6 80.3 43.7 47.8 40.9 32.5 0.5x 85.6 77.7 44.3 43.9 41.4 33.8 1x 85.4 78.8 47.2 44.7 38.2 34.1 X-20 2x 85.4 78.2 51.9 44.0 38.5 34.3 Mean 85.5 78.2 46.1 44.1 39.4 39.4 0.5x 86.1 79.7 47.7 48.8 38.4 30.9 1x 85.8 82.1 48.1 49.6 37.6 32.6 CA 2x 86.1 78.4 48.3 46.3 37.8 32 Mean 86.0 80.1 48.0 48.2 37.9 37.9 0.5x 86.1 79.8 48.7 48.7 37.4 31 1x 85.1 80.7 48.4 48.1 36.7 32.6 A-20 2x 84.9 78.7 49.0 49.4 35.9 29.3 Mean 85.3 79.5 48.7 48.7 36.7 36.7 s.e.m 0.49 0.63 0.63 0.48 0.61 0.91 Polynomial effects P values X-20 level NS NS C* NS C* NS CA level NS Q** NS C** NS NS A-20 level NS NS NS NS NS Q* Contrasts Control vs. X-20 0.011 0.530 0.674 <0.01 0.096 < 0.01 Control vs. CA 0.073 0.171 0039 0.507 < 0.01 0.358 Control vs. A-20 <0.01 0.176 <0.01 0.131 < 0.01 0.892 Control vs. NH3 <0.01 0.082 <0.01 0.997 0.791 0.219 X-20 vs. CA 0.216 <0.01 <0.01 <0.01 < 0.01 < 0.01 X-20 vs. A-20 0.662 <0.01 <0.01 <0.01 < 0.01 < 0.01 CA vs. A-20 0.099 0.512 0.242 0.219 0.016 0.269 NH3 vs. X-20 0.131 <0.01 <0.01 <0.01 0.050 0.149 NH3 vs. CA 0.022 0.719 <0.01 0.507 < 0.01 0.547 NH3 vs. A-20 0.223 0.412 <0.01 0.132 < 0.01 0.172 NDF: neutral detergent fiber, ADF: acid detergent fiber, BE: bermudagrass, BA: bahiagrass, X-20: Biocellulase X-20, CA: Cattle-Ase, A-20: Biocellul ase A-20, L: linear effect, Q: quadratic effect, C: cubic effect, NS: no significant effect, *: P < 0.05, **: P < 0.01 The WSC concentration of BE hays wa s reduced by CA (tendency, P=0.053) and A-20 (P < 0.05) treatment and unaffected by NH3 or X-20 treatment (Table 3.9). However, that of BA hays was increased (P < 0.05) by X-20 treatment.

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59 Table 3.9 Effect of fibrolytic enzyme or ammonia application on the WSC and CP concentrations (%) of tropical hays WSC CP Additive Level BE BA BE BA Control 2.81 1.21 6.5 7.0 NH3 2.82 0.98 16.3 13.0 0.5x 3.39 2.36 6.9 7.2 1x 2.01 1.34 6.7 7.1 X-20 2x 1.75 1.84 6.5 7.0 Mean 2.4 1.8 6.7 7.1 0.5x 1.75 1.26 6.7 7.2 1x 1.19 0.99 7.0 6.9 CA 2x 2.63 2.21 6.5 7.7 Mean 1.9 1.5 6.7 7.3 0.5x 1.64 0.79 6.8 7.1 1x 1.67 0.69 6.8 6.9 A-20 2x 1.74 0.94 6.9 7.3 Mean 1.7 0.8 6.8 7.1 s.e.m. 0.041 0.021 0.019 0.019 Polynomial effects P values X-20 level C* NS L* NS CA level C* L** NS NS A-20 level NS NS NS Q* Contrasts BE BA BE BA Control vs. X-20 0.370 0.019 0.509 0.731 Control vs. CA 0.053 0.281 0.338 0.287 Control vs. A-20 0.025 0.127 0.163 0.286 Control vs. NH3 0.998 0.481 <0.01 <0.01 X-20 vs. CA 0.125 0.056 0.666 0.304 X-20 vs. A-20 0.047 <0.01 0.286 0.303 CA vs. A-20 0.613 <0.01 0.518 0.998 NH3 vs. X-20 0.370 <0.01 < 0.01 < 0.01 NH3 vs. CA 0.053 <0.01 < 0.01 < 0.01 NH3 vs. A-20 0.025 0.060 < 0.01 < 0.01 WSC: water soluble carbohydrates, CP: crude protein, BE: bermudagrass, BA: bahiagrass, X-20: Biocellulase X-20, CA: Cattle-Ase, A-20: Biocellulase A-20, L: linear effect, Q: quadratic effect, C: cubic effect NS: no significant effect, *: P < 0.05, **: P < 0.01,

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60 Ammoniation decreased both ADF and NDF fr actions of BE and did not affect the hemicellulose fraction. All enzyme treatmen ts decreased the NDF concentration of BE by decreasing the hemicellulose concentrati on whereas the only eff ect on BA cell walls was that X-20 treatment increased hemi cellulose concentration by decreasing ADF concentration. This reveals a forage-specifi c response to the treatments which is similar to that found in Experiment 1. It is interesting to note that except for X20 effects on BA, none of the treatments that hydrolyzed forage cel l walls resulted in an increase in the WSC concentration. This may be due to the rela tively low WSC concentr ation of the forages and the conversion of hydrolyzed cell wall fragments into oligosaccharides and disaccharides that are not water soluble, and were therefore undetected in the WSC assay. The results for BE agree with those in Experiment 1, but conflict with those of Colombatto et al. (2003), w ho evaluated the effects of adding a commercial enzyme product on the hydrolysis and fermentation of cellulose, xylan, and a mixture of both substrates. They observed th at addition of enzyme in the absence of ruminal fluid increased (P < 0.01) the release of reduci ng sugars from xylan and the mixture. Similarly, Hristov et al. (1998) observed that enzyme treatment increased the concentration of soluble reduc ing sugars (P < 0.05) and de creased NDF concentration (P < 0.05) in a TMR, consisting of rolled barley grain, corn silage and soybean meal. The results of this study indicate a speci es-related difference in response to enzyme treatment which was consistent across experi ments. The effect of X-20 treatment and ammoniation on the respective ADF concentrat ions of BE and BA show that this treatment was more effective at disrupting li gnocellulosic linkages. However, the reason

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61 why enzymes were generally more effective at hydrolyzing BE despite its higher ADF and lignin concentration is unclear, and is proba bly related to differences in the type of phenolic cross linkages in the cell walls. In addition to differences in lignin concentration, Mandevbu et al. ( 1999) also showed that diffe rences in concentration of ether-linked and ester-linked ferulic acid e xplained digestibility differences between Tifton-85 and coastal bermudagrass. The CP concentration of the hays was unaffected by enzymatic treatments, but increased (P < 0.01) by NH3 treatment. This agrees with Weiss and Underwood (1995), Brown and Adjei (1995) and Barrios-Urdane ta and Ventura (2002) who observed that ammoniation increased the CP in forages, due to the supplemental N provided. Brown (1993) also observed that, compared to a Control treatment, ammoniation (4% DM) increased (P < 0.01) total N concentration (1.0 to 1.4% vs. 1.7 to 2.8%) of stargrass (Cynodon nlemfuensis) hay, and a similar effect (3.26 vs. 4.16% N, P < 0.01) was obtained by Lines et al (1996) for alfalfa hay. Although enzymes are proteins, the small amount of enzyme applied is not enough to effect forage CP concentration. Effect of Enzyme Treatment and Ammoniation on in vitro DM, NDF, and ADF Digestibility in Experiment 2 All of the additives were effective to increase 6-h IVDMD of BE (Table 3.10). However, only NH3 (P < 0.01) and X-20 (tendency: P = 0.088) increased the 6-h IVDMD of BA. Ammoniation was the most effective treatment for increasing the 6-h IVDMD (P < 0.01) in both grasses. The 6-h IVDMD was c onsistently greater (P < 0.01) in BA than in BE hays. This is probably due to the lower NDF concentration and presumably higher soluble fraction of BA hays and which would facilitate the initial degradation of the forage.

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62 Enzyme X-20 increased the 48-h IVDMD of BE (P < 0.05) and BA (P < 0.01) hays, while CA and A-20 tended (P < 0.08) to have similar effects on only BE. However, NH3 treatment was more effective (P < 0.01) th an any of the enzymes at increasing the 48-h IVDMD of both hays. These results suggest that all additive tr eatments can improve the 6-h and 48-h digestion of BE, but only NH3 and X-20 had similar effects on BA. This supports the conclusion that fibrolytic enzyme application can increase the rate of digestion of forages (Wang and McAllister, 2002), but indicates that enzyme effects on rate and extent of digestion depend on the enzyme and forage being tested. The 6-h IVNDFD was higher (P < 0.05) in BA than in BE hays; however, the 48-h IVNDFD was greater (P < 0.01) in BE than in BA hays. The 6-h and 48-h IVNDFD of both hays were unaffected by enzyme trea tment except for a linear increase with increasing A-20 application to BE (Table 3.11). However, NH3 treatment did increase 6 and 48-h IVNDFD (P < 0.01) of BE and 48h IVNDFD of BA (P < 0.01). The 6-h IVADFD of BE hay was improve d (P < 0.01) by X-20, A-20 and NH3 treatment. Only NH3 treatment increased the 6-h IVADFD of BA (P < 0.01); however digestibility of BA hay increased linearly (P < 0.05) with incr easing application of X-20 (Table 3.12). Therefore, the increases in 6-h IVDMD due to X-20 and A-20 treatment were partly due to increases in 6-h IVADFD. Ammoniation was the only treatment that increased (P < 0.01) the 48-h IVADFD in either of the ha ys; though responses al so occurred as the respective rates of CA (linea r) and X-20 (cubic) applicati on to BE and BA increased. None of the enzymes increased the extent of fiber digestion in the hays. Thus the results concur with those of Mandevbu et al. (1999) who observed that treatment of

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63 Table 3.10 Effect of fibrolytic enzyme or ammonia application on the IVDMD (%) of tropical hays After 6 h After 48 h Additive Level BE BA BE BA Control 7.7 12.6 43.6 44.5 NH3 13.7 18.1 56.8 59.9 0.5x 10.6 14.1 49.6 47.5 1x 11.4 13.0 51.1 45.6 X-20 2x 11.3 12.9 51.4 47.3 Mean 11.1 13.3 50.7 46.8 0.5x 9.0 12.1 46.5 42.3 1x 9.9 12.6 47.9 41.9 CA 2x 10.1 12.9 47.6 45.6 Mean 9.7 12.5 47.3 43.3 0.5x 10.2 12.2 47.2 43.9 1x 11.6 11.9 48.1 46.8 A-20 2x 9.4 12.2 46.9 42.4 Mean 10.4 12.1 47.4 44.4 s.e.m. 0.761 0.361 1.789 1.067 Polynomial effects P values X-20 level NS L* NS NS CA level NS NS NS NS A-20 level NS NS NS NS Contrasts Control vs. X-20 <0.01 0.088 <0.01 0.064 Control vs. CA 0.028 0.854 0.076 0.336 Control vs. A-20 <0.01 0.240 0.071 0.939 Control vs. NH3 <0.01 <0.01 <0.01 <0.01 X-20 vs. CA 0.035 <0.01 0.027 <0.01 X-20 vs. A-20 0.300 <0.01 0.026 <0.01 CA vs. A-20 0.261 0.169 0.985 0.215 NH3 vs. X-20 <0.01 <0.01 <0.01 <0.01 NH3 vs. CA <0.01 <0.01 <0.01 <0.01 NH3 vs. A-20 <0.01 <0.01 <0.01 <0.01 DM: dry matter, BE: bermudagrass, BA: bahiagrass, X-20: Biocellulase X-20, CA: Cattle-Ase, A-20: Biocellulase A-20, L: linear effect, NS: no significant effect, *: P < 0.05, **: P < 0.01. bermudagrass forages with fibrolytic enzymes had no effect on in vitro or in situ DM or NDF disappearance of silages. Howeve r, Rode et al. (1999) observed that in vivo

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64 Table 3.11 Effect of fibrolytic enzyme or ammonia application on the IVNDFD (%) of tropical hays After 6-h After 48-h Additive Level BE BA BE BA Control 5.0 6.2 43.1 37.3 NH3 7.4 7.0 52.6 54.4 0.5x 5.4 6.0 46.7 35.8 1x 5.1 5.5 47.5 35.2 X-20 2x 5.1 5.3 43.1 37.9 Mean 5.2 5.6 46.2 36.3 0.5x 4.0 5.8 48.0 33.1 1x 5.4 7.0 46.9 35.7 CA 2x 5.6 4.5 46.4 34.0 Mean 5.0 5.8 47.1 34.2 0.5x 4.9 6.1 47.9 36.2 1x 4.7 6.3 45.0 38.9 A-20 2x 6.1 7.2 47.4 35.4 Mean 5.2 6.5 47.5 36.8 s.e.m. 0.47 0.88 1.74 1.59 Polynomial effects P values X-20 level NS NS NS C* CA level NS NS NS NS A-20 level L* NS NS NS Contrasts Control vs. X-20 0.760 0.542 0.635 0.586 Control vs. CA 0.951 0.683 0.146 0.244 Control vs. A-20 0.714 0.780 0.827 0.312 Control vs. NH3 <0.01 0.546 <0.01 <0.01 X-20 vs. CA 0.605 0.773 0.571 0.162 X-20 vs. A-20 0.931 0.215 0.399 0.716 CA vs. A-20 0.546 0.336 0.779 0.083 NH3 vs. X-20 <0.01 0.185 <0.01 <0.01 NH3 vs. CA <0.01 0.257 0.169 <0.01 NH3 vs. A-20 <0.01 0.645 <0.01 <0.01 NDF: neutral detergent fiber, BE: bermudagrass, BA: bahiagrass, X-20: Biocellulase X-20, CA: Cattle-Ase, A-20: Biocellulase A-20, L: linear effect, Q: quadratic effect, C: cubic effect, NS: no significant effect, *: P < 0.05, **: P < 0.01 digestibility determined using Cr2O3 was increased by a commercial enzyme (Promote) added to the concentrate (DM: 61.7 vs. 69.1%; NDF: 42.5 vs. 51.0%; ADF: 31.7 vs. 41.9%; and CP: 61.7 vs. 69.8%) to a dairy co w diet. This conflicting responseis

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65 Table 3.12 Effect of fibrolyt ic enzyme or ammonia application on the IVADFD (% of DM) of tropical hays After 6 h After 48 h Additive Level BE BA BE BA Control 3.7 5.1 36.8 35.3 NH3 9.3 9.0 53.4 44.2 0.5x 4.1 4.9 36.8 39.9 1x 7.3 5.3 36.0 36.2 X-20 2x 7.2 4.0 39.3 32.7 Mean 6.2 4.7 37.4 36.3 0.5x 2.2 4.0 34.3 37.1 1x 2.8 5.3 36.5 39.2 CA 2x 5.6 3.5 37.7 36.7 Mean 3.5 4.3 36.2 37.7 0.5x 6.0 4.8 37.0 35.9 1x 6.6 4.7 39.2 33.8 A-20 2x 6.6 6.1 36.7 38.3 Mean 6.4 5.2 37.6 36.0 s.e.m. 0.77 0.70 1.22 1.24 Polynomial effects P values X-20 level L** NS NS C** CA level L* NS L* NS A-20 level NS L* NS NS Contrasts Control vs. X-20 <0.01 0.625 0.686 0.508 Control vs. CA 0.902 0.314 0.663 0.120 Control vs. A-20 <0.01 0.902 0.560 0.631 Control vs. NH3 <0.01 <0.01 <0.01 <0.01 X-20 vs. CA <0.01 0.456 0.241 0.196 X-20 vs. A-20 0.741 0.391 0.799 0.795 CA vs. A-20 <0.01 0.117 0.158 0.125 NH3 vs. X-20 <0.01 <0.01 <0.01 <0.01 NH3 vs. CA <0.01 <0.01 <0.01 <0.01 NH3 vs. A-20 <0.01 <0.01 <0.01 <0.01 ADF: acid detergent fiber, BE: bermudagrass, BA: bahiagrass, X-20: Biocellulase X-20, CA: Cattle-Ase, A-20: Biocellulase A-20, L: linear effect, Q: quadratic effect, C: cubic effect, NS: no significant effect, *: P < 0.05, **: P < 0.01 probably attributable to the highe r nutritive value of the dairy cow diet relative to that of C4 grasses, suggesting that hi gher quality diets respond more to enzyme supplementation, presumably due to lower ADF concentra tion.No other studies that simultaneously

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66 compared the effectiveness of chemical and biological treatmen ts at improving the quality of C4 grasses were found in the literature. However, several reports have shown that ammonia treatment is very effective fo r improving the DM and fiber digestibility of low quality forages (Brown, 1993; Vagnoni et al., 1995; Weiss and Underwood, 1995; Barrios-Urdaneta and Ventura, 2002). The bene ficial effect of enzyme treatment on 6-h and 48-h IVDMD were not due to increases in the extent of fiber di gestion. Rather they may have been attributable to increased microbial attachment (Dawson and Tricarico, 1999) and an increased rate of ADF digestion. According to Barrios-Urdaneta and Ventur a (2002), ammoniation improves forage digestibility due to the hydrolytic acti on on linkages between lignin and structural polysaccharides, thus increasing the OM potenti ally available for utilization by ruminal microorganisms. These authors observed th at ammoniation increased (P < 0.01) the in vitro NDF digestibility (from 46.2 to 57.1%) of koroniviagrass. Ammonia treatment also changes the physical characteristics of forage s making them more pliable and increasing their hydration rate. Hydration rate has an important role in digestion rate; the faster a forage particle is hydrated, the faster it is digested (Weiss and Underwood, 1995). Effect of Enzyme Treatments and Ammoniation on in situ DM Degradation The effect of X-20 and NH3 on the kinetics of in situ DM disappearance of BE and BA is presented in Table 3.13. Treatment with X-20 (linear, P < 0.05) and NH3 (P < 0.01) increased the wash loss (a ) fraction of BE, but only NH3 treatment increased that of BA. This result supports the findings obtained in vitro where both of these treatments increased the initial phase of digestion of BE.

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67 Table 3.13 Effect of X-20 or ammonia applic ation on the in situ kinetics of DM disappearance of bermudagrass and bahiagrass Parameter Forage Treatment a, % b, % a + b, % P, % c L1, h Bermudagrass Control 2.65 60.2 62.8 59.1 0.041 9.064 NH3 6.50 70.9 77.4 71.7 0.054 8.341 X-20, 0.5x 2.95 57.5 60.5 57.5 0.041 7.439 X-20, 1x 3.60 57.7 61.4 57.6 0.029 6.226 X-20, 2x 3.85 56.8 60.6 56.0 0.053 9.326 s.e.m. 0.21 1.95 1.99 2.34 0.01 1.00 Contrasts P values Polynomial L* NS NS NS NS NS Control vs. X-20 0.019 0.267 0.425 0.514 0.997 0.287 Control vs. NH3 < 0.01 0.012 < 0.01 0.017 0.346 0.632 X-20 vs. NH3 < 0.01 < 0.01 < 0.01 < 0.01 0.260 0.595 Bahiagrass Control 6.35 58.0 64.4 63.4 0.019 2.232 NH3 7.35 75.4 82.7 81.1 0.026 3.978 X-20, 0.5x 7.50 59.3 66.8 65.2 0.024 2.432 X-20, 1x 5.70 59.6 65.3 63.6 0.025 4.300 X-20, 2x 6.20 60.6 66.8 65.1 0.025 4.158 s.e.m. 0.27 1.04 1.15 1.26 0.003 0.89 Contrasts P values Polynomial Q* NS NS NS NS NS Control vs. X-20 0.727 0.191 0.207 0.419 0.153 0.714 Control vs. NH3 0.049 < 0.01 < 0.01 < 0.01 0.144 0.578 X-20 vs. NH3 0.038 < 0.01 < 0.01 < 0.01 0.679 0.747 DM: dry matter, X-20: Biocellulase X-20, a: soluble fraction, b: insoluble but potentially degradable fraction, a+b= total degradability, P= DM degraded at time t, c: rate of constant degradation, L1: lag phase (period when no net disappearan ce of substrate occurs), L: linear effect, Q: quadratic effect Ammonia treatment was more effective than X-20 treatment at increasing (P < 0.01) the insoluble but potentially degradable (b) fraction, the total de gradable fraction (a + b) and the degradability (P) of both forages. These results partially concur with those

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68 Table 3.14 Effect of A-20 or ammonia applic ation on the in situ kinetics of DM disappearance of bermudagrass and bahiagrass Parameter Forage Treatment a, % b, % a + b, % P, % c L, h Bermudagrass Control 6.95 61.4 68.4 68.2 0.009 1.535 NH3 7.95 62.2 70.2 69.0 0.024 6.849 A-20, 0.5x 7.75 60.0 67.7 67.1 0.015 4.316 A-20, 1x 7.50 59.7 67.2 66.7 0.014 2.778 A-20, 2x 7.25 59.9 67.1 66.2 0.016 3.971 s.e.m. 0.456 0.435 0.351 0.407 0.003 1.572 Contrasts P values Polynomial NS NS NS NS NS NS Control vs. A-20 0.344 0.025 0.052 0.023 0.119 0.289 Control vs. NH3 0.182 0.251 0.015 0.199 0.011 0.062 A-20 vs. NH3 0.432 < 0.01 < 0.01 < 0.01 0.032 0.142 Bahiagrass Control 6.50 56.1 62.6 62.2 0.015 7.583 NH3 5.95 62.1 68.0 67.5 0.016 7.731 A-20, 0.5x 7.00 59.2 66.2 65.8 0.010 7.120 A-20, 1x 6.45 58.9 65.4 64.9 0.014 6.011 A-20, 2x 6.45 58.5 64.9 64.5 0.014 7.366 s.e.m. 0.541 1.644 1.458 1.424 0.003 3.373 Contrasts P values Polynomial NS NS NS NS NS NS Control vs. A-20 0.839 0.209 0.149 0142 0.576 0.855 Control vs. NH3 0.505 0.051 0.047 0.048 0.845 0.976 A-20 vs. NH3 0.324 0.151 0.193 0.207 0.435 0.827 DM: dry matter, A-20: Biocellulase A-20, a: soluble fraction, b: insoluble but potentially degradable fraction, a+b= total degradability, P= DM degraded at time t, c: rate of constant degradation of b, L: lag phase (period when no net disappearance of substrate occurs) obtained in vitro where NH3 was the most effective treatment at increasing the extent of digestion. Only the a fraction of BE wa s affected by X-20, and neither treatment affected the degradation rate or lag phase of the forages. The results presented in Table 3.14 show th at A-20-treated BE hays had lower b, a + b and P values than Control (P < 0.05) and NH3-treated hays (P < 0.01). The A-20

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69 treatment also tended to increase the c value for BE and the b and a + b fraction of BA. In BE hays ammoniation increased the lag phase and the c value and a + b fraction, while in BA it increased b, a+b and P. The NH3 effects concur with results of Vagnoni, et al. (1995), which showed that ammoniation of ma ture bermudagrass increased both the rate (P < 0.05) and the potential extent (P < 0.01) of ruminal forage in situ DM disappearance in lactating cows. The response to A-20 and X-20 treatments partly agre e with Feng et al. (1996) who found that applying cellulase, xy lanase and a mixture of both enzymes at different levels did not affect the in situ DM disappearance of cool-season grasses. Lewis et al. (1996) evaluated a 70% grass hay diet treated with fi brolytic enzymes that were applied at feeding or 24 h befo re feeding and observed that in situ DM disappearance was unaffected by enzyme treatment of samples incubated for 8, 16, and 24 h, but increased after 96-h ( P < 0 .05). The authors proposed that impr oved DM disappearance at 96-h of incubation in enzyme-treated grass may have resulted from enhanced colonization and digestion of the slowly degradable fibe r fraction by ruminal microorganisms. Conclusions This work demonstrates that fibrolytic enzymes had negligible effects on in situ DM degradation of C4 grass hays, though certain enzyme s (X-20 and A-20) did increase the initial and final phases of DM digestion. Such effects were more pronounced in BE than BA. Increasing the enzyme applicat ion rate produced inconsistent effects on nutritive value. However, several key measur es were increased ith increasing X-20 or A20 application rate, suggesting that high (1x an d 2x) application rates were most effective than the low (0.5x) rate. Most of the enzyme-induced enhancem ents in digestibility were not attributable to increased fiber diges tion; therefore other mechanisms such as increased substrate colonization by rumina l microbes may have been involved.

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70 Ammoniation was more effective than any of the enzyme treatments at improving the initial and final phases of digestion, due to in creased fiber hydrolysis Ammoniation also increased the CP concentration and in situ ruminal degradation of the C4 grass hays.

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71 CHAPTER 4 EFFECT OF FIBROLYTIC ENZYMES ON THE FERMENTATION CHARACTERISTICS, AEROBIC STABILITY, AND DIGESTIBILITY OF BERMUDAGRASS SILAGE Introduction Interest in applying fibrolytic enzymes to ruminant diets has increased recently due to enzyme-mediated increases in feed digestion in vitro (Lewis et al., 1996; Kung et al., 2002; Hristov et al., 2000; Bowman et al., 2002) and diet utilization in vivo (Yang et al., 1999; Lewis et al., 1999; Schingoeth e et al., 1999). However, in certain studies (Sheperd and Kung, 1996b; Bowman et al., 2002; Vi cini et al., 2003) exogenous enzyme supplementation did not consistently improve animal performance. Where improved performance was observed, the mechanism was not always confirmed by improved digestion (Mandebvu et al., 1999). These inco nsistencies were due to various factoros such as enzyme type, concentration and act ivity, application method, substrate to which enzyme is added and animal differences (Bowman et al., 2002). Additional factors that may be implicated include prevailing temperature and pH, presence of co-factors and inhibitors, and enzyme and substrate concen tration. Nevertheless, feed enzymes have been used to improve the utilization of a wide range of diets containing legumes, grasses, haylage, straw and other feedstuffs (Beauchem in et al., 2003). The mode of action of these enzymes in ruminants is not fully unders tood. They can enhance feed colonization by increasing the numbers of ruminal fibrol ytic microbes (Morgavi et al., 2000; Nsereko et al., 2000a) and thereby increase the rate of degradation in the rumen (Yang et al., 1999). Enzymes can also partially solubili ze NDF and ADF and release reducing sugars

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72 in the process. Colombatto et al. (2003) observed that fibrolytic enzymes enhanced the fermentation of cellulose and xylan by a combin ation of preand post-incubation effects. These were evident from an increase in the release of reducing sugars during a 20 h preincubation phase and an increase in the hydrolytic activity of the liquid and solid fractions of the ruminal fluid 6-h after in cubation, which led to a higher rate of fermentation. Most of the studies on fibrolyt ic enzyme treatment of ruminant feeds have been done using temperate feedstuffs. Litt le is known about their effectiveness on tropical or subtropical forages which tend to be poorly digested. The objective of this experiment was to evaluate the effect of four proprietary fibrolytic enzyme preparations applied at different rates, at ensiling, on the nutritive va lue of Tifton 85 bermudagrass ( Cynodon spp,) silage. Most of the recent stud ies in this area have involved enzyme application to individual com ponents of the ration or to th e total ration just prior to feeding. There were two reasons for applyi ng the enzymes directly to bermudagrass at the point of ensiling in this study. Firstly, we sought to determine if the sugars produced by enzymatic cell wall hydrolysis would improve the fermentation of bermudagrass which is typically poor and decrease DM losses, which are typically hi gh for this forage. Secondly, we wanted to determine the eff ectiveness of the enzy mes at improving the digestibility of the forage, because although bermudagrass is poorly digested, it is an important digestible fiber source in the rati ons of dairy cows in the Southeast. Materials and Methods Enzyme Application A five-week regrowth of Tifton 85 berm udagrass silage was conserved without treatment (Control) or after treatment with four proprietary fibrolytic enzymes. The enzymes were applied at half (0.5), exactly (1) or twice (2) the rates recommended

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73 by the respective manufacturers for addition at the time of feeding. Because the enzymes were applied at ensiling rather than at feeding as recommended by the manufacturers, this study was not designed to test the effectiven ess of the enzymes as used commercially, and the results should not be misconstrued as doing so. The rationale for the mode of enzyme application employed was to determin e if the fermentation and digestibility of bermudagrass could be improved by enzyme addition. The enzymes used were: (a) Promote (Cargill Corp. St. Louis, MO) applied at: 0.65, 1.3 and 2.6 g/kg DM, (b) Biocellulase X-20 (LodeStar, IL, USA) applied at: 7.3, 14.5 and 29 mg/kg DM, (c) Biocellulase A-20 (LodeStar, IL, USA) a pplied: at 7.3, 14.4 and 29 mg/kg DM,, and (d) Cattle-Ase (CA) applied at 89, 178 and 356 mg/kg DM. Cellulase activity was determined at 39oC and a pH of 5.5 using the filter paper method (Wood and Bhat, 1988) and the values obtained for Pr, X-20, CA and A-20 were 33.7, 22, 0 and 51.3 filter paper units/g, respectively. Xylanase activity was determined at 39oC and a pH of 5.5 using the di-nitro salicylic acid procedure (Bailey et al., 1992) and the values obtained for Pr, X-20, CA and A-20 were 5190, 7025, 0 and 3530 mol/min/ml, respectively. The units of xylanase activity are expressed as mol xylose equivalents released ml-1 min-1 from 1% birchwood xylan (X-0502, Sigma Chemical Company, St. Louis, MO, USA). Each enzyme was dissolved in 400 ml of wa ter and sprayed in a fine mist using a four-liter garden sprayer, over 10 kg of choppe d (5 cm) forage per treatment. The same amount of water was sprayed on the Contro l forages. After thorough mixing, a one-kg representative sample of the treated forage was ensiled within a polythene bag in six, replicate 2.8-L PVC cylindrical mini silos. A hydraulic press was used to compress the forage in the silo to achieve a density of 280 kg/m3. Weights of the empty and full silos

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74 were recorded, and silos were then stored for 145 days at ambient temperature (23oC) in a covered barn. Representative samples (4 kg) of the freshly-treated, unensiled forages were frozen (-20oC) for subsequent laboratory analysis. Laboratory Analysis At silo opening, final silo weights were r ecorded and silages from each of three silos per treatment were sub-sampled for DM determination (200 g) and silage juice extraction (200 g) or freeze-drie d for chemical analysis (200 g) Each of the other three silos was sub-sampled for microbial enumera tion (200 g) and aerobic stability (800 g). Samples destined for microbial analysis were heat-sealed within gas-impermeable bags (Kapak / Scotch Pak, Kapak Corp., Minneapolis, MN), placed in an icebox and transported on the same day to the Amer ican Bacteriological & Chemical (ABC) Research Corporation, Gainesville, Flor ida. Serial dilutions up to 1 1010 were made using 25 g of silage and Butterfields phosphate buffer. Yeast and molds were enumerated by pour plating in Standard Me thods (M124) agar, to which 40 ppm of chloramphenicol and chlortetracycline were added (Tournas et al., 1999). Plates were incubated aerobically at 25oC for 5 days. Aerobic stability was measured by placing thermocouple wires at the center of a bag c ontaining 800 g of silage, within an open-top polystyrene box. The silages were covered w ith two layers of cheesecloth to prevent drying. The thermocouple wires were connect ed to data loggers (Campbell Scientific Inc. North Logan, UT) that recorded the temperature every 30 min for 30 d. Aerobic instability was denoted by the time (h) taken for a 2C rise in sila ge temperature above ambient temperature (23oC). Dry mater losses were estimated using DM concentrations and silo weights measured before and after ensiling. Oven DM concentration was determined in a forced draft oven set at 60oC for 48-h. Ash

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75 concentration was determined in a muffle furnace at 500oC for 5 h. Silage juice was obtained by blending 20 g of sila ge in 200 ml of distilled wa ter for 30 s at high speed and the slurry was filtered through two layers of cheesecloth. The pH was measured using a pH meter (Corning Model 12, Corning Scientif ic Instruments, Medfield, MA). The filtrate was centrifuged at 4oC and 21,500 g for 20 min and the supernatant was frozen (-20oC) in 20 ml vials for subsequent analys is of lactic acid, VFA, WSC, ammonia nitrogen (NH3-N) and water soluble N (WSN). Organic acids were measured using the method of Muck and Dickerson (1988) and a High Performance Liquid Chromatograph (Hitachi, FL 7485, Tokyo, Japan) coupled to a UV Detector (Spectroflow 757, ABI Analyt ical Kratos Division, Ramsey, NJ) set at 210 nm. The column used was a Bio-Rad Aminex HPX-87H (Bio-Rad Laboratories, Hercules, CA 9454) with 0.015M sulfuric acid mobile phase and a flow rate of 0.7 ml/min at 45C. Ethanol was measured by gas chromatography using the procedure of Yomano et al. (1998) with a Varian Star 3400 CX gas chromatograph (Varian, Santa Clarita, CA). The anthrone reaction assay (Ministry of Agriculture, 1986) was used to quantify WSC. Ammonia N was determined us ing an adaptation for the Technicon Auto Analyzer (Technicon, Tarrytown, NY, USA) of the Noel and Hambleton (1976) procedure. Water-soluble N (WSN) was dete rmined by digesting 10 ml of supernatant using micro Kjeldahl apparatus (Labconco Corporation, Kansas City, MO) and the N concentration was determined using a Tec hnicon auto analyzer (Technicon, Tarrytown, NY, USA). Freeze-dried ground (1mm) samples were analyzed for CP, in vitro digestibility, ADF and NDF. In vitro digestibility of DM, NDF and ADF was determined after

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76 incubating forage samples in buffered, ru men fluid for 6 or 48-h using two ANKOMII Daisy Incubators (ANKOM Technology, Fair port, NY). The buffer was prepared according to the ANKOM Technology procedure. The rumen fluid was obtained before feeding from two, non-lactating, fistulated cows, fed 9 kg of Coastal bermudagrass ( Cynodon dactylon ) and 400 g soybean meal daily. The NDF and ADF concentrations (Van Soest et al., 1991) of the samples and di gested residues were determined without amylase pretreatment using an ANKOM200 Fiber Analyzer (ANKOM Technology, Macedon, NY). Hemicellulose was calculated by difference from NDF and ADF concentrations. Statistical Analysis A completely randomized design and a 4 4 factorial arrangement of enzyme types and application rates with 3 replicates per tr eatment was used. The data were analyzed using the GLM procedure of SAS (SAS Inst., In c., Cary, NC). Polynomial contrasts were used to test the effect of increasing enzy me application rate and orthogonal contrasts were used to compare the Control and enzyme treatments. The model used to analyze indi vidual treatment effects was: Yij: + Ti + Eij where: = general mean Ti = effect of treatment (enzyme type enzyme rate) Eij= experimental error. Significance was declared at P < 0.05 and tendencies at P < 0.10

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77 Results and Discussion Chemical Composition of Freshly -treated Bermudagrass before Ensiling Table 4.1 shows the chemical composition of the bermudagrass forages prior to ensiling. The concentrations of the measured chemical components were similar for all treatments. This is probably attributable to the short duration of enzyme action due to placement of the freshly-treated samples on ice after enzyme application. The DM concentration at harvest was typi cal of that at the stage at which bermudagrass is ensiled in Florida, and similar to that (324 g/kg) reported for bermudagrass harvested at a similar maturity stage by Umana et al., ( 1991) and Adesogan et al. (2004). The low WSC and CP concentrations and high NDF a nd ADF concentrations are also typical of bermudagrass (Umana et al., 1991; Adesogan et al., 2004). These re sults indicate that the bermudagrass was representative of those used for dairy production in the southeastern US. Chemical Composition, Microbial Counts and Aerobic Stability of Bermudagrass Silages Neither enzyme type nor a pplication rate affected ( P > 0.05) the DM concentration of the silages. Dry matter values ranged between 296 and 308 g/kg (Table 4.2). The pH of Pr-treated silages was lower (P < 0.01) than that of all other silages, while the other enzymetreated silages had sim ilar pH values to Control s ilages. This suggests that compared to the other forages, Pr was more effective at increasing the availability of WSC for microbial fermentation, through cell wa ll hydrolysis. Though this is not evident from the WSC concentration of the freshlytreated forages due to the short duration allowed for enzyme action, Pr-treated silage s did have greater (P < 0.05) residual WSC concentration

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78 Table 4.1 Chemical composition of bermudagrass forages before ensiling (g/kg DM). Enzyme treatment1 DM (g/kg) pH Ash WSC2 NH3N3 CP NDF ADF Hem4 Control 305 5.97 57 6.93 54 105 786 436 350 Pr 302 6.42 59 6.14 43 99 776 428 348 X-20 305 6.14 55 6.79 56 97 791 440 351 CA 303 5.67 62 5.83 37 97 786 440 346 A-20 306 6.04 57 6.67 44 97 791 438 353 P 0.836 0.795 0.762 0.717 0.758 0.504 0.264 0.236 0.547 S.E. 4.26 0.73 6.67 1.01 18.52 4.01 8.65 6.61 5.50 Contrasts P values Control vs. Pr 0.559 0.607 0.834 0.516 0.631 0.209 0.331 0.325 0.609 Control vs. X-20 0.999 0.846 0.834 0.911 0.939 0.136 0.608 0.614 0.919 Control vs. CA 0.744 0.729 0.508 0.371 0.441 0.136 0.999 0.586 0.514 Control vs. A-20 0.896 0.933 0.999 0.828 0.642 0.110 0.630 0.833 0.614 1 Cellulase-hemicellulase preparations: Pr, Prom ote, X-20, Biocellulase X-20, CA: Cattle-Ase, A-20: Biocellulase A-20, 2 WSC: water-soluble carbohydrates, 3Ammonia-N expressed as g/total N, 4 Hemicellulose than the other silages (Table 4.3). Silage pH also decreased (P < 0.01) linearly as the rate of Pr application increased. The Pr-treated silages had pH values that were similar to or lower than that which is required for achieving stability during the fermentation (Bates et al., 1989a). Similar reductions in pH were obtained when fibrolyt ic enzymes were applied to wheat silage (Adogla-Bessa et al., 1999), corn silage (S heperd and Kung, 1996a; Colombatto et al., 2004) or orchardgrass and alfalfa si lages (Nadeau et al., 2000). Ammonia-N levels were lower in the Pr-treat ed silages (P < 0.01) than in the other silages. This reveals that less proteolysis occurred during ensiling in Pr-treated silages than in other silages, and this was probably due to a faster pH decline in Pr-treated silages. The lower ammonia-N concentrati on of Pr-treated sila ges contrasts with

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79 Table 4.2 Effect of fibrolytic enzymes on pH, concentrations of DM (g/kg) and ammoniaN (g/kg total N), DM losses (%), micr obial counts (log cfu /g) and aerobic stability (h) of bermudagrass silage. L: linear effect, Q: quadratic effect, C: cubic effect, NS: Not significant, P < 0.05, ** P < 0.01. 1 Cellulase-hemicellulase enzyme preparations: Pr: Promote, X-20: Biocellulase X-20, CA: Cattle-Ase, A-20: Biocellulase A-20. Enzyme Treatment1 Application rate pH DM DM loss AmmoniaN Yeasts Molds Aerobic stability Control 4.40 305 8.6 32 1.65 3.24 96 Pr 0.5 4.28 299 4.0 26 2.83 3.70 103 Pr 1 3.93 308 5.9 25 1.50 3.01 102 Pr 2 3.87 299 2.6 24 2.31 2.18 229 Pr Mean 4.03 302 4.2 25 2.21 2.96 138 Pr Rate effect L ** NS NS NS NS NS L** X-20 0.5 4.40 305 7.3 31 1.42 2.37 196 X-20 1 4.49 303 5.8 38 1.59 4.83 210 X-20 2 4.32 307 7.7 38 3.57 4.49 96 X-20 Mean 4.40 305 7.0 35 2.19 3.90 162 X-20 Rate effect NS NS NS L* NS NS L** CA 0.5 4.00 306 8.8 33 2.05 1.93 96 CA 1 4.46 308 7.4 24 1.00 3.37 203 CA 2 4.41 296 5.7 32 2.17 3.12 205 CA Mean 4.40 304 7.3 30 1.74 2.81 168 CA Rate effect NS NS NS Q** NS NS L** A-20 0.5 4.54 306 9.5 39 1.54 3.00 96 A-20 1 4.32 305 6.4 36 1.42 4.63 232 A-20 2 4.12 306 3.6 25 1.00 3.50 261 A-20 Mean 4.33 306 6.5 33 1.32 3.71 196 A-20 Rate effect NS NS L ** C** NS NS L** S.E. 0.09 7.74 1.12 0.03 0.89 0.77 38.00 Contrasts P values Control vs. Pr 0.002 0.703 < 0.01 < 0.01 0.573 0.671 0.011 Control vs. X-20 0.975 0.961 0.218 0.126 0.649 0.472 < 0.01 Control vs. CA 0.992 0.844 0.316 0.248 0.925 0.633 < 0.01 Control vs. A-20 0.516 0.908 0.120 0.630 0.752 0.609 < 0.01 Pr vs. X-20 <0.01 0.639 0.005 < 0.01 0.878 0.121 0.096 Pr vs. CA <0.01 0.794 0.002 < 0.01 0.508 0.939 0.110 Pr vs. A-20 0.005 0.555 0.017 < 0.01 0.225 0.198 < 0.01 X-20 vs. CA 0.977 0.835 0.737 < 0.01 0.609 0.106 0.974 X-20 vs. A-20 0.338 0.903 0.631 0.135 0.285 0.766 0.043 CA vs. A-20 0.353 0.741 0.416 0.026 0.564 0.175 0.059

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80 Table 4.3 Effect of fibrolytic enzymes on the chemical composition of bermudagrass silage (g/kg DM). Enzyme Treatment1 Application rate CP Ash NDF ADF Hem.2 WSC3 WSN4 Control 97 53 753 431 323 4.5 0.84 Pr 0.5 96 49 723 408 315 8.2 0.73 Pr 1 92 50 725 408 317 12.6 0.88 Pr 2 93 54 728 408 319 15.9 0.73 Pr Mean 94 51 725 408 317 12.2 0.78 Pr Rate effect NS NS NS NS NS L ** NS X-20 0.5 98 61 744 420 324 6.1 0.71 X-20 1 96 53 738 426 313 6.1 0.70 X-20 2 97 51 747 426 321 5.9 0.89 X-20 Mean 97 55 743 424 319 6.0 0.77 X-20 Rate effect NS NS NS NS NS NS NS CA 0.5 99 53 750 422 327 5.3 0.86 CA 1 105 53 743 444 299 7.1 0.62 CA 2 89 54 736 442 295 8.5 0.70 CA Mean 98 53 743 436 307 6.9 0.73 CA Rate effect Q ** NS L ** L ** L L NS A-20 0.5 96 54 761 448 313 5.3 0.76 A-20 1 95 54 757 438 319 5.3 0.67 A-20 2 97 57 741 428 312 5.2 0.57 A-20 Mean 96 55 753 438 315 5.3 0.67 A-20 Rate effect NS NS L ** C ** NS NS NS S.E. 1.64 2.29 3.36 2.70 3.84 0.81 0.07 Contrasts P values Control vs. Pr 0.125 0.386 < 0.01 < 0.01 0.204 < 0.01 0.466 Control vs. X-20 0.954 0.590 0.016 < 0.01 0.444 0.109 0.350 Control vs. CA 0.602 0.934 0.015 0.105 0.002 0.014 0.170 Control vs. A-20 0.816 0.508 0.977 0.022 0.078 0.407 0.039 Pr vs. X-20 0.028 0.054 < 0.01 < 0.01 0.463 < 0.01 0.766 Pr vs. CA 0.006 0.269 < 0.01 < 0.01 0.004 < 0.01 0.351 Pr vs. A-20 0.067 0.071 < 0.01 < 0.01 0.463 < 0.01 0.053 X-20 vs. CA 0.513 0.381 0.968 < 0.01 0.001 0.179 0.522 X-20 vs. A-20 0.682 0.860 < 0.01 < 0.01 0.149 0.260 0.097 CA vs. A-20 0.291 0.295 < 0.01 0.299 0.022 0.018 0.293 L: linear effect, Q: quadratic effect, C: cubic effect, NS: Not significant, P < 0.05, ** P < 0.01. 1Cellulase-hemicellulase enzyme preparations: Pr: Promote, X-20: Biocellulase X-20, CA: Cattle-Ase, A-20, Biocellulase A-20. 2 Hem: Hemicellulose. 3 WSC: water-soluble carbohydrates. 4 WSN: water-soluble nitrogen.

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81 previous studies in which ammonia-N c oncentration of silages was unaffected by fibrolytic enzyme application (Sheperd and Kung, 1996a; Adogla-Bessa et al., 1999). Yeast and mold counts were unaffected by enzyme type or rate and the numbers found were less than those (5.0 cfu/g) that pr edispose to rapid deterioration in silage (Kung, 2004). These low yeast and mold counts reflect the antimycotic properties of the VFA produced during the ensiling process (Tab le 4.4). Yeasts usually initiate aerobic deterioration of silages, while molds conti nue the deterioration process because yeast grow faster but tolerate less heat th an do molds (Higginbotham et al., 1998). Aerobic stability was increased (P < 0.05) by enzyme treatment and such increases were linear (P < 0.05) as the ra te of enzyme application in creased except in X-20-treated silages. Silages treated with A-20 enzyme te nded (P < 0.06) to be more stable than other additive-treated silages. Neve rtheless, all the forages were st able for at least four days, such that all of them would be adequately preserved in the feedbunk for several days. This observation is typical of bermudagrass silages which usually undergo heterolactic fermentation, resulting in the production of antimycotic acids li ke acetic acid that ensure the stability of the sila ge (Bates et al., 1989a ; Adesogan et al., 2004). Dry matter losses were lower in the Pr-treated silages than in the other silages (P < 0.05). Though there was no effect of increasi ng Pr application on DM lost, this work demonstrates that Pr can be used to redu ce the losses of DM that usually occur when bermudagrass is conserved as silage. Alt hough DM losses decreased linearly (P < 0.05) as the rate of A-20 application increased, th e mean DM loss for A-20-treated silages was similar to that of Control silages.

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82 Neither enzyme type nor a pplication rate affected ( P > 0.05) the ash concentration of the silages (Table 4.3). Compared to C ontrol silages, NDF con centration was reduced by Pr (P < 0.01), X-20 and CA (P < 0.05). However the lowest NDF values (P < 0.01) were observed in the Pr-treated silages (P < 0.01), indicating that th is treatment was the most effective at reducing the total fiber fractio n. Silages treated with CA had lower (P < 0.05) hemicellulose concentrations than other silages. As the rate of CA application increased, hemicellulose concentration decreased linearly (P < 0.01) while ADF concentration increased linearly (P < 0.01) This suggests that CA hydrolyzed the digestible fiber fraction in the silage but did not affect the less digestible ADF fraction. Silages treated with Pr had lower (P < 0.01) ADF concentrations than Control silages and other enzyme-treated silages, suggesting that this treatment was particularly effective at reducing the concentration of the ADF frac tion which is usually poorly digested by ruminants. Treatment with Pr; therefore, reduced the total and less digestible fiber fractions, and could potentially result in improve d utilization of the fiber fraction in dairy cows fed bermudagrass silage. Although CA tr eatment reduced the total fiber fraction, it also reduced the digestible fi ber concentration, which is an important source of slowlyreleased energy for cattle. The reduction in NDF and ADF concentra tion by Pr and X-20 treatment, and NDF and hemicellulose concentration by CA treatme nt, contradicts the findings of Mandebvu et al. (1999) on bermudagrass but concurs with previous obs ervations on enzyme-treated wheat silage (Adogla-Bessa et al., 1999), co rn silage (Sheperd and Kung, 1996a; Sheperd and Kung, 1996b; Colombatto et al., 2004) and or chardgrass or alfalfa silages (Nadeau et al., 2000). Differences between the effect s of enzymes on cell wall hydrolysis in

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83 bermudagrass silage in this study and that of Mandebvu et al. (1999) may be due to differences in enzyme activity. These results; therefore, provide new evidence that fibrolytic enzymes can enhance cell wall hydrolysis in C4 grasses, as is the case in C3 grasses. Silages treated with Pr (P < 0.01) and CA (P < 0.05) had gr eater residual WSC concentration than Control silages. As the rate of application of both of these enzymes increased, residual WSC concentration increa sed linearly (P < 0.05). However, these enzymes increased residual WSC concentrati on by hydrolyzing different fiber fractions. While Pr hydrolyzed the less digestible fiber fraction, CA reduced the digestible fiber fraction. Therefore, both of these enzymes were effective in increasi ng the availability of fermentation substrates, but Pr was potent ially more beneficial at improving the digestibility of the silages. The WSC values obtained in the Pr-treated silages are also higher than those reported by Adesogan et al. (2004), probably because of gr eater cellulase and xylanase activity in Pr than in the enzyme included in the inoculant used by Adesogan et al. (2004). The increase in WSC c oncentration of enzyme treated silages agrees with results obtained by Sheperd and Kung ( 1996a); Adogla-Bessa et al. (1999) and Nadeau et al. (2000). Colombatto et al. (2003) also obs erved that addition of fibrolytic enzymes increased (P < 0.01) release of reducing sugars from fibrous fractions of forage during a 20-h pre-incubation phase. The concentration of CP was similar in en zyme-treated and Control silages (Table 4.3). However, CP concentration was lower in Pr-treated silages than silages treated with X-20 (P < 0.05) and CA (P < 0.01). This num erically small difference in CP did not

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84 result from greater proteoly sis in Pr-treated silages si nce they had lower (P < 0.05) ammonia-N concentrations than the other silage s. Rather, it may have been due to the higher WSC concentration of Pr treated silages. Organic Acid Concentration of Bermudagrass Silages The lactic acid concentrations (Table 4.4) of the Control and enzyme treated silages were similar ( P > 0.05). These results agree with t hose obtained of Mandebvu et al. (1999) who found that though fi brolytic enzyme treatment of bermudagrass did not increase lactic acid concentr ation, values for enzyme-treat ed forages were 5.4 % higher than those for untreated forage. Acetic acid concentration was lower (P < 0.05) in enzyme-treated silages than in Control silages. Promote-treated silages had the lowest (P < 0.01) acetic acid concentrations and unlike other enzymes increasing the rate of Pr application decreased (P < 0.05) acetic acid concen tration linearly. Th ese results are in contrast with those obtained by Sheperd a nd Kung (1996a) and Mandebvu et al. (1999) These factors are partly res ponsible for the lower (P < 0.05) DM losses in the Prwho found no effect of fibrolytic enzyme treatment on acetic acid concentration of silages. The lower acetic acid concentration (P < 0.01) and nu merically higher lactic acid concentration in Pr-treated si lages suggest that this enzy me enhanced homofermentative processes during ensiling, which reduce CO2 formation; and therefore, minimize DM and energy losses.treated silages relative to those in the other silages. However, the tendency towards greater homofermentative processes in enzyme-treated forages conflicts with their greater aerobic stability as homofermentative silages are often more susceptible to aerobic spoilage. The reason for this anomaly is not clear. Nevertheless, the extent of enzyme-induced homofermentation was not sufficient to override the inherent heterofermentation and aerobic stability of bermudagrass silage.

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85 Table 4.4 Effect of fibrolytic enzymes on th e organic acid concentration (g/kg DM)of bermudagrass silage. Enzyme Treatment1 Application rate Lactic acid Acetic acid Isobutyric acid Butyric acid Isovaleric acid Control 50 46 54 7.1 11 Pr 0.5 83 29 63 7.7 17 Pr 1 54 13 37 0.57 8 Pr 2 60 15 46 0 10 Pr Mean 66 19 48 2.8 11 Pr Rate effect NS L ** NS L ** L X-20 0.5 32 22 34 3.9 8 X-20 1 49 38 42 5.9 9 X-20 2 64 38 53 5.9 13 X-20 Mean 48 33 43 5.3 10 X-20 Rate effect L L ** NS NS NS CA 0.5 57 38 46 5.8 11 CA 1 54 36 68 0 18 CA 2 57 32 58 4.9 14 CA Mean 56 35 57 3.6 14 CA Rate effect NS NS NS Q Q A-20 0.5 44 42 54 8.1 12 A-20 1 62 35 59 9.2 14 A-20 2 64 33 76 0 14 A-20 Mean 56 37 63 5.7 13 A-20 Rate effect NS NS L C ** NS S.E. 10.61 3.36 7.41 1.96 1.97 Contrasts P values Control vs. Pr 0.211 < 0.01 0.504 0.064 0.735 Control vs. X-20 0.899 < 0.01 0.213 0.414 0.915 Control vs. CA 0.635 0.012 0.690 0.129 0.126 Control vs. A-20 0.612 0.028 0.287 0.551 0.210 Pr vs. X-20 0.057 < 0.01 0.372 0.130 0.531 Pr vs. CA 0.267 < 0.01 0.154 0.607 0.091 Pr vs. A-20 0.287 < 0.01 0.022 0.071 0.194 X-20 vs. CA 0.398 0.366 0.025 0.307 0.024 X-20 vs. A-20 0.373 0.159 0.003 0.752 0.060 CA vs. A-20 0.963 0.600 0.342 0.185 0.677 L: linear effect, Q: quadratic effect, C: cubic effect, NS: Not significant, P < 0.05, ** P < 0.01. 1 Cellulase-hemicellulase enzyme preparations: Pr: Promote, X-20: Biocellulase X-20, CA: Cattle-Ase, A-20, Biocellulase A-20

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86 Butyric acid was found in a ll the silages except those treated with Pr and A-20 at twice the recommended rate and CA at the recommended rate. Butyric acid concentration decreased (P < 0.05) with increasing applicatio n of Pr (linear), A-20 (cubic effect), and CA (quadratic effect). Howe ver, only Pr treatment produced butyric acid concentrations that tended to be less ( P = 0.064) than those in Control silages. The decrease in butyric acid concentration followi ng Pr treatment supports the observations of Adogla-Bessa et al. (1999) for wheat silage but contradicts thos e of Mandebvu et al. (1999) for bermudagrass silage. This discrepanc y is attributable to the high cellulase and xylanase activities in Pr which resulted in subs tantial hydrolysis of ce ll walls into WSC. When the concentration of such WSC is adequate, and moisture is not excessive, homofermentative lactic acid bacteria proliferate rather than heterofermenters and clostridia, such that lactic acid ac cumulates instead of butyric acid. The isobutyric acid concentrations of the treated and untreated silages were similar (P > 0.05). Neither propionic acid nor ethano l was found in the silages. The absence of ethanol in the silages may be explained by th e low yeast counts and relatively low WSC concentrations in the silages, because y easts are primarily responsible for ethanol production from the fermentation of sugars in silages. In vitro DM and Fiber Digestibility of Bermudagrass Silages Unlike silages treated with the other enzyme s, Pr-treated silages had greater (P < 0.05) 6-h and 48-h IVDMD values as well as greater 48-h IVNDFD and IVADFD than Control silages (Table 4.5). S ilages treated with Pr consiste ntly had greater 6-h and 48-h IVDMD, and 48-h IVNDF values than those treated with the ot her enzymes. The increase in IVDMD at 6-h and 48-h by Pr tr eatment suggests that application of this enzyme can increase both the rate and extent of digestion of bermudagrass silage.

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87 Table 4.5 Effect of fibrolytic enzymes on in vitro digestibility of DM (g/kg), NDF, ADF and hemicellulose (Hem) in bermudagrass sila ge after 6 or 48-h of digestion (g/kg DM). Enzyme Treatment1 Application rate DM 6-h DM 48h NDF 48h ADF 48h Hem 48h Control 200 501 402 427 368 Pr 0.5 227 561 461 489 425 Pr 1 223 546 442 457 423 Pr 2 225 554 448 442 456 Pr Mean 225 554 450 462 435 Pr Rate effect NS NS NS C NS X-20 0.5 208 498 393 414 365 X-20 1 195 498 370 426 344 X-20 2 209 495 385 405 360 X-20 Mean 204 497 383 415 356 X-20 Rate effect Q NS NS NS Q ** CA 0.5 198 487 385 401 363 CA 1 199 536 447 512 342 CA 2 205 521 416 479 321 CA Mean 201 515 416 464 342 CA Rate effect NS L ** Q ** L ** NS A-20 0.5 183 502 424 462 369 A-20 1 190 491 399 436 349 A-20 2 184 538 464 504 411 A-20 Mean 186 510 429 467 376 A-20 Rate effect NS C ** C ** Q ** C* S.E. 4.398 8.290 13.102 13.295 13.134 Contrasts P values Control vs. Pr < 0.01 <0.01 0.004 0.032 0.007 Control vs. X-20 0.401 0.638 0.215 0.425 0.206 Control vs. CA 0.897 0.176 0.371 0.026 0.264 Control vs. A-20 0.009 0.350 0.085 0.017 0.724 Pr vs. X-20 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 Pr vs. CA < 0.01 < 0.01 0.003 0.895 < 0.01 Pr vs. A-20 < 0.01 < 0.01 0.059 0.686 < 0.01 X-20 vs. CA 0.317 0.014 0.005 < 0.01 0.826 X-20 vs. A-20 < 0.01 0.054 0.000 < 0.01 0.027 CA vs. A-20 < 0.01 0.538 0.224 0.785 0.044 L: linear effect, Q: quadratic effect, C: cubic effect, NS: Not significant, P < 0.05, ** P < 0.01, 1 Cellulase-hemicellulase enzyme preparations: Pr: Promote, X-20: Biocellulase X-20, CA: Cattle-Ase, A-20: Biocellulase A-20.

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88 Furthermore, Pr treatment was effective at improving the digestibility of the total, digestible and less digestible cell wall fract ions, corroborating the ea rlier suggestion that Pr treatment will increase the utilization of bermudagrass silage by ruminants. Previous studies did not detect such benefits of enzyme treatment on the in vitro digestibility of DM and NDF of bermudagrass sila ge (Mandebvu et al., 1999) or the in vivo digestibility of orchardgrass silage (Nadeau et al., 2000). The IVADFD was greater in the Pr, CA and A-20 -treated silages compared to Contro l silages (P < 0.05) and X-20 (P < 0.01). Therefore, these enzymes increased the suscep tibility of the typically indigestible cell wall fraction to ruminal digestion. Hemicellulose digestibility was also greater in the Prtreated silages (P < 0.01) than Control silages or other enzy me-treated silages, while A20 showed higher values (P < 0.05) than X-20. The superior effect of Pr treatment in this study is partly attributab le to the fact that in accordance with the each of the enzyme manufacturers guidelines, a greater amount of enzyme was applied to the forages in the Pr treatment, than in the other enzyme treatments. This is partly because Pr is supplied in liquid form, while the other enzymes were supplied in solid form. Though Pr did not have the highest xylanase or cellulase activity, it had greater combined cellulase and xylanase activity than any of the other enzymes. The synergistic, complementary effects of these enzymes in Pr, probably accounted for its superior e ffects on the silages. T hough no cellulase or xylanase activity was detected in CA, the results of this study i ndicate that it had fi brolytic activity. It probably; therefore, cont ained different enzymes to those that were analyzed. Conclusions This study shows that the nutritive value and fermentation of bermudagrass silage can be improved by treating it with fibrolytic enzymes. Compared to Control silages,

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89 enzyme-treated silages had greater WSC concen tration due to hydrolysis of different cell wall fractions. However, Pr was the most promising enzyme for increasing residual WSC concentration enhancing homofermenta tion, and reducing the pH, DM losses and proteolysis.

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90 CHAPTER 5 EFFECT OF METHOD OF DIETARY A DDITION OF A FIBROLYTIC ENZYME ON THE PERFORMANCE OF LACTATING DAIRY COWS Introduction One of the main problems limiting livestoc k production in the southeastern United States is the seasonal variation in forage quality and quantity. Dairy producers have reduced the winter forage deficiency by ha rvesting and conserving forages during their peak growth periods in the summer for feedi ng in the winter. Howe ver since the nutritive value of the tropical forage s is usually low due to th eir high indigestible fiber concentration, ideal methods of conservation sh ould also aim to improve forage quality. Several recent studies have evaluated the pot ential of improving forage quality and diet utilization with fibrolytic enzymes (Rode and Beauchemin 1998; Colombatto et al., 2003a). Milk yield has been increased in so me studies in which dairy cow diets were supplemented with exogenous fibrolytic en zymes (Rode et al., 1999; Yang et al., 2000, Kung et al., 2002), but not in others (She perd and Kung, 1996; Lewis et al., 1999; Beauchemin et al., 2000; Vicini et al., 2003). Dry matter intake (DMI) either increased (Beauchemin et al., 2000) or was unaffected (Beauchemin et al., 1999; Kung et al., 2000) when enzymes were added to the diet. Feed intake increases have been generally small and inconsistent (Yang et al., 1999 and 2000; R ode et al., 1999; Schi ngoethe et al., 1999; Vicini et al., 2003). Similarly, effects of supplemental enzymes on digestibility have been inconsistent. Use of enzyme products comprised mainly of xylanases and cellula ses have increased

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91 digestibility (Rode et al., 1999; Yang et al., 2000 ) or did not affect di gestibility (Lewis et al., 1999). Attempts to improve feed efficiency for milk production in dairy cows by the use of direct-fed fibrolytic enzymes applied at or shortly before feeding have also yielded variable production responses (S utton et al., 2003). Changes in milk fat and protein also have been inconsistent (Beauchemin et al ., 1999; Lewis et al., 1999; Schingoethe et al., 1999; Rode et al., 1999; Yang et al., 1999; 2000; Kung et al., 2000; Vicini et al., 2003). Although a few studies demonstrated that enzyme application to bermudagrass silage improved DM recovery (Dean et al., 2005 ) and feed intake by beef cows (Bates et al., 1989b), there has been little concerted effort aimed at de termining the efficacy of using commercial enzymes to improve milk production in cows fed enzymesupplemented tropical grasses. The objective of this study was to determine the effects of applying an enzyme to bermudagrass at ensiling, or to different components of the diet at feeding on feed intake, milk production a nd composition, digestion kinetics and blood metabolites of lactating dairy cows. The enzy me selected for this study was found to be more effective than three other commercial enzymes at reducing DM losses and fiber concentration, increasing water soluble ca rbohydrate concentration, promoting a more homolactic fermentation and increasing fiber digestibility in bermudagrass silage (Dean et al., 2005). Material and Methods Two experiments were carried out at the Da iry Research Unit of the University of Florida from November 2004 to March 2005. In the first experiment, 30 lactating Holstein cows (peimiparous and multiparous) in mid-lactation (129 6 days in milk) were allocated randomly to 5 dietary trea tments for two, 28-d periods. Each period consisted of 14 d for adaptation to a new diet and 14 d for sample collection. A cellulase,

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92 xylanase enzyme complex (Promote, Cargil l, Minnetonka, MN, US A) was applied to different fractions of the diet. The manufactur er-stipulated main activ ity of this product is 1200 cellulase units/g of the product, where one unit is the amount of enzyme that releases 1 mol of glucose from cellulose in 1 min at 40C. Cellulase activity also was determined at 39oC and pH 5.5 using the filter pa per method (Wood and Bhat, 1988). The activity was found to be 38.4 filter paper units/g, where one unit of activity is the amount of enzyme that releases exactly 2 mg of glucose from 50 g of filter paper in 60 min. Diets For both experiments, the diets contained Tifton 85 bermudagrass ( Cynodon spp.) silage (BS), corn silage (CS) and concen trate mixed at 35, 10 and 55% of dietary DM, respectively (Table 5.1). Th e dietary treatments evaluate d were the following: 1) no enzyme added (Control), 2) enzyme applied to the concentrate (EC), 3) enzyme applied to the TMR at feeding (ETMR), 4) enzyme app lied to the forage at feeding (EF) and 5) enzyme applied at a rate of 1.3 g/kg of DM to bermudagrass at ensiling (TS). Each cow in Treatments 2, 3 and 4 received four g of enzyme/per d. Cows were individually fed twice daily (at 0700 and 1430 h), using Calan gates (American Calan Inc., Northwood, NH). Feed refusals were collected daily at 0600 h. Cows were trained to use calan gates for 10 days before the beginning of the trial Diets were mixed prior feeding using 250 kg Calan Data Rangers (American Calan Inc ., Northwood, NH). Three data rangers were used for mixing the diets. One was reserved for the Control diet, another for the TS diet and a third for diets to which enzyme applicati on occurred just before feeding. The latter was washed between feeding Treatments 2, 3 and 4 to avoid cross contamination.

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93 Table 5.1 Ingredient and chemical com position of the basal untreated diet. Ingredient composition % Diet DM Bermudagrass silage 35.0 Corn silage 10.0 Ground corn 27.0 Citrus pulp 5.1 Whole Cottonseed 2.8 Mineral mix1 4.4 Biophos (Calcium phosphate)2 0.4 SoyPlus3 6.6 Soybean meal 8.6 Chemical composition DM, % 46.4 CP, % of DM 16.1 ADF, % of DM 27.2 NDF, % of DM 46.5 TDN, % of DM 66.0 NEL, mcal/kg of DM 1.57 1 Mineral mix contained 26.4% CP, 10.2 Ca, 0.9% P, 3.1% Mg, 1.5% S, 5.1% K, 8.6% Na, 11698 mg/kg of Zn, 512 mg/kg of Cu, 339 mg/kg of Fe, 2231 mg/kg of Mn, 31 mg/kg of Co, 26 mg/kg of I, 7.9 mg/kg of Se, 147,75 IU of vitamin A/kg, 787 IU of vitamin E/kg (DM basis). 2 Biophos contained 15.9% Ca, 21.2% P. 3 West Central Soy, Ralston, IA. Tifton 85 bermudagrass (T-85) was mowed after 35 d of regrowth using a CLAAS Disco 3000 TC forage mower (CLAAS of Amer ica, Omaha, NE). After wilting for 2 h, the forage was chopped (5-cm particle size) using a CLAAS Jaguar 9000 (CLAAS of America, Omaha, NE) forage harvester. For the TS treatment, the enzyme was applied to bermudagrass as it was packed at a rate of 3 ton/min into an Ag Bag (AG Bag International, Warrenton, OR ) using a Versa Bagger (Versa Corporation, Astoria, OR) model ID 1012. Two 62-ton untreated bags were prepared followed by one 46-ton enzyme-treated bag. The forage was ensi led for 35 days before the experiments commenced.

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94 The enzyme-treated concentrate was prepar ed weekly by dissolving the enzyme in water (1:5 ratio v/v) and spraying the solution on 140 kg of ground corn using a 3.75-l hand sprayer, while the corn was being mixe d in a Marion Mixer (Rapids Machinery Co., Marion, IA). The rest of the concentrate ingredients were subsequently mixed with the treated corn in a 900 kg New Holland 355 mixer. Untreated and treated concentrates were stored in metal grain bins (5.5 ton cap acity). For the EF and ETMR treatments, the enzyme was dissolved in water (1:10 ratio v/ v) and sprayed on the forage and the TMR, respectively, while loaded in a Calan Data Ranger. The enzyme-treated feedstuff was subsequently mixed for 5 min to ensure pr oper distribution. For EF, the enzyme was applied in the morning to the entire untreate d bermudagrass silage that was to be offered during that day. Sample Collection and Analysis In Experiment 1, cows were balanced for parity, milk production and DIM and assigned to each treatment at the beginning of the first period. At the end of Period 1, cows were randomly assigned to another treatm ent with the requirement that no treatment follow the same treatment. Cows were milked thrice daily at 0200, 1000 and 1800 h and milk production (MP) was measured on the last 14 d of each period. Milk samples were collected twice daily on two days during each week in the last 14 d of each period, preserved with potassium dichromate and stored at 4C. Milk samples were analyzed by Southeast Milk lab (Belleview, FL) for concen tration of fat (MCF), true protein (MCP) and somatic cell counts (SCC) using a Bentle y 2000 NIR analyzer (Bentley Instruments Inc., Chaska, MN). Feed efficiency wa s calculated based on milk production and DMI (kg milk/kg DM fed). Body we ight and BCS ranging from 1 (thin) to 5 (obese) were measured on three consecutive days after the 1000 h milking at the beginning and end of

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95 each period. Blood samples (10 ml) were taken using vacutainers by caudal arteriovenipuncture on the last day of each period. Samples were centrifuged at 2500 g for 20 min and the plasma was frozen at -20 C. Concentration of plas ma glucose (Glc) was determined using a Technicon Autoanalyzer II (Bran-Luebbe, Elinsford, NY) and method modified from Gochman and Schmitz (1972). In this modification the specificity of glucose oxidase is combined with the peroxidase indication couple (3-methyl-2benzothiazolinone hydrazone-HCl, MBTH and N-N-dimethylaniline, DMA method N38). Glucose oxidase initiate s reactions which generate hydr ogen peroxide that reacts with the peroxidase indicator to form an intensely-colo red indamine dye. Blood urea nitrogen (BUN) was determined using an autoanalyzer method (Technicon Industrial systems Autoanalyzer II; Industrial met hod # 339-01; Tarrytown, NY), which is a modification of the carbamido-diacetyl reac tion, described by Coulombe and Favreau (1963). Plasma concentration of -hydroxybutyrate acid ( HBA) was determined using the procedure described by Williamson et al. (1962). Chromic oxide (Cr2 O3) was used for determination of apparent digestibility. Chromium oxide powder weighed into gelati n capsules and was dosed twice daily via balling gun (10 g/dose at 0700 and 1900 h) for 10 consecutive days in each experimental period. Fecal samples (approximately 100 g) we re collected during th e last 5 days of each period at the time of dosing. Feces were dried to constant weight at 55C in a convection oven, ground to pa ss through a 1-mm screen and a composite sample was made from all ten fecal samples per cow per period. Chromium concentration in feces was determined using a Perkin Elmer 5000 (Wellesley, MA) Atomic Absorption Spectrometer, according to the procedure described by Williams et al. (1962).

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96 Quadruplicate samples of the concentrat e, forages and untreated TMR collected during each week of each colle ction period were composited, s ub-sampled and sent to the Dairy One Forage Testing Laboratory (Ithaca, NY) for CP, NDF and ADF analysis. In addition silages were analyzed fo r non-fiber carbohydrates (NFC), NH3-N, lactic acid, VFA, pH and TDN. In Experiment 2, five ruminally fistulated co ws were used to evaluate the effect of the dietary treatments on ruminal pH, VFA and ammonia-N concentration and in situ rumen degradation, during thr ee consecutive 15-d periods. Each period consisted of 12 d of adaptation to a new diet, 2 days of in situ rumen degradability measurements, and 1 d of rumen fluid collection. Rumen fluid wa s collected (200 ml) by aspiration and filtered through two layers of cheesecloth at 0, 2, 4, 6, 8 and 10 h after feeding on the last day of each period. The pH was measured within 20 min of rumen fluid collection using a pH meter (Accumet, model HP-71, Fisher Scientif ic, Pittsburgh, PA). The rumen fluid was acidified with 3 ml/sample of H2SO4 (50% v/v). Samples we re centrifuged at 11,500 g for 20 min, after which the supernatant was collected and frozen (-20oC) in 20-ml vials. Volatile fatty acids were measured using the method of Muck and Dickerson (1988) and a High Performance Liquid Chromatograph (Hitachi, FL 7485, Tokyo, Japan) coupled to a UV Detector (Spectroflow 757, ABI Analyt ical Kratos Division, Ramsey, NJ) set at 210 nm. The column used was a Bio-Rad Aminex HPX-87H (Bio-Rad Laboratories, Hercules, CA 9454) column with 0.015M sulfuric acid mobile phase and a flow rate of 0.7 ml/min at 45C. Ammonia N was determined with a Technicon Auto Analyzer (Technicon, Tarrytown, NY, USA) and adapta tion of the Noel and Hambleton (1976) procedure.

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97 During the second and third periods, th e rumen degradation kinetics of the experimental diets were measured in situ by incubating TMR samples within nylon (50 m pore size) bags for 0, 2, 4, 6, 8, 24, and 48 h. Dried (65C for 48 h), ground (4 mm) diet samples were weighed (5 g as is) into nylon bags in triplicate and incubated in cows fed the same diet during d 13 and 14 of each period. At each incubation time, bags were removed from the rumen and rinsed with cool water and frozen. At the end of each period, all bags were thawed and washed tr ough a rinse cycle without soap in a washing machine and dried for 48 h at 60C. The in situ degradation parameters were described with the method of McDonald (1981): P = a + b (1-e-c (t-L)) where P= DM degraded at time t, a = wash lo ss of DM at time zero, b = potentially degradable DM fraction, a+b = total degrad able fraction, c = the rate at which b is degraded, t = ruminal incubation duration, and L = lag time in hours. The constants a, b, c, and L were estimated using the nonlinea r regression procedures of SAS (1995) and analyzed using the GLM procedure (SAS, 1995). Statistical Analysis Both experiments involved cross-over desi gns and the data were analyzed with Proc Mixed (SAS, 1995). The model used for analyzing the results from Experiment 1 and the in situ degradability results in Experiment 2 was: Yijk = + Ti + Pj + Ck + Rl + Eijkl Where; : general mean Ti: treatment effect (fixed effect)

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98 Pj: period effect (fixed) Ck: cow effect (random effect) Rl: residual effect Eijkl: experimental error For analysis of milk produc tion, each cows pretrial m ilk production (129 days in milk) was used as a covariate. Orthogonal contrasts were used to compare each of the enzyme treatments to the Control. The model used for analyzing rumen VFA and ammonia data in Experiment 2 was: Yijk = + Ti + Pj + Hk + Cl + Eijkl where : general mean Ti: treatment effect (fixed effect) Pj: period effect (fixed) Hk: time effect (rep eated measurement) Cl: cow effect (random effect) Eijkl: experimental error The covariance structure that was used was AR(1), and a slice statement was used to detect differences among treatments at ea ch incubation time. Significance was declared at P < 0.05 and tendencies at P < 0.15. Results and Discussion Chemical Composition of the Dietary Ingredients The chemical composition of the enzyme-treated and untreated forages and concentrate are shown in Table 5.2. Crude protein, NFC, TDN and organic acid concentrations were greater and fiber a nd NH3-N concentrations were lower in

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99 enzyme-treated than in untreated bermudagr ass silage. This sugge sts that the enzyme improved the fermentation indices of the bermudagrass silage Voluntary Intake Promote addition did not affect the intake of DM, NDF (NDFI) or CP (CPI) (Table 5.3). The DMI response is consistent with th e results of Rode et al. (1999) who fed Promote-supplemented diets to lactating cows in early lactation. The lack of effect enzyme supplementation on DMI also concurs with those observed by Kung et al. (2000), Yang et al. (2000), Kung et al. (2002) a nd Sutton et al. (2003) Nevertheless, Beauchemin et al. (2000) observed that adding a low or high amount of an enzyme supplement to the diet increased (P < 0.01) DM I, and OM digestibilit y, and also tended to increase intake of NDF (P = 0.17) and ADF (P = 0.14). These increases were relatively small (20 and 8% for NDF and ADF, respectiv ely) and they may be attributable to increased palatability or rate of passage. More conclusive results were obtained by Lewis et al. (1999) who detected increased (P < 0.05) DMI due to increasing amounts of enzyme supplementation. Knowlt on et al. (2002) found that DMI was numerically higher in enzyme-supplemented cows in early lactation but not in late-lactation cows. In vivo Digestibility of DM, NDF, and CP Apparent total tract digestibilities of DM, NDF and CP were unaffected by enzyme treatment (Table 5.3). Digestibility is one of the main determinants of voluntary intake; therefore, the ineffectiveness of the enzyme at improving digestibility helps explain the lack of enzyme effect on voluntar y intake. These results agree with those obtained by Hristov et al. (1998) and Lewis et al. (1999). Knowlton et al. (2002) also observed that digestibility of DM was not different between Control and enzymesupplemented diets fed to cows in early or late lactation. Beauchemin et al. (2000) found

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100 that digestion of the TMR measured in situ was not affected by enzyme supplementation. However, Rode et al. (1999) observed that digestibility of a TMR consisting of corn silage (24%), alfalfa hay (15%) and c oncentrate (47%), determined using Cr2O3, was increased dramatically by enzyme treatment (DM, 61.7 vs. 69.1%; NDF, 42.5 vs. 51.0%; ADF, 31.7 vs. 41.9%; CP, 61.7 vs. 69.8%). Similarly Beauchemin et al. (1999) reported that applying enzymes to the TMR befo re feeding increased digestibility in the total tract due to grea ter post-ruminal digestion. Sutton et al. (2003) observed that rumen digestibility of DM and OM were unaffected by the enzyme addition; however, total tract digestibility of DM and OM were higher when enzyme was applied to the TMR. In the latter study, enzyme addition reduced NDF di gestibility in the rumen but increased it postruminally, and did not affect total tract NDF digestibility. This was probably because the enzymes were not degraded in the rumen and therefore they exerted their fibrolytic action in the small intestine. The NDF and li gnin concentrations of bermudagrass silage are greater than that of the corn silage or the temperate forages used in the studies cited above. The negative effect of lignin on the digestibility of th e fiber fraction may partly explain ineffectiveness of the enzyme at im proving fiber digestion in this experiment. Some fiber particles cannot be ruminally digested irresp ective of rumen retention time due to physical barriers to digestion imposed by ferulate cross linkages between lignin and polysaccharides (Buxton and Redfern, 1997). Effect of Promote on Milk Production and Composition Production of milk and 4% FCM were not different among diets, though cows fed the Control diet tended (P < 0.15) to produce more milk than those fed EC or EF (Table 5.4). This result agrees with that of Beauch emin et al. (1999). However, others have reported a tendency for greater milk pr oduction when fibrolytic enzymes were

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101Table 5.2 Chemical composition of the enzyme -treated and untreated forages and concen trate (% DM) (n= 4 re plicates per mean) Chemical composition Item DM, % CP NH3N1 NDF ADF NFC TDN pH Lactic Acid Acetic Acid Butyric Acid Corn silage 28.1 8.8 12.0 45.2 27.0 38.1 79.0 4.8 3.08 6.68 0.07 Pre-ensiled bermudagrass 23.4 12.7 76.1 45.3 7.85 43.0 Untreated bermudagrass silage 29.9 9.3 38.0 81.8 49.9 5.3 52.7 8.4 0.10 0.05 0 Treated bermudagrass silage 29.6 11.4 13.7 76.2 45.2 8.6 55.0 4.6 1.77 3.08 0.18 Untreated concentrate 88.3 21.9 15.8 8.55 84.5 Treated concentrate 88.4 21.5 15.6 8.15 84.0 1 As percentage of total N,

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102 Table 5.3 Effect of method of en zyme addition on diet digest ibility and voluntary intake Variable Treatment DMI, kg/d DMI, % BW NDFI, kg/d CPI, kg/d DMD, % NDFD, % CPD, % FE, kg milk/kg DMI Control 20.9 3.35 9.7 3.4 66.4 50.7 65.6 1.64 EC 21.6 3.46 9.9 3.5 64.2 51.0 65.7 1.46 ETMR 22.4 3.65 10.0 3.5 66.3 50.4 66.9 1.42 EF 19.9 3.18 9.0 3.1 64.3 51.6 65.7 1.64 TS 21.8 3.41 9.5 3.3 68.3 48.7 67.4 1.59 s.e 0.92 0.18 0.61 0.21 1.37 2.29 2.11 0.13 P values Treatment effect 0.298 0.397 0.369 0.370 0.139 0.924 0.866 0.256 Contrasts Control vs. EC 0.560 0.632 0.621 0.621 0.930 0.918 0.970 0.153 Control vs. ETMR 0.223 0.209 0.215 0.215 0.777 0.926 0.551 0.082 Control vs. EF 0.404 0.481 0.440 0.440 0.797 0.756 0.959 0.962 Control vs. TS 0.447 0.797 0.717 0.717 0.498 0.545 0.402 0.697 EC: enzyme applied to concentrate, ETMR: enzyme applied to the TMR, EF: enzyme applied to forage at feeding, TS: enzyme-treated silage: DMD: dry matter digestibility, NDFD: neutral detergent fiber digestibility, CPD: crude protein digestibility, DMI: dry matter digestibility, RIN: relative dry matter intake, FE: feed efficiency. applied to the concentrate (Yang et al., 2000) or to the TMR (Sutt on et al., 2003). Kung et al. (2002) observed that milk produc tion was unaffected (P < 0.05) by enzyme treatment of a diet consisting of corn silage (30%), alfalf a hay (15%) ans a concentrate (55%), but cows fed forage treated with a mixture of cellulase and xylanase produced 2.5 kg more 3.5% FCM (P < 0.12) than those fed untreated forage. Lewis et al. (1999) found that cows assigned to an enzyme suppl emented diet produced more (P < 0.01) milk than did cows fed the Control diet (27.2 vs. 25.9 kg/d), indicating that enzyme treatment increased nutrient availability for milk production.

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103 Table 5.4 Effect of method of enzyme a ddition on milk production and composition Variable Treatment Milk, kg/d 4% FCM, kg/d Milk fat, % Milk fat, kg/d Milk protein % Milk protein, kg/ d SCC 103 cells/ml Control 33.1 31.8 3.67 1.23 2.91 0.956 339 EC 30.9 29.9 3.78 1.16 3.07 0.948 488 ETMR 32.3 32.4 3.99 1.29 3.07 0.997 581 EF 31.2 30.0 3.77 1.64 3.03 0.933 817 TS 32.3 30.6 3.72 1.19 2.90 0.923 458 s.e 1.282 1.387 0.124 0.064 0.092 0.034 263 P values Treatment effect 0.432 0.265 0.417 0.195 0.257 0.240 0.531 Contrasts Control vs. EC 0.102 0.171 0.533 0.282 0.076 0.821 0.264 Control vs. ETMR 0.521 0.677 0.073 0.330 0.081 0.235 0.530 Control vs. EF 0.131 0.175 0.587 0.273 0.187 0.496 0.101 Control vs. TS 0.512 0.404 0.787 0.503 0.917 0.345 0.581 EC: enzyme applied to concentrate, ETMR: enzyme applied to the TMR, EF: enzyme applied to forage at feeding, TS: enzyme-treated silage, SCC: somatic cell counts. Furthermore, Jurkovich et al (2002) obtained an increase of between 5 to 10% in milk production when lactating cows in ear ly lactation were supplemented with a lignolytic enzyme. Schingoethe et al. (1999) also found that production of milk (P = 0.12) and FCM (P < 0.01) increased in enzyme supplemented cows, and the responses to enzyme-treated forages occurred 2 to 4 wk af ter the cows started to consume the treated forages. In the same study, cows that starte d to receive enzyme-treated forage during the first 100 d postpartum produced 9 to 15% more milk than cows fed the untreated diet in the same time frame. However, milk produc tion was unaffected when cows were in midlactation at the start of the experiment. Th erefore, the duration of the adaptation period (2 wk) and the stage of lactation (129 DIM) of cows in this study may have affected the milk response to enzyme addition. Efficiency of feed conversion in to milk tended (P = 0.082) to be lower in cows fed ETMR compared to those fed the Control diet. Kung et al.

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104 (2000) observed that treatment with enzymes di d not improve feed efficiency relative to cows fed the Control diet. Milk fat and true protein yields were unaffected (P >0.05) by enzyme supplementation, however, cows fed ETMR tended (P < 0.09) to have greater milk fat and protein concentration than cows fed the C ontrol diet. Cows fed EC also tended (P = 0.076) to have greater concentr ation of milk protein than those fed the Control diet. Tendencies for lower milk fat percentage from cows fed enzyme-treated diets have been reported by Rode et al. (1999). In contrast, Lewis et al. (1999), Zheng et al. (2000), Jurkovich et al. (2002), and Kung et al. ( 2002) observed that milk composition was similar between Control and enzyme-supplemen ted cows. Sutton et al. (2003) observed that compared to values in cows fed a C ontrol diet, milk protein concentration was greater (P < 0.05) when enzyme was applied to the concentrate, whereas milk protein yield was greater when the enzyme was app lied to the TMR or to the concentrate. Schingoethe et al. (1999) found that milk production was 10.8% greater (P=0.12) in enzyme supplemented cow diets relative to Control diets and production of milk fat and protein increased by 20 and 13%, respectively. The tendency for greater milk constituent concentrations in cows fed ETMR vs. Control cows, suggests that this mode of enzyme addition is promising and warrants further study. Somatic cell counts were unaffected (P >0.05) by enzyme treatment, in agreement w ith results of Schingoethe et al. (1999). Body Weight Gain and Body Condition Score The BWG and BCS of the cows were unaff ected by enzyme treatment (Table 5.5). Knowlton et al. (2002) found that enzyme addition did not affect BW, but noted a tendency (P < 0.07) for an interaction between the effects of stage of lactation and enzyme treatment. This tendency was due to a numerical increase in BW in early

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105 lactation cows fed diets containing the enzyme compared to those fed the Control diet, whereas BW in late lactation cows was unaff ected by enzyme treatment. Kung et al. (2002) observed that BW and ADG over a 12wk treatment period were unaffected by treatment of diet with mixtur es of xylanases and cellulases. However, Jurkovich et al. (2002) observed that body condition loss in co ws fed enzyme-treated diets was lower than in those fed Control diets. According to these researchers, this was probably due to the enhanced ruminal VFA concentration of cows supplemented with fibrolityc enzymes which implies greater energy availability. Cows assigned to the EC treatment tended (P < 0.015) to have greater BW gain than Control, probably because they partitione d more nutrients away from MP to BWG. However this did not result in improved BCS. Blood Glucose, Urea-N and -Hydroxybutyrate Plasma glucose concentration (Table 5.5) wa s similar across treatm ents, in agreement with the results of Hristov et al. (1998) and (2000). The mean glucose concentration of the cows in this experiment was similar to the basal concentrati on reported by Lemosquet et al. (1996) in Holstein co ws in mid-late lactation that were producing 34 kg of milk daily. However, cows fed EF and TS had lower (P < 0.05) BUN concentrations than those fed the Control diet and cows fed ETMR had a similar tendency (P = 0.123). This suggests that there was greater ruminally fe rmentable metabolizable energy availability from these diets, leading to improved mi crobial efficiency of N utilization. Enzyme treatment tended (P < 0.112) to reduce HBA concentration. The concentration of this blood metabolite was particularly lower (P < 0.01) in cows fed ETMR, which indicates that this treatment decreased fat mobilization in the cows.

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106 Table 5.5 Effect of method of enzyme addi tion on body weight and condition score, and blood metabolites Variable Treatment BW, kg BW gain, kg/d BCS BUN, mg/dl Glc, mg/dl HBA, mg/dl Control 633 0.198 2.82 16.9 64.5 0.931 EC 635 0.638 2.61 16.0 62.9 0.877 ETMR 624 0.421 2.77 15.6 64.6 0.732 EF 618 0.205 2.64 15.2 64.5 0.826 TS 623 0.310 2.83 15.2 64.5 0.833 s.e 21.47 0.204 0.157 0.625 1.096 0.074 P values Treatment effect 0.961 0.462 0.477 0.232 0.774 0.112 Contrasts Control vs. EC 0.938 0.138 0.175 0.282 0.294 0.467 Control vs. ETMR 0.723 0.451 0.737 0.123 0.967 0.010 Control vs. EF 0.565 0.981 0.251 0.049 0.994 0.154 Control vs. TS 0.716 0.696 0.962 0.047 0.982 0.195 EC: enzyme applied to concentrate, ETMR: enzyme applied to the TMR, EF: enzyme applied to forage at feeding, TS: enzy me-treated silage, BW: body weight, BCS: body condition score, BUN: blood urea nitrogen, Glc: blood glucose, HBA: beta hydroxy butyrate. This result concurs with that obtained by Hristov et al. (2000) who observed that concentration of plasma HBA was reduced (P < 0.01) in cows supplemented with enzymes. Jurkovich et al. (2002) also f ound a lower incidence of hyperketonaemia, and lower concentrations of acetoa cetic acid and non-esterified fatty acid (NEFA) in the blood of cows supplemented with a mi xture of fibrolytic enzymes. Ruminal pH and Concentration of VFA and NH3-N Mean ruminal pH was more acidic in cows fed EC (P < 0.01) than in those fed the Control diet and a similar tendency (P < 0.08) was evident in cows fed EF or TS. The pH decreased (P < 0.01) after f eeding (Figure 5.1 and Table 5.6), and the lowest (P < 0.05) values were observed at 8 and 10 h after feed ing in all cows. This result concurs with that of Hristov et al. (2000) who observed that ruminal pH decreased linearly (P < 0.01)

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107 to a greater degree due to enzyme supplemen tation and the response depended on time of sampling However, Bowman et al. (2003) and Sutton et al. (2003) did not detect enzyme effects on daily mean pH, though Sutton et al. ( 2003) found that pH was numerically lower in cows fed enzyme-treated diets. Ruminal pH fell below 6 within 6-h of feeding in cows fed EC and remained at 5.5 between 8 and 10 h af ter feeding. Values for cows fed EF and TS also fell below 6 by 7 h after feeding and dropped to a nadir of 5.75 10 h after feeding. A pH of 5 5.8 is used often to indicate sub-clinical ruminal acidosis in dairy cows (Oetzel et al., 1999). Therefore, cows fed EC and to a lesser extent FF and EC were most likely to have experienced sub-clinical ruminal acidosis. This partially explains the tendency for lower MP of cows fed EC and EF. Generally, NH3-N concentration increased after f eeding and subsequently decreases progressively, presumably due to N uptake by ruminal microbes for microbial protein synthesis (Figure 5.2 and Table 5.7). Ruminal NH3-N concentration was lower in cows fed ETMR (P < 0.05) than in cows fed th e Control diet. The reduced ruminal NH3-N concentration in cows fed ETMR suggests that there was enhanced uptake of NH3-N by the ruminal microbes probably due to gr eater fermentable metabolisable energy availability. Hristov et al (2000) reported that rumina l ammonia N was below 25 mg/L in their study and responded quadratically (P < 0.01) to enzyme supplementation. Ammonia-N concentration decreased (P < 0.01) after feeding in the study of Sutton et al. (2003) but was numerica lly greater in cows fed enzyme supplemented diets instead of Control diets.

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108 5.0 5.5 6.0 6.5 7.0 7.5 0246810 Time after feeding, hpH Control EC ETMR EF TS Figure 5.1 Effect of method of enzyme addition on ruminal fluid pH P < 0.01 P < 0.05 Table 5.6 Effect of method of enzy me addition on ruminal fluid pH Treatment Mean Control 6.35 EC 6.08 ETMR 6.32 EF 6.23 TS 6.22 P value Treatment effect < 0.01 Time effect < 0.01 Contrasts Control vs. EC < 0.01 Control vs. ETMR 0.514 Control vs. EF 0.075 Control vs. TS 0.052 ** * **

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109 0 5 10 15 20 25 30 35 40 0246810 Time after feeding, hAmmonia-N, mg/dL Control EC ETMR EF TS Figure 5.2 Effect of method of enzyme a ddition on ruminal NH3-N concentration P < 0.01 Table 5.7 Effect of method of enzyme addition on ruminal NH3-N concentration Treatment Mean Control 15.4 EC 13.3 ETMR 10.7 EF 16.3 TS 14.8 P value Treatment effect 0.041 Time effect < 0.01 Contrasts Control vs. EC 0.356 Control vs. ETMR 0.023 Control vs. EF 0.615 Control vs. TS 0.814 ** **

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110 Mean ruminal concentration of acetic aci d (Figure 5.3 and Table 5.8) was lower in cows fed ETMR (P < 0.05) and EF (P <0.01) than in Control cows However, propionic (Figure 5.4 and Table 5.9) and butyric acid s (Figure 5.5 and Table 5.10) concentrations were unaffected (P > 0.05) by dietary trea tment or time after feeding. Ruminal fluid acetate:propionate ratio was lower in cows fed ETMR diets (P <0.01) rather than the Control diet (Figure 5.6 and Table 5.11), whic h indicates that the ETMR diet promoted a more efficient fermentation in the rumen, wh ich probably was due to a higher release of soluble carbohydrates by the ETMR treatment. Rapidly fermenta ble carbohydrates yield relatively higher ruminal propionate as comp ared to acetate, and thereby lower the acetate:propionate ratio. The highest (P <0.01) acetate:propionate ratio was observed in Control diets at 10 h. This re sult disagrees with that of Dawson and Tricarico (1999) who found that treating tall fescue hay with prepar ations high in either xylanase or cellulase activity increased the acetate:propionate ratio. Mean total VFA concentration was lower (P < 0.05) in cows fed the ETMR or in TS diets rather than the Cont rol diet (Figure 5.7 and Table 5.12). This result contradicts that of Dawson and Tricarico (1999) and Pi nos-Rodriguez et al. (2002), who found that enzyme supplementation increased ruminal VFA concentration. Hr istov et al. (2000) found that enzyme supplementation produced a cubic effect on acetate and total VFA concentrations and these measures were elevated ( P < 0.01) after feeding, while Sutton et al. (2003) did not detect treatment ef fects on total VFA concentration. Jurkovich et al. (2002) found that en zyme supplementation increased VFA concentration in the rumen from about 32 da ys after calving leading to improved milk

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111 50 53 56 59 62 65 0246810 Time after feeding, hAcetic acid, molar % Control EC ETMR EF TS Figure 5.3 Effect of method of enzyme addition on ruminal acetic acid molar percentage Table 5.8 Effect of method of enzy me addition on ruminal acetic acid molar percentage Treatment Mean Control 58.2 EC 57.3 ETMR 54.8 EF 54.2 TS 57.8 P value Treatment effect < 0.01 Time effect < 0.05 Contrasts Control vs. EC 0.398 Control vs. ETMR 0.017 Control vs. EF 0.004 Control vs. TS 0.792

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112 18 19 20 21 22 23 24 25 0246810 Time after feeding, hPropionic acid, molar % Control EC ETMR EF TS Figure 5.4 Effect of method of enzyme addition on ruminal propionic acid molar percentage x: P = 0.071, P < 0.01 Table 5.9 Effect of method of enzyme a ddition on ruminal propionic acid molar percentage Treatment Mean Control 20.4 EC 20.3 ETMR 21.7 EF 20.6 TS 19.9 P value Treatment effect 0.534 Time effect 0.626 Contrasts Control vs. EC 0.970 Control vs. ETMR 0.170 Control vs. EF 0.506 Control vs. TS 0.922 ** x **

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113 9 10 11 12 13 14 15 16 0246810 Time after feeding, hButyric acid, molar % Control EC ETMR EF TS Figure 5.5 Effect of method of enzyme additi on on ruminal butyric acid molar percentage Table 5.10 Effect of method of enzyme addition on ruminal butyric acid molar percentage Treatment Mean Control 12.8 EC 11.5 ETMR 12.3 EF 11.8 TS 12.3 P value Treatment effect 0.164 Time effect 0.069 Contrasts Control vs. EC 0.278 Control vs. ETMR 0.866 Control vs. EF 0.956 Control vs. TS 0.960

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114 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 0246810 Time after feeding, hC2:C3 ratio Control EC ETMR EF TS Figure 5.6 Effect of method of enzyme add ition on ruminal acetic:propionic acid ratio P < 0.01 Table 5.11 Effect of method of enzyme add ition on ruminal acetic:propionic acid ratio Treatment Mean Control 2.9 EC 2.8 ETMR 2.5 EF 2.8 TS 2.9 P value Treatment effect < 0.01 Time effect 0.106 Contrasts Control vs. EC 0.796 Control vs. ETMR <0.01 Control vs. EF 0.025 Control vs. TS 0.984 ** **

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115 13 18 23 28 33 38 43 0246810 Time after feeding, hmg/100 ml Control EC ETMR EF TS Figure 5.7 Effect of method of enzyme a ddition on total VFA concentration X: P < 0.10 Table 5.12 Effect of method of enzyme addition on total VFA concentration Treatment Mean Control 28.5 EC 26.4 ETMR 22.8 EF 23.6 TS 21.1 P value Treatment effect 0.139 Time effect < 0.05 Contrasts Control vs. EC 0.466 Control vs. ETMR 0.041 Control vs. EF 0.192 Control vs. TS 0.410 x

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116 yield and butterfat concentrations. This was because the greater VFA concentration improved energy balance in the experimental cows as indicated by lower incidence of hyperketonaemia and acetoacetic acid and lo wer non-esterified fatty acid (NEFA) concentration in the blood. In this study, th e lower acetate:propionate ratio in cows fed ETMR, partly explains their lower HBA values and higher (P < 0.15) milk fat concentrations relative to cows fed the Control diets. The lower total VFA (P > 0.05) concentra tion of cows fed ETMR is al so attributable to lower acetate production and similar or greater propion ate production than that in cows fed the Control diet. Branched-chain VFA concentra tions were unaffected (P > 0.05) by enzyme treatment, except for greater isovaleric acid concentrations in cows fed EC, TS or EF rather than the Control diet (Figures 5.8 and 5.9, and Tabl es 5.13 and 5.14, respectively). In situ DM disappearance The kinetics of in situ feed DM disappearance of the experimental diets were unaffected (P > 0.05) by enzyme supplementation (Table 5.16) though the rate of degradation of TS tended to be greater (P = 0.107) than th at of the Control diets. Feng et al. (1996) observed that in situ disappearance of DM of cool-s eason grasses was not altered by treatment with cellulase, xylan ase and a mixture of both enzymes. Similarly, Adesogan et al. (2005) observed that in creasing the application rate of an esterase enzyme to bermudagrass hays did not affect the ferm entation rate of the hays using the gas technique, but enzyme treatment linearly incr eased (P < 0.05) the lag phase. Hristov et al. (1998) observed that the soluble, read ily degradable fraction of DM was greater

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117 0.0 1.0 2.0 3.0 4.0 5.0 0246810 Time after feeding, h Isobutyric acid, molar % Control EC ETMR EF TS Figure 5.8 Effect of method of enzyme a ddition on ruminal isobutyric acid molar proportion Table 5.13 Effect of method of enzyme a ddition on ruminal isobutyric acid molar proportion Treatment Mean Control 3.06 EC 2.66 ETMR 2.75 EF 2.85 TS 3.19 P value Treatment effect 0.827 Time effect 0.123 Contrasts Control vs. EC 0.449 Control vs. ETMR 0.418 Control vs. EF 0.317 Control vs. TS 0.212

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118 Figure 5.9 Effect of method of enzyme a ddition on ruminal isovaleric acid molar percentage Table 5.14 Effect of method of enzyme a ddition on ruminal isovaleric acid molar percentage Treatment Mean Control 2.99 EC 3.22 ETMR 3.13 EF 4.25 TS 3.71 P value Treatment effect 0.067 Time effect 0.057 Contrasts Control vs. EC < 0.05 Control vs. ETMR 0.192 Control vs. EF < 0.01 Control vs. TS < 0.05 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 0 2 4 6 8 10 Time after feeding, h Isovaleric acid, molar %, Control EC ETM R EF TS

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119 Table 5.15 Effect of method of enzyme add ition on kinetics of in situ feed DM disappearance in lactating Holstein Cows Parameters Treatments a, % b, % a + b, % P, % c, per h L, h Control 40.3 38.9 79.2 74.7 0.061 2.6 EC 36.2 38.5 74.7 72.5 0.058 3.6 ETMR 39.3 38.4 77.7 73.2 0.063 2.3 EF 36.2 36.6 72.8 59.0 0.079 4.2 TS 36.4 40.9 77.3 68.3 0.112 5.6 s.e.m. P values Treatment effect 0.544 0.936 0.721 0.685 0.336 0.589 Contrasts Control vs. EC 0.307 0.987 0.486 0.854 0.930 0.647 Control vs. ETMR 0.723 0.947 0.765 0.899 0.924 0.907 Control vs. EF 0.318 0.578 0.236 0.236 0.507 0.494 Control vs. TS 0.161 0.829 0.751 0.606 0.107 0.216 EC: enzyme applied to concentrate, ETMR: en zyme applied to the total mixed ration, EF: enzyme applied to forage at feeding, TS: en zyme-treated silage, DM: dry matter, a: soluble fraction, b: insoluble but potentially degradable fraction, a + b= total degradability, P: DM degraded at time t, c: rate of constant degradation, L: lag phase (P <0.05) in an enzyme-treated TMR, than in the untreated TMR (29.6 vs. 24.0%), but the insoluble potentially degrad able fraction was similar in both diets (56.6 vs. 56.2%). These results contradict those of Colombatto et al. (2001) who observed that Promote treatment did not increase the in vitro DM digestibility of corn silage that was incubated for durations of 0 to 30 h in buffered rumen fluid, but did increase DM digestibility after 48-h of incubation. The values for the soluble, readily degradable and potentially degradab le fractions in this study are higher than those of Hristov et al. (1998) probably due to differences in dietary composition. Promote supplementation was not effective at improving the degradation of the bermudagrass silage. However, the numeri cal improvements in degradation rate and lag phase of ETMR vs. Control are co nsistent with tendencies for lower

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120 acetate:propionate ratio, HBA and BUN concentrations, and greater milk fat and protein concentrations in cows fed ETM R instead of the Control diet. According to Russell and Wilson (1996) wh en ruminal pH falls below 6, fiber digestion declines for two reasons: firstly, th e enzymes necessary fo r fiber degradation do not function effectively, and secondly, the growth rate of fibrolytic bacteria declines markedly. Therefore the occurrence of periods of low (< 6) pH in cows on EC, EF and TS partly explain the lack of degr adation responses to enzyme addition. Conclusions Enzyme supplementation was ineffective (P > 0.05) at improving rumen function, in situ degradability and voluntary intake of the diets. Therefore, milk production and composition, FCM, BW gain, BCS and blood gl ucose and urea-N, were unaffected by enzyme supplementation. However, the ETM R treatment merits further study because compared to the Control treatment it resulted in numerically greater FCM and tendencies for greater concentrations of milk fat a nd milk protein, lower BHBA, BUN and lower acetate:propionate ratios.

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121 CHAPTER 6 GENERAL SUMMARY, CONCLUSI ONS AND RECOMMENDATIONS Many studies have shown that the nutritive va lue of tropical grasses is less than that of temperate perennial grasses, mainly due to differences in composition of cell wall polysaccharides and anatomy. Different chemi cal and biological treatments have been used to try to enhance the nutritive value of tropical forages. Biological treatments have been less successful than chemical treatme nts at increasing cell wall hydrolysis and improving nutritive value, but the hazards involv ed have limited the adoption of chemical treatment methods. The aim of these experi ments was to determine whether fibrolytic enzyme application can improve the nutritive value of tropical forages, and therefore enhance the performance of livestock fed such forages. Five experiments were conducted in the study. The objective of Experiments 1 and 2 was to evaluate the effect of applying ammonia or four fibrolytic enzymes at diffe rent rates on the nutritive value of two C4 grass hays. In the first experiment, NH3 or a fibrolytic enzyme (Promote) were evaluated, and in the second experiment, ammonia or three fibrolytic enzymes (Biocellulase X-20, Cattle-Ase and Biocellulase A-20) were evaluated. The effects of these treatments on chemical composition and DM, NDF and ADF digestibility of 12 week-regrowths of Coastal bermudagrass ( Cynodon dactylon ) hay (BE) and Pensacola bahiagrass ( Paspalum notatum ) hay (BA) were determined. The ammonia was applied at 40 g/kg DM and the enzymes were applied at 0 (Control) 0.5, 1 and 2 times the rates

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122 recommended by the respective manufacturers. The hays were stored for three weeks after enzyme application. The forages had low CP and high NDF, ADF, hemicellulose and lignin concentrations as usual in mature tropical grasses. Ammonia and Promote application decreased the NDF concentra tion of BA, however Promote treatment increased the NDF concentration of BE, probably because th is product contained other non-fibrolytic enzymatic activities. The reason why both tr eatments reduced the NDF fraction of BA but not BE is probably due to lower ADF a nd lignin concentrations of BA, as well as anatomical differences between the two grasse s. The hemicellulose concentration of BE was increased by Promote and NH3 treatment, while that of BA was decreased by NH3 treatment. Concentration of WSC was increased by Pr and NH3 treatment in BE hay, and NH3 treatment in BA hay. Therefore the res ponse to treatment was dependent on forage type. Ammoniation was more effective than enzyme treatment at hydrolyzing the fiber fraction of the forages. In both forages, CP concentration was unaffected by enzyme treatment, but higher values were observed in the NH3-treated hays, due to fixation of the supplemental N from the NH3. Promote and NH3 treatments increased the 6-h IVDMD, but only NH3 increased the 48-h IVDMD of both forages. Treatment with NH3 increased the 6 and 48-h IVNDFD and IVADFD of both fo rages. Promote treatment reduced most of the 6 and 48-h IVADFD values of the hays. These results indicate that the treatm ents had different effects on cell wall components. In BE, total cell wall content was unaffected by treatment, but the NDF fraction was hydrolyzed, thus in creasing the digestible fiber fraction. Whereas in BA, the NDF fraction was unaffected by treatment, but the total and digestible cell wall fractions

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123 were hydrolyzed into sugars. Ammoniat ion increased 6 and 48-h DM and fiber digestibility but Promote treatment did not. In Experiment 2, ammoniation decreased both ADF and NDF concentrations of BE and hence did not affect the hemicellulose concentration. All enzyme treatments decreased the NDF concentra tion of BE by decreasing the hemicellulose concentration whereas the only effect on BA cell walls was that X-20 treatment increased the hemicellulose concentration by decreasing ADF concentration. This confirms the forage specific-response to the treatments th at was observed in Experiment 1. The WSC concentration of BE hays had a tendency to be reduced by CA and A-20 treatment and unaffected by NH3 or X-20 treatment. However, that of BA hays was increased by X-20 treatment. None of th e treatments that hydrolyzed BE cell walls resulted in an increase in WSC concentrati on. This may be due to the relatively low WSC concentration of the forages and the conversion of hydrolyzed cell wall fragments into oligosaccharides and disaccharides that are not water soluble, and were therefore undetected in the WSC assay. Unlike enzy me treatment, ammoniation increased CP concentration of both hays as in Experiment 1. All treatments increased 6-h IVDMD of BE but only ammoniation and X-20 treatment tended to increase the 6-h IVDMD of BA. Enzyme X-20 increased the 48-h IVDMD of BE and BA hays, while CA and A-20 tended to have a similar effect. However, NH3 treatment was more effective than any of the enzymes at increasing the 48-h IVDMD of both hays. These results s uggest that all additive treatments can improve the initial and final phases of DM digestion in BE, but only NH3 and X-20 had similar effects on BA. The 6-h IVADFD of BE hay was improved by X-20, A-20 and

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124 NH3 treatment, but only NH3 treatment increased the 6-h IV ADFD of BA. Therefore, the increases in 6-h IVDMD due to X-20 and A-20 treatment were partly due to increases in 6-h IVADFD. Ammoniation was the only treat ment that increased the 48-h IVADFD in either of the hays. Ammoniation and X-20 treatments were th erefore more effective at disrupting lignocellulosic linkages in the forages than the other enzyme treatments. Nevertheless, treatment effects varied with forage type pr obably because of differences in the type and concentration of phenolic cross li nkages in the cell walls. Treatment with X-20 and NH3 increased the wash loss (a ) fraction of BE, but only NH3 treatment increased that of BA. Ammoni ation was more effective than X-20 at increasing the insoluble but potentially degr adable (b) fraction, the total degradable fraction (a + b) and the degrad ability (P) of both forages. The A-20-treated BE hays had lower b, a + b and P values th an Control (P < 0.05) and NH3-treated hays (P < 0.01). In BE hays ammoniation increased the lag phase and the c value and a + b fraction, while in BA it increased b, a + b and P. Therefore, Experiments 1 and 2 demonstrate that fibrolytic enzy mes had negligible effects on in situ DM degradation of C4 grass hays, though certain enzymes (X-20 and A20) did increase the ini tial and final phases of in vitro DM digestion. Such effects were more pronounced in BE than BA for reasons th at are not clear. Most of the enzymeinduced enhancements in DM digestibility were not attributable to increased fiber digestion; therefore other mechanisms such as increased substrate colonization by ruminal microbes may have been involved. A mmoniation was more effective than any of the enzyme treatments at improving the initia l and final phases of digestion, and these

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125 effects were due to increased fiber hydrol ysis. Ammoniation al so increased the CP concentration and in situ ru minal degradation of the C4 grass hays. Therefore although ammoniation is hazardous, it is a more e ffective method of improving the nutritive value of the hays than enzyme application. Furthe r studies need to be done to determine if enzyme application is more effective on le ss mature forages, which contain less lignin and ferulic acid cross linkages. Furthermore, potentially more potent enzymes such as ferulic acid esterases should al so be investigated as thes e are more likely to hydrolyze phenolic cross linkages that impede digestion. The objective of the Experiment 3 was to determine the eff ectiveness of the fibrolytic enzymes examined in Experiment s 1 and 2 on nutritive value of bermudagrass silage, because although bermudagrass is poorly digested, it is an important digestible fiber source in the rations of dairy cows in the southeastern US. A five-week regrowth of Tifton 85 bermudagrass was conserved for 145 days in mini-silos without treatment (Control), or after treatment w ith the same fibrolytic enzyme s evaluated in Experiments 1 and 2. The resulting silage was analyzed for chemical composition, in vitro digestibility, fermentation products and aerobic stability. The Tifton-85 bermudagrass used had low WSC and CP concentrations and high NDF and ADF concentration, at harvest. Theref ore it was representative of bermudagrass used for dairy production in the southeast. Promote was more effective than the rest of the enzymes at reducing the pH of the silage This was a consequence of improved cell wall hydrolysis in Pr-treated silages, which in creased the availability of sugars that are used as fermentation substrates by silage bact eria. Promote was also more effective at decreasing the ammonia-N levels in the silage than the other enzymes, which reveals that

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126 less proteolysis occurred during en siling in Pr-treated silages th an in other silages. This was probably due to a faster pH decline in Pr-treated silages, which probably reduced undesirable microbial activ ity during ensiling. Although yeast and mold counts were unaffected by enzyme type or rate, the numbers found were less than those (1 x 105 cfu/g) that predispose to rapid deterioration in silage. Except for X-20, all the other enzy mes increased the aerobic stability of the silages, and A-20 proved to be the most effec tive enzyme at improving the stability of the silage. Treatment with Promote and to a lesser extent X-20, was more effective at reducing DM losses and fiber concentration than the other enzymes. Therefore both products could potentially re sult in improved fiber utili zation in dairy cows fed bermudagrass silage. Promote treatment linear ly reduced acetic aci d concentration and numerically increased lactic aci d concentration in treated silages, s uggesting that this enzyme enhanced homolactic fermentation wh ich is more efficient than the typical heterolactic fermentation of bermudagrass silage. The Promote-treated silages also had gr eater 6-h and 48-h IVDMD values as well as greater 48-h IVNDFD and IVADFD than Cont rol silages. They also had greater 6-h and 48-h IVDMD, and 48-h IVNDF values than silages treated with the other enzymes. These results indicate that Promote wa s the most promising enzyme. Promote application reduced proteolysis during ensiling, improved the fermentation of bermudagrass and increased the nutritive value of the silage. Therefore feeding Promotetreated bermudagrass silage to dairy co ws may improve their performance. The objective of Experiments 4 and 5 was to determine the effects of applying Promote to bermudagrass at ensiling, or to different components of dairy cow diets at

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127 feeding on feed intake, milk production a nd composition, blood metabolites and ruminal fermentation parameters. A ration consisti ng of Tifton 85 bermudagrass silage, corn silage, and concentrate (35, 10 and 55% DM basis respectively) was fed ad libitum as a total mixed ration (TMR), twi ce daily. Cows were randomly assigned to the following five treatments: 1) Control (no enzyme additi on), 2) enzyme applied to the concentrate at feeding (EC), 3) enzyme applied to the TMR at feeding (ETMR), 4) enzyme applied to bermudagrass silage at feeding (EF), and 5) enzyme applied to bermudagrass at ensiling (TS). Cows received approximately 4 g enzyme/cow per day when added at feeding and 1.3 g/kg DM when added at ensiling. In Experi ment 4, thirty Holstein cows (129 days in milk, DIM) were used in an experiment with a partially balanced, completely randomized design consisting of two-28 d periods, with 14 d for adaptation and 14 d for sample collection. In Experiment 5, fi ve fistulated cows were fed the five same diets as in Experiment 4, for three consecutive 15-day periods. A completely randomized design consisting of 12 d for adaptation, 1 d for rumen fluid sampling and 2 d for in situ degradability analysis was used. Promote application at ensiling improved th e nutritive value of the treated silage, by increasing CP and non-fiber carbohydrate concentration and re ducing pH and fiber and ammonia-N concentration in agreement w ith the results of Experiment 3. Promote treatment also increased TDN concentration revealing the positive effect of this enzyme on the energy concentration of the silage. Enzyme treatment did not affect DMI. The digestibility of the feed fractions were unaffected by Promote treatment; however the DM digestibility of TS was approximately 3, 6, 3 and 6% higher than Contro l, EC, ETMR and EF diets, respectively.

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128 Milk production was also unaffected by treatm ent. Cows fed ETMR and EC rather than the Control diet tended to ha ve higher milk protein concentrations. Cow BW gain, BCS and plasma glucose concentration were unaffected by enzyme treatment. However, concentrations of HBA and BUN were lower in cows fed ETMR instead of the Control diet. This indicates that the TMR treatment decreased fat mobilization and increased the efficiency of protein ut ilization in the cows. In Experiment 5, ruminal pH was decreased by EC diet, and EF and TS diets had a similar tendency. Ruminal pH fell below 6 af ter 6-h of feeding in cows fed EC, EF and TS diets, This indicates that these cows may have experienced subclinical ruminal acidosis, which probably compromised their performance. Ruminal NH3-N, propionic acid and butyric acid concentrations were unaffected by enzyme treatment, but ruminal acetic acid concentration was lower in cows fed ETMR and EF than those fed Control diets. Consequently, acetate: propionate ratio was lower in cows fed ETMR diets rather than Control diets. This indicates that the ETMR diet promoted a more efficient fermentation in the rumen, which was probably due to a better balance in ruminal supply of readily fermentable carbohydrates and rumen degradable protein. Enzyme supplementation did not affect the kinetics of in situ DM disappearance of the experimental diets. However the rate of degradation of TS diet tended to be greater than that of the Control diets. The ETMR di et also had a numerically greater degradation rate and numerically shorte r lag phase than the Cont rol diet, which support the numerically higher DM intake of the cows fe d the ETMR diet. Compared to the Control diet, the numerical improvements in degr adation rate and lag phase of ETMR are consistent with numerical improvements in DMI and FCM, lower HBA and BUN

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129 concentrations and greater milk fat and milk protein concentrations in cows fed ETMR instead of the Control diet. Therefore this study shows that enzyme supplementation did not improve voluntary intake, BW gain, BCS, blood gl ucose, milk production and in situ degradability. However cows fed the ETMR diet had numeri cally greater DMI, FCM, tended to have greater milk fat and protein concentrations and lower BHBA, BUN and rumen acetate to propionate ratios. Therefore the ETMR treatme nt was more effective than any of the other treatments. Future experiments shoul d validate the potential of this mode of Promote application with early lactation co ws which have greater energy requirements and are therefore more likely to respond to enzyme supplementation than cows used in this study. Such experiments should be conducte d for the entire lactation to establish the stage of the lactation that be nefit the most from enzyme application. Furthermore, a greater number of cows per treatment should be used to facilita te identification of treatment effects and to allow more definitive conclusions to be drawn. Fibrolytic enzyme application to mature tropical grass hays di d not prove to be effective at increasing nutritive value. This is probably due to the high lignin concentration of the grasses, and the inabi lity of the enzymes to hydrolyze the ferulate linkages in the hays. Although Promote a pplication was effective at improving the nutritive value of bermudagrass ensiled in mini silos it had only a fe w beneficial effects on the performance of cows fed diets c ontaining the enzyme. Therefore these experiments dont support the use of commerc ial fibrolytic enzymes for enhancing the performance of dairy cows.

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130 APPENDIX A ABSTRACT FOR CHAPTER 3 In the first of two experiments, the effect of applying NH3 or a fibrolytic enzyme complex (Promote, Pr, Cargil, Minnentoka, MN) on the chemical composition, DM, NDF, ADF and hemicellulose concentrations an d digestibility of two tropical grass hays was measured. In the second experiment, the effects of applying ammonia or three fibrolytic enzymes (Biocellulase X-20 (X -20) (LodeStar, IL, USA), Cattle-Ase (CA) (Loveland Industries Inc, Greel ey, CO, USA) and Biocellula se A-20 (A-20) (LodeStar, IL, USA) on the same variables as in the previous experiment were measured. The forages were 12 week-regrowth of Coastal bermudagrass hay ( Cynodon dactylon ) (BE) and Pensacola bahiagrass hay ( Paspalum notatum ) (BA). The ammonia was applied at 40 g/kg DM and the enzymes were applied at 0 (Control) 0.5, 1 and 2 times the rates recommended by the respective manufacturers. The hays were stored for three weeks after enzyme application. Calculations of IVDMD, IVNDFD, and IVADFD were made after digesting the hays in buffered rumen fluid for 6 or 48-h in two ANKOMII Daisy Incubators. Treatments were analyzed using a 2 x 4 factorial design w ith 3 replicates per treatment for each digestion period. Low CP and high NDF, ADF, hemicellulose and lignin concentrations were observed in both forages. Bahiagrass had lower (P < 0.01) NDF, ADF and lignin concentration than BE. In Experiment 1, Pr and NH3 decreased (P < 0.01) the NDF concentration of BA. Both treatments decreased (P < 0.01) ADF concentration of BE but not BA. The hemice llulose concentration of BE was increased (P < 0.01) by enzyme (quadratic) and NH3 treatment, while that of BA was decreased (P

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131 < 0.01) by NH3 treatment. Concentration of WSC was greater (P < 0.01) in BA than in BE hays and that of BA was increased (P < 0.01) by Pr and NH3 treatment (P < 0.01). The CP concentration of ammoniated hays was consistently greater (P < 0.01) than those of enzyme treated or Control hays. Promote and NH3 treatments increased the 6-h IVDMD of both hays, but only NH3 increased their 48-h IVDMD. Treatment with NH3 increased the 6-h and 48-h I VNDFD and IVADFD in both forage s. Pr treatment reduced most of the 6 and 48-h IVADFD values of the hays. In Experiment 2, NH3 treatment decreased both ADF and NDF fractions of BE and hence did not affect the hemicellulose fraction. All enzyme treatments decreased the NDF concentration of BE. The WSC concentration of BA hays was increased (P < 0.05) by X-20 treatment. All treatments increased 6-h IVDMD of BE but only NH3 (P < 0.01) increased th e 6-h IVDMD of BA. Enzyme X-20 increased the 48-h IVDMD of BE (P < 0.05) and BA (P < 0.01) hays, while CA and A-20 tended (P < 0.08) to have similar effects. Ammoniation increased the 6 and 48-h IVNDFD (P < 0.01) of BE and 48-h IVNDFD of BA (P < 0.01). The 6-h IVADFD of BE hay was improve d (P < 0.01) by X-20, A-20 and NH3 treatment. Treatment with X-20 (linear, P < 0.05) and NH3 (P < 0.01) increased the wash loss (a) fraction of BE, but only NH3 treatment increased that of BA. Ammoniation was more effective than X-20 at increasing (P < 0.01) th e insoluble but potentially degradable (b) fraction, the total degradable fr action (a + b) and the degradab ility (P) of both forages. The A-20-treated BE hays had lower b, a + b and P values than Control (P < 0.05) and NH3-treated hays (P < 0.01). In BE hays, ammoniati on increased the lag phase and the c value and a + b fraction, while in BA it increased b, a + b and P. This work demonstrates that fibrolytic enzymes had negligible effects on in situ DM degradation of C4 grass hays,

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132 though certain enzymes (X-20 and A-20) did in crease the initial and final phases of in vitro DM digestion. Ammoniation was more eff ective than any of the enzyme treatments at improving the initial and fina l phases of digestion, due to increased fiber hydrolysis.

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133 APPENDIX B ABSTRACT FOR CHAPTER 4 The aim of this study was to determine if th e nutritive value and aerobic stability of bermudagrass ( Cynodon dactylon ) silage can be improved by addition of proprietary, exogenous cellulase/hemicellulase enzyme preparations at ensiling. A five-week regrowth of Tifton 85 bermudagrass was conser ved without treatment (Control), or after treatment with exogenous fibrol ytic enzymes including Promote NET (Cargill Corp. St. Louis, MO), Biocellulase X-20 (LodeStar, IL, USA), Biocellulase A-20 (LodeStar, IL, USA), and Enzyme CA. The respectiv e enzymes were applied at half the recommended rate, the recommended rate or twice the recommended rate corresponding to 0.65, 1.3 and 2.6 g/kg DM, 7.3, 14.5 and 29 mg/kg DM, at 7.3, 14.4 and 29 mg/kg DM 89, 178 and 356 mg/kg DM, for Promote, X20, A-20 and CA, respectively. The enzymes were sprayed on the bermudagrass at ensiling, and not a dded at feeding as suggested by the manufacturers in order to test the objectives of the study. Six replicates of 1 kg of chopped (5 cm) forage were ensile d for 145 days in 2.8 L mini silos. Three silos per treatment were used for chemical analysis and three for aerobic stability monitoring. The silage juice was analyzed for organic acids, pH, water soluble carbohydrates (WSC), ammonia-N and soluble N. Freeze-dried samples were analyzed for crude protein (CP), NDF and ADF. In vitro digestibility of DM (IVDMD), NDF (IVNDFD) and ADF (IVADFD) were determined after digesting the silages in bufferedrumen fluid for 6 or 48-h in two ANKOMII Daisy Incubators. Compared to the other silages, those treated with Pr had lowe r DM losses, and lower pH and ammonia-N

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134 concentration than Control silages. Residua l WSC concentration was greater in Pr (P < 0.01) and CA (P < 0.05) treated silages than in Control silages, and greater (P < 0.01) in Pr-treated silages than CA treated sila ges. Compared to Control silages, NDF concentration was lower in silages treated w ith Pr (P < 0.01), X-20 (P < 0.05), and CA (P < 0.05) while ADF concentration was lower (P < 0.05) in silages treated with Pr, X-20 and A-20. Nevertheless, Pr-treated silage s contained lower (P < 0.01) ADF and NDF concentrations than silages treated with th e other enzymes. Enzyme-treated silages contained less (P < 0.05) acetic acid than Cont rol silages, and Pr-treated silages had the lowest concentrations of acetic acid. Aerobic stability (A S) and microbial counts were unaffected by treatment. The 6-h IVDMD was increased (P < 0.01) by treatment with Pr and A-20, however only Pr increased (P < 0.01) the IVDMD and I VNDFD at 48-h. The 48-h IVADFD (P < 0.05) was also increased by treatment with Pr, CA and A-20. These results show that when applied at ensiling, certain fibrolytic enzymes, particularly Promote can improve the digestibility, fermentation and aerobic stability of bermudagrass silage.

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135 APPENDIX C ABSTRACT FOR CHAPTER 5 Two experiments were carried out to invest igate the effect of applying a cellulase enzyme (Promote ; Cargill; Minnetonka, MN ) on the performance of lactating dairy cows. A ration consisting of Tifton 85 bermuda grass silage, corn silage, and concentrate (35, 10 and 55% DM basis respectively) was fed ad libitum as a total mixed ration (TMR) twice daily. Cows were randomly assigned to the following five treatments: 1) Control (no enzyme addition), 2) enzyme app lied to the concentrate at feeding (EC), 3) enzyme applied to the TMR at feeding ( ETMR), 4) enzyme applied to bermudagrass silage at feeding (EF), and 5) enzyme applie d to bermudagrass at ensiling (TS). Cows received approximately 4 g enzyme/cow per day when added at feeding and the application rate at ensiling was 1.3 g/kg DM. In Experiment 1, thirty Holstein cows (129 days in milk, DIM) were used in a partially balanced, completely randomized design consisting of two-28 d periods, with 14 d for adaptation and 14 d for sample collection. Voluntary DMI, digestibility of DM, NDF and CP, milk producti on and composition and blood glucose were not affected (P>0.05) by enzyme supplementation. Cows fed ETMR had lower (P <0.01) blood -hydroxybutyrate concentration and tended to have greater milk fat (P=.073) and protein (P=0.081) concentrations and lower blood urea-N concentration (P=0.123) than cows fed the Contro l diet. In Experiment 2, five fistulated cows were fed the five same diets as in Experiment 1, for three consecutive 5-day periods. A completely randomized design c onsisting of 12 d for adaptation, 1 d for rumen fluid sampling and 2 d for in situ degradability analysis was used. Ruminal pH,

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136 acetic, propionic and butyric acid molar proportions were unaffected by enzyme treatments. The kinetics of in situ DM disappearance were also unaffected by enzyme treatment. Therefore applying the enzyme to the TMR reduced fat mobilization and tended to increase milk fat and protein cont ents and decreased B UN concentration and ruminal acetate:propionate ratio. However, other animal performance and ruminal fermentation parameters were unaffected by fibrolytic enzyme supplementation.

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149 BIOGRAPHICAL SKETCH The author was born in Ciuda d Ojeda, Venezuela. He got his B.S. in Animal Science from the University Rafael Urdaneta in Maracaibo, Venezuela. After working for two years as a Farms Supervisor for a da iry company and as Di rector and Technical Consultant for beef and dairy farmers for two more years, he pursued an M.S. program in Animal Nutrition at the University of Zulia During the last two years of his M.S. program and one year afterwards he was em ployed as an Assistant Professor in the Maracaibo Technological College Subsequently he joined Protinal, as Manager of Nutrition and almost two years later, he b ecame employed as an Associate Professor in the Veterinary School of the Univ ersity of Zulia. This Univer sity is his sponsor and they have covered all of the expenses for his P h.D. program in Ruminant Nutrition in the Animal Science Department of the University of Florida.


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Title: Effect of fibrolytic enzymes on the nutritive value of tropical grasses and dairy cattle performance
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Title: Effect of fibrolytic enzymes on the nutritive value of tropical grasses and dairy cattle performance
Physical Description: Mixed Material
Copyright Date: 2008

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EFFECT OF FIBROLYTIC ENZYMES ON THE NUTRITIVE VALUE OF
TROPICAL GRASSES AND DAIRY CATTLE PERFORMANCE















By

DERVIN BARTOLO DEAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005



























Copyright 2005

by

Dervin B. Dean
































To my lovely wife Domenicchella, and my dear kids, Sheryll, Homer, and Stephanie















ACKNOWLEDGMENTS

I would like to give thanks to my supervisory committee chair (Dr. Adegbola

Adesogan) for his valuable guidance during my Ph.D. program and to the rest of my

committee (Dr. Charles Staples, Dr. Lynn Sollenberger, Dr. Ramon Littell and Dr. Ann

Wilkie) for their time and dedication to my research activities.

I thank my sponsor (the University of Zulia) for covering the expenses required to

complete my program, the Department of Animal Sciences of the University of Florida,

for giving me the opportunity to improve my knowledge and the crew of the Dairy

Research Unit for their help during my in vivo trial.

I would also like to thank to all of my lab supervisors and partners (Nathan

Krueger, Sam-Churl Kim, Kathy Arriola, Susan Chikagwa-Malunga, John Funk, Jamie

Foster, Nancy Wilkinson, Pam Miles, Max Huisden, Alvin Boning, Bruno Amaral,

Ashley Hughes, Tolu Ososanya, Mustapha Salawu and Sergei Sennikov) for their help

during my field and lab activities. I thank Dr. Dario Colombatto for helping me to

determine the enzyme activities.

Finally I thank my friends (German Portillo, Maria Padua, Tomas Belloso, Andres

Kowalski, Carlos Lucena, Lucia Holsthausen, Carlos Rodriguez, and Carlos Vargas) for

their support during the last five years.
















TABLE OF CONTENTS





A C K N O W L E D G M E N T S ................................................................................................. iv

TA B LE O F C O N TEN T S................................................................. ........................... v

L IST O F TA B LE S ....................................................................... ..... ........ viii

LIST OF FIGURES ......... ........................................... ............ xi

LIST O F A BREV IA TION S .................................................. ............................... xii

A B STR A C T ..................... ................................... ........... ................. xiii

CHAPTER

1 INTRODUCTION ............... ................. ........... ................. ... ..... 1

2 LITERA TURE REVIEW .......................................................... ..............4

Cell Wall Differences between Tropical and Temperate Forages.............................4
Methods for Improving Forage Nutritive Value..................................... ................6
Ammoniation ...................................... ................................... ......... 7
Am m onia Treatm ent M ethods................................................................. ...... 10
Animal Response to Feeding Ammoniated Forages ...........................................11
Feed Enzym es ................. .................... ................................. ..........13
Definition, Types, and Classification .............. ............................................. 13
Commercial Exogenous Fibrolytic Enzymes ............................................... 17
M ode of Enzym e Action .................................. .....................................18
Factors Affecting Enzyme Action .............................................. ...............19
Enzyme Stability in the Digestive Tract............... ............................................21
Methods of Determining Enzyme Activity ...............................................24
Effect of Enzyme Treatment on Chewing Behavior .........................................26
Effect of Enzyme Treatment on the Ruminal Microbial Population...................27
Effect of Enzyme Treatment on Ruminal Fibrolytic Capacity.........................29
Effect of Enzyme Treatment on Fiber Concentration Before Ingestion..............31
Effect of Enzyme Treatment on DM and Fiber Digestibility Post Ingestion......32
Effect of Enzyme Treatment on Silage Fermentation .................................34









Effect of Enzyme Treatment on Hay Nutritive Value........................................35
Effect of Enzyme Treatment on Animal Performance.............. ................. 36
Effect of Enzyme Treatment on Blood Metabolites................ .............. ....42
Effects of Combining Enzyme and Chemical Treatments ................................42

3 EFFECT OF TREATMENT WITH AMMONIA OR FIBROLYTIC ENZYMES
ON THE NUTRITIVE VALUE OF HAYS PRODUCED FROM TROPICAL
G R A SSE S ..........4. .. ............... ........................... ................ 46

Introduction ............. .... ... ......... .............. ............................. 46
M materials and M methods ....................................................................... ..................47
E nzym e A application ........................ .. ...................... .. .. .... ........... 47
L laboratory A analysis .......................... .................. ... ...... .. .... ...........48
Statistical A analysis ...................................... ................. .......... 50
R results and D discussion .......................................... ......... ........ .................... 51
Chemical Composition of Tropical Hays........................................................51
Effect of Promote and Ammoniation on Chemical Composition in
E xperim ent 1 ........................................................................52
Effect of Promote and Ammonia Application on in vitro DM, NDF, and ADF
D igestibility in Experim ent 1.................. ...................... ............... ... 53
Effect of Fibrolytic Enzyme and Ammonia Application on Chemical
Concentration of C4 Forages in Experiment 2.............................................57
Effect of Enzyme Treatment and Ammoniation on in vitro DM, NDF, and
A D F D igestibility in Experim ent 2.................................................................61
Effect of Enzyme Treatments and Ammoniation on in situ DM Degradation....66
C o n clu sio n s..................................................... ................ 6 9

4 EFFECT OF FIBROLYTIC ENZYMES ON THE FERMENTATION
CHARACTERISTICS, AEROBIC STABILITY, AND DIGESTIBILITY OF
BERM UDAGRASS SILAGE .............................................................................71

Introduction .............. ..... ... .............. .................................. 71
M materials and M methods ....................................................................... ..................72
E nzym e A application ........................ .. ...................... .. .. .... ........... 72
L laboratory A analysis .......................... .................. ... ...... .. .... ...........74
Statistical A analysis .......................... .......... ............... .... ..... .. 76
R results and D discussion ...................... .. ........ .... ........ .. ..... ................. 77
Chemical Composition of Freshly-treated Bermudagrass before Ensiling .........77
Chemical Composition, Microbial Counts and Aerobic Stability of
B erm udagrass Silages ............................................................................... 77
Organic Acid Concentration of Bermudagrass Silages................. ..............84
In vitro DM and Fiber Digestibility of Bermudagrass Silages ............................86
C o n c lu sio n s..................................................... ................ 8 8









5 EFFECT OF METHOD OF DIETARY ADDITION OF A FIBROLYTIC
ENZYME ON THE PERFORMANCE OF LACTATING DAIRY COWS ............90

In tro d u ctio n ............................ ....................................... ................ 9 0
M material and M methods ........................................... ......................... ............... 91
Diets ............... .. ..... ..................................................... 92
Sam ple Collection and A nalysis...................................... ........................ 94
Statistical A analysis .......................... .......... ............... .... ..... .. 97
R results and D discussion ................. ................................ ........ .........................98
Chemical Composition of the Dietary Ingredients.................... ..................98
V voluntary Intake .......... .... .......... ................... ................. ........ .......... ....99
Effect of Promote on Milk Production and Composition ..............................100
Body Weight Gain and Body Condition Score ...........................................104
Blood Glucose, Urea-N and P-Hydroxybutyrate..............................................105
Ruminal pH and Concentration of VFA and NH3-N......................................106
In situ D M disappearance .... ... ..................................................................... 116
C o n c lu sio n s......................................................................................................... 12 0

6 GENERAL SUMMARY, CONCLUSIONS AND RECOMMENDATIONS .........121

APPENDIX

A ABSTRACT FOR CHAPTER 3 .............. ............ ............................... .......130

B ABSTRACT FOR CHAPTER 4 ...................................... .............................. .. 133

C ABSTRACT FOR CHAPTER 5 .............. ............ ............................... .......135

L IST O F R E FE R E N C E S ......................................................................... .......... .......... 137

BIOGRAPHICAL SKETCH .............. ................................................ 149
















LIST OF TABLES

Table p

2.1 Some fibrolytic enzyme-producing microorganisms and the enzymes they
produce ............... ... ..... .. ................ ............................. 15

2.2 Effect of spraying enzymes onto feeds prior to feeding on milk production in
recent studies .................. ......... ............... .... ........... .............38

3.1 Actual enzym e application rates used ........................................... ............... 49

3.2 M anufacturer-stipulated enzyme activities. .................................. .................49

3.3 Chemical composition of the untreated hays ............. ............................................53

3.4 Effect of Promote or ammonia treatment on chemical composition (% DM) of
tropical grass hay s ......................................................................54

3.5 Effect of Promote or ammonia application on the IVDMD of tropical grass hays..55

3.6 Effect of Promote or ammonia application on the IVNDFD of tropical grass
h ay s ............................................................................. 5 6

3.7 Effect of Promote or ammonia application on the IVADFD of tropical grass
h ay s ............................................................................. 5 7

3.8 Effect of fibrolytic enzyme or ammonia application on the NDF, ADF and
hemicellulose concentrations (%) of tropical hays ...............................................58

3.9 Effect of fibrolytic enzyme or ammonia application on the WSC and CP
concentrations (% ) of tropical hays............... ............. .................................. 59

3.10 Effect of fibrolytic enzyme or ammonia application on the IVDMD (%) of
tro p ic al h ay s ....................................................... ................ 6 3

3.11 Effect of fibrolytic enzyme or ammonia application on the IVNDFD (%) of
tro p ic al h ay s ....................................................... ................ 6 4

3.12 Effect of fibrolytic enzyme or ammonia application on the IVADFD (% of DM)
of tropical hays .................................................................... .........65









3.13 Effect of X-20 or ammonia application on the in situ kinetics of DM
disappearance of bermudagrass and bahiagrass ..................................................67

3.14 Effect of A-20 or ammonia application on the in situ kinetics of DM
disappearance of bermudagrass and bahiagrass ..................................................68

4.1 Chemical composition of bermudagrass forages before ensiling (g/kg DM). ........78

4.2 Effect of fibrolytic enzymes on pH, concentrations of DM (g/kg) and ammonia-
N (g/kg total N), DM losses (%), microbial counts (log cfu /g) and aerobic
stability (h) of bermudagrass silage. ............................................. ............... 79

4.3 Effect of fibrolytic enzymes on the chemical composition of bermudagrass
silage (g/kg D M ). ................................................... ................. 80

4.4 Effect of fibrolytic enzymes on the organic acid concentration (g/kg DM)of
bermudagrass silage. ................................... .. .......... .. ............. 85

4.5 Effect of fibrolytic enzymes on in vitro digestibility of DM (g/kg), NDF, ADF
and hemicellulose (Hem) in bermudagrass silage after 6 or 48-h of digestion
(g/kg D M )................................................................................................87

5.1 Ingredient and chemical composition of the basal untreated diet. ...........................93

5.2 Chemical composition of the enzyme-treated and untreated forages and
concentrate (% DM ) (n= 4 replicates per mean)....................................................101

5.3 Effect of method of enzyme addition on diet digestibility and voluntary intake... 102

5.4 Effect of method of enzyme addition on milk production and composition.......... 103

5.5 Effect of method of enzyme addition on body weight and condition score, and
blood m etabolites ......................... ........................ .. .. ....... .... ........... 106

5.6 Effect of method of enzyme addition on ruminal fluid pH.................................. 108

5.7 Effect of method of enzyme addition on ruminal NH3-N concentration ...............109

5.8 Effect of method of enzyme addition on ruminal acetic acid molar percentage.... 111

5.9 Effect of method of enzyme addition on ruminal propionic acid molar
percentage............................................................... ... .... ......... 112

5.10 Effect of method of enzyme addition on ruminal butyric acid molar percentage.. 113

5.11 Effect of method of enzyme addition on ruminal acetic:propionic acid ratio........114

5.12 Effect of method of enzyme addition on total VFA concentration ........................115









5.13 Effect of method of enzyme addition on ruminal isobutyric acid molar
proportion ............................................................... ... .... ......... 117

5.14 Effect of method of enzyme addition on ruminal isovaleric acid molar
percentage............................................................... ... .... ......... 118

5.15 Effect of method of enzyme addition on kinetics of in situ feed DM
disappearance in lactating Holstein Cows...................................................... 119
















LIST OF FIGURES


Figure page

5.1 Effect of method of enzyme addition on ruminal fluid pH................................ 108

5.2 Effect of method of enzyme addition on ruminal NH3-N concentration............. 109

5.3 Effect of method of enzyme addition on ruminal acetic acid molar percentage.... 111

5.4 Effect of method of enzyme addition on ruminal propionic acid molar
percentage............................................................... ... .... ......... 112

5.5 Effect of method of enzyme addition on ruminal butyric acid molar percentage.. 113

5.6 Effect of method of enzyme addition on ruminal acetic:propionic acid ratio........ 114

5.7 Effect of method of enzyme addition on total VFA concentration ........................115

5.8 Effect of method of enzyme addition on ruminal isobutyric acid molar
proportion ............................................................... ... .... ......... 117

5.9 Effect of method of enzyme addition on ruminal isovaleric acid molar
percentage............................................................... ... .... ......... 118














LIST OF ABREVIATIONS


A-20 Biocellulase A-20
ADF acid detergent fiber
ADFD acid detergent fiber digestibility
BA bahiagrass
BCS body condition score
BE bermudagrass
BHBA beta hydroxybutyrate
BUN blood urea nitrogen
BW body weight
BWG body-weight gain
CA Cattle-Ase
CP crude protein
CPP crude protein production
CPD crude protein digestibility
DM dry matter
DMD dry matter digestibility
DMI dry matter intake
FCM fat-corrected milk
FP fat production
Glc glucose
IVADFD in vitro acid detergent fiber digestibility
IVDMD in vitro dry matter digestibility
IVNDFD in vitro neutral detergent fiber digestibility
MCF milk crude fat
MCP milk crude protein
NDF neutral detergent fiber
NDFD neutral detergent fiber digestibility
NFC non-fiber carbohydrates
NH3 ammonia
NH3-N ammonia nitrogen
Pr Promote
RIN relative intake
SCC somatic cell counts
TDN total digestible nutrients
TMR total mixed ration
VFA volatile fatty acids
WSC water soluble carbohydrates
WSN water soluble nitrogen
X-20 Biocellulase X-20














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


EFFECT OF FIBROLYTIC ENZYMES ON THE NUTRITIVE VALUE OF
TROPICAL GRASSES AND DAIRY CATTLE PERFORMANCE
By

Dervin Bartolo Dean

December 2005

Chair: Adegbola T. Adesogan
Major Department: Animal Sciences

Five experiments were conducted to determine whether the nutritive value of hay or

silage made from tropical grasses and animal performance can be improved by addition

of fibrolytic enzymes. Experiments 1 and 2 determined the effect on the digestibility of

Coastal bermudagrass and Pensacola bahiagrass hays of applying NH3 or four fibrolytic

enzymes: Promote (Pr), Biocellulase X-20 (X-20), Biocellulase A-20 (A-20), and Cattle-

Ase (CA) at 0, 0.5, 1, and 2x the rates recommended by the respective manufacturers.

Biocellulase X-20 and A-20 improved the 6-h and 48-h digestion of the forages, but

ammoniation was more effective. In Experiment 3, Tifton 85 bermudagrass was ensiled

without treatment (Control), or after treatment with the enzymes used in Experiments 1

and 2. Compared to Control silages, Promote-treated silages had lower pH and dry

matter (DM) losses, and lower concentrations of ammonia-N, neutral (NDF) and acid

detergent fiber (ADF), and greater concentrations of residual water soluble carbohydrates

(WSC), in vitro DM, NDF, and ADF digestibility. The other enzymes also increased









fiber hydrolysis, but did not improve indices of fermentation quality. Therefore, Promote

was the most promising enzyme for improving the fermentation and nutritive value of

silages. Experiments 4 and 5 tested the effect of Promote on the performance of thirty

Holstein lactating dairy cows fed a ration consisting of bermudagrass silage, corn silage,

and concentrate ad libitum for two 28-d periods. Treatments were the following: Control,

enzyme applied at ensiling to the bermudagrass (TS), or at feeding to the concentrate

(EC), the total mixed ration (ETMR) or the bermudagrass silage (EF). Voluntary intake,

apparent digestibility, milk production, and blood glucose concentration were unaffected

by treatment. Cows fed ETMR tended to have lower beta hydroxybutyrate and blood

urea-N concentrations and greater milk fat and protein concentrations than cows fed the

control diet. In Experiment 5, five ruminally fistulated lactating cows were fed the same

diets as in Experiment 4 for three, 15-d periods. Ruminal pH was decreased by feeding

EC, whereas acetate:propionate ratios were reduced by feeding ETMR. In situ DM

disappearance was unaffected by enzyme treatment. These experiments suggest that

ETMR was the most promising treatment.














CHAPTER 1
INTRODUCTION

Forages represent the most important, cost effective feed resource in ruminant

nutrition (Jung and Allen, 1995). However, the relatively low quality of tropical forages

militates against their use as the sole feed for actively growing or high-performing

ruminants. Several attempts have been made to improve forage quality genetically or by

chemical or biological treatments. One of the most important goals in this regard is to

improve the fiber digestibility of the forages. Some chemical and biological treatments

have been effective at achieving this objective.

Ammoniation is one of the most studied chemical treatments for enhancing fiber

digestibility and several reports have described its effectiveness for improving both

forage quality and animal performance. Ammoniation increases forage crude protein

(CP) concentration and substantially reduces the concentration of neutral detergent fiber

(NDF) in forages. Most of the loss of NDF is due to hydrolysis of hemicellulose, though

the disruption of chemical linkages between lignin and hemicellulose also occurs (Weiss

and Underwood, 1995; Barrios-Urdaneta and Ventura, 2002). Additional benefits of

ammoniation include reduced yeast and mold growth, and less aerobic deterioration of

high moisture hay and silage (Woolford and Tetlow, 1984; Bates et al., 1989b).

Consequently, feeding ammoniated forage often results in increased daily gain and dry

matter (DM) intake in beef cattle (Vagnoni et al., 1995; Brown and Pate, 1997).

However, the use of ammonia for improving forage quality has been limited because of

the corrosive nature of the alkali which can be hazardous to operators and their









equipment (Lalman et al., 2005). Ammoniation also contributes to N importation to

farms and therefore represents a small but important threat to air quality because of its

contribution to surface water eutrophication and nitrate contamination of ground water

(Ishler, 2005). In developing countries, the labor required for ammoniation and problems

of delivering anhydrous ammonia have limited adoption of the technique.

Fibrolytic enzyme application is one of the most studied biological treatments for

improving forage quality and animal performance. Such enzymes have been effective at

improving the utilization of a wide range of diets containing roughages (Rode and

Beauchemin, 1998) due to improved fiber hydrolysis (Colombatto et al., 2003b) which

often results in increased digestibility (Christensen, 1997; Rode et al., 1999) and

voluntary intake (Pinos-Rodriguez et al., 2002). Nevertheless, another study has shown

that exogenous enzymes did not consistently improve forage utilization by ruminants

(Lewis et al., 1999). This inconsistency is attributable to several factors such as

differences in enzyme type and activity, treatment duration, application method, diet

composition and level of animal performance.

Additional factors that may be implicated include suboptimal prevailing

temperature and pH for enzyme action, presence of inhibitors or absence of cofactors and

inadequate enzyme to substrate ratios. Nevertheless, feed enzymes have been used to

improve the utilization of a wide range of diets containing legumes, grasses, haylage,

straw and other feedstuffs (Beauchemin et al., 2003). The mode of action of these

enzymes in ruminants is not fully understood. In addition to increasing fiber hydrolysis,

they can enhance feed colonization by increasing the numbers of ruminal fibrolytic

microbes (Morgavi et al., 2000; Nsereko et al., 2000a) and thereby increase the rate of









degradation of feeds in the rumen (Yang et al., 1999). Additionally, modes of action

include improved palatability (Adesogan, 2005), changes in gut viscosity (Officer, 2000),

and changes in the site of digestion (Rode and Beauchemin, 1998; Hristov et al., 2000).

Most of the studies of fibrolytic enzyme treatment of ruminant feeds have been

done using feedstuffs grown under temperate conditions. Little is known about their

effectiveness on tropical or subtropical forages which tend to be less digestible. Yet there

is greater scope for improving the quality of tropical forages than there is for temperate

forages due to the greater nutritive value of the latter. The aim of this series of

experiments was to evaluate the effect of ammonia and proprietary fibrolytic enzyme

application on the nutritive value of tropical forages and animal performance.














CHAPTER 2
LITERATURE REVIEW

Cell Wall Differences between Tropical and Temperate Forages

Even under the intensive concentrate feeding systems of ruminant animal

production in developed countries, forages continue to represent the single most

important feed resource; however, cell-wall concentration and digestibility limit the

intake potential and energy availability from forage crops in beef and dairy production

systems (Jung and Allen, 1995). Depending on the stage of maturity of the plant, cell

walls represent between 30 and 80% of plant dry matter (DM) in grasses so that under

some circumstances (high forage diets) the bulk of carbohydrate fermented to

metabolizable volatile fatty acids (VFA) by rumen micro-organisms may be derived from

cell wall polysaccharides and in senescent grasses, almost all fermentable carbohydrate

arises from wall polysaccharides (Stone, 1994). Cellulose is the predominant wall

polysaccharide. The cellulosic microfibrils are embedded in a matrix composed of non-

cellulosic polysaccharides and some proteins. The major matrix polysaccharides in

grasses are glucuronoarabinoxylans, together with smaller amounts of heteroglucans

(xyloglucans) and glucans (Stone, 1994).

The main reason why the digestibility of tropical grasses is less than that of

temperate perennial grasses is because of differences in cell wall composition. There are

differences in polysaccharide composition in cell walls of different types and also

considerable quantitative differences in components, e.g., mesophyll cells which are more

abundant in temperate forages are relatively cellulose-rich (Gordon et al., 1977, cited by









Stone, 1994) and readily degraded by rumen micro-organisms; but tropical grasses are

rich in lignified and secondarily thickened cells of xylem (Akin and Burdick, 1975).

Differences in leaf anatomical structure between Panicoid tropical and Festucoid

temperate grasses associated with the C4 and C3 photosynthetic pathways have been

known to botanists for many years (Wilson and Hacke, 1987). These researchers

determined that leaves of C4 (tropical) grasses consistently had less mesophyll and more

of the less-digestible epidermis, bundle sheath, sclerenchyma and vascular tissues than

leaves of C3 temperate and legumes grasses. Wilson and Hattersley (1989) also found

that anatomical differences between the leaf structural groups were consistently

expressed, with C3 species having higher proportions of mesophyll (53-67 vs. 28-47%)

and lower proportions of bundle sheath (5-20 vs. 12-33%) and vascular tissue (3-9 vs. 6-

12%) than the C4 species.

According to Wilson and Hacke (1987) the anatomy associated with either C4

tropical or C3 temperate grass genera clearly contributes to difference in DM digestibility

between leaves. Comparisons of C4 or C3 leaf anatomy in a wide range of summer-

growing Panicum species grown under the same environmental conditions determined

that the C4 anatomy of tropical grass genera causes their leaves to have lower digestibility

and higher cell wall concentration than grasses with C3 anatomy (Akin et al., 1983).

Quantitative analysis of leaf anatomy of a number of grasses indicated that leaves of the

tropical species had 25 percentage units more of the slowly digested cell tissues than had

the temperate grasses (Akin and Burdick, 1975).

According to Buxton and Redfearn (1997), energy availability from forages is

limited by fiber concentration because fiber is slowly and incompletely digested, whereas









cell soluble are almost completely digested. Thus, the proportion of fiber to cell soluble

is a major determinant of energy availability in forages. Ruminants digest about 40 to

70% of grass fiber, and some fiber fragments cannot be digested no matter how long they

remain in the rumen. Lignin interferes with microbial degradation of fiber

polysaccharides by acting as a physical barrier and by being cross-linked to

polysaccharides by ferulate bridges (Moore and Jung, 2001). Lignin and ferulate cross

linkages are more abundant in C4 than C3 grasses. This is the chemical basis for the lower

digestibility of C4 grasses (Ramalho, 1991).

Voluntary intake of forages is a critical determinant of animal performance and cell

wall concentration is negatively related to intake of ruminants consuming high-forage

diets. Cell walls affect intake by contributing to ruminal fill. Cell wall concentration and

rate of passage are the most critical parameters determining ruminal fill (Jung and Allen,

1995). Ruminal fill is typically greater in ruminants consuming C4 grasses than in those

consuming C3 grasses because of the poorer digestibility of C4 grasses.

Methods for Improving Forage Nutritive Value

Different chemical and biological treatments have been used for enhancing the

nutritive value of low quality forages and roughages. The main effect of such treatments

is due to modifications of cell wall components. The changes that take place when low

quality roughages are treated with alkali (e.g., ammonium hydroxide, NaOH) are of a

physical as well as of a chemical nature (Ramalho, 1991). It is well known that the

roughages are normally softer after chemical treatment and this may be one of the reasons

for the higher intake found for treated forage (Ramalho, 1991). Another important

change that takes place during alkali treatment is a swelling of the plant cell wall. This is

probably most pronounced for forage treated with NaOH solution (Harbers et al., 1982).









There are a number of chemical reactions taking place during alkali treatment of forages.

Saponification of ester linkages between acetic acid and phenolic acids, and

polysaccharides and or lignin as well as such linkages between uronic acid residues of

xylans in hemicelluloses and lignin occur during the alkali treatment of straw (Harbers et

al., 1982). If the temperature is high enough in the presence of alkali, lignin undergoes

cleavage of other linkages between phenyl propane units and free phenolic groups are

formed. As a result of the accompanying decrease in the molecular weight and cleavage

of linkages to hemicellulose, an increased solubility of lignin in the alkaline solution will

occur (Theander and Aman, 1984; cited by Ramalho, 1991). Sundstizil (1998) reported

that the OM digestibility of alkali-treated rye straw increased from about 46 to 71%.

Disruptions of ferulate bridges by ammoniation have been also associated with improving

fiber digestion (Brown and Adjei, 1995, Barrios-Urdaneta and Ventura, 2002), voluntary

intake (Glenn, 1990; Vagnoni et al., 1995; Lines and Weiss, 1996) and animal

performance (Rasby and Ward, 1989; Brown, 1993; Brown and Pate, 1997).

Ammoniation

Ammoniation is one of the most studied chemical treatments for improving forage

digestion in the past few years (Chaudhry, 1998). Chemical treatments of low quality

forages, such as ammoniation, have been shown to economically extend the use of such

forages into more nutritionally challenging periods of the production cycle, such as late

gestation and early lactation (Wiedmeier et al., 2003). Low quality forages are treated

with ammonia for the two following reasons: 1) ammonia is an effective preservative for

hay containing up to 30% moisture and 2) treatment of mature grass hays and poorer

quality crop residues is a cost-effective way for improving their feeding value (Weiss and

Underwood, 1995). Ammoniation increases crude protein (CP) in the treated forages by









adding about 50 to 80% of the N in NH3 to the forage. Some of the retained nitrogen is

converted by microbes present on the forage into microbial protein and another fraction

of the retained nitrogen is bound in an unknown manner to the forage fiber components

(Weiss and Underwood, 1995). Barrios-Urdaneta and Ventura (2002) observed that their

dry ammoniation method improved the CP of koroniviagrass (Brachiaria humidicola)

from 3.2 to 8.3%. Brown (1993) observed that ammoniation (4% of DM) of stargrass

(Cynodon nlemfuensis) hay increased (P < 0.01) total N concentration (from 1.0 to 1.4%).

A similar increase (0.9% N, P < 0.01) was obtained by Lines et al. (1996) in alfalfa hay.

Ammoniation also improves forage digestibility. This is due to hydrolytic action

on linkages between lignin and structural polysaccharides, thus increasing organic matter

(OM) potentially available for utilization by the ruminal microorganisms (Barrios-

Urdaneta and Ventura, 2002). Ammonia treatment substantially reduces the

concentration of neutral detergent fiber (NDF) in forages and most of the loss of NDF is

due to hydrolysis of hemicellulose and disruption of chemical linkages between lignin

and hemicellulose, making the hemicellulose more digestible (Weis and Underwood,

1995). Cellulose digestibility also increases since lignified hemicellulose encases

cellulose (Chaudhry, 1998). Ammoniation partially breaks down the structure of

cellulose by disrupting hydrogen bonds. This reaction causes a swelling of the fiber and

allows cellulase better access to the fiber for digestion (Church, 1988). Lines et al.

(1996) reported lower NDF (58.8 vs. 56.2%, P < 0.01) and hemicellulose (13.2 vs. 9.4%,

P < 0.01) concentrations in ammoniated alfalfa hay compared to the untreated hay. This

agrees with results obtained by Brown and Adjei (1995) who found lower NDF (-5%, P <









0.05) and hemicellulose (-17%, P < 0.01) concentrations in urea-treated (6% DM)

guineagrass (Panicum maximum) hay compared to the untreated hay.

Ammonia treatment also changes the physical characteristics of forages making

them more pliable and increases their hydration. Hydration rate has an important role in

digestion rate; the faster a forage particle is hydrated, the faster it is digested (Weiss and

Underwood, 1995). Barrios-Urdaneta and Ventura (2002) showed that ammoniation

increased the in vitro NDF digestibility (by 10.9%) of koroniviagrass. Brown (1993)

observed that ammoniation increased in vitro OM, NDF, and ADF digestibility and

decreased (P < 0.01) NDF concentration in stargrass hay. Vagnoni et al. (1995) showed

that ammoniation of mature bermudagrass increased both the in situ rate (P < 0.05) and

the potential extent (P < 0.01) of forage DM and NDF disappearance in lactating cows.

Zorrila-Rios et al. (1991) found that the in vitro DM digestibility (IVDMD) of wheat

straw was increased by 54% due to ammoniation. In that study, ammoniation also almost

doubled the CP concentration of the straw compared to untreated straws.

Woolford and Tetlow (1984) observed that ammoniation of high-moisture hay

reduced the growth of yeasts and molds, and decreased the rate of aerobic deterioration.

Bates et al. (1989b) found a substantial reduction of external molding when ammonia

was metered into the sealed plastic container of round bale silage; however, they

observed that ammoniation was associated with undesirable fermentation characteristics,

especially when direct-cut, low DM tropical forages were ensiled. Dry matter recovery

and intake of ammoniated, direct-cut, bermudagrass round bale silage was very poor.

Although application of ammonia to bermudagrass wilted to 40 to 50% DM improved the

quality of round bale silage, these authors did not recommend this practice because of the









high level of management required for success, and because treatment of silage and hay

with ammonia has, on occasion, been toxic to cattle.

Ammonia Treatment Methods

Gaseous anhydrous ammonia has been used for forage treatment in developed

countries while, in developing countries, spraying ammonia solutions and dipping hay in

urea solutions are preferred (Chenost and Kayouli, 1997). According to these authors,

using anhydrous ammonia is more effective, but its high cost and requirement for special

delivery and storage facilities have hindered its utilization by farmers. The use of a urea

solution is a simple, low cost technique; however, it has not become widely accepted.

The labor involved in handling the material and the appearance of molds as a

consequence of the water added has limited adoption by commercial producers (Barrios-

Urdaneta and Ventura, 2002). The latter researchers recently developed a method that

they called "dry ammoniation" by adding water and urea into plastic containers (19-1) and

suspending 1 kg hay bales 5-8 cm over the perforated cover of the container. Thereafter

the hay and container were hermetically sealed with a plastic tarp and stored for 14 or 21

days. The method was found to increase CP concentration and in vitro NDF digestibility

by 190% and 37%, respectively.

Barrios-Urdaneta and Ventura (2002) evaluated the effects of storage time (14 and

21 days), water volume (200 and 400 ml/kg DM of hay) and urea quantity (20 and 40

g/kg DM of hay) on the CP concentration and in vitro digestibility of NDF of

koroniviagrass of 1 kg hay bales. The best increase in nutritive value was obtained when

the hay was stored for 21 days and treated with 200 ml of water and 40 g of urea/kg.

According to Dolberg (1992), ammoniation treatment time may vary from one to

five weeks. However, temperature and treatment time are inversely related; and









therefore, more time is required in the winter or cold weather. Simple tests of successful

treatment of straw are a browning in the color of the forage, a strong smell of ammonia,

and absence of rotten and molded straw (Lalman et al., 2005). The amount of anhydrous

ammonia necessary to improve digestibility is between 2 and 4% of DM (Weiss and

Underwood, 1995). The reaction between ammonia and fiber is dependent on

temperature, so if forages are treated with ammonia during cold weather a five-week

treatment period is recommended (Dolberg, 1992). After this treatment period, forage

can remain covered for extended periods without problems. It is recommended that the

forage be left uncovered for at least 3 to 5 days prior to feeding to allow free ammonia to

escape (Lalman et al., 2005). This may not be necessary, but sometimes animal

acceptance may be poor initially if ammoniated bales are not aired out prior to feeding

(Weiss and Underwood, 1995).

Brown and Adjei (1995) applied a urea solution (0, 4, 6, or 8% of the forage DM)

to guineagrass hay harvested at different moisture concentrations (25 or 40%) and

observed that CP concentration and in vitro OM digestibility (IVOMD) increased linearly

(P < 0.01), whereas concentrations of hemicellulose (P < 0.01) and ADL (P < 0.05)

decreased linearly with increasing amount of urea applied. The same researchers treated

guineagrass hays with urea at 0, 4, or 6% of the forage DM. The urea solution was

sprayed onto the flat sides of the bales, or applied by low pressure (10 psi) injection. The

greatest improvements in CP and NDF concentration and IVOMD were obtained at the

25% forage moisture concentration using the low pressure injection method.

Animal Response to Feeding Ammoniated Forages

Several reports show that ammoniation of low quality forages can improve animal

performance. Weiss and Underwood (1995) stated that ammonia treatment increases the









DMI and DMD of low to medium quality grass hay by 5 to 10 percentage units. The

increase in both variables results in a substantial increase in consumption of digestible

energy by animals fed ammoniated forages as compared to those fed untreated forage.

Consequently, ammoniated straw can provide adequate energy and protein to maintain

lactating beef cows and ewes under most conditions while untreated straw can not.

Vagnoni et al. (1995) fed crossbred beef steers anhydrous ammoniated (3% of hay DM)

mature bermudagrass hay or supplemented their diets with urea and observed that

ammoniation, unlike urea supplementation, increased ADG and DMI (P < 0.05), which

suggests that ammoniation resulted in greater growth of ruminal microorganisms. In two

digestion and growth trials, round bales of hay were sprayed with solutions of 0, 4, or 6%

urea and fed to beef steers. Hay intake increased in a quadratic (P < 0.05) manner with

increasing urea concentration. Apparent NDF and ADF digestibility increased linearly (P

< 0.05) with increasing urea concentration and linear improvements in ADG (P < 0.05)

and gain/feed (P < 0.07) were observed (Brown and Adjei, 1995).

According to Rasby and Ward (1989), when animal requirements for protein are

high, as during lactation, the N needed by rumen bacteria can be supplied using

ammoniated forages and by supplementing with a source of ruminally undegraded

protein (RUP) but is digested in the small intestine. The latter meets the remaining

protein need and may enhance animal performance because of the improved amino acid

profile reaching the small intestine from dietary RUP. Because the energy requirements

of lactating dairy cows are quite high, the amount of ammoniated low quality forages

included in the diet should be limited (Weiss and Underwood, 1995).









The only dried forages that should be considered for 3% ammoniation are straws,

mature grass hays and corn stover. No more than 1% of NH3 should be applied to high

quality forages such as alfalfa (Medicago sativa), immature orchardgrass (Dactylis

glomerata), fescue (Festuca arundinacea), sudangrass (Sorghum sudanense), cereal grain

hays or any moderate to early harvested grass hay (including both cool and warm season

species) because the resulting product is often toxic to livestock (Weiss and Underwood,

1995). Ammoniation of high quality roughages can lead to toxicity problems known as

"crazy cow syndrome" or "bovine bonkers." Symptoms include hyperexcitability,

circling, convulsions, and even death. Toxicity is caused when cattle consume sufficient

quantities of the toxic compound, 4-methylimidazole, which is formed when soluble

sugars in the roughage react with ammonia. This compound passes through the milk to

affect nursing calves, which seem to be more susceptible to the toxicity than mature

animals. Mature roughages have low soluble sugar content and present little NH3 toxicity

risk (Lalman et al., 2005).

The foregoing indicates that ammonia treatment is a viable method of increasing

the nutritive value of low quality forages and improving the animal performance of

ruminant livestock fed such grasses. However, the use of NH3 is limited due to the high

investment in infrastructure required for delivering NH3, treating the forages, and storing

the treated forage and concerns about the hazardous nature of the alkali.

Feed Enzymes

Definition, Types, and Classification

Enzymes are naturally occurring globular protein molecules that catalyze specific

chemical reactions in biological systems. Two mechanisms have been propounded to

explain enzyme action. The first, the lock and key mechanism, was proposed by Emil









Fischer in the late 1800s, which postulates that enzymes accommodate substrates with

specific shape that complement the enzyme active site (Scrutton, 1999). However, in

1958 Daniel Koshland proposed the induced fit theory, which postulates that the substrate

can induce conformational changes in an enzyme structure to bring the catalytic groups

of the enzyme into the proper alignment for binding the substrate (Koshland, 1994). Both

theories are now accepted mechanisms for enzyme action.

Enzymes are involved in the digestion of complex feed molecules into their

chemical constituents (e.g., glucose, amino acids) in both bacteria and the host animal.

Digestive enzymes are essential to animals because complex feeds are not readily

absorbed from the digestive tract unless they are degraded to simpler molecules (Kung,

2001)

Enzymes are classified broadly by the substrate on which they act and by their

specificity. Commercial enzyme products are fermentation extracts of bacterial (Bacillus

spp.) or fungal (Trichoderma and Aspergillus spp.) origin (Beauchemin et al., 2004a),

and contain a unique array of enzymatic activities (Table 2.1). Enzyme activity can be

assayed using in vitro methods by measuring end products of hydrolysis (i.e., reducing

sugars, amino acids or peptides) per unit time, using a specified substrate under defined

conditions. These substrates are often purified or modified to simplify measurements of

activity (Kung, 2001).

Cellulose is hydrolyzed through a complex process involving cellulases. Numerous

specific enzymes contribute to cellulase activity, including endocellulase, exocellulase,

and B-glucosidase. In general, endoglucanases hydrolyze cellulose chains at random to









Table 2.1 Some fibrolytic enzyme-producing microorganisms and the enzymes they
produce
Microorganism Enzymes
Aspergillus niger 1, a-amylase, endoxylanase, 0 -xylosidase,
acetylxylan esterase, a-L-arabinofuranosidase
Aspergillus ficuum 3 P-glucanase
Aspergillus candidus 3 Cellulase
Aspergillus sydowi 4 Phytase, 0 -D-fructofuranosidase
Microorganism Enzymes
Aspergillus oryzae 1,2 a-amylase, protease
Bacillus licheniformis 3 a-amylase
Bacillus subtilis 3 Phytase, a-amylase
Trichoderma viridae 3 Xylanase, P-glucanase, protease, cellulase
Saccharomyces cerevisae 1, 3 a-galactosidase
Humicola insolens 6 P-glucanase
1 Beauchemin et al., 2004a
2 Carlsen et al., 1996
SHutcheson, 2001
4Muramatsu and Nakakuki, 1995
5Noel et al., 1998
6 Schulein, 1997


produce cellulose oligomers of varying degrees of polymerization; exoglucanases

hydrolyze the cellulose chain from the non-reducing end, producing cellobiose, and 3-

glucosidases hydrolyze short-chain cellulose oligomers and cellobiose to glucose

(Beauchemin et al., 2003).

The main enzymes involved in degrading the xylan core polymer to soluble sugars

are xylanases and 1-1,4 xylosidase (Bhat and Hazlewood, 2001). The xylanases include

endoxylanases, which yield xylooligomers and 3-1,4-xylosidases, which in turn yield

xylose. Other hemicellulase enzymes involved primarily in the digestion of side chains

include 1-mannosidase, d-L-arabinofuranosidase, 6 -D-glucuronidase, 6 -D-

galactosidase, acetyl xylan esterases, and ferulic acid esterase (White et al., 1993, cited

by Beauchemin et al., 2003; Bhat and Hazlewood, 2001).









According to Fanutti et al. (1995), endo-P-1,4-xylanase hydrolyzes the 0-1,4-

linked polysaccharide backbones of xylans, which form the major hemicellulose

component of forages. Some studies on the structure of xylanases have revealed that

some enzymes are comprised of single catalytic domains while other xylanases are

modular, consisting of single or multiple catalytic domains fused via linker sequences to

noncatalytic sequences, some of which constitute cellulose binding domains (Fanutti et

al., 1995). Hemicellulases derived from aerobic microorganisms do not appear to

associate into multi-enzyme complexes, while anaerobic organisms often synthesize

multi enzyme cellulase-hemicellulase complexes (Fanutti et al., 1995). These researchers

have focused their studies on plant cell wall-degrading enzymes of anaerobic fungi that

are particularly active against the more recalcitrant plant structural polysaccharides and

have observed that these organisms produce cellulases and hemicellulases that associate

into large molecular weight, multi-enzyme complexes and bind tightly to cellulose,

exerting their cellulolytic effect.

Murashima et al. (2003) noted that plant cell walls are comprised of cellulose and

hemicellulose and other polymers that are intertwined, and this complex structure presents

a barrier to degradation by pure cellulases or hemicellulases. They determined the

synergistic effects on corn (Zea mays) cell wall degradation by the action of xylanases and

cellulases from Clostridium cellulovorans. Xylanase and cellulase were found to degrade

corn cell walls synergistically but not purified substrates such as xylan and crystalline

cellulose. The mixture ofxylanases and cellulases at a molar ratio of 1: 2 gave the

highest synergistic effect on corn cell wall degradation. The amounts both of









xylooligosaccharides and cellooligosaccharides liberated from corn cell walls were

increased by the synergistic action of xylanases and cellulases.

Pectin, a minor component of grass cell walls, is digested in the rumen either by

strictly pectinolytic species or by species possessing a combination of pectinases (e.g.,

pectin lyase, polygalacturonase, pectin methyl esterase) and xylanases (Cheng et al.,

1996).

Commercial Exogenous Fibrolytic Enzymes

Commercial enzyme products are relatively concentrated and purified, and they

contain specific enzyme activities (Beauchemin et al., 2004a). Use of exogenous

enzymes can be beneficial when the enzyme preparation and the feed are complimentary.

The use of fibrolytic enzymes as additives for ruminant diets has been the focus of

considerable research recently following positive responses to enzyme supplementation

in feeding trials (Beauchemin et al., 1995; Kung et al., 2000). However, in contrast to the

case in non-ruminants (Bedford and Schulze, 1998), the mode of action of these enzyme

additives in ruminants is not fully understood. As an alternative to costly in vivo trials,

several in vitro studies have been conducted to examine the effects of enzymes on the

degradation of feedstuffs, but the complexity of these feeds makes it difficult to identify

which feed fractions are most influenced by enzymatic action. The use of purified xylans

and cellulose can minimize this complexity and provide a more informative method of

evaluating the mode of action of enzymes. However, the results of such studies may not

always correlate to the enzyme effects on feedstuffs.

Several feed enzyme products that contain a blend of enzymes have been shown to

be effective at enhancing the utilization of ruminant diets (Rode and Beauchemin, 1998).

Nevertheless, the enzyme levels and activities that will effectively improve dietary









nutrients will vary with the diet being considered and the nature of the enzyme. Types of

cellulases and hemicellulases in commercial enzyme products differ substantially, and

differences in the relative proportions and activities of these individual enzymes

determine the efficacy of cell wall degradation by these products (Beauchemin et al.,

2003). In addition to fiber-degrading enzymes, these products also have secondary

enzyme activities, including amylases, proteases, and pectinases, which contribute to their

hydrolytic capacity. Various factors such as enzyme type and method of preparation and

application, amount of enzyme applied and fraction of the diet targeted, and animal

differences have lead to inconsistencies in results of trials in which enzymes have been

added to ruminant feeds (Bowman et al., 2002).

Rode and Beauchemin (1998) evaluated commercial enzyme preparations in vitro

using alfalfa hay or barley (Hordeum vulgare) silage as a substrate. Effectiveness of

enzyme products differed for the two substrates, indicating that an enzyme product that

elicits a positive response in one diet may not be effective if evaluated using a different

diet. According to Newbold (1995, cited by McAllister et al., 2001) destruction of the

multi-enzyme complexes during the extraction process may explain why enzymes from

mixed ruminal microorganisms failed to release much soluble sugar from hay and straws.

Mode of Enzyme Action

The mode of action of exogenous enzymes is generally to hydrolyze some plant

components that impede digestion, thereby increasing the nutritive value of the feed. A

number of different mechanisms of enzyme action have been postulated, including direct

hydrolysis (Sheperd and Kung, 1996b; Colombatto et al., 2003b), stimulation of

microbial numbers and attachment to substrate (Morgavi et al., 2000a), improvements in

palatability (Adesogan, 2005), changes in gut viscosity (Officer, 2000), and changes in









the site of digestion (Rode and Beauchemin, 1998; Hristov et al., 2000). Some of these

factors increase the hydrolytic capacity of the rumen, which indirectly reduces gut fill,

and hence enhances feed intake (Adesogan, 2005). Morgavi et al. (2000a) suggested that

synergy between ruminal fibrolytic enzymes and added enzymes may also be responsible

for improvements in animal production when ruminants are fed enzyme-supplemented

feeds.

Lack of information about enzyme products used and method of providing the

product to animals makes it difficult to compare the results from early studies to more

recent studies. Inconsistent results seem to be caused by a number of factors including

diet composition, type of enzyme preparation used, complement of enzyme activities,

level of enzyme provided, enzyme stability and method of application (Rode and

Beauchemin, 1998).

Factors Affecting Enzyme Action

It is essential to determine the conditions necessary for optimizing effects of

supplemental fibrolytic enzymes on animal performance. When viewed across a variety

of enzyme products and experimental conditions, the response to feed enzymes by

ruminants has been variable. This variation can be attributed to differences in the

lactation stage of cows (Lewis et al., 1999; Rode et al., 1999), enzyme type, activity and

characteristics (Dawson and Tricarico, 1999), under or over-supplementation with

enzymes (Beauchemin et al., 1995; Yang et al., 1999; Beauchemin et al., 2000; Kung et

al., 2000), and inappropriate method of supplying the enzyme product to the animal

(Bowman et al., 2002; Sutton et al., 2003). According to Beauchemin et al. (2003)

animal responses to fibrolytic enzymes are also greater at times when fiber digestion is

compromised and when energy is the first-limiting nutrient in the diet.









Different experiments have examined the impact of delivery method on the

effectiveness of exogenous enzymes. Bowman et al. (2002) compared a Control diet to

diets in which a fibrolytic enzyme product (Promote) was applied to the concentrate

(45% of TMR), or to a pelleted portion of the supplement (4% of TMR), or to a premix

(0.2% of TMR). All diets that were supplemented with the enzyme product delivered

about 1.0 g per cow per day. Digestibility of OM, NDF and ADF in the total tract was

increased in comparison to the Control when enzymes were added to the entire

concentrate. Enzyme application to smaller portions of the diet produced only numerical

increases in digestibility over the Control. However, there was an increase in microbial

N synthesis in cows fed the enzyme-supplemented premix. Enzyme supplementation did

not affect milk production and composition, but cows receiving the enzyme-

supplemented concentrate had numerically higher fat-corrected milk (FCM) production

compared to the Control cows. These results indicate that the proportion of the diet to

which the enzyme is applied must be maximized to ensure a beneficial response.

Lewis et al. (1996) examined the effect of a solution containing cellulases and

xylanases on the digestion of a forage-based diet. Ruminally cannulated beef steers were

assigned randomly to a Control diet (70:30 grass hay: barley ratio DM basis) or diets in

which an enzyme was added to the forage 24 h before feeding (F-24), to the forage 0 h

before feeding (F-0), to the barley 0 h before feeding (B-0), or infused ruminally 2 h after

feeding (RI). Dry matter and NDF intakes were not different across treatments. In situ

rate of NDF disappearance of the enzyme-treated barley or forage was greater (P < 0.05)

than that of the untreated diet. Ruminal infusion of enzymes compared with F-24 and F-0

produced lower disappearance of DM and NDF at 96-h (P < 0.05). In situ rate of DM









disappearance of enzyme-treated grass tended to be greater (P < 0.10) in steers fed B-0

and Control than in those fed F-24 and F-0. Total tract digestibilities of DM, NDF, and

ADF were greater (P < 0.10) in cows fed F-24 and F-0 than those fed the Control diet.

Forage transit time was shorter (P < 0.10) for B-0 than for F-24 and F-0; however, all

other contrasts for particulate passage did not differ (P > 0.10). Results from this study

indicate that direct application of enzymes to forage is capable of improving forage

digestion.

Enzyme Stability in the Digestive Tract

Several digestive enzymes have been successfully used to enhance poultry and

swine performance, but they have not been used traditionally in diets fed to ruminants.

The primary reason for this practice was due to the fact that enzymes are proteins and

thus would be subject to degradation by microbial proteases in the rumen and/or

inactivated by proteases in the small intestine (Kung, 2001). Stability in the rumen is

critical for enzyme effectiveness. Considerable variation exists among fibrolytic

enzymes in their ability to maintain activity in the ruminal environment. Some enzymes

lose their activity rapidly when incubated in ruminal fluid due to proteolysis or adverse

pH and temperature conditions that limit enzyme activity, whereas other enzymes show

little or no loss in activity even after 12 hours of ruminal incubation. This is partially

because enzymes also have pH and temperature optima at which they are most effective.

Kopecny et al., 1994 (cited by Kung, 2001) reported that a cellulase enzyme complex

was rapidly degraded by ruminal bacterial proteases and its addition to ruminal fluid had

no effect on in vitro fiber digestion.

According to Morgavi et al. (2001) the cellulase enzyme complex from

Trichoderma spp. has a pH and temperature optima of 4.5 and 500C, respectively.









Colombatto et al. (2004a) found that the xylanase activity of two fibrolytic enzymes

(Depol 40 and Liquicell 2500) showed optimal activity at pH of 5.6 and both products

retained at least 70% of their xylanase activity after 48-h incubation at 15 or 390C in

ruminal fluid. Vicini et al. (2003) analyzed the in vitro activities of two commercial

fibrolytic enzymes and observed that all major cellulose and hemicellulose-degrading

activities were present; however, the optimal pH range was more acidic, and the optimal

temperature (approximately 500C) was greater than the normal pH and temperature in the

rumen. The authors concluded that it appears that a considerable part of the potential

activity of these enzyme preparations was lost due to conditions in the rumen.

Kung et al. (2002) indicated that the activity of similar fibrolytic enzymes may be

optimized under different conditions. They evaluated two different xylanases (B and C)

and reported that at 400C, the activity of xylanase C was greatest at a pH of 6.5 but was

substantially reduced as the pH decreased. In contrast, xylanase B showed greatest

activity at pH 5 and activity of xylanase C was twice that of xylanase B at pH 5.5 and 6.

Fontes et al. (1995) reported that several xylanases were resistant to several proteases but

only one cellulase from a mesophilic organism was resistant to proteolytic attack.

Hristov et al. (2000) observed that increasing ruminal doses of exogenous

polysaccharide-degrading enzymes in heifers increased ruminal fluid

carboxymethylcellulase and xylanase activities linearly (P < 0.01) and that elevated levels

of fibrolytic activities in the rumen resulted in increased (quadratic, P < 0.01)

carboxymethylcellulase, xylanase and P-glucanase activities in duodenal digesta.

Duodenal amylase activity and reducing sugar concentration also were increased

(quadratic response, P < 0.01, and P < 0.05, respectively) by polysaccharidase enzyme









supplementation. Xylanase activity of fecal DM was increased linearly (P < 0.05) with

increasing ruminal exogenous polysaccharidases enzyme levels.

Xylanases have been shown to be much more stable in the rumen than cellulases

(Hristov et al., 1996, cited by Rode and Beauchemin, 1998). This may due to the

relatively large and more complex structure of cellulases compared to xylanases.

Morgavi et al. (2001) observed that polysaccharidase activities of commercial

preparations from T. longibrachiatum incubated for up to 6-h within ruminal fluid were

remarkably stable. Cellulase and cellulose 1, 4-beta-cellobiosidase activities were least

stable, followed by xylanase, whereas beta-glucanase activity was not affected.

Feed enzyme supplements may exert their effect on feed digestibility in the small

intestine as well as in the rumen (Rode and Beauchemin, 1998). Therefore, stability is

very important if these enzymes are to remain active in the intestines as well as in the

rumen. According to Fontes et al. (1995), the stability of xylanases and cellulases in the

rumen may be related to glycosylation, which may protect them from inactivation from

temperature and proteases. Many xylanases and cellulases from bacteria and fungal

sources are glycosylated. Glycosylation involves covalent bonding of monosaccharides

to specific amino acid side chains in enzymes and glycosylation has been shown to confer

resistance to proteolysis in monogastrics and ruminal fluid (Fontes et al., 1995).

The survival of exogenous enzyme activities in the rumen may also depend upon

the proteolytic environment of the host animal, which can be variable (Falconer and

Wallace, 1998). For example, stability of exogenous enzymes varied depending upon the

donor cow, and ruminal fluid obtained from cows before feeding inactivated

polysaccharidases to a greater extent than ruminal fluid taken after feeding (Morgavi et









al., 2001). This was probably due to the higher ruminal feed content after feeding, which

allowed the proteolytic microorganisms to colonize the feed particles and exert their

enzymatic effect, thereby decreasing the residual proteolytic activity in the rumen fluid.

The activity of enzymes derived from mesophilic (e.g. Trichoderma and

Aspergillus spp) or thermophilic (e.g., Thermoascus aurantiacus) sources will not be

optimized when used as ruminant feed additives (Beauchemin et al., 2004a). These

authors suggested that enzymes should be selected that work at lower temperatures. The

organisms that produce these enzymes are psychrophilic (Beauchemin et al., 2004a), and

their potential to improve the initial rate of OM degradation of corn has been

demonstrated by Colombatto et al. (2004b). Cummings and Black (1999) reported that a

psychrophilic, gram-negative bacterium has been isolated and has abundant xylanolytic

activity. Crude enzyme activity was measured in the supernatant at temperatures ranging

from -5 to 500C. The bacterium gave faster growth at 150C, however optimal enzyme

temperature was observed at 37C. The isolation of the enzymes secreted by these

microorganisms is potentially promising for improving ruminal fiber degradation.

Some researchers have suggested that feeding unprotected enzymes may be more

useful in immature ruminants where ruminal microbial populations are not fully

developed. For example, Baran and Kmet (cited by Kung, 2001) reported that a

pectinase-cellulase enzyme additive improved ruminal fermentation in newly weaned

lambs but not in adult sheep (with established ruminal microflora).

Methods of Determining Enzyme Activity

According to Beauchemin et al. (2003) fiber-degrading enzyme activities are

generally determined by measuring the rate of release of reducing sugars from pure

substrates, with enzyme units expressed as the quantity of reducing sugars released per









unit of time per unit of enzyme. Reducing sugars, which include monosaccharides,

disaccharides and some oligosaccharides, can be measured colorimetrically using the

Nelson/Somogyi copper method (Somogyi, 1952) or the dinitrosalicyclic acid method

(Miller, 1959). The most commonly used substrate for measuring cellulase activity

(endo-B-1,4-glucanase activity) is carboxymethyl cellulose (Wood and Bhat, 1988).

Exoglucanase activity can be measured using crystalline cellulose preparations, such as

Avicel. 1-glucosidase activity is determined by measuring the release of glucose from

cellobiose, or the release ofp-nitrophenol from p-nitrophenyl-1-D-glucoside (Bhat and

Hazlewood, 2001). Xylanase activity is most commonly measured by determining the

release of reducing sugars from prepared xylan, such as oat (Avena sativa) spelt or

birchwood xylan. Xylanases are specific for the internal 1-1,4 linkages within the xylan

backbone, and are generally considered endoxylanases (Bhat and Hazlewood, 2001).

Endoxylanases can be considered to be debranching or non-debranching based on their

ability to release arabinose in addition to hydrolyzing the main xylan chain. 3-xylosidase

activity can be determined by using p-nitrophenyl derivatives (Bhat and Hazlewood,

2001).

Enzyme activity measurements must be conducted under conditions closely defined

with respect to temperature, pH, ionic strength, substrate concentration, and substrate

type, since all of these factors affect the enzyme activity. The optimal temperature and

pH for most commercial fibrolytic enzymes is approximately 60C and optimal pH is

between 4 and 5 (Coughlan, 1985). However, these optima are not representative of the

conditions in the rumen, which are closer to a pH of 6.0 to 6.7 and 390C (Van Soest,

1994).









Wallace and Hartnell (2001) evaluated enzymatic and tracer methods for detecting

and measuring the quantity of fibrolytic enzyme preparations added to corn silage,

ryegrass (Lolium multiflorum) silage and a total mixed ration and observed that the

quantity of enzyme preparations added to the feeds could not be detected using their

enzymatic activities. Glycosidase activities of soluble washed from the feed were more

than an order of magnitude greater than glycosidase in the added enzymes.

Carboxymethylcellulase and xylanase activity determinations which used reducing sugar

release as the measurement, were subject to interference from reducing sugars present in

the feed. A fluorescent tracer method, using fluorescein added at a rate of 1 g/L of feed

enzymes, or 2 g/t of feed, was developed that enabled sensitive detection of liquid

enzyme additions to feeds (Wallace and Hartnell, 2001).

Effect of Enzyme Treatment on Chewing Behavior

Alterations in mechanical processing (Beauchemin and Rode, 1997) and chemical

properties (Beauchemin and Buchanan-Smith, 1989; cited by Rode and Beauchemin,

1998) of feeds can significantly alter chewing behavior, and consequently saliva

production. Therefore, the use of exogenous fibrolytic enzymes in dairy cow diets may

alter feeding behavior and saliva production. Increasing the rate of fermentation within

the rumen leads to a decrease in ruminal pH, which can decrease fiber digestion (Russell

and Wilson, 1996).

Supplemental fibrolytic enzymes have been shown to increase fiber digestion

(Rode et al., 1999; Yang et al., 2000), and ruminal pH has been lowered in some cases

(Lewis et al., 1996), but not others (Yang et al., 1999). Thus, applying fibrolytic

enzymes to feed before feeding may decrease both chewing time and saliva output and

increases the risk of acidosis (Bowman et al., 2003). The latter researchers investigated









the effects of enzyme supplementation on the chewing and feeding behavior, saliva

secretion, and ruminal pH in lactating dairy cows fitted with ruminal cannulas. Enzyme

supplementation did not alter daily time spent eating or ruminating, but increased saliva

production, with no difference among enzyme application treatments. These results

indicate that application of this fibrolytic enzyme product did not alter the physical

structure of the feed measured by feeding and chewing variables. The increase in total

saliva production observed in cows fed enzyme-supplemented diets may be attributed to a

physiological response to compensate for the increase in fermentation products produced

during digestion.

Beauchemin et al. (2000) evaluated two doses of a fibrolytic enzyme fed to dairy

cows in a diet containing 45% forage and 50% concentrate. They observed that the time

spent eating each day was similar for cows regardless of diet, even though cows fed the

enzyme-treated diets ate more than the cows fed the Control diet. Thus, adding enzyme

to the diet decreased the time spent eating per unit of DM, NDF, or ADF, with no

difference between the low and high amount of enzyme supplementation. According to

the authors, decreased time spent eating per unit of fiber suggests the enzyme mixture

may have had a pre-ingestive effect on the feed that enhanced the ease of ingestive

mastication, which contradicts the conclusion of Bowman et al. (2003). Beauchemin et

al. (2000) also measured rumination activity and did not detect effect of an enzyme

supplementation on this variable.

Effect of Enzyme Treatment on the Ruminal Microbial Population

The enzyme activities that exist in the rumen are diverse, and include those that

degrade cellulases, xylanases, a -glucanases, pectinases, amylases, proteases, phytases

and those that degrade specific plant toxins (e.g., tanninases) (Wang and McAllister,









2002). The variety of enzymes present in the rumen arises from the diversity of the

microbial community and the multiplicity of fibrolytic enzymes produced by individual

microorganisms. Efficient digestion of complex substrates in the rumen requires the

coordinated activities of these enzymes. Limitations to cell wall digestion in the rumen

can result from insufficient quantities or types of enzymes produced by ruminal microbes

or from an inability of degradative enzyme(s) to interact with target substrates, or from an

unconducive environment for optimal enzyme activity (e.g., low ruminal pH) (McAllister

et al., 2001)

According to Morgavi et al. (2000), feed enzyme additives used to improve

digestion in ruminants interact not only with the feed but also with ruminal

microorganisms. These authors reported overall increases in the rumen microbial

population due to the addition of an exogenous fibrolytic enzyme to different substrates.

However, it is not clear whether this effect was due directly to microbial growth

stimulation or indirectly by modifying feed structure. Morgavi et al. (2000) studied the

effect of an enzyme preparation from T longibrachiatum (TE) on growth ofF.

succinogenes in a medium containing cellobiose, crystalline cellulose or corn silage fiber.

Fiber disappearance and fermentation products were evaluated. The growth rate ofF.

succinogenes on cellobiose was not affected by TE (P > 0.05), but growth on cellulose

was increased by TE though substrate disappearance and gas production were unaffected.

When corn silage fiber was used, the addition of TE increased NDF disappearance (P <

0.05) at 24 and 48-h (33 and 52% in Controls versus 53% and 65% in TE treatments,

respectively). These results suggest that the Trichoderma enzyme preparation did not

supply nutrients or growth factors to F. succinogenes. Fibrobacter succinogenes digests









cellulose efficiently and addition of exogenous cellulases did not further increase

cellulose disappearance. However, TE increased corn silage fiber degradation probably

by providing an enzyme(s) that limited degradation, but was not secreted by F.

succinogenes. Enzyme additives have been shown to enhance colonization of feed by

ruminal microorganisms and increase the rate of degradation in the rumen (Yang et al.,

1999). Morgavi et al. (2000a) found that an enzyme product derived from Trichoderma

longibrachiatum worked in synergy with ruminal enzymes to release sugars from corn

silage, xylan, and cellulose, thereby enhancing ruminal hydrolytic activity.

Nsereko et al. (2000a) supplemented two dairy cow diets with 0, 1, 2, 5 or 10 L of

enzyme per ton of DM. Incremental levels of this enzyme stimulated numbers of total

viable ruminal bacteria (P < 0.05) by 100, 330, 390 and 250% (quadratic effect, P <

0.05). Of the rumen bacteria, the most notable increases in numbers were for cellobiose-

utilizing (P < 0.01), xylanolytic (P < 0.05) and amylolytic (P < 0.05) subgroups. The

numbers of cellulolytic bacteria were unaffected (P < 0.05). Increasing concentrations of

the enzyme had a convex, quadratic effect on protozoal numbers (P < 0.05), and the

lower protozoa numbers partially explain the increased number of bacteria. These data

suggest that exogenous enzymes can enhance feed digestion at least, in part, by

increasing numbers of rumen bacteria that utilize hemicellulose and secondary products

of cellulose digestion.

Effect of Enzyme Treatment on Ruminal Fibrolytic Capacity

The inclusion rate of exogenous enzymes in ruminant diets is usually in the range

of 0.01 to 1% of the diet, contributing about 10 to 100-times greater fibrolytic activity per

gram of feed than when silage additives are used (Christensen, 1997). Based on the

estimated average fibrolytic activity normally present in the fluid fraction in the rumen, it









has been estimated that supplemental enzymes may contribute up to 15% of the total

fibrolytic activity (Rode and Beauchemin, 1998). However, the activity of commercial

enzymes is measured at pH and temperature ranges that generally differ from that of

rumen fluid. Thus, once ingested, exogenous enzymes likely contribute considerably less

fibrolytic activity than calculated. Furthermore, fibrolytic enzyme activity associated

with particulate matter is notably higher than in the ruminal fluid (Wang and McAllister,

2002; Rode and Beauchemin, 1998). This is probably because the attachment of the

microbes to the feed particle allows the enzyme to act directly on the substrate, thereby

increasing the catalytic action of the enzyme. Thus, the contribution of exogenous

enzymes to ruminal fibrolytic activity is difficult to estimate and is probably less than that

commonly indicated on commercial enzyme containers.

Colombatto et al. (2003a) observed that enzyme addition to rumen fluid in vitro

increased (P < 0.05) the initial (up to 6-h) xylanase, endoglucanase, and a-D-glucosidase

activities in the liquid fraction by an average of 85%. Xylanase and endoglucanase

activities in the solid fraction also were increased (P < 0.05) indicating an increase in

fibrolytic activity by ruminal microbes. Furthermore, incremental addition of enzyme

increased (P < 0.05) the rate of gas production of various substrates, suggesting that

fermentation of cellulose and xylan was enzyme-limited. However, adding the enzyme at

levels higher than 2.55 tL/g of DM failed to further increase the rate of gas production,

indicating that the maximal level of stimulation was already achieved at lower enzyme

concentrations. Authors concluded that enzymes enhanced the fermentation of cellulose

and xylan by a combination of pre and post-incubation effects (i.e., an increase in the

release of reducing sugars during the pretreatment phase and an increase in the hydrolytic









activity of the liquid and solid fractions of the ruminal fluid), which resulted in a higher

rate of fermentation.

Effect of Enzyme Treatment on Fiber Concentration Before Ingestion

Previous findings indicate that application of exogenous fibrolytic enzyme products

to diets have pre-ingestive effects. The adsorption of enzyme onto the substrate is an

important prerequisite for hydrolysis (Pell and Schofield, 1993). Applying exogenous

enzymes directly to feeds releases reducing sugars (Hristov et al., 1998), and in some

cases, partially solubilizes NDF and ADF (Krause et al., 1998). Colombatto et al.

(2003a) evaluated the effects of adding a commercial enzyme product on the hydrolysis

and fermentation of cellulose, xylan, and a mixture of both substrates. They reported that

addition of enzyme in the absence of ruminal fluid increased (P < 0.01) the release of

reducing sugars from xylan and the mixture after 20 h of incubation at 200C. Hydrolysis

of the fiber pre-feeding may indicate a modification of the plant cell wall structure, which

could decrease the physical effectiveness of the fiber in the diet. When inadequate

effective fiber is fed, chewing activity decreases, which leads to less salivary buffer

secretion, resulting in a more acidic ruminal pH, altered ruminal fermentation patterns

and low ratios of acetate to propionate that ultimately result in modified animal

metabolism and reduced milk fat synthesis (Mertens, 1997).

Recent studies also have shown that enzyme preparations containing high ferulic

acid esterase activity as well as xylanase and cellulase activity reduced the NDF and ADF

concentrations and increased the digestion of hays made from 12-week regrowth of

Tifton 85 bermudagrass, Coastal bermudagrass and Pensacola bahiagrass (Krueger et al.,

2003). This enzyme also increased the rate and extent of in situ degradation of the

forages and reduced the lag time before forage degradation commenced (Krueger et al.,









2004). These studies suggest that enzyme treatment can improve the nutritive value of

tropical grasses.

Sheperd and Kung (1996b) observed that applying an enzyme additive containing

cellulase and hemicellulase reduced NDF and ADF concentration of corn silage during

the ensiling period. However, Mandevbu et al. (1999) observed that treatment of

bermudagrass forages harvested after 3 or 6 wk regrowth periods with a mixture of

cellulase, amylase and hemicellulase had no effect on silage fiber concentration or cell

wall carbohydrate fraction. This discrepancy is probably due to differences in activity of

the enzyme products used in both experiments and to differences in cell wall components

of corn and bermudagrass silage.

Effect of Enzyme Treatment on DM and Fiber Digestibility Post Ingestion

There is increasing evidence that exogenous fibrolytic enzymes improve fiber

digestion within the rumen, thereby increasing feed utilization in ruminants (Lewis, 1999;

Rode et al., 1999). According to Beauchemin et al. (2003) the focus of most enzyme-

related research for ruminants has been on plant cell-wall degrading enzymes. Cellulose

and hemicellulose, the major structural polysaccharides in plants (Van Soest, 1994), are

converted to soluble sugars by enzymes collectively referred to as cellulases and

hemicellulases.

More than thirty years ago, various studies showed significant improvements in

average daily gain (ADG) and feed conversion rate (FCR) of cattle when fed diets

supplemented with enzymes containing amylolytic, proteolytic and cellulolytic activities

(Rode and Beauchemin, 1998). Improvements in animal performance were due to

increased DM and fiber digestibility. Christensen (1997) found an increase (P < 0.05) in

DM digestibility when fibrolytic enzymes were added to rations of steers at feeding time









or 24 h prior to feeding. The NDF and ADF digestibilities of the rations increased

numerically by approximately three percentage units. In the same study, a positive effect

on rumen degradation of forage was observed, when a mixed cellulase, hemicellulase and

pectinase enzyme additive was applied to alfalfa hay, grass hay, oat straw and barley

silages at different rates. Christensen (1997) also observed that application of 600 IU/kg

DM of xylanase had a positive effect on both in vitro and in situ degradation of both

high-fiber and low-fiber forages.

Applying fibrolytic enzymes prior to feeding can alter the structure of the cell wall,

thereby making it more amenable to degradation (Beauchemin et al., 2004b). Nsereko et

al. (2000b) applied an enzyme product to alfalfa hay that was then autoclaved to

inactivate enzyme activities and washed to remove any product of the hydrolysis,

eliminating the possibility of chemotactic enhancement of digestion or synergy between

microbial enzymes and exogenous enzymes. In vitro NDF digestibility was higher at 12

and 48-h for treated than for untreated hay and generally this effect was enhanced by

longer pre-incubation with enzymes. Since these effects were observed in the absence of

active ruminal enzymes and soluble hydrolysis products, the exogenous enzymes

probably caused structural changes to the forages that improved digestion.

Rode et al. (1999) evaluated the effect of exogenous fibrolytic enzyme (Promote)

on DMI and digestibility in cows fed Control diets or diets in which an enzyme was

added to the concentrate at a rate of 1.3 g/kg (DM basis). Enzyme addition did not affect

DMI. However, total tract digestibility of nutrients as determined using Cr203, was

increased by enzyme treatment (DM, 61.7 vs. 69.1%; NDF, 42.5 vs. 51.0%; ADF, 31.7

vs. 41.9%; CP, 61.7 vs. 69.8%). Nevertheless, effects of supplemental enzymes on









digestibility have been inconsistent. Enzyme products comprised mainly of xylanases

and cellulases have increased digestibility (Rode et al., 1999; Yang et al., 2000), or not

affected digestibility (Lewis et al., 1999). Other studies have shown that exogenous

enzymes did not consistently improve animal performance, and the mechanism for

improved growth was not always confirmed by digestibility trials (Rode and

Beauchemin, 1998). Mandevbu et al. (1999) observed that treatment ofbermudagrass

forages with fibrolytic enzymes had no effect on in vitro or in situ DM or NDF

disappearance of silages. Hristov et al. (2000) observed that the ruminally soluble

fraction and effective degradability of feed DM in situ were increased (quadratic

response, P < 0.01) by enzyme treatment in ruminally cannulated heifers, but apparent

digestibility of DM, CP, and NDF were not affected.

Effect of Enzyme Treatment on Silage Fermentation

Applying cell-wall degrading enzymes during the ensiling process can increase the

release of fermentable sugars from the structural polysaccharides thereby providing extra

substrate for the microbial fermentation. This often increases the production of lactic

acid, which reduces the risk of clostridial fermentation (Van Vuuren et al., 1989). When

used as silage additives, fibrolytic enzymes predigest plant cell walls and this can

increase the extent and rate of degradation of silage in the rumen, and consequently,

improve digestibility and nutritive value (McHan, 1986). Rodrigues et al. (2001)

reported that application of a mixture of cellulase and endoxylanase to ryegrass before

ensiling reduced NDF, ADF and acetic acid (P < 0.01) concentration and increased lactic

acid and sugar concentration (P < 0.01). Similar results were obtained by Clavero and

Razz (2002) with dwarf elephantgrass (Pennisetum purpureum) silage treated with a

cellulase mixture. Selmerolsen (1993) showed that the fermentation of crops with low









sugar concentration, such as tropical grasses, was improved more by enzyme addition,

while that of crops with high sugar concentration were improved more by lactic acid

bacteria inoculation. In agreement, recent results also have shown that treatment of

bermudagrass, which is low in sugars, with fibrolytic enzymes alone or with an enzyme-

inoculant blend (Adesogan et al., 2004) improved the fermentation, but contradictory

results exist (Mandevbu et al., 1999). Clearly enzyme application at ensiling to forage

containing low sugar contents is logical because of potential sugar release from enzyme-

induced fibrolysis, but the response depends on the enzyme activities and treatment

conditions (Adesogan, 2005).

Kung and Ranjit (2001) compared whole-plant barley treated with L. buchneri and

enzymes, or a mixture ofL. plantarum, P. pentosaceus, P. freudenreichii and enzymes

or a buffered propionic acid-based additive. They observed that silages treated with L.

buchneri and enzymes had lower pH and higher concentrations of acetic and propionic

acids and improved aerobic stability when compared with untreated silage. These results

indicate that enzymatic treatments can represent a viable strategy for improving the

quality of silages, though they don't directly affect aerobic stability.

Effect of Enzyme Treatment on Hay Nutritive Value

Direct effects of fibrolytic enzyme treatment on chemical composition of treated

hays before ingestion have not been studied previously. Instead, research has focused on

the effect of such enzymes on ruminal fermentation or voluntary intake of the treated

hays. Pinos-Rodriguez et al. (2002) observed that application of an exogenous fibrolytic

enzyme to alfalfa or ryegrass hays increased intake of DM (P < 0.01), OM and CP (P <

0.05) in lambs; however, NDF and ADF intake were not affected. The enzyme increased

apparent digestibility of CP, hemicellulose (P < 0.05), and NDF (P < 0.10) in alfalfa. The









enzyme also improved N balance (P < 0.05), and total VFA (P < 0.05) concentration in

the rumen (after 3 and 6-h of incubation) for both hays. According to the authors, these

results indicate that directly fed exogenous fibrolytic enzymes may change ruminal

fermentation, intake, and digestibility of forages. Dawson and Tricarico (1999) showed

that when fescue hay was not treated or treated with preparations high in xylanase or

cellulase activity, xylanase addition increased carbohydrate utilization and VFA

production, cellulase addition altered VFA proportions, and addition of a mixture of the

enzymes increased carbohydrate digestion and the acetate: propionate ratio.

Novak et al. (2003) evaluated the effect of a fibrolytic enzyme containing

carboxymethyl cellulase and xylanase on ruminal disappearance of DM, NDF and ADF,

and intestinal DM digestibility of wheat straw. Enzyme addition had no effect (P >0.05)

on the effective degradability of DM, NDF and ADF, but increased DM, NDF and ADF

disappearance after 4 and 6-hours of incubation and decreased these measures after

incubation for 12 and 24 hours. Differences in enzyme activity and stability in rumen

fluid, application methods, and characteristics of rumen fluid due to donor animal diet

partly explain the discrepancies between these studies. The conflicting results highlight

the merits of further evaluation of the benefits of fibrolytic enzyme application to hays.

Effect of Enzyme Treatment on Animal Performance

Dry matter intake has been increased (Beauchemin et al., 2000) or unchanged

(Beauchemin et al., 1999; Kung et al., 2000) by dietary supplementation with enzymes.

Feed intake responses to enzyme supplementation have generally been small and

inconsistent (Yang et al., 1999; Rode et al., 999; Schingoethe et al., 1999; Vicini et al.,

2003) with only occasional significant (P < 0.05) increases (Lewis et al., 1999).

Attempts to improve feed efficiency in dairy cows by the use of direct-fed fibrolytic









enzymes applied at or a few hours before feeding have yielded variable production

responses (Sutton et al., 2003).

Milk production also has been increased in some studies in which dairy cow diets

were supplemented with fibrolytic enzymes (Rode et al., 1999; Yang et al., 2000; Kung et

al., 2002), but it has been unaffected in other studies (Sheperd and Kung, 1996b;

Beauchemin et al., 2000; Vicini et al., 2003). Table 2.2 summarizes the results of

supplementation with fibrolytic enzymes on milk production from several experiments.

Milk yield responses have been generally positive but often not significant, while

changes in milk fat and protein concentration have been both positive and negative and

are often not significant (Beauchemin et al., 1999; Lewis et al., 1999; Schingoethe et al.,

1999; Yang et al., 1999; 2000; Kung et al., 2000; Phipps et al., 2000; Rode et al., 1999;

Vicini et al., 2003). Rode et al. (1999) used lactating Holstein cows in early lactation to

investigate effects of exogenous fibrolytic enzyme (Promote) supplementation on DMI,

milk production and digestibility. Enzyme addition did not affect DMI (P > 0.05) but

tended (P < 0.1) to increase milk yield (35.9 vs. 39.5 kg/d) as a consequence of increased

digestibility. Percentage of milk fat was lower (P < 0.05) and percentage of milk protein

tended to be lower (P < 0.1) in cows fed the enzyme-supplemented diet, such that

component yields were similar (P > 0.05) for cows fed either diet. Energy deficiency was

numerically lower (P > 0.05) for cows fed the enzyme-supplemented diet than for cows

fed the Control diet (-3.33 vs. -3.62 Mcal/d). Consequently, the authors concluded that

supplementing dairy cow diets with Promote has the potential to enhance milk yield and

nutrient digestibility by cows in early lactation without changing feed intake.









Table 2.2 Effect of spraying enzymes onto feeds prior to feeding on milk production in
recent studies
Increase in milk Dietary forage P
Study production1, kg/d type values
Beauchemin et al., 1999 +0.3, +1.5 Barley silage + NS
alfalfa haylage (45%)
Lewis et al., 1999 +1.2, +6.3, +1.6 Alfalfa hay + alfalfa < 0.05
silage (41.6%)
Rode et al., 1999 +3.6 Corn silage + alfalfa < 0.11
hay (38.5%)
Schingoethe et al., 1999 Expt. 1: + 1.2, + 0.9, + 2.7 Corn silage + alfalfa NS
Expt. 2: + 1.3 hay (55%) < 0.01
Yang et al., 1999 + 0.9, + 1.9, + 1.6 Barley silage + <0.05
alfalfa cubes (52.8%)
Beauchemin et al., 2000 0.5, 0.5 Barley silage + NS
alfalfa haylage (45%)
Kung et al., 2000 Expt. 1: + 2.5, -0.8 Corn silage + alfalfa < 0.10
Expt. 2: + 0.7, + 2.5 hay (45%) < 0.10
Yang et al., 2000 + 0.1, + 2.1 Corn silage + alfalfa < 0.05
hay (38%)
Zheng et al., 2000 + 2.0, + 4.1, + 1.5 Corn silage + alfalfa < 0.07
hay (50%)
Bowman et al., 2002 + 0.6, 0.6, -1.5 Barley silage + <0.10
alfalfa silage (55%)
Knowlton et al., 2002 + 1.8, 1.2 Corn silage + alfalfa NS
silage (53%)
SThe increase in milk is relative to milk production by Control cows, NS: no significant effect


Studies have shown that application of low or high amounts of enzymes to forages

or diets produced different responses. Yang et al. (1999) examined the effect of two

doses of a cellulase-xylanase enzyme mixture applied to the forage or concentrate

component of dairy cow diets. They observed that milk production increased in

cows fed a high dosage of the enzyme compared with cows fed the Control diet, but

effects on milk composition were minimal. The response to enzyme supplementation

was affected more by amount of enzyme applied than by the dietary component treated

with the enzyme. The authors claimed that the results demonstrated the benefits of using

a fibrolytic enzyme to enhance feed digestion and milk production by dairy cows.









However, Beauchemin et al. (2000) found that a high level of enzyme application was

less effective than a low level at increasing total tract digestibility.

Lewis et al. (1999) carried out two experiments to evaluate the effectiveness of

adding a mixture of cellulases and xylanases to dairy cow diets. In Experiment 1, cows

were assigned to diets containing forages that had or had not been treated with the

enzyme between 8 and 24 h prior to feeding. They observed that cows consuming the

enzyme-treated forage produced more milk (27.2 vs. 25.9 kg/d, P < 0.05) and digested

more DM per day than did cows fed the Control forage. In Experiment 2, early lactation

cows were assigned to one of four treatments for 16 wk: 1) no enzyme treatment, 2) a low

(1.25 ml/kg of forage DM) enzyme treatment, 3) a medium (2.5 ml/kg of forage DM)

enzyme treatment, or 4) a high (5.0 ml/ kg of forage DM) enzyme treatment. Dry matter

intake was similar across enzyme treatments and intake was greater than for cows fed the

Control forage. Yield of milk, 3.5% fat-corrected milk, and energy-corrected milk were

greater by cows on Treatment 3 than by cows on Treatment 1. Therefore, applying

fibrolytic enzymes to the forage portion of the rations improved lactational performance

of early and mid-lactation cows.

Schingoethe et al. (1999) evaluated the response to a direct-fed (applied at feeding

time) cellulase and xylanase enzyme mixture applied at 0, 0.7, 1.0 or 1.5 L/ton of DM to

the forage portion (60% corn silage and 40% alfalfa hay) of a TMR for lactating cows

just prior to feeding. Over the 12-wk trial period, milk production from cows assigned to

the 1.5-L enzyme treatment increased by 10.8% relative to those in the Control (no

enzyme addition) group, while fat and protein production increased by 20 and 13%,

respectively. The lowest enzyme rate accounted for approximately one-half of the milk









production increases that occurred with the highest enzyme application rate. The

responses to enzyme-treated forages were initially evident 2 to 4 wk after experiment

started and they were maintained throughout the remainder of the experiment. Cows that

started to receive enzyme-treated forage during the first 100 d postpartum produced 9 to

15% more milk and 16 to 23% more energy-corrected milk than did cows fed the Control

diet. However, milk production was not increased when cows were in mid-lactation (121

d postpartum) at the start of the experiment (Schingoethe et al., 1999).

The reason for the general poor response to low levels of enzyme application is

obvious, but a lack of benefit for the high levels is less apparent. Such occurrences may

be attributed partly to negative feedback inhibition which is one of the classical modes of

regulation of enzyme action (Adesogan, 2005). This feedback mechanism occurs when

enzyme action is inhibited by production of a critical concentration of a product of the

enzyme-substrate interaction. For instance, fermentation of sugars produced by cell wall

hydrolysis may reduce ruminal pH to levels that inhibit cell wall digestion (Adesogan,

2005) by the negative effect of low pH on ruminal fibrolytic microorganisms. An

alternative hypothesis is that excessive enzyme application blocks the binding sites for

enzymes or may prevent substrate colonization (Beauchemin et al., 2003).

Sutton et al. (2003) used multiparous cows fitted with rumen and proximal

duodenal cannulas in early lactation to investigate the effect of method of application of a

fibrolytic enzyme product on digestive processes and milk production. The enzyme was

not applied (Control), sprayed on the TMR before the morning feed (TMR-E), or on the

concentrate the day before feeding (Conc-E), or infused into the rumen for 14 h/d

(Rumen-E). There was no treatment effect on either feed intake or milk yield but values









were numerically higher in cows fed TMR-E than in the rest of the cows. Ruminal

digestibility of DM, OM and starch were unaffected by the enzyme. Ruminal NDF

digestibility was lowest in cows fed TMR-E, but these cows also had the greatest post-

ruminal NDF digestibility. Total tract digestibility of starch, DM and OM were highest

in cows fed TMR-E. Ruminal retention time was reduced by all enzyme treatments but

postruminal transit time was increased so the decline in total tract retention time with

enzymes was not significant. It was suggested that the reduction in ruminal particle

retention time would reduce time available for fibrolysis to occur; and therefore, partly

explain the variability in the reported responses to enzyme treatment.

Bowman et al. (2002) also investigated the effect on dairy cows (averaged 111 +

32 DIM) of a fibrolytic enzyme (Promote) added at 1.0 g/cow/d to the concentrate

portion (45% of the dietary DM) of the TMR, to the pelleted supplement portion (4% of

the dietary DM) of the TMR, or to a premix (0.2 % of the dietary DM). The effects of

enzyme supplementation on milk production and composition were not significant (P

>0.05), but cows receiving the enzyme-supplemented concentrate had numerically higher

FCM compared to the Control cows. Knowlton et al. (2002) evaluated the effect of a

fibrolytic enzyme formulation on the intake, partitioning, and excretion of N and P by

dairy cows in early and late lactation. Cows fed diets containing the enzyme formulation

gained more weight than those fed the enzyme-free diet, particularly in early lactation.

Enzyme treatment did not affect apparent digestibility, excretion of N and P, or retention

of these nutrients in body tissues. Interactions observed between the effects of stage of

lactation and treatment indicated that the nature of the milk yield and manure excretion

responses differed between early and late-lactation cows. Milk yield, fecal output and N









excretion in cows fed enzyme-supplemented diets were greater than those of Control

cows in early lactation, but lesser in late-lactation. Energy requirements of early lactation

cows are higher that those in late lactation; therefore, the enzyme supplementation in

early lactation is potentially more promising than in mid-lactation or late-lactation

because it can improve energy balance (Jurkovich et al., 2002).

Effect of Enzyme Treatment on Blood Metabolites

Urea is the primary form of excretory N in mammals, and greater concentrations of

blood urea N (BUN) have long been known to reflect inefficient utilization of dietary CP

by ruminants (Broderick and Clayton, 1997). Few papers have reported the effect of

fibrolytic enzymes on blood metabolites. Hristov et al. (1998) found that blood glucose

and urea concentration in lactating dairy cows were not affected by enzyme treatments.

Hristov et al. (2000) observed that plasma beta hydroxybutyrate (BHBA) concentration

was reduced (P < 0.01) in cows supplemented with fibrolytic enzymes. Jurkovich et al.

(2002) also found a lower incidence of hyperketonaemia and lower acetoacetic acid and

non-esterified fatty acid (NEFA) concentrations in the blood of cows supplemented with

a mixture of fibrolytic enzymes, which indicates that enzyme supplementation can

improve energy balance in lactating cows.

Effects of Combining Enzyme and Chemical Treatments

Though chemical treatments also have been successfully used to disrupt ferulate

bridges and hydrolyze cell walls in tropical forages, little is known about the

effectiveness of biological treatment at achieving these objectives. Opportunities exist to

improve overall utilization of lignocellulosic materials as ruminant feeds by using

organisms or their secreted enzymes with the capacity to attack the most refractory fiber

components that have lignin-carbohydrate bonds (Varga and Kolver, 1997). The









prospects for improved use of fibrous residues relies on enhancing the rate of

fermentation of the more readily fermented cell wall constituents and increasing the

extent of digestion of poorly degraded constituents.

Combinations of chemical and biological treatments have been applied to low

quality forages, and the results show that they can act synergistically for improving the

nutritive value of such roughages. Wang et al. (2004) carried out four experiments to

study the effects of pre-treating wheat straw with alkali (5% of NaOH, wt/wt, or 3%,

wt/wt of NH3) and then spraying it with an enzyme mixture (xylanase, B-glucanase,

carboxymethylcellulase, and amylase) on in vitro, in situ, and in vivo digestibility. In

Experiment 1 enzymes increased (P < 0.01) gas production and the incorporation of 15N

into microbial N at 4 h from NaOH-treated wheat straw (P < 0.01 for gas; P < 0.05 for

15N) compared to untreated wheat straw. In Experiment 2, untreated and alkali-treated

wheat straw were sprayed with enzymes at 0, 0.15, or 1.5 mg/g DM and incubated

ruminally in nylon bags for up to 80 h to determine the in situ DM disappearance

(ISDMD). Interactive effects (P < 0.05) of pretreatment and enzymes were observed on

all ruminal degradation parameters. Alkali increased the rate (P < 0.01) and extent (P <

0.01) ofISDMD irrespective of enzymes. Enzyme application to untreated straw did not

affect the extent of ISDMD, but increased (P < 0.01) that of alkali-treated straw.

In Experiment 3, substrates from Experiments 1 and 2 were incubated in acetate

buffer for 24 h to measure the hydrolytic loss of DM and release of reducing sugars and

phenolic compounds. Alkali pretreatment and enzymes each increased (P < 0.01) DM

loss and the release of reducing sugars, and in combination, exerted additive effects (P <

0.01). Enzymes did not affect the release of phenolic compounds from the straw. In









Experiment 4, wrapped straw bales were injected with NH3 four months before the study,

and enzymes were applied immediately before feeding. Applying enzymes to

ammoniated straw increased (P < 0.05) digestibility ofDM, OM, and total N but did not

affect the intake of DM or digestibility of ADF by crossbred beef cows in late gestation.

According to the authors, pretreatment of straw with alkali enhanced the efficacy of

exogenous enzymes, presumably by breaking esterified bonds and releasing phenolic

compounds and/or by swelling the crystalline cellulose and enhancing enzyme

penetration. Adogla-Bessa et al. (1999) also found that adding urea and fibrolytic

enzymes to wheat silage was more effective than either treatment alone. However, using

enzymes and chemicals for forage improvement is probably not economically viable.

Including enzymes that mimic alkali hydrolysis (e.g., esterases) in commercial feed

additives could improve substantially the effectiveness of enzyme products for ruminants.

The conflicting results on the effectiveness of fibrolytic enzymes for enhancing

forage nutritive value and animal performance highlight the need for more concerted

investigation of this subject. The fact that even less is known about the extent to which

enzymes can improve the quality of tropical forages and enhance animal performance

from such forages, emphasizes the importance of future studies in this area.

The aim of this series of experiments was to evaluate the effect of ammoniation and

proprietary fibrolytic enzyme application on the nutritive value of tropical grasses and on

animal performance.

The specific objectives were:

To evaluate the effect of applying ammonia or four commercial fibrolytic enzymes on the
nutritive value of two C4 grass hays (Chapter 3).






45


To evaluate the effect of applying different rates of four proprietary fibrolytic enzyme
preparations at different rates, at ensiling, on the nutritive value of Tifton-85
bermudagrass silage (Chapter 4).

To determine the effects of applying an enzyme to bermudagrass at ensiling, or to
different components of the diet at feeding 8 in feed intake, milk production and
composition, blood metabolites and digestion kinetics of dairy cows (Chapter 5).














CHAPTER 3
EFFECT OF TREATMENT WITH AMMONIA OR FIBROLYTIC ENZYMES ON
THE NUTRITIVE VALUE OF HAYS PRODUCED FROM TROPICAL GRASSES

Introduction

Feed enzymes have been shown to be effective in a wide range of diets containing

roughages (Rode and Beauchemin, 1998). Their effectiveness is partly due to improved

hydrolysis of the fiber fraction (Colombatto et al., 2003b) which increases digestibility

(Christensen, 1997; Rode et al., 1999) and voluntary intake (Pinos-Rodriguez et al.,

2002). Nevertheless, other studies have shown that exogenous enzymes do not

consistently improve forage utilization. This inconsistency is attributable to factors such

as differences in enzyme type and activity, treatment duration, application method, diet

composition and level of animal performance.

Fibrolytic enzymes seem to work by increasing the rate, but not the extent of fiber

digestion (Feng et al., 1996; Yang et al., 1999). This suggests that the fibrolytic enzyme

products currently on the market for ruminants may not be introducing novel enzyme

activities into the rumen (Wang and McAllister, 2002). One of the few studies on

fibrolytic enzyme treatment of tropical grasses showed that enzyme treatment had no

effect on silage fiber concentration, cell wall carbohydrate fraction and in vitro or in situ

DM or NDF disappearance of silages (Mandevbu et al., 1999). Yet due to the widespread

use of C4 grasses which intrinsically have low nutritive values, it is important to

determine if modifying treatment conditions will lead to enzyme-mediated enhancements

in their quality.









Ammoniation is one of the most studied chemical treatments for improving forage

quality. Ammoniation improves forage digestibility due to the hydrolytic action of the

ammonia on linkages between lignin and structural polysaccharides, thus increasing the

organic matter potentially available for utilization by the ruminal microorganisms

(Barrios and Ventura, 2002). Ammoniation also increases the crude protein (CP)

concentration of the treated forages, and this improvement is through fixation of the

applied nitrogen (Weiss and Underwood, 1995). The objective of this experiment was to

evaluate the effect of applying ammonia or four commercial fibrolytic enzymes on the

nutritive value of two C4 grass hays.

Materials and Methods

Enzyme Application

In the first of two experiments, the effects of applying NH3 or a fibrolytic enzyme

complex (Promote, Pr) (Cargill, Minnetonka, MN) were measured on the DM and

chemical composition and in vitro and in situ digestibility of two tropical grass hays. The

forages tested were 12-week regrowth of Coastal bermudagrass hay (Cynodon dactylon)

(BE) and Pensacola bahiagrass hay (Paspalum notatum) (BA). The ammonia was

applied at 40 g/kg DM and the enzymes were applied at 0 (Control), 0.5, 1 and 2 times

the rates recommended by the respective manufacturers. This was done because the

optimal application rate for C4 grass hays was unknown. The actual application rates are

shown in Table 3.1. The enzymes were dissolved in 500 ml of water and applied in a fine

spray to 3 replicates of 2 kg of each hay. Treated hays were stored for 3 weeks in plastic

bags (30 bags for Experiment 1 and 66 bags for Experiment 2) and then chemically

characterized. The manufacturer-stipulated activities of the enzymes are shown in Table

3.2. Cellulase activity was also determined at 390C and pH 5.5 using the filter paper









method (Wood and Bhat, 1988) and the values obtained for Pr, X-20, CA and A-20 were

33.7, 22, 0 and 51.3 filter paper units/g, respectively, where one unit of activity is the

amount of enzyme that releases exactly 2 mg of glucose from 50 g of filter paper in 60

min. Xylanase activity was determined at 390C and pH 5.5 using the di-nitro salicylic

acid procedure (Bailey et al., 1992) and the values obtained for Pr, X-20, CA and A-20

were 5190, 7025, 0 and 3530 ptmol of xylose released/min/ml, respectively. In the

second experiment, the effects of applying NH3 or three fibrolytic enzymes were

measured on the same variables as in the previous experiment. The enzymes studied in

Experiment 2 were Biocellulase X-20 (X-20) (LodeStar, IL, USA), Cattle-Ase (CA)

(Loveland Industries Inc, Greeley, CO, USA) and Biocellulase A-20 (A-20) (LodeStar,

IL, USA). Two separate experiments were conducted because the enzymes were not

simultaneously available.

Laboratory Analysis

The NDF and ADF concentrations (Van Soest et al., 1991) of the samples and

digested residues were determined without amylase pretreatment using an ANKOM200

Fiber Analyzer (ANKOM Technology, Macedon, NY). Hemicellulose was calculated by

difference from NDF and ADF concentrations. Water soluble carbohydrates (WSC) were

determined with the anthrone reaction assay (Ministry of Agriculture Fisheries and Food,

1986). Crude protein (CP) was determined by digesting 0.5 g of sample using a micro

Kjeldahl apparatus (Labconco Corporation, Kansas City, MO) and the N concentration

was determined (Noel and Hambleton, 1976) using a Technicon Auto Analyzer

(Technicon, Tarrytown, NY, USA).









Table 3.1 Actual enzyme application rates used
Application rate
Enzyme 0.5x Ix 2x

Promote1 (mg/kg DM) 650 1300 2600
Biocellulase X-202 (mg/kg DM) 7.3 14.5 29
Biocellulase A-202 (mg/kg DM) 7.3 14.5 29
Cattle-Ase3 (mg/kg DM) 89 178 356
'Cargill, Minnentoka, MN
2 LodeStar, Channahon, IL, USA
3 Loveland Industries Inc, Greeley, CO, USA


Table 3.2 Manufacturer-stipulated enzyme activities.
Enzymatic activity
Enzyme Cellulase Xylanase B-Glucanase Amylase
(Units/g) (Units/g) (Units/g) (Units/g)
Promote1 1,200-
Biocellulase X-202 5,700 16,000 600 1,200
Biocellulase A-202 6,000 400 4,300 3,100
Cattle-Ase3 15,000--
SCargill, Minnentoka, MN, USA
2 LodeStar, Channahon, IL, USA
3 Loveland Industries Inc, Greeley, CO, USA


The in vitro digestibility of DM (IVDMD), NDF (IVNDFD) and ADF (IVADFD)

were determined in duplicate runs after incubating forage samples in buffered rumen

fluid for 6 or 48-h using two ANKOM" Daisy Incubators (ANKOM Technology,

Macedon, NY). The buffer was prepared according to the ANKOM Technology

procedure. The rumen fluid was obtained before feeding from two, non-lactating,

fistulated cows, fed 9 kg of Coastal bermudagrass hay and 400 g of soybean (Glycine

max) meal daily.

In situ rumen degradability was measured only in hays treated with NH3, X-20 and

A-20, because these treatments were found to be more effective at increasing in vitro

digestibility than the others. Five g of ground (4 mm screen) hay samples were weighed

into nylon bags (50 mrn pore size) in triplicate and placed into the two fistulated, non-









lactating Holstein cows for 0, 3, 6, 9, 12, 24, 48, 72, 96, and 120 h. At each incubation

time, bags were removed and rinsed with cool water and frozen. At the end of each

period, all bags were washed in a washing machine and dried for 48-h at 60 oC. The

cows used for this study were the same as those used as rumen fluid donors for the in

vitro study. In order to avoid placing too many substrate-filled bags in the rumen, only

bags for 3 treatments (Control, ammonia and X-20 or A-20) and one forage were

simultaneously incubated (60 bags maximum incubated at the same time/cow).

Statistical Analysis

A completely randomized design with 3 replicates per treatment was used to

quantify the effects of enzyme or NH3 application on chemical components.

The model used was:

Yijk: { + Ti + Ei

Where:

Yij: dependent variable

i: general mean

Ti: treatment effect (enzyme*level) and NH3

Ei: experimental error

A completely randomized design with 3 replicates per treatment was used to

quantify the effects of enzyme or NH3 application on digestibility after 6-h and 48-h.

Data from 6-h and 48-h incubations were analyzed separately.

The model used was:

Yijk: t + Ti + Rj + Eij

Where:

Yij: dependent variable









i: general mean

Ti: treatment effect (enzyme*level) and NH3

Rj: run effect

Eij: experimental error

Data were analyzed using the GLM procedure of SAS (1995). Orthogonal

contrasts were used to compare additive treatment means, and polynomial contrasts were

used to determine the effect (linear, quadratic and cubic) of increasing the amount of

enzyme application. Treatment significance was declared at the 5% level and tendencies

were declared at the 15% level.

The interaction treatment forage was not included in the previous models because

the in vitro and in situ trial were done separately.

The in situ ruminal degradation parameters were estimated using the model

described by McDonald (1981):

P= a + b (-e-c(tL))

where

P = DM degraded at time t, a = wash fraction, b = potentially degradable fraction,

a+b= total degradable fraction, c = the rate at which b is degraded, t = time

incubated in the rumen, and L = lag phase. The constants a, b, c, and L were estimated

using the nonlinear regression (NLIN) procedure of SAS (1995) and analyzed using the

GLM procedure of SAS (1995).

Results and Discussion

Chemical Composition of Tropical Hays

The chemical composition of untreated bermudagrass and bahiagrass hays is

shown in Table 3.3. The low CP and high NDF, ADF, hemicellulose and lignin









concentrations are typical of mature tropical grasses. These values agree with Jung and

Allen (1995) who concluded, that depending on the stage of maturity, cell walls represent

between 30 and 80% of plant DM in grasses so that under most circumstances, the bulk

of carbohydrates in mature grasses are from cell wall polysaccharides.

Effect of Promote and Ammoniation on Chemical Composition in Experiment 1

Enzyme treatment increased (P < 0.05) the NDF concentration of BE hay, suggesting that

Pr also contained non-fibrolytic enzymes (Table 3.4). However, the NDF concentration

of BA was decreased (P < 0.01) by both Pr and NH3 treatment, though NH3 treatment

was more effective (P < 0.01) at hydrolyzing the NDF fraction than Pr. Bahiagrass hay

had a lower (P < 0.01) concentration of ADF than BE. The ADF concentration of BE

was decreased (P < 0.01) by NH3 and Pr (linear response) treatments, but that of BA was

not. Promote tended (P = 0.052) to be more effective at hydrolyzing the ADF of BE than

NH3. The hemicellulose concentration of BE was increased (P < 0.01) by enzyme

(quadratic response) and NH3 treatment, whereas that of BA was decreased (P < 0.01) by

NH3 treatment.

The WSC concentration of BA was increased (P < 0.01) by Pr and NH3 treatment

(P < 0.01) and NH3 was more effective in this respect. Both treatments also numerically

(P > 0.05) increased the WSC concentration of BE. The higher WSC concentration

observed in the NH3-treated BA hay compared to other hays, shows that the chemical

treatment was more effective at hydrolyzing the fiber fraction of this forage. Enzyme

treatment did not affect the CP concentration of either of the hays; however NH3

treatment produced greater (P < 0.01) values than those of Control and enzyme-treated

hays.









Table 3.3 Chemical composition of the untreated hays
Forage
Nutrient Bahiagrass Bermudagrass
CP, g/kg DM 66 69
NDF, g/kg DM 792 821
ADF, g/kg DM 431 485
Lignin, g/kg DM 55 67
Hemicellulose, g/kg DM 336 354
Ash, g/kg DM 57 50
CP: crude protein, NDF: neutral detergent fiber, ADF: acid detergent fiber


These results indicate that the treatments, particularly NH3, had different effects on

cell wall components of the forages and forage type affected the response. In BE, total

cell wall concentration was not reduced by additive treatment but the NDF fraction was

hydrolyzed by other additives, thus increasing the ADF. Whereas in BA, the ADF

fraction was unaffected by treatment, but NDF and hemicellulose fractions were

hydrolyzed into sugars, which is in agreement with Colombatto et al. (2003), and Hristov

et al. (1998). Promote treatment was less effective than NH3 treatment at cell wall

hydrolysis. This result agrees with those of Brown (1993), who observed that

ammoniation decreased (P < 0.01) the NDF concentration of stargrass (Cynodon

nlemfuensis) hay.

Effect of Promote and Ammonia Application on in vitro DM, NDF, and ADF
Digestibility in Experiment 1

After 6-h of digestion, BA had greater (P < 0.01) IVDMD than BE, but this

difference was no longer evident after 48-h of digestion. Promote-treatment increased (P

< 0.01) the 6-h IVDMD of BE; however, ammoniation was more effective (Table 3.5).

Only NH3 treatment increased (P < 0.01) the 6-h IVDMD of BA or the 48-h IVDMD of

both forages. The effectiveness of NH3 at increasing the 6-h and 48-h digestibility of the

hays concurs with the results of Zorrila-Rios et al. (1991), who observed that the












Table 3.4 Effect of Promote or ammonia treatment on chemical composition (% DM) of tropical grass hays
NDF ADF Hemicellulose WSC CP
Additive Level BE BA BE BA BE BA BE BA BE BA


Control
Promote


0.5x
lx
2x
Mean


Ammonia


82.1
85.2
85.1
86.2
85.5
83.6


79.2
78.1
78.1
78.5
78.2
75.4


48.5
46.7
46.8
44.5
46.0
46.7


43.1
43.8
42.6
44.3
43.6
43.7


33.6
38.4
38.3
41.7
39.5
37.0


35.4
35.0
34.4
34.2
34.5
31.7


6.9
6.7
6.8
7.1
6.9
12.8


0.964 0.354 0.293 0.491 0.872 0.737


0.039


0.019 0.258 0.139


P values


Polynomial effects
Promote level

Promote vs. Control
Control vs. Ammonia
Promote vs. Ammonia


L** NS


0.015
0.289
0.128


<0.01
<0.01
<0.01


<0.01
<0.01
0.052


0.452
0.962
0.485


C* NS


<0.01
0.021
0.593


0.339
<0.01
0.098


0.232
0.234
0.785


<0.01
<0.01
<0.01


0.477
<0.01
<0.01


s.e.m.


Contrasts


NDF: neutral detergent fiber, ADF: acid detergent fiber, WSC: water soluble carbohydrates, CP: crude protein, BE: bermudagrass,
BA: bahiagrass, L: linear effect, Q: quadratic effect, C: cubic effect, NS: no significant effect, *: P < 0.05, **: P < 0.01, 'Bahiagrass vs.
bermudagrass


0.285
<0.01
<0.01









Table 3.5 Effect of Promote or ammonia application on the IVDMD of tropical grass
hays
DM digestibility, %
After 6-h After 48-h
Additive Level BE BA BE BA
Control 10.1 14.2 49.8 47.9
0.5x 10.6 14.2 49.7 48.8
Promote lx 11.4 14.9 49.0 49.3
2x 11.1 14.4 49.9 48.3
Mean 11.0 14.5 49.5 48.8
Ammonia 16.5 18.7 61.4 61.7
s.e.m. 0.237 0.308 0.463 1.582

Polynomial effects P values
Promote level NS NS NS NS

Contrasts
Promote vs. Control <0.01 0.418 0.686 0.633
Control vs. Ammonia <0.01 <0.01 <0.01 <0.01
Promote vs. Ammonia <0.01 <0.01 <0.01 <0.01
DM: dry matter, BE: bermudagrass, BA: bahiagrass, NS: non significant effect


IVDMD of the wheat (Triticum aestivum) straw was increased by approximately 54% by

ammoniation.

Dawson and Tricarico (1999) suggested that the most active period for exogenous

enzyme is the first 6 12 h of digestion, which transpires prior to bacterial colonization

of feed substrates or action of endogenous enzymes. This partly explains why Pr

increased the 6-h IVDMD and not the 48-h IVDMD of BE.

The 6 and 48-h IVNDFD of both hays were similar (Table 3.6), despite the lower

NDF and lignin concentrations of BA. Treatment with NH3 increased (P < 0.01) the 6

and 48-h IVNDFD in both forages. Pr treatment tended (P < 0.15) to reduce the 6-h and

48-h IVNDFD of BE but did not affect the corresponding values for BA. In contrast to

the 6-h IVADFD, the 48-h IVADFD was higher (P < 0.01) in BE than in BA hay









Table 3.6 Effect of Promote or ammonia application on the IVNDFD of tropical grass
hays
NDF digestibility, %
Additive Level After 6-h After 48-h
BE BA BE BA
Control 6.4 5.5 43.6 40.7
0.5x 5.1 4.9 44.5 42.9
Promote lx 3.5 4.1 38.2 37.1
2x 6.3 5.8 40.2 38.9
Mean 5.0 4.9 41.0 39.6
Ammonia 8.1 7.9 58.4 59.4
s.e.m. 0.723 0.473 1.305 1.032

Polynomial effects P values
Promote level Q* NS Q* Q*

Contrasts
Promote vs. Control 0.149 0.349 0.105 0.383
Control vs. Ammonia <0.01 <0.01 <0.01 <0.01
Promote vs. Ammonia <0.01 0.080 <0.01 <0.01
NDF: neutral detergent fiber, BE: bermudagrass, BA: bahiagrass, Q: quadratic effect,
NS: no significant effect, *: P < 0.05, **: P < 0.01,


(Table 3.7). The 6-h and 48-h IVADFD were higher (P < 0.01) in NH3-treated hays than

the other hays for both forage types. Pr treatment reduced (P < 0.15) most of the 6-h and

48-h IVADFD values of the hays.

These results suggest that except for slightly increasing the 6-h IVDMD of BE, Pr

treatment did not improve DM or cell wall digestion in the forages. The fact that some of

these measures were decreased by Pr treatment is surprising. In contrast, NH3 treatment

increased 6-h and 48-h digestibility estimates, which reflect the rate and extent of

digestion, respectively. Since intake is constrained by the rate at which the diet is

digested (Romney and Gill, 2000), ammoniation is more likely to increase intake and

thereby increase animal performance than Pr treatment. Incremental addition of Pr did

not have consistent beneficial effects on the nutritive value of BA or BE.









Table 3.7 Effect of Promote or ammonia application on the IVADFD of tropical grass
hays
ADF digestibility, %
Additive Level After 6-h After 48-h
BE BA BE BA
Control 3.7 5.4 48.0 41.9
0.5x 1.5 4.2 46.7 41.8
Promote lx 3.1 3.1 45.0 37.2
2x 1.8 5.1 48.2 39.2
Mean 2.1 4.1 46.6 39.4
Ammonia 9.3 7.4 65.5 58.3
s.e.m. 0.855 0.511 1.345 0.880

Polynomial effects P values
Promote level Q* NS Q* Q*

Contrasts
Promote vs. Control 0.148 0.052 0.413 0.032
Control vs. Ammonia <0.01 0.021 <0.01 <0.01
Promote vs. Ammonia <0.01 <0.01 <0.01 <0.01
ADF: acid detergent fiber, BE: bermudagrass, BA: bahiagrass, Q: quadratic effect,
NS: no significant effect, *: P < 0.05, **: P < 0.01


Effect of Fibrolytic Enzyme and Ammonia Application on Chemical Concentration
of C4 Forages in Experiment 2

Unlike responses with BA, treating BE with NH3 (P < 0.01), X-20 (P < 0.05) or A-

20 (P < 0.01) decreased the NDF concentration and the CA enzyme gave the same

tendency (P=0.073) (Table 3.8). Ammonia treatment was more effective than CA

treatment. The ADF concentration of BE was decreased (P < 0.01) by NH3 treatment and

increased (P < 0.05) by CA and A-20 treatment, but that of BA was only decreased by X-

20 treatment (P < 0.01). The hemicellulose concentration of BE was decreased (P <

0.01) by treatment with X-20 (cubic response), CA and A-20, but not NH3. Only X-20

treatment increased (P < 0.01) the hemicellulose concentration of BA, other treatments

had no effect.











Table 3.8 Effect of fibrolytic enzyme or ammonia application
hemicellulose concentrations (%) of tropical hays


on the NDF, ADF and


NDF ADF Hemicellulose
Additive Level BE BA BE BA BE BA


Control
NH3


X-20


CA



A-20


0.5x
lx
2x
Mean

0.5x
Ix
2x
Mean
0.5x
lx
2x
Mean


s.e.m.
Polynomial effects
X-20 level
CA level
A-20 level


87.1
84.6
85.6
85.4
85.4
85.5

86.1
85.8
86.1
86.0
86.1
85.1
84.9
85.3
0.49


NS
Q**
NS


40.6 30.9
40.9 32.5
41.4 33.8
38.2 34.1
38.5 34.3
39.4 39.4


47.7
48.1
48.3
48.0
48.7
48.4
49.0
48.7
0.63
P values
C*
NS
NS


30.9
32.6
32
37.9
31
32.6
29.3
36.7
0.91


Contrasts
Control vs. X-20 0.011 0.530 0.674 <0.01 0.096 <
0.01
Control vs. CA 0.073 0.171 0039 0.507 < 0.01 0.358
Control vs. A-20 <0.01 0.176 <0.01 0.131 <0.01 0.892
Control vs. NH3 <0.01 0.082 <0.01 0.997 0.791 0.219
X-20 vs. CA 0.216 <0.01 <0.01 <0.01 <0.01 <
0.01
X-20 vs. A-20 0.662 <0.01 <0.01 <0.01 <0.01 <
0.01
CA vs. A-20 0.099 0.512 0.242 0.219 0.016 0.269
NH3 vs. X-20 0.131 <0.01 <0.01 <0.01 0.050 0.149
NH3 vs. CA 0.022 0.719 <0.01 0.507 <0.01 0.547
NH3 vs. A-20 0.223 0.412 <0.01 0.132 < 0.01 0.172
NDF: neutral detergent fiber, ADF: acid detergent fiber, BE: bermudagrass, BA: bahiagrass, X-20:
Biocellulase X-20, CA: Cattle-Ase, A-20: Biocellulase A-20, L: linear effect, Q: quadratic effect,
C: cubic effect, NS: no significant effect, *: P < 0.05, **: P < 0.01


The WSC concentration of BE hays was reduced by CA (tendency, P=0.053) and

A-20 (P < 0.05) treatment and unaffected by NH3 or X-20 treatment (Table 3.9).

However, that of BA hays was increased (P < 0.05) by X-20 treatment.










Table 3.9 Effect of fibrolytic enzyme or ammonia application on the WSC and CP
concentrations (%) of tropical hays
WSC CP


Additive
Control
NH3

X-20





CA





A-20


Level


0.5x
lx
2x
Mean

0.5x
lx
2x
Mean

0.5x
lx
2x
Mean


s.e.m.

Polynomial effects
X-20 level
CA level
A-20 level
Contrasts
Control vs. X-20
Control vs. CA
Control vs. A-20
Control vs. NH3
X-20 vs. CA
X-20 vs. A-20
CA vs. A-20
NH3 vs. X-20
NH3 vs. CA
NH3 vs. A-20


BE
2.81
2.82
3.39
2.01
1.75
2.4

1.75
1.19
2.63
1.9

1.64
1.67
1.74
1.7
0.041


C*
C*
NS
BE
0.370
0.053
0.025
0.998
0.125
0.047
0.613
0.370
0.053
0.025


BA
1.21
0.98
2.36
1.34
1.84
1.8

1.26
0.99
2.21
1.5

0.79
0.69
0.94
0.8
0.021


P values
NS
L**
NS
BA
0.019
0.281
0.127
0.481
0.056
<0.01
<0.01
<0.01
<0.01
0.060


WSC: water soluble carbohydrates, CP: crude protein, BE: bermudagrass,
BA: bahiagrass, X-20: Biocellulase X-20, CA: Cattle-Ase, A-20: Biocellulase A-20,
L: linear effect, Q: quadratic effect, C: cubic effect, NS: no significant effect, *: P < 0.05,
**: P < 0.01,


BE
6.5
16.3
6.9
6.7
6.5
6.7

6.7
7.0
6.5
6.7

6.8
6.8
6.9
6.8
0.019


L*
NS
NS
BE
0.509
0.338
0.163
<0.01
0.666
0.286
0.518
< 0.01
<0.01
< 0.01


BA
7.0
13.0
7.2
7.1
7.0
7.1

7.2
6.9
7.7
7.3

7.1
6.9
7.3
7.1
0.019


NS
NS
Q*
BA
0.731
0.287
0.286
<0.01
0.304
0.303
0.998
< 0.01
<0.01
< 0.01









Ammoniation decreased both ADF and NDF fractions of BE and did not affect the

hemicellulose fraction. All enzyme treatments decreased the NDF concentration of BE

by decreasing the hemicellulose concentration whereas the only effect on BA cell walls

was that X-20 treatment increased hemicellulose concentration by decreasing ADF

concentration.

This reveals a forage-specific response to the treatments which is similar to that

found in Experiment 1. It is interesting to note that except for X-20 effects on BA, none

of the treatments that hydrolyzed forage cell walls resulted in an increase in the WSC

concentration. This may be due to the relatively low WSC concentration of the forages

and the conversion of hydrolyzed cell wall fragments into oligosaccharides and

disaccharides that are not water soluble, and were therefore undetected in the WSC assay.

The results for BE agree with those in Experiment 1, but conflict with those of

Colombatto et al. (2003), who evaluated the effects of adding a commercial enzyme

product on the hydrolysis and fermentation of cellulose, xylan, and a mixture of both

substrates. They observed that addition of enzyme in the absence of ruminal fluid

increased (P < 0.01) the release of reducing sugars from xylan and the mixture.

Similarly, Hristov et al. (1998) observed that enzyme treatment increased the

concentration of soluble reducing sugars (P < 0.05) and decreased NDF concentration (P

< 0.05) in a TMR, consisting of rolled barley grain, corn silage and soybean meal.

The results of this study indicate a species-related difference in response to enzyme

treatment which was consistent across experiments. The effect of X-20 treatment and

ammoniation on the respective ADF concentrations of BE and BA show that this

treatment was more effective at disrupting lignocellulosic linkages. However, the reason









why enzymes were generally more effective at hydrolyzing BE despite its higher ADF

and lignin concentration is unclear, and is probably related to differences in the type of

phenolic cross linkages in the cell walls. In addition to differences in lignin

concentration, Mandevbu et al. (1999) also showed that differences in concentration of

ether-linked and ester-linked ferulic acid explained digestibility differences between

Tifton-85 and coastal bermudagrass.

The CP concentration of the hays was unaffected by enzymatic treatments, but

increased (P < 0.01) by NH3 treatment. This agrees with Weiss and Underwood (1995),

Brown and Adjei (1995) and Barrios-Urdaneta and Ventura (2002) who observed that

ammoniation increased the CP in forages, due to the supplemental N provided. Brown

(1993) also observed that, compared to a Control treatment, ammoniation (4% DM)

increased (P < 0.01) total N concentration (1.0 to 1.4% vs. 1.7 to 2.8%) of stargrass

(Cynodon nlemfuensis) hay, and a similar effect (3.26 vs. 4.16% N, P < 0.01) was

obtained by Lines et al. (1996) for alfalfa hay. Although enzymes are proteins, the small

amount of enzyme applied is not enough to effect forage CP concentration.

Effect of Enzyme Treatment and Ammoniation on in vitro DM, NDF, and ADF
Digestibility in Experiment 2

All of the additives were effective to increase 6-h IVDMD of BE (Table 3.10).

However, only NH3 (P < 0.01) and X-20 (tendency: P = 0.088) increased the 6-h IVDMD

ofBA. Ammoniation was the most effective treatment for increasing the 6-h IVDMD (P

< 0.01) in both grasses. The 6-h IVDMD was consistently greater (P < 0.01) in BA than

in BE hays. This is probably due to the lower NDF concentration and presumably higher

soluble fraction of BA hays and which would facilitate the initial degradation of the

forage.









Enzyme X-20 increased the 48-h IVDMD of BE (P < 0.05) and BA (P < 0.01)

hays, while CA and A-20 tended (P < 0.08) to have similar effects on only BE. However,

NH3 treatment was more effective (P < 0.01) than any of the enzymes at increasing the

48-h IVDMD of both hays.

These results suggest that all additive treatments can improve the 6-h and 48-h

digestion of BE, but only NH3 and X-20 had similar effects on BA. This supports the

conclusion that fibrolytic enzyme application can increase the rate of digestion of forages

(Wang and McAllister, 2002), but indicates that enzyme effects on rate and extent of

digestion depend on the enzyme and forage being tested.

The 6-h IVNDFD was higher (P < 0.05) in BA than in BE hays; however, the 48-h

IVNDFD was greater (P < 0.01) in BE than in BA hays. The 6-h and 48-h IVNDFD of

both hays were unaffected by enzyme treatment except for a linear increase with

increasing A-20 application to BE (Table 3.11). However, NH3 treatment did increase 6

and 48-h IVNDFD (P < 0.01) of BE and 48-h IVNDFD of BA (P < 0.01). The 6-h

IVADFD of BE hay was improved (P < 0.01) by X-20, A-20 and NH3 treatment. Only

NH3 treatment increased the 6-h IVADFD of BA (P < 0.01); however digestibility of BA

hay increased linearly (P < 0.05) with increasing application of X-20 (Table 3.12).

Therefore, the increases in 6-h IVDMD due to X-20 and A-20 treatment were partly due

to increases in 6-h IVADFD. Ammoniation was the only treatment that increased (P <

0.01) the 48-h IVADFD in either of the hays; though responses also occurred as the

respective rates of CA (linear) and X-20 (cubic) application to BE and BA increased.

None of the enzymes increased the extent of fiber digestion in the hays. Thus the

results concur with those of Mandevbu et al. (1999) who observed that treatment of









Table 3.10 Effect of fibrolytic enzyme or ammonia application on the IVDMD (%) of
tropical hays
After 6 h After 48 h
Additive Level BE BA BE BA
Control 7.7 12.6 43.6 44.5
NH3 13.7 18.1 56.8 59.9
0.5x 10.6 14.1 49.6 47.5
X-20 lx 11.4 13.0 51.1 45.6
2x 11.3 12.9 51.4 47.3
Mean 11.1 13.3 50.7 46.8

0.5x 9.0 12.1 46.5 42.3
CA lx 9.9 12.6 47.9 41.9
2x 10.1 12.9 47.6 45.6
Mean 9.7 12.5 47.3 43.3

0.5x 10.2 12.2 47.2 43.9
A-20 lx 11.6 11.9 48.1 46.8
2x 9.4 12.2 46.9 42.4
Mean 10.4 12.1 47.4 44.4
s.e.m. 0.761 0.361 1.789 1.067

Polynomial effects P values
X-20 level NS L* NS NS
CA level NS NS NS NS
A-20 level NS NS NS NS
Contrasts
Control vs. X-20 <0.01 0.088 <0.01 0.064
Control vs. CA 0.028 0.854 0.076 0.336
Control vs. A-20 <0.01 0.240 0.071 0.939
Control vs. NH3 <0.01 <0.01 <0.01 <0.01
X-20 vs. CA 0.035 <0.01 0.027 <0.01
X-20 vs. A-20 0.300 <0.01 0.026 <0.01
CA vs. A-20 0.261 0.169 0.985 0.215
NH3 vs. X-20 <0.01 <0.01 <0.01 <0.01
NH3 vs. CA <0.01 <0.01 <0.01 <0.01
NH3 vs. A-20 <0.01 <0.01 <0.01 <0.01
DM: dry matter, BE: bermudagrass, BA: bahiagrass, X-20: Biocellulase X-20,
CA: Cattle-Ase, A-20: Biocellulase A-20, L: linear effect, NS: no significant effect,
*: P < 0.05, **: P < 0.01.



bermudagrass forages with fibrolytic enzymes had no effect on in vitro or in situ DM or

NDF disappearance of silages. However, Rode et al. (1999) observed that in vivo










Table 3.11 Effect of fibrolytic enzyme or ammonia application on the IVNDFD (%) of
tropical hays
After 6-h After 48-h
Additive Level BE BA BE BA


Control
NH3

X-20





CA





A-20


0.5x
lx
2x
Mean

0.5x
lx
2x
Mean

0.5x
lx
2x
Mean


s.e.m.

Polynomial effects
X-20 level
CA level
A-20 level


4.0
5.4
5.6
5.0

4.9
4.7
6.1
5.2
0.47


5.8
7.0
4.5
5.8

6.1
6.3
7.2
6.5
0.88


43.1
52.6
46.7
47.5
43.1
46.2

48.0
46.9
46.4
47.1

47.9
45.0
47.4
47.5
1.74


37.3
54.4
35.8
35.2
37.9
36.3

33.1
35.7
34.0
34.2

36.2
38.9
35.4
36.8
1.59


P values


Contrasts
Control vs. X-20 0.760 0.542 0.635 0.586
Control vs. CA 0.951 0.683 0.146 0.244
Control vs. A-20 0.714 0.780 0.827 0.312
Control vs. NH3 <0.01 0.546 <0.01 <0.01
X-20 vs. CA 0.605 0.773 0.571 0.162
X-20 vs. A-20 0.931 0.215 0.399 0.716
CA vs. A-20 0.546 0.336 0.779 0.083
NH3 vs. X-20 <0.01 0.185 <0.01 <0.01
NH3 vs. CA <0.01 0.257 0.169 <0.01
NH3 vs. A-20 <0.01 0.645 <0.01 <0.01
NDF: neutral detergent fiber, BE: bermudagrass, BA: bahiagrass, X-20: Biocellulase X-20,
CA: Cattle-Ase, A-20: Biocellulase A-20, L: linear effect, Q: quadratic effect, C: cubic effect,
NS: no significant effect, *: P < 0.05, **: P < 0.01


digestibility determined using Cr203 was increased by a commercial enzyme (Promote)

added to the concentrate (DM: 61.7 vs. 69.1%; NDF: 42.5 vs. 51.0%; ADF: 31.7 vs.

41.9%; and CP: 61.7 vs. 69.8%) to a dairy cow diet. This conflicting responses










Table 3.12 Effect of fibrolytic enzyme or ammonia application on the IVADFD (% of
DM) of tropical hays
After 6 h After 48 h
Additive Level BE BA BE BA


Control
NH3

X-20





CA





A-20


0.5x
lx
2x
Mean

0.5x
lx
2x
Mean

0.5x
lx
2x
Mean


s.e.m.

Polynomial effects
X-20 level
CA level
A-20 level


2.2
2.8
5.6
3.5

6.0
6.6
6.6
6.4
0.77


L**
L*
NS


4.0
5.3
3.5
4.3

4.8
4.7
6.1
5.2
0.70


36.8
53.4
36.8
36.0
39.3
37.4

34.3
36.5
37.7
36.2

37.0
39.2
36.7
37.6
1.22


P values


35.3
44.2
39.9
36.2
32.7
36.3

37.1
39.2
36.7
37.7

35.9
33.8
38.3
36.0
1.24


C**
NS
NS


Contrasts
Control vs. X-20 <0.01 0.625 0.686 0.508
Control vs. CA 0.902 0.314 0.663 0.120
Control vs. A-20 <0.01 0.902 0.560 0.631
Control vs. NH3 <0.01 <0.01 <0.01 <0.01
X-20 vs. CA <0.01 0.456 0.241 0.196
X-20 vs. A-20 0.741 0.391 0.799 0.795
CA vs. A-20 <0.01 0.117 0.158 0.125
NH3 vs. X-20 <0.01 <0.01 <0.01 <0.01
NH3 vs. CA <0.01 <0.01 <0.01 <0.01
NH3 vs. A-20 <0.01 <0.01 <0.01 <0.01
ADF: acid detergent fiber, BE: bermudagrass, BA: bahiagrass, X-20: Biocellulase X-20,
CA: Cattle-Ase, A-20: Biocellulase A-20, L: linear effect, Q: quadratic effect, C: cubic effect,
NS: no significant effect, *: P < 0.05, **: P < 0.01


probably attributable to the higher nutritive value of the dairy cow diet relative to that of

C4 grasses, suggesting that higher quality diets respond more to enzyme supplementation,

presumably due to lower ADF concentration.No other studies that simultaneously









compared the effectiveness of chemical and biological treatments at improving the

quality of C4 grasses were found in the literature. However, several reports have shown

that ammonia treatment is very effective for improving the DM and fiber digestibility of

low quality forages (Brown, 1993; Vagnoni, et al., 1995; Weiss and Underwood, 1995;

Barrios-Urdaneta and Ventura, 2002). The beneficial effect of enzyme treatment on 6-h

and 48-h IVDMD were not due to increases in the extent of fiber digestion. Rather they

may have been attributable to increased microbial attachment (Dawson and Tricarico,

1999) and an increased rate of ADF digestion.

According to Barrios-Urdaneta and Ventura (2002), ammoniation improves forage

digestibility due to the hydrolytic action on linkages between lignin and structural

polysaccharides, thus increasing the OM potentially available for utilization by ruminal

microorganisms. These authors observed that ammoniation increased (P < 0.01) the in

vitro NDF digestibility (from 46.2 to 57.1%) of koroniviagrass. Ammonia treatment also

changes the physical characteristics of forages making them more pliable and increasing

their hydration rate. Hydration rate has an important role in digestion rate; the faster a

forage particle is hydrated, the faster it is digested (Weiss and Underwood, 1995).

Effect of Enzyme Treatments and Ammoniation on in situ DM Degradation

The effect of X-20 and NH3 on the kinetics of in situ DM disappearance of BE and

BA is presented in Table 3.13. Treatment with X-20 (linear, P < 0.05) and NH3 (P <

0.01) increased the wash loss (a) fraction of BE, but only NH3 treatment increased that of

BA. This result supports the findings obtained in vitro, where both of these treatments

increased the initial phase of digestion of BE.









Table 3.13 Effect of X-20 or ammonia application on the in situ kinetics of DM
disappearance of bermudagrass and bahiagrass
Parameter
Forage Treatment a, % b, % a + b, % P, % c L1, h


Bermudagrass
Control
NH3
X-20, 0.5x
X-20, lx
X-20, 2x
s.e.m.


Contrasts
Polynomial
Control vs. X-20
Control vs. NH3
X-20 vs. NH3


2.65
6.50
2.95
3.60
3.85
0.21


L*
0.019
< 0.01
< 0.01


Bahiagrass


Control
NH3
X-20, 0.5x
X-20, lx
X-20, 2x
s.e.m.


Contrasts
Polynomial
Control vs. X-20
Control vs. NH3
X-20 vs. NH3


6.35
7.35
7.50
5.70
6.20
0.27


Q*
0.727
0.049
0.038


60.2
70.9
57.5
57.7
56.8
1.95


NS
0.267
0.012
< 0.01


58.0
75.4
59.3
59.6
60.6
1.04


NS
0.191
< 0.01
< 0.01


62.8
77.4
60.5
61.4
60.6
1.99


59.1
71.7
57.5
57.6
56.0
2.34


P values
NS NS
0.425 0.514
< 0.01 0.017
< 0.01 < 0.01


64.4
82.7
66.8
65.3
66.8
1.15


63.4
81.1
65.2
63.6
65.1
1.26


P values
NS NS
0.207 0.419
< 0.01 < 0.01
< 0.01 < 0.01


0.041
0.054
0.041
0.029
0.053
0.01


NS
0.997
0.346
0.260


0.019
0.026
0.024
0.025
0.025
0.003


NS
0.153
0.144
0.679


9.064
8.341
7.439
6.226
9.326
1.00


NS
0.287
0.632
0.595


2.232
3.978
2.432
4.300
4.158
0.89


NS
0.714
0.578
0.747


DM: dry matter, X-20: Biocellulase X-20, a: soluble fraction, b: insoluble but potentially
degradable fraction, a+b= total degradability, P= DM degraded at time t, c: rate of constant
degradation, L': lag phase (period when no net disappearance of substrate occurs), L: linear
effect, Q: quadratic effect


Ammonia treatment was more effective than X-20 treatment at increasing (P <

0.01) the insoluble but potentially degradable (b) fraction, the total degradable fraction (a

+ b) and the degradability (P) of both forages. These results partially concur with those









Table 3.14 Effect of A-20 or ammonia application on the in situ kinetics of DM
disappearance of bermudagrass and bahiagrass
Parameter
Forage Treatment a, % b, % a + b, % P, % c L, h


Bermudagrass
Control
NH3
A-20, 0.5x
A-20, lx
A-20, 2x
s.e.m.


Contrasts
Polynomial
Control vs. A-20
Control vs. NH3
A-20 vs. NH3


Bahiagrass


Control
NH3
A-20, 0.5x
A-20, lx
A-20, 2x
s.e.m.


Contrasts
Polynomial
Control vs. A-20
Control vs. NH3
A-20 vs. NH3


6.95
7.95
7.75
7.50
7.25
0.456


NS
0.344
0.182
0.432


6.50
5.95
7.00
6.45
6.45
0.541


NS
0.839
0.505
0.324


61.4
62.2
60.0
59.7
59.9
0.435


NS
0.025
0.251
< 0.01


56.1
62.1
59.2
58.9
58.5
1.644


NS
0.209
0.051
0.151


68.4
70.2
67.7
67.2
67.1
0.351


68.2
69.0
67.1
66.7
66.2
0.407


P values
NS NS
0.052 0.023
0.015 0.199
< 0.01 < 0.01


62.6
68.0
66.2
65.4
64.9
1.458


62.2
67.5
65.8
64.9
64.5
1.424


P values
NS NS
0.149 0142
0.047 0.048
0.193 0.207


0.009
0.024
0.015
0.014
0.016
0.003


NS
0.119
0.011
0.032


0.015
0.016
0.010
0.014
0.014
0.003


NS
0.576
0.845
0.435


1.535
6.849
4.316
2.778
3.971
1.572


NS
0.289
0.062
0.142


7.583
7.731
7.120
6.011
7.366
3.373


NS
0.855
0.976
0.827


DM: dry matter, A-20: Biocellulase A-20, a: soluble fraction, b: insoluble but potentially
degradable fraction, a+b= total degradability, P= DM degraded at time t, c: rate of constant
degradation of b, L: lag phase (period when no net disappearance of substrate occurs)


obtained in vitro, where NH3 was the most effective treatment at increasing the extent of

digestion. Only the 'a' fraction of BE was affected by X-20, and neither treatment

affected the degradation rate or lag phase of the forages.

The results presented in Table 3.14 show that A-20-treated BE hays had lower b, a

+ b and P values than Control (P < 0.05) and NH3-treated hays (P < 0.01). The A-20









treatment also tended to increase the c value for BE and the b and a + b fraction of BA. In

BE hays ammoniation increased the lag phase and the c value and a + b fraction, while in

BA it increased b, a+b and P. The NH3 effects concur with results of Vagnoni, et al.

(1995), which showed that ammoniation of mature bermudagrass increased both the rate

(P < 0.05) and the potential extent (P < 0.01) of ruminal forage in situ DM disappearance

in lactating cows. The response to A-20 and X-20 treatments partly agree with Feng et al.

(1996) who found that applying cellulase, xylanase and a mixture of both enzymes at

different levels did not affect the in situ DM disappearance of cool-season grasses. Lewis

et al. (1996) evaluated a 70% grass hay diet treated with fibrolytic enzymes that were

applied at feeding or 24 h before feeding and observed that in situ DM disappearance was

unaffected by enzyme treatment of samples incubated for 8, 16, and 24 h, but increased

after 96-h (P < 0.05). The authors proposed that improved DM disappearance at 96-h of

incubation in enzyme-treated grass may have resulted from enhanced colonization and

digestion of the slowly degradable fiber fraction by ruminal microorganisms.

Conclusions

This work demonstrates that fibrolytic enzymes had negligible effects on in situ

DM degradation of C4 grass hays, though certain enzymes (X-20 and A-20) did increase

the initial and final phases of DM digestion. Such effects were more pronounced in BE

than BA. Increasing the enzyme application rate produced inconsistent effects on

nutritive value. However, several key measures were increased ith increasing X-20 or A-

20 application rate, suggesting that high (lx and 2x) application rates were most effective

than the low (0.5x) rate. Most of the enzyme-induced enhancements in digestibility were

not attributable to increased fiber digestion; therefore other mechanisms such as

increased substrate colonization by ruminal microbes may have been involved.






70


Ammoniation was more effective than any of the enzyme treatments at improving the

initial and final phases of digestion, due to increased fiber hydrolysis. Ammoniation also

increased the CP concentration and in situ ruminal degradation of the C4 grass hays.














CHAPTER 4
EFFECT OF FIBROLYTIC ENZYMES ON THE FERMENTATION
CHARACTERISTICS, AEROBIC STABILITY, AND DIGESTIBILITY OF
BERMUDAGRASS SILAGE

Introduction

Interest in applying fibrolytic enzymes to ruminant diets has increased recently due

to enzyme-mediated increases in feed digestion in vitro (Lewis et al., 1996; Kung et al.,

2002; Hristov et al., 2000; Bowman et al., 2002) and diet utilization in vivo (Yang et al.,

1999; Lewis et al., 1999; Schingoethe et al., 1999). However, in certain studies (Sheperd

and Kung, 1996b; Bowman et al., 2002; Vicini et al., 2003) exogenous enzyme

supplementation did not consistently improve animal performance. Where improved

performance was observed, the mechanism was not always confirmed by improved

digestion (Mandebvu et al., 1999). These inconsistencies were due to various factors

such as enzyme type, concentration and activity, application method, substrate to which

enzyme is added and animal differences (Bowman et al., 2002). Additional factors that

may be implicated include prevailing temperature and pH, presence of co-factors and

inhibitors, and enzyme and substrate concentration. Nevertheless, feed enzymes have

been used to improve the utilization of a wide range of diets containing legumes, grasses,

haylage, straw and other feedstuffs (Beauchemin et al., 2003). The mode of action of

these enzymes in ruminants is not fully understood. They can enhance feed colonization

by increasing the numbers of ruminal fibrolytic microbes (Morgavi et al., 2000; Nsereko

et al., 2000a) and thereby increase the rate of degradation in the rumen (Yang et al.,

1999). Enzymes can also partially solubilize NDF and ADF and release reducing sugars









in the process. Colombatto et al. (2003) observed that fibrolytic enzymes enhanced the

fermentation of cellulose and xylan by a combination of pre- and post-incubation effects.

These were evident from an increase in the release of reducing sugars during a 20 h pre-

incubation phase and an increase in the hydrolytic activity of the liquid and solid

fractions of the ruminal fluid 6-h after incubation, which led to a higher rate of

fermentation. Most of the studies on fibrolytic enzyme treatment of ruminant feeds have

been done using temperate feedstuffs. Little is known about their effectiveness on

tropical or subtropical forages which tend to be poorly digested. The objective of this

experiment was to evaluate the effect of four proprietary fibrolytic enzyme preparations

applied at different rates, at ensiling, on the nutritive value of Tifton 85 bermudagrass

(Cynodon spp,) silage. Most of the recent studies in this area have involved enzyme

application to individual components of the ration or to the total ration just prior to

feeding. There were two reasons for applying the enzymes directly to bermudagrass at

the point of ensiling in this study. Firstly, we sought to determine if the sugars produced

by enzymatic cell wall hydrolysis would improve the fermentation of bermudagrass

which is typically poor and decrease DM losses, which are typically high for this forage.

Secondly, we wanted to determine the effectiveness of the enzymes at improving the

digestibility of the forage, because although bermudagrass is poorly digested, it is an

important digestible fiber source in the rations of dairy cows in the Southeast.

Materials and Methods

Enzyme Application

A five-week regrowth of Tifton 85 bermudagrass silage was conserved without

treatment (Control) or after treatment with four proprietary fibrolytic enzymes. The

enzymes were applied at half (0.5x), exactly (1 x) or twice (2x) the rates recommended









by the respective manufacturers for addition at the time of feeding. Because the enzymes

were applied at ensiling rather than at feeding as recommended by the manufacturers, this

study was not designed to test the effectiveness of the enzymes as used commercially,

and the results should not be misconstrued as doing so. The rationale for the mode of

enzyme application employed was to determine if the fermentation and digestibility of

bermudagrass could be improved by enzyme addition. The enzymes used were: (a)

Promoted (Cargill Corp. St. Louis, MO) applied at: 0.65, 1.3 and 2.6 g/kg DM, (b)

Biocellulase X-20 (LodeStar, IL, USA) applied at: 7.3, 14.5 and 29 mg/kg DM, (c)

Biocellulase A-20 (LodeStar, IL, USA) applied: at 7.3, 14.4 and 29 mg/kg DM,, and (d)

Cattle-Ase (CA) applied at 89, 178 and 356 mg/kg DM. Cellulase activity was

determined at 390C and a pH of 5.5 using the filter paper method (Wood and Bhat, 1988)

and the values obtained for Pr, X-20, CA and A-20 were 33.7, 22, 0 and 51.3 filter paper

units/g, respectively. Xylanase activity was determined at 390C and a pH of 5.5 using the

di-nitro salicylic acid procedure (Bailey et al., 1992) and the values obtained for Pr, X-20,

CA and A-20 were 5190, 7025, 0 and 3530 [t mol/min/ml, respectively. The units of

xylanase activity are expressed as [imol xylose equivalents released ml-1 min1 from 1%

birchwood xylan (X-0502, Sigma Chemical Company, St. Louis, MO, USA).

Each enzyme was dissolved in 400 ml of water and sprayed in a fine mist using a

four-liter garden sprayer, over 10 kg of chopped (5 cm) forage per treatment. The same

amount of water was sprayed on the Control forages. After thorough mixing, a one-kg

representative sample of the treated forage was ensiled within a polythene bag in six,

replicate 2.8-L PVC cylindrical mini silos. A hydraulic press was used to compress the

forage in the silo to achieve a density of 280 kg/m3. Weights of the empty and full silos









were recorded, and silos were then stored for 145 days at ambient temperature (23-27C)

in a covered barn. Representative samples (4 kg) of the freshly-treated, unensiled forages

were frozen (-20C) for subsequent laboratory analysis.

Laboratory Analysis

At silo opening, final silo weights were recorded and silages from each of three

silos per treatment were sub-sampled for DM determination (200 g) and silage juice

extraction (200 g) or freeze-dried for chemical analysis (200 g). Each of the other three

silos was sub-sampled for microbial enumeration (200 g) and aerobic stability (800 g).

Samples destined for microbial analysis were heat-sealed within gas-impermeable bags

(Kapak / Scotch Pak, Kapak Corp., Minneapolis, MN), placed in an icebox and

transported on the same day to the American Bacteriological & Chemical (ABC)

Research Corporation, Gainesville, Florida. Serial dilutions up to 1 x 1010 were made

using 25 g of silage and Butterfields' phosphate buffer. Yeast and molds were

enumerated by pour plating in Standard Methods (M124) agar, to which 40 ppm of

chloramphenicol and chlortetracycline were added (Tournas et al., 1999). Plates were

incubated aerobically at 250C for 5 days. Aerobic stability was measured by placing

thermocouple wires at the center of a bag containing 800 g of silage, within an open-top

polystyrene box. The silages were covered with two layers of cheesecloth to prevent

drying. The thermocouple wires were connected to data loggers (Campbell Scientific

Inc. North Logan, UT) that recorded the temperature every 30 min for 30 d. Aerobic

instability was denoted by the time (h) taken for a 2C rise in silage temperature above

ambient temperature (23-27C). Dry mater losses were estimated using DM

concentrations and silo weights measured before and after ensiling. Oven DM

concentration was determined in a forced draft oven set at 600C for 48-h. Ash









concentration was determined in a muffle furnace at 5000C for 5 h. Silage juice was

obtained by blending 20 g of silage in 200 ml of distilled water for 30 s at high speed and

the slurry was filtered through two layers of cheesecloth. The pH was measured using a

pH meter (Corning Model 12, Coming Scientific Instruments, Medfield, MA). The

filtrate was centrifuged at 40C and 21,500 x g for 20 min and the supernatant was frozen

(-20C) in 20 ml vials for subsequent analysis of lactic acid, VFA, WSC, ammonia

nitrogen (NH3-N) and water soluble N (WSN).

Organic acids were measured using the method of Muck and Dickerson (1988) and

a High Performance Liquid Chromatograph (Hitachi, FL 7485, Tokyo, Japan) coupled

to a UV Detector (Spectroflow 757, ABI Analytical Kratos Division, Ramsey, NJ) set at

210 nm. The column used was a Bio-Rad Aminex HPX-87H (Bio-Rad Laboratories,

Hercules, CA 9454) with 0.015M sulfuric acid mobile phase and a flow rate of 0.7

ml/min at 450C. Ethanol was measured by gas chromatography using the procedure of

Yomano et al. (1998) with a Varian Star 3400 CX gas chromatograph (Varian, Santa

Clarita, CA). The anthrone reaction assay (Ministry of Agriculture, 1986) was used to

quantify WSC. Ammonia N was determined using an adaptation for the Technicon Auto

Analyzer (Technicon, Tarrytown, NY, USA) of the Noel and Hambleton (1976)

procedure. Water-soluble N (WSN) was determined by digesting 10 ml of supernatant

using micro Kjeldahl apparatus (Labconco Corporation, Kansas City, MO) and the N

concentration was determined using a Technicon auto analyzer (Technicon, Tarrytown,

NY, USA).

Freeze-dried ground (1mm) samples were analyzed for CP, in vitro digestibility,

ADF and NDF. In vitro digestibility of DM, NDF and ADF was determined after









incubating forage samples in buffered, rumen fluid for 6 or 48-h using two ANKOM"

Daisy Incubators (ANKOM Technology, Fairport, NY). The buffer was prepared

according to the ANKOM Technology procedure. The rumen fluid was obtained before

feeding from two, non-lactating, fistulated cows, fed 9 kg of Coastal bermudagrass

(Cynodon dactylon) and 400 g soybean meal daily. The NDF and ADF concentrations

(Van Soest et al., 1991) of the samples and digested residues were determined without

amylase pretreatment using an ANKOM200 Fiber Analyzer (ANKOM Technology,

Macedon, NY). Hemicellulose was calculated by difference from NDF and ADF

concentrations.

Statistical Analysis

A completely randomized design and a 4 x 4 factorial arrangement of enzyme types

and application rates with 3 replicates per treatment was used. The data were analyzed

using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). Polynomial contrasts were

used to test the effect of increasing enzyme application rate and orthogonal contrasts

were used to compare the Control and enzyme treatments.

The model used to analyze individual treatment effects was:

Yij: i + Ti + Eij

where:

9= general mean

Ti = effect of treatment (enzyme type x enzyme rate)

Eij= experimental error.

Significance was declared at P < 0.05 and tendencies at P < 0.10









Results and Discussion

Chemical Composition of Freshly-treated Bermudagrass before Ensiling

Table 4.1 shows the chemical composition of the bermudagrass forages prior to

ensiling. The concentrations of the measured chemical components were similar for all

treatments. This is probably attributable to the short duration of enzyme action due to

placement of the freshly-treated samples on ice after enzyme application. The DM

concentration at harvest was typical of that at the stage at which bermudagrass is ensiled

in Florida, and similar to that (324 g/kg) reported for bermudagrass harvested at a

similar maturity stage by Umana et al., (1991) and Adesogan et al. (2004). The low

WSC and CP concentrations and high NDF and ADF concentrations are also typical of

bermudagrass (Umana et al., 1991; Adesogan et al., 2004). These results indicate that the

bermudagrass was representative of those used for dairy production in the southeastern

US.

Chemical Composition, Microbial Counts and Aerobic Stability of Bermudagrass
Silages

Neither enzyme type nor application rate affected (P > 0.05) the DM concentration

of the silages. Dry matter values ranged between 296 and 308 g/kg (Table 4.2). The pH

of Pr-treated silages was lower (P < 0.01) than that of all other silages, while the other

enzyme- treated silages had similar pH values to Control silages. This suggests that

compared to the other forages, Pr was more effective at increasing the availability of

WSC for microbial fermentation, through cell wall hydrolysis. Though this is not evident

from the WSC concentration of the freshly-treated forages due to the short duration

allowed for enzyme action, Pr-treated silages did have greater (P < 0.05) residual WSC

concentration









Table 4.1 Chemical composition of bermudagrass forages before ensiling (g/kg DM).
Enzyme DM pH Ash WSC2 NH3- CP NDF ADF Hem4
treatment' (g/kg) N3
Control 305 5.97 57 6.93 54 105 786 436 350
Pr 302 6.42 59 6.14 43 99 776 428 348
X-20 305 6.14 55 6.79 56 97 791 440 351
CA 303 5.67 62 5.83 37 97 786 440 346
A-20 306 6.04 57 6.67 44 97 791 438 353

P 0.836 0.795 0.762 0.717 0.758 0.504 0.264 0.236 0.547
S.E. 4.26 0.73 6.67 1.01 18.52 4.01 8.65 6.61 5.50

Contrasts P values
Control vs. Pr 0.559 0.607 0.834 0.516 0.631 0.209 0.331 0.325 0.609
Control vs. X-20 0.999 0.846 0.834 0.911 0.939 0.136 0.608 0.614 0.919
Control vs. CA 0.744 0.729 0.508 0.371 0.441 0.136 0.999 0.586 0.514
Controlvs. A-20 0.896 0.933 0.999 0.828 0.642 0.110 0.630 0.833 0.614
1Cellulase-hemicellulase preparations: Pr, Promote, X-20, Biocellulase X-20, CA: Cattle-Ase,
A-20: Biocellulase A-20, 2 WSC: water-soluble carbohydrates, 3Ammonia-N expressed as g/total N,
4 Hemicellulose


than the other silages (Table 4.3). Silage pH also decreased (P < 0.01) linearly as the

rate of Pr application increased.

The Pr-treated silages had pH values that were similar to or lower than that which

is required for achieving stability during the fermentation (Bates et al., 1989a). Similar

reductions in pH were obtained when fibrolytic enzymes were applied to wheat silage

(Adogla-Bessa et al., 1999), corn silage (Sheperd and Kung, 1996a; Colombatto et al.,

2004) or orchardgrass and alfalfa silages (Nadeau et al., 2000).

Ammonia-N levels were lower in the Pr-treated silages (P < 0.01) than in the other

silages. This reveals that less proteolysis occurred during ensiling in Pr-treated silages

than in other silages, and this was probably due to a faster pH decline in Pr-treated

silages. The lower ammonia-N concentration of Pr-treated silages contrasts with










Table 4.2 Effect of fibrolytic enzymes on pH, concentrations of DM (g/kg) and ammonia-
N (g/kg total N), DM losses (%), microbial counts (log cfu /g) and aerobic
stability (h) of bermudagrass silage.

Enzyme Application DM Ammonia- Aerobic
Treatment' rate pH DM loss N Yeasts Molds stability
Control 4.40 305 8.6 32 1.65 3.24 96


0.5x
lx
2x
Mean
Rate effect

0.5x
lx
2x
Mean
Rate effect

0.5x
lx
2x
Mean
Rate effect

0.5x
lx
2x
Mean
Rate effect


X-20
X-20
X-20
X-20
X-20

CA
CA
CA
CA
CA

A-20
A-20
A-20
A-20
A-20


4.28
3.93
3.87
4.03
L**

4.40
4.49
4.32
4.40
NS

4.00
4.46
4.41
4.40
NS

4.54
4.32
4.12
4.33
NS


4.0
5.9
2.6
4.2
NS

7.3
5.8
7.7
7.0
NS

8.8
7.4
5.7
7.3
NS

9.5
6.4
3.6
6.5
L**


26
25
24
25
NS

31
38
38
35
L*

33
24
32
30
Q**

39
36
25
33
C**


2.83
1.50
2.31
2.21
NS

1.42
1.59
3.57
2.19
NS

2.05
1.00
2.17
1.74
NS

1.54
1.42
1.00
1.32
NS


3.70
3.01
2.18
2.96
NS

2.37
4.83
4.49
3.90
NS

1.93
3.37
3.12
2.81
NS

3.00
4.63
3.50
3.71
NS


103
102
229
138
L**

196
210
96
162
L**

96
203
205
168
L**

96
232
261
196
L**


S.E. 0.09 7.74 1.12 0.03 0.89 0.77 38.00
Contrasts P values
Control vs. Pr 0.002 0.703 < 0.01 < 0.01 0.573 0.671 0.011
Control vs. X-20 0.975 0.961 0.218 0.126 0.649 0.472 < 0.01
Control vs. CA 0.992 0.844 0.316 0.248 0.925 0.633 < 0.01
Control vs. A-20 0.516 0.908 0.120 0.630 0.752 0.609 < 0.01
Pr vs. X-20 <0.01 0.639 0.005 < 0.01 0.878 0.121 0.096
Prvs. CA <0.01 0.794 0.002 < 0.01 0.508 0.939 0.110
Pr vs. A-20 0.005 0.555 0.017 < 0.01 0.225 0.198 < 0.01
X-20 vs. CA 0.977 0.835 0.737 < 0.01 0.609 0.106 0.974
X-20 vs. A-20 0.338 0.903 0.631 0.135 0.285 0.766 0.043
CA vs. A-20 0.353 0.741 0.416 0.026 0.564 0.175 0.059
L: linear effect, Q: quadratic effect, C: cubic effect, NS: Not significant, P < 0.05, ** P < 0.01.
1 Cellulase-hemicellulase enzyme preparations: Pr: Promote, X-20: Biocellulase X-20,
CA: Cattle-Ase, A-20: Biocellulase A-20.










Table 4.3 Effect of fibrolytic enzymes on the chemical composition of bermudagrass
silage (g/kg DM).
Enzyme Application
Treatment' rate CP Ash NDF ADF Hem.2 WSC3 WSN4
Control 97 53 753 431 323 4.5 0.84


X-20
X-20
X-20
X-20
X-20


0.5x
lx
2x
Mean
Rate effect

0.5x
1x
2x
Mean
Rate effect

0.5x
lx
2x
Mean
Rate effect

0.5x
lx
2x
Mean
Rate effect


A-20
A-20
A-20
A-20
A-20


96
92
93
94
NS

98
96
97
97
NS

99
105
89
98
Q **

96
95
97
96
NS


723
725
728
725
NS

744
738
747
743
NS

750
743
736
743
L**

761
757
741
753
L**


408
408
408
408
NS

420
426
426
424
NS

422
444
442
436
L**

448
438
428
438
C **


8.2
12.6
15.9
12.2
L**

6.1
6.1
5.9
6.0
NS

5.3
7.1
8.5
6.9
L*

5.3
5.3
5.2
5.3
NS


0.73
0.88
0.73
0.78
NS

0.71
0.70
0.89
0.77
NS

0.86
0.62
0.70
0.73
NS

0.76
0.67
0.57
0.67
NS


S.E.
Contrasts
Control vs. Pr
Control vs. X-20
Control vs. CA
Control vs. A-20
Pr vs. X-20
Pr vs. CA
Pr vs. A-20
X-20 vs. CA
X-20 vs. A-20
CA vs. A-20


1.64 2.29 3.36 2.70
P values
0.125 0.386 < 0.01 < 0.01
0.954 0.590 0.016 < 0.01
0.602 0.934 0.015 0.105
0.816 0.508 0.977 0.022
0.028 0.054 < 0.01 < 0.01
0.006 0.269 < 0.01 < 0.01
0.067 0.071 < 0.01 < 0.01
0.513 0.381 0.968 < 0.01
0.682 0.860 < 0.01 < 0.01
0.291 0.295 < 0.01 0.299


3.84 0.81 0.07


0.204
0.444
0.002
0.078
0.463
0.004
0.463
0.001
0.149
0.022


< 0.01
0.109
0.014
0.407
< 0.01
< 0.01
< 0.01
0.179
0.260
0.018


0.466
0.350
0.170
0.039
0.766
0.351
0.053
0.522
0.097
0.293


L: linear effect, Q: quadratic effect, C: cubic effect, NS: Not significant, P < 0.05, ** P < 0.01.
1Cellulase-hemicellulase enzyme preparations: Pr: Promote, X-20: Biocellulase X-20, CA: Cattle-Ase,
A-20, Biocellulase A-20. 2 Hem: Hemicellulose. 3WSC: water-soluble carbohydrates.
4WSN: water-soluble nitrogen.









previous studies in which ammonia-N concentration of silages was unaffected by

fibrolytic enzyme application (Sheperd and Kung, 1996a; Adogla-Bessa et al., 1999).

Yeast and mold counts were unaffected by enzyme type or rate and the numbers

found were less than those (5.0 cfu/g) that predispose to rapid deterioration in silage

(Kung, 2004). These low yeast and mold counts reflect the antimycotic properties of the

VFA produced during the ensiling process (Table 4.4). Yeasts usually initiate aerobic

deterioration of silages, while molds continue the deterioration process because yeast

grow faster but tolerate less heat than do molds (Higginbotham et al., 1998).

Aerobic stability was increased (P < 0.05) by enzyme treatment and such increases

were linear (P < 0.05) as the rate of enzyme application increased except in X-20-treated

silages. Silages treated with A-20 enzyme tended (P < 0.06) to be more stable than other

additive-treated silages. Nevertheless, all the forages were stable for at least four days,

such that all of them would be adequately preserved in the feedbunk for several days.

This observation is typical ofbermudagrass silages which usually undergo heterolactic

fermentation, resulting in the production of antimycotic acids like acetic acid that ensure

the stability of the silage (Bates et al., 1989a; Adesogan et al., 2004).

Dry matter losses were lower in the Pr-treated silages than in the other silages (P <

0.05). Though there was no effect of increasing Pr application on DM lost, this work

demonstrates that Pr can be used to reduce the losses of DM that usually occur when

bermudagrass is conserved as silage. Although DM losses decreased linearly (P < 0.05)

as the rate of A-20 application increased, the mean DM loss for A-20-treated silages was

similar to that of Control silages.









Neither enzyme type nor application rate affected (P > 0.05) the ash concentration

of the silages (Table 4.3). Compared to Control silages, NDF concentration was reduced

by Pr (P < 0.01), X-20 and CA (P < 0.05). However the lowest NDF values (P < 0.01)

were observed in the Pr-treated silages (P < 0.01), indicating that this treatment was the

most effective at reducing the total fiber fraction. Silages treated with CA had lower (P <

0.05) hemicellulose concentrations than other silages. As the rate of CA application

increased, hemicellulose concentration decreased linearly (P < 0.01) while ADF

concentration increased linearly (P < 0.01). This suggests that CA hydrolyzed the

digestible fiber fraction in the silage but did not affect the less digestible ADF fraction.

Silages treated with Pr had lower (P < 0.01) ADF concentrations than Control silages and

other enzyme-treated silages, suggesting that this treatment was particularly effective at

reducing the concentration of the ADF fraction which is usually poorly digested by

ruminants. Treatment with Pr; therefore, reduced the total and less digestible fiber

fractions, and could potentially result in improved utilization of the fiber fraction in dairy

cows fed bermudagrass silage. Although CA treatment reduced the total fiber fraction, it

also reduced the digestible fiber concentration, which is an important source of slowly-

released energy for cattle.

The reduction in NDF and ADF concentration by Pr and X-20 treatment, and NDF

and hemicellulose concentration by CA treatment, contradicts the findings of Mandebvu

et al. (1999) on bermudagrass but concurs with previous observations on enzyme-treated

wheat silage (Adogla-Bessa et al., 1999), corn silage (Sheperd and Kung, 1996a; Sheperd

and Kung, 1996b; Colombatto et al., 2004) and orchardgrass or alfalfa silages (Nadeau et

al., 2000). Differences between the effects of enzymes on cell wall hydrolysis in









bermudagrass silage in this study and that ofMandebvu et al. (1999) may be due to

differences in enzyme activity. These results; therefore, provide new evidence that

fibrolytic enzymes can enhance cell wall hydrolysis in C4 grasses, as is the case in C3

grasses.

Silages treated with Pr (P < 0.01) and CA (P < 0.05) had greater residual WSC

concentration than Control silages. As the rate of application of both of these enzymes

increased, residual WSC concentration increased linearly (P < 0.05). However, these

enzymes increased residual WSC concentration by hydrolyzing different fiber fractions.

While Pr hydrolyzed the less digestible fiber fraction, CA reduced the digestible fiber

fraction. Therefore, both of these enzymes were effective in increasing the availability of

fermentation substrates, but Pr was potentially more beneficial at improving the

digestibility of the silages.

The WSC values obtained in the Pr-treated silages are also higher than those

reported by Adesogan et al. (2004), probably because of greater cellulase and xylanase

activity in Pr than in the enzyme included in the inoculant used by Adesogan et al.

(2004). The increase in WSC concentration of enzyme treated silages agrees with results

obtained by Sheperd and Kung (1996a); Adogla-Bessa et al. (1999) and Nadeau et al.

(2000). Colombatto et al. (2003) also observed that addition of fibrolytic enzymes

increased (P < 0.01) release of reducing sugars from fibrous fractions of forage during a

20-h pre-incubation phase.

The concentration of CP was similar in enzyme-treated and Control silages (Table

4.3). However, CP concentration was lower in Pr-treated silages than silages treated with

X-20 (P < 0.05) and CA (P < 0.01). This numerically small difference in CP did not









result from greater proteolysis in Pr-treated silages since they had lower (P < 0.05)

ammonia-N concentrations than the other silages. Rather, it may have been due to the

higher WSC concentration of Pr treated silages.

Organic Acid Concentration of Bermudagrass Silages

The lactic acid concentrations (Table 4.4) of the Control and enzyme treated silages

were similar (P >0.05). These results agree with those obtained ofMandebvu et al.

(1999) who found that though fibrolytic enzyme treatment of bermudagrass did not

increase lactic acid concentration, values for enzyme-treated forages were 5.4 % higher

than those for untreated forage. Acetic acid concentration was lower (P < 0.05) in

enzyme-treated silages than in Control silages. Promote-treated silages had the lowest (P

< 0.01) acetic acid concentrations and unlike other enzymes increasing the rate of Pr

application decreased (P < 0.05) acetic acid concentration linearly. These results are in

contrast with those obtained by Sheperd and Kung (1996a) and Mandebvu et al. (1999)

These factors are partly responsible for the lower (P < 0.05) DM losses in the Pr-

who found no effect of fibrolytic enzyme treatment on acetic acid concentration of

silages. The lower acetic acid concentration (P < 0.01) and numerically higher lactic acid

concentration in Pr-treated silages suggest that this enzyme enhanced homofermentative

processes during ensiling, which reduce CO2 formation; and therefore, minimize DM and

energy losses.treated silages relative to those in the other silages. However, the tendency

towards greater homofermentative processes in enzyme-treated forages conflicts with

their greater aerobic stability as homofermentative silages are often more susceptible to

aerobic spoilage. The reason for this anomaly is not clear. Nevertheless, the extent of

enzyme-induced homofermentation was not sufficient to override the inherent

heterofermentation and aerobic stability ofbermudagrass silage.









Table 4.4 Effect of fibrolytic enzymes on the organic acid concentration (g/kg DM)of
bermudagrass silage.
Enzyme Application Lactic Acetic Isobutyric Butyric Isovaleric
Treatment rate acid acid acid acid acid


Control


50


46


54


7.1


11


Pr 0.5x 83 29 63 7.7 17
Pr lx 54 13 37 0.57 8
Pr 2x 60 15 46 0 10
Pr Mean 66 19 48 2.8 11
Pr Rate effect NS L ** NS L ** L*

X-20 0.5x 32 22 34 3.9 8
X-20 lx 49 38 42 5.9 9
X-20 2x 64 38 53 5.9 13
X-20 Mean 48 33 43 5.3 10
X-20 Rate effect L L ** NS NS NS

CA 0.5x 57 38 46 5.8 11
CA lx 54 36 68 0 18
CA 2x 57 32 58 4.9 14
CA Mean 56 35 57 3.6 14
CA Rate effect NS NS NS Q Q *

A-20 0.5x 44 42 54 8.1 12
A-20 x 62 35 59 9.2 14
A-20 2x 64 33 76 0 14
A-20 Mean 56 37 63 5.7 13
A-20 Rate effect NS NS L C ** NS

S.E. 10.61 3.36 7.41 1.96 1.97
Contrasts P values
Control vs. Pr 0.211 < 0.01 0.504 0.064 0.735
Control vs. X-20 0.899 < 0.01 0.213 0.414 0.915
Control vs. CA 0.635 0.012 0.690 0.129 0.126
Control vs. A-20 0.612 0.028 0.287 0.551 0.210
Pr vs. X-20 0.057 < 0.01 0.372 0.130 0.531
Pr vs. CA 0.267 < 0.01 0.154 0.607 0.091
Pr vs. A-20 0.287 < 0.01 0.022 0.071 0.194
X-20 vs. CA 0.398 0.366 0.025 0.307 0.024
X-20 vs. A-20 0.373 0.159 0.003 0.752 0.060
CA vs. A-20 0.963 0.600 0.342 0.185 0.677
L: linear effect, Q: quadratic effect, C: cubic effect, NS: Not significant, P < 0.05, ** P < 0.01.
1 Cellulase-hemicellulase enzyme preparations: Pr: Promote, X-20: Biocellulase X-20,
CA: Cattle-Ase, A-20, Biocellulase A-20









Butyric acid was found in all the silages except those treated with Pr and A-20 at

twice the recommended rate and CA at the recommended rate. Butyric acid

concentration decreased (P < 0.05) with increasing application of Pr (linear), A-20 (cubic

effect), and CA (quadratic effect). However, only Pr treatment produced butyric acid

concentrations that tended to be less (P = 0.064) than those in Control silages. The

decrease in butyric acid concentration following Pr treatment supports the observations of

Adogla-Bessa et al. (1999) for wheat silage, but contradicts those ofMandebvu et al.

(1999) for bermudagrass silage. This discrepancy is attributable to the high cellulase and

xylanase activities in Pr which resulted in substantial hydrolysis of cell walls into WSC.

When the concentration of such WSC is adequate, and moisture is not excessive,

homofermentative lactic acid bacteria proliferate rather than heterofermenters and

clostridia, such that lactic acid accumulates instead of butyric acid.

The isobutyric acid concentrations of the treated and untreated silages were similar

(P > 0.05). Neither propionic acid nor ethanol was found in the silages. The absence of

ethanol in the silages may be explained by the low yeast counts and relatively low WSC

concentrations in the silages, because yeasts are primarily responsible for ethanol

production from the fermentation of sugars in silages.

In vitro DM and Fiber Digestibility of Bermudagrass Silages

Unlike silages treated with the other enzymes, Pr-treated silages had greater (P <

0.05) 6-h and 48-h IVDMD values as well as greater 48-h IVNDFD and IVADFD than

Control silages (Table 4.5). Silages treated with Pr consistently had greater 6-h and 48-h

IVDMD, and 48-h IVNDF values than those treated with the other enzymes. The

increase in IVDMD at 6-h and 48-h by Pr treatment suggests that application of this

enzyme can increase both the rate and extent of digestion of bermudagrass silage.