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1 USING BIOLOGICAL ADDITIVES TO IMPROVE DIETARY NUTRIENT CONSERVATION AND UTILIZATION BY LACTATING DAIRY COWS By KATHY GISELA ARRIOLA 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 2010
2 2010 Kathy Gisela Arriola
3 To my lovely children, Wilhelm and Melanie
4 ACKNOWLEDGMENTS I would like to thank my supervisory committee chair, Dr. Adegbola Adesogan, for his invaluable guidance and patience duri ng my Ph.D. program. I will always be thankful to him for being such a wonderful per son, professor, m entor, and friend. I appreciate his trust in me and everything he did to develop my critical thinking skills and professionalism. Thanks to Dr. Charles Staples and Dr. Lokenga Badinga for all the advice and time that they dedi cated to my Ph.D. program. I appreciate Dr. Carlos Riscos long-standing trust in my abilities. His advice an d inspiration helped me to persist with my dream. I thank my laboratory partners and supervi sors (Sam-Churl Kim, Jamie Foster, Oscar Queiroz, Taewon Kang, Juan Rome ro, Max Huisden, Jan Kivipelto, Nancy Wilkinson, and Sergji Sennikov) for their hel p during my laboratory and field activities. Special thanks to my dear friends Sam and Jami e; you were the best friends I had at UF and our friendship will continue even though we are far apart. Thanks for her support to Miriam Garcia, who has been my friend sinc e we were undergraduate students at the Universidad Nacional Agraria la Molina in Peru. I would also like to thank my parents, Martha and Jackson Arriola for their continuous help, patience, and dedi cation through every important event in my life. I am grateful to my brothers, Jackson and Ricardo for their s upport and I specially appreciate my sister, Ines for her enc ouragement during tough times. Special thanks to my husband, Julio Alber to, for being there unconditionally for me. Finally, I thank my children Wilhelm and Me lanie for being my daily inspiration, my strength, and my reason for per sisting with my studies.
5 TABLE OF CONTENTS page ACKNOWLEDG MENTS .................................................................................................. 4LIST OF TABLES ............................................................................................................ 9LIST OF FIGURES ........................................................................................................ 11LIST OF ABBR EVIATIONS ........................................................................................... 12ABSTRACT ................................................................................................................... 14CHAPTER 1 INTRODUC TION .................................................................................................... 192 LITERATURE REVIEW .......................................................................................... 22Importance of Forages in Dairy Nu trition ................................................................ 22Plant Cell Wall Structure in C3 and C4 Forages ...................................................... 23Photosynthetic Pathways of C4 and C3 Forages............................................... 23Anatomical Differences between C3 and C4 Forages ....................................... 24Cell Wall Chemical Composition ............................................................................. 25Cellulose ........................................................................................................... 25Hemicell ulose ................................................................................................... 26Lignin and Pheno lic Acids ................................................................................ 26Chemical Methods of Cell Wall Degradation ........................................................... 29Alkali Hydrolysis of Cell Walls .......................................................................... 29Enzymatic Hydrolysi s of Cell Walls ......................................................................... 32Fibrolytic Enzymes ........................................................................................... 32Cellulase .................................................................................................... 33Xylanase .................................................................................................... 34Synergistic Action of Ce llulases and Xylanases ............................................... 35Estera se ..................................................................................................... 36Etheras e..................................................................................................... 37Ligni nase.................................................................................................... 38Modes of En zyme Ac tion .................................................................................. 39Preingestive effects .................................................................................... 39Post-ingestion effects ................................................................................. 41Factors Affecting Enzyme Effi cacy in Dairy Cattle Ra tions............................... 42Effect of the form or component of the diet to which the enzyme is applied .................................................................................................... 43Effect of the site of enzyme delivery .......................................................... 45Effect of time of enzyme application to the diet .......................................... 46Effect of the enzym e applicati on rate ......................................................... 47Effects of stage of lactation and parity of dairy ca ttle ................................. 48
6 Silage Ferm entation ................................................................................................ 50Factors Affecting Silage Ferment ation .................................................................... 50Moisture Concentrati on and Matu rity ................................................................ 50Epiphytic Ba cteria ............................................................................................. 51Water-Soluble Ca rbohydra tes .......................................................................... 53Buffering C apacity ............................................................................................ 53Climatic Fa ctors ................................................................................................ 54Factors Affecting Ae robic Stability .......................................................................... 55Yeasts .............................................................................................................. 55Molds and Mycotoxins ...................................................................................... 56Bacilli ................................................................................................................ 58Moisture ............................................................................................................ 58Packing De nsity ................................................................................................ 59Silage Addi tives ...................................................................................................... 59Chemical A dditives ........................................................................................... 60Organic ac ids ............................................................................................. 60Ammonia .................................................................................................... 61Biological A dditives .......................................................................................... 62Homolactic Bacter ial Inocul ants ....................................................................... 62Heterolactic Bact erial Inoc ulants ...................................................................... 65Lactobacillus buchneri ............................................................................... 65Propionibac teria ......................................................................................... 67Inoculants containing Homolactic and Heterolactic Bacteria ............................ 68Enzymes ........................................................................................................... 69Nutrient A dditives ............................................................................................. 703 EFFECT OF FIBROLYTIC ENZYME APPL ICATION TO DIETS DIFFERING IN CONCENTRATE PROPORTION ON TH E PERFORMANCE OF LACTATING DAIRY CA TTLE ...................................................................................................... 73Introduc tion ............................................................................................................. 73Materials and Methods ............................................................................................ 74Cows, Treatments and Desi gn ......................................................................... 74Enzyme Acti vity ................................................................................................ 75Sampling and Analysis ..................................................................................... 76In Situ Ruminal Degradabi lity ........................................................................... 77Statistical Analysis ............................................................................................ 79Results and Discussion ........................................................................................... 80Conclusi ons ............................................................................................................ 844 EFFECTS OF FIBROLYTIC ENZYME APPL ICATION ON THE DIGESTIBILITY OF CORN SILAGE, ALFALFA H AY, TWO CONCENTRATES, AND COMPLETE DIETS UNDER SIMULATED RUMINAL AND PRERUMINAL CONDITIONS ....................................................................................................... 100Introduc tion ........................................................................................................... 100Materials and Methods .......................................................................................... 102
7 Dietary Substrates .......................................................................................... 102Enzyme Acti vity .............................................................................................. 102In vitro Fermentati on and Degra dability .......................................................... 103Statistical Analysis .......................................................................................... 105Results and Discussion ......................................................................................... 106Conclusi on ............................................................................................................ 1115 EFFECT OF APPLYING BACTERIAL INOCULANTS ON THE FERMENTATION AND QUAL ITY OF CORN SILAGE ......................................... 121Introduc tion ........................................................................................................... 121Material and Methods ........................................................................................... 122Forage and Tr eatment s .................................................................................. 122Chemical Analysis .......................................................................................... 123Phenol-chloroform Extrac tion of To tal DNA .................................................... 124Conventional P CR Conditi ons ........................................................................ 124Statistical Analysis .......................................................................................... 125Results and Discussion ......................................................................................... 126Conclusi ons .......................................................................................................... 1296 EFFECT OF APPLYING BACTERIAL INOCULANTS TO CORN SILAGE ON THE PERFORMANCE OF DAIRY CATTLE ......................................................... 133Introduc tion ........................................................................................................... 133Material and Methods ........................................................................................... 134Forage and Tr eatment s .................................................................................. 134Diets, Cows, and Managem ent ...................................................................... 135Sampling and Analysis ................................................................................... 135Statistical Analysis .......................................................................................... 137Results and Discussion ......................................................................................... 138Conclusi ons .......................................................................................................... 1417 GENERAL SUMMARY A ND CONCLU SIONS ...................................................... 147APPENDIX A METHOD FOR MEASURING HE MICELLULASE AC TI VITY ............................... 153Xylanase Assay .................................................................................................... 153Xylose St andard ............................................................................................. 153Reagent s ........................................................................................................ 153Assay ............................................................................................................. 154
8 B METHODS FOR MEASURING CELLULASE AC TIVITY ...................................... 155Endoglucanas e Assay .......................................................................................... 155Glucose Standard ........................................................................................... 155Reagent s ........................................................................................................ 155Assay ............................................................................................................. 155Exoglucanas e Assay ............................................................................................ 157Glucose Standard ........................................................................................... 157Reagent s ........................................................................................................ 157Assay ............................................................................................................. 157LIST OF RE FERENCES ............................................................................................. 159BIOGRAPHICAL SKETCH .......................................................................................... 186
9 LIST OF TABLES Table page 3-1 Ingredient and chemical composition of the untreated experimental diets ......... 863-2 Effect of adding a fibrolytic enzyme1 (Enz) to diets cont aining low (33%) or high (48%) amounts of concentrate (conc) on intake and digestibility by dairy cows. .................................................................................................................. 873-3 Effect of adding a fibrolytic enzyme1 (Enz) to diets cont aining low (33%) or high (48%) amounts of concentrate (conc) on body weight, average daily gain (ADG), body condition score (BCS), and plasma metabolites by dairy cows. .................................................................................................................. 883-4 Effect of adding a fibrolytic enzyme1 (Enz) to diets cont aining low (33%) or high (48%) amounts of concentrate (conc ) on milk production, composition, and efficiency of feed utiliz ation by dairy cows. .................................................. 893-5 Effect of adding a fibrolytic enzyme1 (Enz) to diets cont aining low (33%) or high (48%) amounts of concentrate (conc) on ruminal fermentation characteristics by dairy cows. ............................................................................. 903-6 Effect of adding a fibrolytic enzyme1 (Enz) to diets cont aining low (33%) or high (48%) amounts of concentrate (conc) on the in situ ruminal degradability of dry matter by dairy cows. ................................................................................ 914-1 Ingredient compositio n of concentrate and TMR substrates and chemical composition of all substr ates ............................................................................ 1134-2 Effect of enzyme application to diffe rent substrates on concentrations of NDF, ADF, hemicellulose, and WSC (Experiment 1). ....................................... 1144-3 Effect of enzyme application on di sappearance of DM from substrates incubated in a buffer fo r 24 h (Exper iment 2).................................................... 1164-4 Effect of incubation medium on t he dry matter (DMD) and neutral detergent fiber (NDFD) digestibility of enzymetreated substrates (Experiment 3). .......... 1174-5 Effect of enzyme application and in cubation medium on the dry matter digestibility (DMD) of subs trates incubated for 24 h in water or ruminal fluid after incubation for 24 h in a buffer (Experiment 4). .......................................... 1184-6 Effect of enzyme application and in cubation medium on neutral detergent fiber digestibility (NDFD) of substrat es of substrates incubated for 24 h in water or ruminal fluid after incubation for 24 h in a buffer (Experiment 4) ........ 119
10 4-7 Effect of enzyme (Enz) application on pH, VFA and ammonia-N concentrations of substrates incubated for 24 h in ruminal fluid after incubation for 24 h in a buffer (Exper iment 4) ................................................... 1205-1 Chemical composition of corn fo rages treated with or without bacterial inoculants befor e ensi ling ................................................................................. 1305-2 Effect of bacterial inoculants on the chemical composition of corn silages ....... 1305-3 Effect of bacterial inoculants on DM losses and fermentation indices of corn silages .............................................................................................................. 1315-4 Effect of bacterial inoculants on f ungal counts and aerobic stability of corn silages .............................................................................................................. 1316-1 Ingredient and chemic al composition of t he experimenta l diets........................ 1426-2 Chemical composition and ferment ation indices of corn s ilages....................... 1436-3 Effect of applying different inoculants1 to corn silage at ensiling on feed intake and digestibility in dairy cows. ................................................................ 1446-4 Effect of applying different inoculants1 to corn silage at ensiling on growth performance and plasma metabo lites in dai ry cows. ........................................ 1456-5 Effect of applying different inoculants1 to corn silage at ensiling on milk yield, milk composition, and effici ency of feed ut ilizatio n. .......................................... 146
11 LIST OF FIGURES Figure page 2-1 Chemical structural characteristics of typical lig nin precursors ........................... 282-2 Phenylpropanoid pathway lead ing to lignin bi osynthesis .................................... 282-3 Schematic representat ion of the major enzymes involved in cellulose hydrolysis ........................................................................................................... 342-4 Schematic diagram showing possi ble covalent cross links between polysaccharides and li gnin in wa lls. .................................................................... 493-1 Effect of adding a fibrolytic enzyme application to dairy cow diets with low or high levels of concentra tes on dry matter intake. ................................................ 923-2 Effect of esterase-xylanase enzyme app lication to dairy cow diets with low or high levels of concentra tes on milk yield. ........................................................... 933-3 Effect of adding a fibrolytic enzyme to dairy cow diets with low or high levels of concentrates on ruminal pH. ........................................................................... 943-4 Effect of adding a fibrolytic enzyme to dairy cow diets with low or high levels of concentrates on ruminal amm onia nitrogen c oncentrati on. ............................ 953-5 Effect of adding a fibrolytic enzyme to dairy cow diets with low or high levels of concentrates on ruminal to tal VFA concen tration.. ......................................... 963-6 Effect of adding a fibrolytic enzyme to dairy cow diets with low or high levels of concentrates on ruminal acetate proporti on.................................................... 973-7 Effect of adding a fibrolytic enzyme to dairy cow diets with low or high levels of concentrates on rumi nal propionate proporti on .............................................. 983-8 Effect of adding a fibrolytic enzyme to dairy cow diets with low or high levels of concentrates on ruminal ac etate to propi onate rati o.. ..................................... 995-1 Gel electrophoresis analysis after polym erase chain reaction amplification of DNA from silages treated with or without inoculants ......................................... 132
12 LIST OF ABBREVIATIONS ADF Acid-detergent fiber ADG Average daily gain BCS Body condition score BHBA Beta-hydroxybutyrate BUC Buchneri 40788 inoculant BUN Blood urea nitrogen BW Body weight B2 Biotal Plus II inoculant B500 Buchneri 500 inoculant CP Crude protein DM Dry matter DMD Dry matter digestibility DMI Dry matter intake FA Ferulic acid FAE Ferulic acid esterase FCM Fat-corrected milk HC High concentrate HCE High concentrate enzyme HPLC High-performance liquid chromatograph LC Low concentrate LCE Low concentrate enzyme Meq milliequivalent NDF Neutral-detergent fiber NDFD Neutral detergent fiber dibestibility
13 NH3 Ammonia NH3-N Ammonia nitrogen PCR Polymerase chain reaction PUN Plasma urea nitrogen PEP Phosphoenolpyruvate carboxylase RF Rumen fluid SCC Somatic cell counts TMR Total mixed ration TMRH High concentrate Total mixed ration TMRL Low concentrate Total mixed ration W Water WSC Water-soluble carbohydrate
14 Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy USING BIOLOGICAL ADDITIVES TO IMPROVE DIETARY NUTRIENT CONSERVATION AND UTILIZATION BY LACTATING DAIRY COWS By Kathy Gisela Arriola May 2010 Chair: Adegbola Adesogan Major: Animal Sciences Four experiments were conducted to evaluate the effect of adding biological additives to the diet or feeds of lactating dairy cows on their performance or the quality of the feeds or diets. The objective of Experiment 1 was to determine the effect of dietary addition of a fibrol ytic enzyme preparation containing cellulase, xylanase and esterase activities on the performance of dairy cows fed low or high concentrate diets. Sixty lactating Holstein cows in early lactation (22 + 3 days in milk) were assigned to the following treatments: 1) Low concentrate (33%) diet (LC); 2) LC plus enzyme (LCE); 3) High concentrate (48%) diet (HC); 4) HC plus enzyme (HCE). The enzyme was sprayed at a rate of 3.4 mg/g of dry matt er (DM) on the total mixed ration (TMR) daily for 63 d. The first 14 d were used for adaptation to diets and the last 49 d for measurements. In addition, four ruminally-fistulated cows were used to determine dietary treatment effects on i ndices of ruminal fermentati on and in situ DM degradation in the rumen. Enzyme application did not af fect milk yield or intake of DM, but increased digestibility of DM, CP, NDF, and ADF,and increased the efficiency of milk production. Increasing the concentrate leve l reduced ruminal pH but increased intakes of DM and CP, digestibility of DM (DMD) and CP, and milk yiel d and milk protein yield.
15 Cows fed LCE instead of HC had less DMI, sim ilar milk yield and greater efficiency of milk production. Enzyme application did not a ffect ruminal pH or ruminal degradation of the diets. However, increas ing the level of concentrate supplementation decreased the pH, increased the immediatel y soluble dietary fraction, and tended to decrease the potentially degradable fraction. In conclusion, application of the enzyme increased nutrient digestion and the efficiency of milk production by the cows. Experiment 2 was designed to determine if the enzyme used in Experiment 1 primarily exerted its hy drolytic effect prior to ingestion or within the rumen. A second objective was to determine if the enzyme was more effective on specific components of the diet. Substrates were incubated in a buffe r or a buffer enzyme solution in triplicate for up to 24 h and chemical composition and DM disappearance were measured. In addition, DMD and NDFD were determined after untreated or enzyme-treated substrates were incubated in water (W) or ruminal fluid (RF) for a further 24 h after the initial incubation in the buffer or buffer-enz yme solution. Applic ation of the enzyme reduced concentrations of NDF and hemicellulose, and increased water-soluble carbohydrate (WSC) concentration and DM disappearance. Incubation of enzymetreated substrates in RF resulted in greater DMD than incubation in W except for AH, which had similar DMD in both media. Enzyme addition increased DMD and NDFD in W by 10 and 84% respectively, but had no effect on DMD and NDFD in RF; suggesting that preingestive effects of the enzyme were greater than ruminal effects. Enzyme effects on NDF, WSC, and hemicellulose conc entration or DMD and NDFD in W or RF did not depend on the substrate. Therefore, this study provided no evidence that the enzyme preferentially hydrolyzed specific substrates and it suggested that preingestive
16 effects of the enzyme were greater than ruminal effects under the conditions of the study. Experiment 3 determined the effect of bac terial inoculants on the fermentation and quality of corn silages. A corn hybrid Vi goro 61R36 (Royster Clark Inc.) was grown and harvested at 35% DM. Chopped corn forage was treated with 1) deionized water (CON); 2) Biotal Plus II (B2) inoculant containing Pediococcus pentosaceus and Propionibacteria freudenreichii ); 3) Buchneri 40788 (BUC) inoculant containing Lactobacillus buchneri ; and 4) Buchneri 500 (B500) inoculant containing P. pentosaceus and L. buchneri Four replicates of each treatment were weighed into polyethylene bags within 20-L mini silos, which were stored for 575 d at ambient temperature (25C) in a covered barn. After silos were opened, aerobic stability, chemical composition, and yeast and mold counts were determined. The DNA from treated and untreated silages wa s isolated using a lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform extraction method. The DNA was used as a template for a conventional PCR with primers designed on the 16S rRNA gene to detect the presence of L. buchneri in silage samples. The WSC concentra tions of all silages were reduced during the fermentation. However, B500 had the greatest residual WSC concentration, suggesting that plant sugars we re less extensively fermented by the bacteria in this inoculant compared to those in other treat ments. Dry matter loss was lower in BUC silages compared with Control and B2 silage s. Control and B2 silages had higher pH and propionic acid concentration and lower lactic acid concentrations than other treatments. The greater lactate concentration and lower pH of BUC silages explain the lower DM loss from this silage. Acetat e concentration was greatest in B2 silage,
17 intermediate in Control and BUC silages, and lowest in B500 silage. However, aerobic stability was generally high (> 250 h) and was not improved by inoculant application. The PCR analysis confirmed the pr esence of similar populations of L. buchneri in all treatments, perhaps explaining why aerobic stability was high in all silages. The inoculants had differing effects on the ferm entation of the silages with BUC producing the most desirable fermentation and least DM losses. However, none of the inoculants improved aerobic stability, probably because all treatment s had high populations of L. buchneri. Experiment 4 determined the effect of appl ying three different bacterial inoculants to corn silage on the performance of lactati ng dairy cows. Corn plants were harvested, chopped, and ensiled in 2.4-m wide bag silos after application of the same treatments as in Experiment 1. Each of the 4 silages was mixed into separate TMR consisting of 44% corn silage, 50% concentrate, and 6% alfa lfa hay (DM basis). Fifty-two lactating Holstein cows in early lactation (22 DIM) we re fed for 49 d. Chem ical composition and yeast and mold counts of silages did not di ffer among treatments. Treatment with BUC improved silage aerobic stability by 200% and numerically resulted in the least losses compared with other treatm ents. Inoculant treatment did not affect DMI or digestibility of DM or CP. However, cows fed B2 had lower NDF and ADF digestibility than cows fed the control diet. Consequently, cows fed B2 had lower digestible NDF and ADF intake than cows fed the control diet. Neve rtheless, milk yield, milk composition, and feed efficiency were not affected by treatment. Therefore, the inoculants did not affect the performance of the cows, but application of L. buchneri improved the aerobic stability of corn silage.
18 These experiments indicate that fibrolytic enzyme applic ation can improve nutrient digestion and efficiency of milk production by lactating dairy cows. Application of the bacterial inoculants improved the fermentat ion of silages in one study and improved aerobic stability in another study, but feeding inoculated silages did not affect the performance of lactating dairy cows.
19 CHAPTER 1 INTRODUCTION Forages provide ener gy and nutrients that ar e vital for the growth and productivity of cattle and therefore repr esent an important feed resource for cattle production. However, the relatively high lignin and fiber concentrations in forages limit the extent to which they are digested by cattle. Ther efore, various methods of improving the digestibility of forages hav e been explored. One of the most common and successful forage treatment methods is ammonia treat ment. Ammoniation typically increases forage crude protein concentration (CP), reduce s the concentration of neutral detergent fiber (NDF), and increases the digestibility of NDF in forages. However, the use of ammonia and other alkalis for forage improv ement has been limited by their corrosive nature and the hazards they pose to humans. Recently, interest in using fibrolytic enzymes to improve forage quality has increased. Fibrolytic enzymes increase fiber hydrolysis and enhance feed colonization by increasing the number of ruminal fibrolyt ic microbes (Nsereko et al., 2000a), such that the rate of degradation of feeds in the rumen is increased (Yang et al., 1999). However, results of applying such enzym es to feeds have been equivocal. Some studies have reported that diet ary addition of exogenous fibr olytic enzymes increased dry matter (DM) digestion and DM intake (DMI; Feng et al., 1996; Yang et al., 1999), but others found no improvements in animal perfo rmance (ZoBell et al., 2000). Some authors reported an increase in milk yield w hen enzymes were added to the diet of dairy cattle (Lewis et al., 1999; Beauchemin et al., 2000; Zheng et al., 2000) but others did not (Kung et al., 2003a; Sutton et al., 2003; Vici ni et al., 2003). The principal enzymes that have been used in such animal perform ance trials are cellulases and xylanases.
20 Little is known about potential benefits on animal performance of incorporating other fibrolytic activities like estera ses in such enzyme preparations. Silage is a forage conservation method widely used in temperate livestock production systems. In many US dairy systems, silage represents up to 45% of the total mixed ration. Ensiling involves anaerobic bacterial fermentation of plant sugars to primarily lactic and acetic acids (Muck, 1988) Rapid achievement of a low pH during the process is vital because inadequate fermentation can increase the population of undesirable microorganisms in s ilage leading to deterioration, nutrient and DM losses, and growth of spoilage and pat hogenic organisms. Several types of silage additives such as acids, alkalis, and microbial in oculants have been evaluated to improve the fermentation and nutritive value of forages. Though effective, chemicals such as sulfuric acid, ammonia and unbuffered propionic acid are not widely used because of their caustic nature (Kung, 2009). Bacterial inoculants are the most common type of additive in the US. They supplement the natural lactic acid bacteria on the crop in order to guarantee a fast and efficient fermentati on in the silo (Muck, 1993). The species most widely used include Lactobacillus plantarum L. acidophilus Enterococcus faecium Pediococcus acidilacti P. pentosaceus (McAllister and Hristov, 2000), and L. buchneri (Weinberg and Muck, 1996). However, these bacteria have different roles and effects on the fermentation. So me rapidly increase the rate of acidification at the onset of the fermentation, others increase acidification in a more gradual manner whereas others increase aerobic stability after the silo is opened. Various inoculants containing one type or multiple types of these bacte ria exist, but few studies have compared effects of different types of inoculants on the fermentation, nutri tive value and aerobic
21 stability of silage. Even fewer studies have examined inoculant effects on animal performance. The objectives of the first two experiments were to determine effects of adding a fibrolytic enzyme containing esterase, cellu lase, and xylanase activity on the hydrolysis of various dietary ingredients and the per formance of lactating dairy cows. The objectives of the last two ex periments were to compare effe cts of different bacterial inoculants on the fermentation, nutritive val ue, and aerobic stability of silages and the performance of lactating dairy cows.
22 CHAPTER 2 LITERATURE REVIEW Importance of Forages in Dairy Nutrition Forages represent an important cost effe ctive feed resource in ruminant nutrition (Jung and Allen, 1995). Legumi nous forages like alfalf a ( Medicago sativa ) provide an important source of protein for ruminant liv estock, whereas grass and small-grain cereal silages are important sources of dietary energy. However, fiber is perhaps the most important contribution of forages to animal feeds. An adequate fiber supply is necessary to maintain the rumen mat, whic h slows the passage of feed through the digestive tract and increases the amount of nutrients that can be digested and absorbed from the feed. Forage fiber also incr eases chewing and rumination and therefore increases the production of saliva, which maintains normal ruminal pH and reduces the incidence of ruminal acidosis. National Research Council (2001) recommends that dairy cow rations should contain at leas t 25% of neutral detergent fiber (NDF) and at least 19% of NDF from forage when the diet is fed as a TMR providing the forage has adequate particle size and the predominant starch source is ground corn ( Zea mays). Numerous studies have shown the import ance of an optimal ratio of forage to concentrate for increasing the productivity of dairy cows (Miller and ODell, 1969; Weiss and Shockey, 1991). Diets with low forage to concentrate ratios generally have high starch concentrations that decrease milk fat yield because they reduce the acetate to propionate ratio in the rumen (Nocek and Tamminga, 1991). Appropriate levels of forages in the diets of dairy cows promot e production of ruminal acetate and butyrate, which are the major carbon sources for de novo synthesis of milk fatty acids.
23 Plant Cell Wall Structure in C3 and C4 Forages Cell walls are the main plant fraction that is resistant to en zymatic degradation in mammalian gastrointestinal tracts. Cell wa ll concentration and chemical composition differs between forage species and parts but for most species, stems have a greater concentration of cell walls than do leaves and cell walls of stems usually contain a greater lignin concentration (Albrecht et al., 1987; Buxton and Hornstein, 1986). Photosynthetic Pathways of C4 and C3 Forages Photosynthesis occurs mainly in leaf ch loroplasts, but the pathway involved varies between C3 and C4 forages. Ribulose biphosphate ca rboxylase/oxygenase (rubisco) is an abundant photosynthetic enzyme in plants, accounting for 20 to 30% of leaf N in cool-season or C3 plants (Long, 1999). During the Ca lvin Benson photosynthetic pathway in C3 forages, atmospheric CO2 enters the leaf through the stoma and diffuses into the mesophyll cells. Rubisco, a carboxylase adds CO2 to ribulose 1,5 phosphate (RuBP) to form a highly unstable 6-carbon compound that immediately forms two molecules of the 3-carbon compound, 3phosphoglycerate, which is ultimately converted into glucose. However, rubisco is also an oxygenase with a high affinity for O2. Photorespiration occu rs when rubisco fixes O2, leading to a substantial reduction in the efficiency of C fixation (Moore et al., 2004). The uni que anatomical features of C4 plants reduce photorespiration and make the C4 photosynthetic pathway more efficient. In such plants, photosynthesis occurs in mesophyll cells as well as the bundle sheath within the Kranz st ructure. The CO2 entering the leaf through the stomata diffuses into the mesophyll cells where it is fixed by phosphoenolpyruvate carboxylase (PEP) into oxaloacetate, a C4 compound, which is converted to malate or aspartate, and transported to the bundle sheath cells where it is decarboxylated The CO2 released is
24 refixed by rubisco in the bundle sheath and t he Calvin Benson photosynthetic pathway proceeds as in C3 plants. The presence of the CO2 shuttle in C4 plants allows them to increase the concentration of CO2 around rubisco, which reduces the occurrence of photorespiration and increases the efficiency of C fixation relative to that in C3 plants. Consequently, C4 species grow at a faster rate than C3 species particularly under warm temperature and full sunlight (Long, 1999). Anatomical Differences between C3 and C4 Forages The Kranz anatomy is a unique characteristic of C4 grasses which consists of many vascular bundles surrounded by specialized parenchyma bundle sheath cells (Dengler and Nelson, 1999). This thick-wa lled, lignified parenc hyma bundle sheath occupies about 18% of the leaf volume of C4 plants, with a corresponding decrease in proportion of thin-walled mesophyll tissue (Coleman et al., 2004). Consequently, the Kranz structure resists bacterial digestion and slows access to the digestible content within parenchyma bundle sheath ce lls (Coleman et al., 2004). In legumes and most cultivated C3 grasses, the epidermis is lost from the leaf quickly with chewing and digestion. This proc ess is much slower and less complete in tropical C4 grasses (Pond et al., 1984) because their epidermis is attached to the leaf at the major and intermediate-sized vascular bundles through thi ck-walled sclerenchyma cells (Wilson et al., 1989). This attachment c an be either directly to the vascular bundle or indirectly to thick-walled bundle sheath cells and then to th e vascular tissue. In both cases, the attachment reduces epidermal d egradation during mastic ation thus limiting microbial access to other anatomical features in the cell wall (Wilson et al., 1989). In leaves of legumes and C3 grasses, the mesophyll cells are more loosely arranged than in C4 grasses. Byott (1976) stated that the percentage of intercellular air
25 space in C3 grasses (10 35%) and legumes (41 51%) is much higher than in C4 grasses (3 12%). This allows more rapid penetration of bacteria into the C3 leaf and hence quicker digestion of leaves (Hanna et al. 1973) than occurs in C4 grasses. In addition, Wilson and Hattersle y (1989) reported that C3 grasses have greater proportions of mesophyll (53 to 67 versus 28 to 47%), lower proportions of bundle sheath cells (5 to 20 versus 12 to 33%), and lower portions of vascular tissue (3 to 9 versus 6 to 12%) than C4 grasses. Consequently, greater proportions of thin-walled, non-lignified easily digestible cells are present in C3 grasses. The foregoing illustrates anatom ical characteristics of C4 forages that make them less susceptible to physical and microbial breakdown in animals than C3 forages (Wilson et al., 1989). However, the magnitu de of these differences varies between species, maturity stage, and growth conditions. Cell Wall Chemical Composition Cellulose Cellulos e and hemicellulose are the main structural polysaccharides in plants. Cellulose, hemicelluloses, and lignin occupy about 40 to 45%, 30 to 35%, and 20 to 30% of the plant cell wall (Ladish et al., 1983). Cellulose is a linear polymer of glucose linked by -1,4 glycosidic bonds with a simple prim ary structure and a complex tertiary structure (Bhat and Hazlewood, 2001). The primary structure reflects the pattern of covalent bonding in cellulose molecules. The secondary structure is the conformation of individual molecules, which defines the re lative organization in space of the repeating units of an individual molecule. The tertiary structure reflects the arrangement of the molecules relative to each other in a parti cular state of aggregatio n (Atalla, 1990). In some regions, cellulose chains are highly ordered and linked by strong hydrogen bonds
26 to form crystallites, whereas loosely arr anged cellulose molecules form the amorphous regions (Bhat and Hazlewood, 2001). Pure cellu lose is quickly and completely degraded ruminally as is cellulose cross-linked to hemicellulose alone (Hatfield et al., 1999). Hemicellulose Hemicellulose is the second most abundant plant structural polysaccharide. Hemicellulose is composed of a range of heteropolysac charides, which contain different types of linkages and sugars (Hatfield et al., 2007). Xylans are the main heteropolysaccharides and their backbone structure is comprised of -1,4-linked xylose residues (Chesson et al., 1983). Xylan po lymers may be cross-linked to other hemicellulose backbones ( -1,4-linked-Dpyranosyl residues such as glucose, mannose and xylose) or to lignin through ferulic acid or 4O -methyl-D-glucoronic acid residues (Hatfield and Ford, 1989; Lam et al., 1992). Grasses produce xylans with more complex structures than legumes and contain substitutions of arabinose, glucuronic acid, or both (Hatfield et al ., 2007). In addition, some of the arabinose residues contain ferulic acid and to a much lesser extent p -coumaric acid ester linked to the C5 hydroxyl group (Hartley 1972). Hemicellulose is soluble in dilute alkali and is linked to cellulose by multiple hydrogen bonds (Albersheim et al., 1984). Hemicellulose is completely digestible when removed from t he cell wall matrix but its association with lignin in the cell wall limits its digestion (Hatfield et al., 2007). Lignin and Phenolic Acids Lignin is a key component of plant cell walls and its functions include imparting strength to cell walls, facilitating water tr ansport, and acting as a major line of defense against pathogens, insects, and other herbivores (H atfield and Vermerris, 2001). Lignin also acts as a physical barrier to microbi al digestion of fiber polysaccharides. Sarkanen
27 and Ludwig (1971) described ligni n as a polymeric natural product arising from an enzyme initiated dehydrogenative pol ymerization of three primar y precursors: coniferyl, sinapyl, and p -coumaryl alcohols (Figure 2-1). The shikimic acid pathway and phenylpropanoid metabolism lead to synthesis of lignin intermediates like p -coumaric acid, ferulic acid, diferulic acid, sinapic acid, cinnamic acid, and p -hydroxybenzoic acid (Bidlack et al., 1992; Humphreys and Chap ple, 2002), which are converted into coniferyl, sinapyl, and p -coumaryl alcohols and ultimate ly to guaiacyl, syringyl, or p hydroxyphenyl lignin, respectively (Figure 22). The hydroxycinnama tes are structurally related to lignin precursors and they may be a ttached to lignin, playing an important role during regulation of wall matr ix organization (Hatfield et al., 1999). Ferulate and p coumarate molecules are esterified to arabinox ylan in grasses; however, the majority of p -coumarates are ester linked to lignin (Ral ph et al., 1994). As forages mature and lignin concentration increases, ferulates that were esterified to arabinoxylan become etherified to lignin via cross-links between lignin and the cell wall polysaccharides (Iiyama et al., 1990). The degr ee of lignin/arabi noxylan cross-linking by ferulates influences cell wall digestibilit y (Grabber et al., 1998). Increas es in the ratio of syringyl to guaiacyl lignin has also been associated wi th poorer cell wall digestibility due to accumulation of poorly digested, lignified secondary cell walls that are intrinsically higher in syringyl lignin content (Jung and Engels, 2002). Akin (1986) reported that C4 grasses contained greater concentrations of phenolic compounds like p -coumaric acid and greater ratios of p -coumaric acid to ferulic acid than did C3 grass species. The authors suggest ed that these compounds might be responsible for limiting the extent of degradation in C4 grasses.
28 Figure 2-1. Chemical structural characterist ics of typical lignin precursors (coniferyl, sinapyl, and p-coumaryl alcohols) and hy droxycinnamic acids (ferulate and pcoumarate) found in forage cell walls (Hatfield et al., 1999). Figure 2-2. Phenylpropanoid pathway leadin g to lignin biosynthesis (adapted from Humphreys and Chapple, 2002).
29 Others have also attributed the poorer digestibility of C4 grasses to greater concentrations of phenolic acid precursors of lignin (Chaves et al ., 1982; Burritt et al., 1984; Akin and Benner, 1988). Chemical Methods of Cell Wall Degradation Several chemical treatment methods have been developed to improve the nutritive value of low quality forages by reducing the concentration of cell walls or increasing their digestion. Alkali Hydrolysis of Cell Walls Alkali treatment is the most widely us ed method for increasing the degradation of forage cell walls and the main alk alis used are ammonia and NaOH. The underlying principle of the method is partial solubiliz ation of hemicellulose, lignin, and silica and hydrolysis of uronic and acetic acid esters by the alkali (Chesson, 1981; Chesson et al., 1983). The alkalis disrupt intermolecula r H bonding within cellulose and hydrolyze covalent ester linkages between arabinoxylans and phenolic acids that are cross-linked to lignin (Chesson et al., 1983; Mueller-Harve y et al., 1986). Alkali treatment reduces concentrations of phenolic compounds, hemicellu lose, and acetyl groups in forages and thereby improves digestibility (Sawai et al., 1983). Several studies have demonstrated increased performance of animals due to forage ammoniation. Kunkle et al. (1983) r eported that treating poor quality, tropical forages with anhydrous ammonia (3 to 5%) increased the crude protein (CP) concentration (4.2 to 11.7%) and in vitro dr y matter (DM) digestibi lity (41 to 52%). Horton and Steacy (1979) reported an increase in apparent digestibility of DM, organic matter (OM), crude fiber and CP when steers were fed ammoniated barley ( Hordeum vulgare), oat ( Avena sativa) and wheat ( Triticum aestivum ) cereal straws. In addition,
30 Solaiman et al. (1979) report ed that treating wheat stra w with ammonium hydroxide increased in vitro DM digestibility Ammoniation of bermudagrass ( Cynodon dactylon ) increased intake (Wyatt et al., 1989) and average daily gain (ADG) of heifers (Ocumpaugh et al., 1984). Brown (1988) c onducted laboratory, di gestion and growth trials to evaluate ammoniatio n effects on Ona stargrass (Cynodon nlemfuensis Vanderyst) hay. Ammoniation increased in vitr o total N and in vitro OM digestibility (IVOMD) of stargrass hay, but only increased in vivo apparent OM, NDF, and acid detergent fiber (ADF) digestibilities when the hay was mature (6 versus 12 week regrowth). During the growth trial, ammoniation improved ADG and intake by cattle fed the more mature hay. Vagnoni et al. (1995) reported that ADG was increased when steers were fed ammoniated bermudagrass hay. In a second study, treating mature bermudagrass hay with ammonia increased both the in situ rate and the potential extent of forage DM and NDF disappearance in Holstein steers. Ther efore, the literature clearly demonstrates the nutriti onal benefits of ammoniation. Treating hay with ammonia also has nonnutritional benefits. Cattle waste less ammoniated hay than non-treated hay (10% vs. 25% loss, respectively) and ammoniation allows proper conservation and utilization of wet (25 to 60% moisture) forages that would otherwise deteriorate rapi dly when stored as hay due to mold growth (Brown and Kunkle, 1992). Despite the many benefits of ammoniati on, the use of this forage improvement method has been limited due to the hazards it poses to humans and its corrosive effect on equipment. Another widely used chemical method for im proving forage nutritive value is NaOH treatment. Two treatment methods are available. In the we t or Beckmann method, the
31 forage is soaked in a dilute solution of NaOH for several days and then washed to remove the residual chemical. Sundstol (1988) reported that using this method increased the in vivo OM digestibility of rye ( Secale cereal L.) straw from 460 to 760 g/ kg. However, there are two major drawbacks wit h this procedure. Firstly, the wastewater was contaminated with residual NaOH, which could pollute the environment. Secondly, there is considerable loss of DM due to ri nsing the treated material prior to feeding. Consequently, a dry treatment method was developed. This involves spraying the forage with a solution of NaOH without rinsi ng the treated forage prior to feeding. The disadvantage of the dry method is the possibilit y of increasing toxicity if the samples are not uniformly treated. Various studies have demonstrated the effectiveness of dry and wet NaOH treatment processes at improving the nutritive value of low quality forages and crop residues (Wanapat et al., 1985; Moss et al., 1990). However, NaOH has the similar caustic properties to ammonia, which make it hazardous to handle. Other chemicals such as hydroxides of calcium, ammonium and potassium have also been used to treat low quality forages Two experiments were conducted by Haddad et al. (1994) to com pare different alkali treatment s. In Experiment 1, ground wheat straw was treated with NaOH, NH4OH, urea, or Ca (OH)2 or certain mixtures of these alkalis. The NDF concentration of wheat st raw was decreased by all alkali treatments whereas ADF concent ration was not affected. The greatest in vitro NDF digestibility was obtained when wheat stra w was treated with 5% NaOH or with 2.5% NaOH and 2.5% Ca (OH)2. In Experiment 2, chopped wheat straw was fed to Holstein heifers after treatment wit h 1) nothing (control); 2) 2.5% NaOH; 3) 5% Ca(OH)2; 4) 2.5%
32 Ca(OH)2 + 2.5% NaOH; and 5) 5% Ca (OH)2. The greatest in vivo NDF digestibility occurred when NaOH alone or NaOH plus Ca(OH)2 was applied. Calcium hydroxide is safer to handle than NaOH or NH3. However, few studies have used it to treat low quality forages (Owen et al., 1984) because it is a weaker alkali than NaOH and is therefore less effective at improving digestibility (Wilkinson and Gonzalez-Santillana, 1978). In addition, Ca(OH)2 treatment has increased mold growth in some forages (Patterson et al., 1980; Bass, et al., 1982). Enzymatic Hydrolysis of Cell Walls Fibrolytic Enzymes Ruminant feed enzyme additives, primar ily xylanases and cellulases, are concentrated extracts resulting from bacterial or fungal fermentations that have specific enzymatic activities (Beauchemin et al., 2003). Several digestive enzymes are used regularly and successfully to enhance the performance of poultry and swine diets but enzymes have not routinely been used in diet s fed to ruminants (Kung, 2001). This was partly due to the perception that enzymes are proteins that are subject to degradation by microbial proteases in the rumen and or inactivation by proteases in the small intestine. Kopecny et al. (1987) reported that a cellulase enzyme complex was rapidly degraded by ruminal bacterial proteases and ther efore had no effect on in vitro fiber digestion when added to substrates incubated in ruminal fluid. However, Fontes et al. (1995) compared the resistance of cellulase s and xylanases to proteolysis and reported that several xylanases were resistant to se veral proteases but only one cellulase from a mesophilic organism was resistant to proteol ytic attack. Other studies have also demonstrated that exogenous polysaccharide-degrading en zymes resist microbial proteolysis in the rumen long enough to escape the reticulorumen and some have been
33 active in duodenal fluid (Hrist ov et al., 1997, 1998). When applied to feeds, the close association of enzymes to feeds may also confer resistance to ruminal proteolysis (Beauchemin et al., 2004). The next section describes the main enzymes involved in cell wall digestion by ruminants. Cellulase Cellulos e and hemicellulose are the main st ructural polysaccharides in plants (Van Soest, 1994) and they are converted to soluble sugars by cellulases and hemicellulases, respectively. Such enzymes ar e sourced from cultures of fungi such as Trichoderma longibrachiatum Aspergillus niger or A. oryzae or bacteria such as Bacillus subtilis and Streptococcus faecium (Pendleton, 2000, as cited by Beauchemin et al., 2004). The main enzymes involved in cellulose hydrolysis are as follows: 1) endoglucanase (EC 3.2.1. 4), which hydrolyzes cellulose chains at random to produce cellulose oligomers of varying degrees of polymerization; 2) exoglucanase (EC 22.214.171.124.1) which hydrolyze s the cellulose chain from the non-reducing end producing cellobiose; and 3) -glucosidase (EC 126.96.36.199) which re leases glucose from cellobiose and hydrolyzes short chain cell o-oligosaccharides from bot h reducing and non-reducing ends (Bhat and Hazlewood, 2001; Beauchemin et al., 2003; Figure 2-3). Bhat et al. (1990) stated that microorganisms secrete multiple endoglucanas es (I, II, II, IV, and V) with a wide range of substratespecificity that allow efficient hydrolysis of complex cellulosic substrates. Likewise, two classe s of exoglucanase (cellobiohydrolases; CBH) have been described (Barr et al., 1996). The first class includes one enzyme (CBH-I) from T. reesei and two exoglucanases (E4 and E6) from T. fusca These hydrolyze the cellulose chain from the reducing end. The second class includes CBH-II from T. reesei
34 and E3 from T. fusca These hydrolyze the cellulose chain and release cellobiose from the non-reducing end (Barr et al., 1996). Figure 2-3. Schematic repres entation of the major enzym es involved in cellulose hydrolysis (Beauchemin et al., 2004) Xylanase The core polymer of xylan is degr aded to soluble sugars by two main enzymes: xylanase (EC 188.8.131.52) and -1,4 xylosidase (EC 184.108.40.206) (Bhat and Hazlewood, 2001). Xylanases are commonly called endoxylanases and they yiel d xylooligomers when they hydrolyze xylan whereas, -1,4-xylosidases yields xylose (White et al., 1993). In addition, other hemicellulase enzymes involved primarily in the digestion of side chains include -mannosidase (EC 220.127.116.11), -L-arabinofuranosi dase (EC 18.104.22.168), -Dglucuronidase (EC 22.214.171.124), -D-galactosidase (EC 126.96.36.199), acetylxylan esterases (188.8.131.52) and ferulic acid esterase (EC 3.1. 1.73) (Biely et al., 1992). Xylans containing
35 branching involving linkages wit h arabinose and glucuronic acid are more soluble than those that do not (Van Soest 1994). Xylanas es efficiently degrade linear xylan but branched chains are more slowly or incomp letely degraded (Van Soest, 1994). Xylans from annual plants are more heterogeneous than xylans from perennial plants (Bhat and Hazlewood, 2001). Some arabinoxylans such as those in cereal endosperm are highly branched and lack uronic acid substitution for xylose, whereas others found in lignified tissues, have much less branching and have substitutions of uronic acid, 4O methyl ether and galactose for xylose (Bhat and Hazlewood, 2001). Synergistic Action of Cellulases and Xy lanases Some authors have demonstrated the ex istence of synergy between endo and exoglucanases during solubilization of crystal line cellulose (Wood et al., 1989; Klyosov, 1990). The most common types of synergy are between endoglucanase and exoglucanase (CBH) or different types of exoglucanases (CBH) or endoglucanases. Complete and efficient hydrolysis of xylan requires the synergistic action of main and side-chain-cleaving enzymes with different specificities (Coughlan et al., 1993). These authors described the following types of sy nergy between enzymes: 1) Homeosynergy, the synergistic interaction between two or mo re different types of side-chain-cleaving enzymes or between two or more types of main-chain-cleaving enzymes; 2) Heterosynergy, the synergistic interacti on between side-chain and main-chain cleaving enzymes; 3) Antisynergy, the action of one type of enzyme that prevents the action of a second enzyme (Coughlan et al., 1993). Combin ing cellulases and xylanases has led to synergistic degradation of corn fiber (Kos ugi et al., 2002; Mura shima et al., 2003; Koukiekolo et al., 2005) oat hulls (Yu et al., 2005), and bahiagrass ( Paspalum notatum )
36 hay (Krueger and Adesogan, 2008) but the spec ific types of synergistic interaction involved in these studies was not disclosed. Esterase Polysaccharides may be linked to lignin by glycosidic, ether, and ester crosslinkages, or cinnamic acid bridges (Figure 24; Lam et al., 1990). Yet xylanas es and cellulases can only hydrolyze glycosidic link ages. Ferulic acid (FA), the most abundant hydroxycinnamic acid in the cell wall of ce real grains (Kroon et al., 1996; Bartolome et al., 1997a, b) is covalently cross-linked to polysaccharides by ester bonds and to components of lignin mainly by ether bonds (Scalbert et al., 1985; Borneman et al., 1991). Ferulic acid esterase (FAE), can hydrolyze the ester linkage between FA and the attached sugar and release FA from comple x cell walls like wheat bran (Bartolome et al., 1997a), sugar beet pulp (Kroon et al., 1996), and spent barley grain (Bartolome et al., 1997b) thus opening the remaining cell wall polysaccharides to further hydrolytic attack by other enzymes (Yu et al., 2005). Xylan exists in many plants in an ac etylated, poorly digestible form, a circumstance largely neglected in studies of its breakdown by microbial enzymes (Biely et al., 1986). Biely et al. (1986) demonstr ated that fungal es terase and xylanase activities act cooperatively to hydrolyze ac etyl xylan. In the absence of esterase, xylanases break only a limited nu mber of glycosidic bonds in xylans. Both the extent and rate of breakdown increase when esterase is present. The two activities also act synergistically to liberate acetyl residues. Yu et al. (2002) studied the ability of FAE from Aspergillus to release FA from oat hulls in the presence or absence of xylanase from Trichoderma xylanase. Little release of FA by FAE occurred in the absence of the
37 xylanase, whereas a significant release in occurred in the presence of the xylanase, indicating a synergistic interaction between the enzymes. Several authors have suggested a possible role for supplemental esterases in enhancing forage degradation in the rumen (N sereko et al., 2000b; Tricarico and Dawson, 2001; Dhiman et al., 2002). However, es terase activities of fibrolytic enzymes have not been reported in most enzyme studies and few studies have examined supplementary esterase effects on anima l performance. Dhiman et al. (2002) compared adding xylanase and cellu lase enzymes with or without FAE to a diet fed to lactating dairy cows, but no effect on DMI or milk production res ponse was reported. Therefore, beneficial responses of esterase addition have only been demonstrated in vitro. For instance, Eun and Beauchemin (2006) reported that combining xylanase, cellulase, and esterase enzymes improved DM and NDF degradability of alfalfa hay and corn silage by 25% relative to the untreat ed control. Krueger et al. (2008a) applied an esterase enzyme at different rates to three tropical gr asses and reported that enzyme application enhanced NDF hydrolysis and 96 h IVDMD. Increasing the rate of application also increased the in situ was hout fraction and enhanced release of watersoluble carbohydrates (WSC) and ether-linked ferulic acid from Tifton 85 bermudagrass, and ester-linked ferulic and p -coumaric acids from Pensacol a bahiagrass. These in vitro studies reveal the important contribution of FAE to cell wall hydrolysis and emphasize the need for in vivo experiments to evaluat e performance responses of ruminants to dietary esterase addition. Etherase As forages mature, and become increasingly li gnified, ferulates that were esterified to arabinoxylan become etherified via cro ss-links between lignin and the cell wall
38 polysaccharides (Iiyama et al., 1990). Ether linkages are highly resistant to biodegradation due to their high bond energy and chemical inertness (White et al., 1996). Etherases are a group of enzymes that catalyze the scission of such linkages by the following mechanisms: oxygenative cleavage via monooxygenases, oxidation of the C atom linked to the ether bond followed by hydrolysis of the resultant ester, cleavage of methyl-aryl ethers, direct hydrolysis of the C O bond, and C O lyase-mediated cleavage (Cain, 1981). Ruminal fungi such as Neocallimastix patriciarum may solubilize lignin via dissolution of the xylan in the lignin xylan matrix (Mc Sweeney et al., 1994) but they do not cleave the ether linkage that cross links lignin and polysaccharides in hydroxycinnamic acid bridges. Actual lig nin depolymerization and ether linkage scission involves cleavage of the arylglycerolaryl ether linkage, which has been achieved with soil bacteria such as Pseudomonas paucimobilis SYK-6 (Masai et al., 1989) but not with ruminal or commercial ex ogenous fibrolytic enzymes. Ligninase Ligninase is a fungal enzyme discovered in the extracellular fluid o f lignolytic cultures of White-rot fungi (Phanerochaete chrysosporium; Tien and Kirk, 1983). Some species of White-rot fungi have improved the biodegradation of grasses by attacking the aromatic constituents of ligni n (Akin, 1993). These fungi ar e capable of degrading lignin due to the presence of several enzymes like ligninases, phenol oxidases, Mndependent peroxidases, and H2O2-producing enzymes (Kirk and Farrell, 1987). To test if ligninase enzymes from white rot fungi can be used to improve forage quality, Khazaal et al. (1993) examined the effe ct of treating barley straw with P. chrysosporium with or without addition of ligninase. No effect on chemical composition and digestibility was reported. In contrast, severa l studies have shown that additi on of strains of white-rot
39 fungi to forage increases in vi tro digestibility (Agosin and Od ier, 1985; Gamble et al., 1994; Zadrazil et al., 1995). However, the grow th substrates of the fungi are C sources like carbohydrates (Fahey et al., 1993), which no longer available to animals after fungal hydrolysis. This has limited the use of wh ite-rot fungi for forage improvement. Further research on optimizing ligni n degradation by cell-free ligninases is required. Modes of Enzyme Action Mechanism s of fibrolytic enzyme acti on on forages that have been postulated include direct hydrolysis (Sheperd and K ung, 1996; Colombatto et al., 2003a), stimulation of microbial number s and microbial attachment to substrates (Morgavi et al., 2000), and improvement of palatability (Ades ogan, 2005). These effects could be initiated before or after ingestion of the forage. Preingestive effects The preconsumptive effect of exogenous enzymes on feeds may be as simple as release of soluble carbohydrates or as complex as removal of structural barriers that limit the microbial digestion of feed in the rumen (McAllister et al., 2001). Cell wall hydrolysis in the rumen proceeds in an eros ive manner (White et al., 1993) and the major restriction to digestion is limited colonization and penetration by cellulolytic microbes and their hydrolytic enzymes onto the exposed surfaces of feed particles (Beauchemin et al., 2003). Applying exogen ous enzymes directly to feed causes a release of reducing sugars (Hristov et al. 1996a), and in some cases, partial solubilization of neutral detergen t fiber and acid detergent fiber (Krause et al., 1998). Forsberg et al. (2000) sugges ted that the sugars releas ed would provide sufficient additional available carbohydrates to encour age rapid microbial growth, shortening the lag time required for microbi al colonization. The degree of sugar release depends on
40 both the type of feed and the type of enzyme. Hristov et al (1996a) reported that only two of 11 fibrolytic enzyme preparations tested released significant amounts of reducing-sugars from barley silage. The aut hors suggested that t he release of sugars arises at least partially from the solub ilization of NDF and ADF. Some studies have attempted to simulate preingestive effects of enzymes by examining their hydrolytic action on substrates in the absence of ruminal fl uid or in buffers or water. Colombatto et al. (2003a) examined the effect of adding a commercial fibrolytic enzyme on hydrolysis and fermentation of cellulose, xy lan and a mixture of both polymers. They reported increased release of reducing sugars after 20 h of incubation in the absence of ruminal fluid and concluded their enzym e released sugars from cell wall polysaccharides prior to ruminal digesti on. Krueger and Adesogan (2008) noted that combinations of ferulic acid esterase cellulase and xylanase improved 24 h DM disappearance of mature bahiagrass in the abs ence of ruminal fluid. Yu et al. (2005) also demonstrated that xylanase and cellulase enzymes synergistically increased the DM disappearance of oat hulls in the absence of ruminal fluid. To determine if activity of exogenous enzym es in the rumen is a prerequisite for ruminal fiber digestion, Nsereko et al. (2000b) pretreated alfalfa hay with buffer alone or with fibrolytic enzymes diluted in the buffer for 0 or 2 h. The hay was then autoclaved to denature enzymic activities, washed to re move hydrolysis products, dried, and incubated in ruminal fluid for 12 or 48 h. Pretreatment with t he enzyme increased NDF degradation at 12 and 48 h in the absence of active enzymes or soluble hydrolysis products. This indicated that hydrolytic action prior to incubation with ruminal microorganisms was the prevailing mode of action of the en zyme. The authors
41 concluded that activity of exogenous enzymes in the rumen is not a prerequisite for improved ruminal fiber degradation but interactions between exogenous enzymes and ruminal bacteria or endogenous could further enhance fiber degradation. These studies demonstrate the existence of pr eingestive enzyme effects but their relative importance compared to ruminal effects has not been documented. Post-ingestion effects Within the rumen, exogenous enzymes could directly hydrolyze feeds or indirectly stimulate digestive activity through syner gistic effects with ruminal microorganisms (McAllister et al., 2001). Exoge nous enzymes have increased the hydrolytic capacity of the rumen by exhibiting synergy with rumina l microbial enzymes (M orgavi et al., 2000) and increasing bacterial attachment (Morgavi et al., 2000; Wang et al., 2001) and numbers (Nsereko et al., 2002). Postconsum ptive effects of exogenous enzymes can potentially increase the rate (Adesogan, 2005) and extent of digestion or passage (McAllister et al., 2001; Adesogan, 2005). Enzym es can change the viscosity of ruminal fluid (Hristov et al., 2000; McAllister et al ., 2001) and modify the site of digestion of nutrients (Hristov et al., 2000) Beauchemin et al. (2003) suggested that supplementing the diet with exogenous en zymes should increase total enzyme activity within the rumen. Based on the enzymic activity of ruminal fluid and the amount of enzyme product consumed daily, Beauchemin and Rode (1996) calculated that adding exogenous enzymes to feed could potentially increase cellulase activity in the rumen by up to 15%. Recent studies revealed that enzyme addition (0.5 and 2.55 L/g of DM) increased ( P < 0.05) the initial (up to 6 h) xylanase, endoglucanase, and -Dglucosidase activities in the liquid fraction of ruminal fluid by an average of 85% (Colombatto et al., 2003c). Hr istov et al. (2000) reported t hat once daily intraruminal
42 dosing of exogenous polysaccharide-degrading enzymes into heifers increased xylanase and cellulase activity in the rumen. However, over ti me, the activity of exogenous enzymes can decline due to inacti vation and outflow with the fluid phase of ruminal contents (Hristov et al., 1996b). Several studies have been conducted to examine the existence of synergy between ruminal and exogenous enzymes. Colombatto et al. (2003b) examined the effect of two commercial fibrolytic enzyme products on the hydrolysis of alfalfa stems and leaves in ruminal fluid after a pretreatment period consisting of incubation in an enzyme solution or water alone for 20 h before ruminal fluid was added. Solubilization of substrates by the enzymes or water alone during the pret reatment period was separately quantified. The enzymes increased OM D in ruminal fluid of stems and leaves by up to 8 and 15%, respectively. However, direct hydrolysis of forage fractions during the pretreatment period did not fully account for the magnitu de of the increases in OMD, suggesting that synergy betw een the exogenous and endogeno us enzymes occurred. Wood et al. (1994) reported synergy in the so lubilization of crysta lline cellulose between cellulase from Trichoderma koningii and the cellulosome-type complex of the ruminal anaerobic fungus Neocallimastix frontalis Morgavi et al. (2000) demonstrated synergy between exogenous and ruminal enzymes, by s howing that the combined hydrolytic effect of both types in the rumen was much greater than that estimated from the individual enzymes. These studies confirm t hat exogenous enzymes act synergistically with endogenous ruminal enzymes to increase t he hydrolytic capacity of the rumen. Factors Affecting Enzyme Effi cac y in Dairy Cattle Rations Responses of dairy cattle to dietary addition of exogenous enzymes have been variable and in many cases small (Rode et al., 1999; Schingoethe et al., 1999; Yang et
43 al., 1999). Beauchemin et al. (1999) examined the effect of applying a cellulase xylanase enzyme to barley and hull-less barleybased diets of dairy cows. Fibrolytic enzyme supplementation tended to improve 4% FCM and increased total tract digestion of OM, starch, N, NDF, and ADF. In contra st, Elwakeel et al. (2007) reported that a cellulase xylanase enzyme increased in vitro DM digestibility but did not affect DMI and milk production by dairy cows. Several fact ors contribute to the variability in animal response to fibrolytic enzyme addition to diets. These include the co mposition, activity and application rate of the enzyme (Daws on and Tricarico, 1999; Siciliano-Jones, 1999), the time and method of enzyme applic ation (Sutton et al., 2003), and the prevailing temperature, pH, ionic strengt h, and substrate concentration and type (Beauchemin et al., 2003). In addition, the effectiveness of a particular enzyme depends on the composition of the diet (B eauchemin et al., 1995). For instance, enzymes that increased the DM degradation of alfalfa had minimal effects on corn silage and vice versa (Colombatto et al., 2003 a, b). Some of these factors are discussed in the ensuing sections. Effect of the form or component of the diet to which the enzyme is applied Several studies show that effects of enzyme application depend on the part or portion of the diet to which the enzyme is applied, reflecting enzym e-feed specificity. Application of enzymes to different form s of the same ingredient has also produced different results. Feng et al. (1996) reported that cellulase and xylanase enzymes were more effective when applied to dried smooth bromegrass ( Bromus inermis ) at feeding than to freshly cut wilted grass at harvest or to untreated grass. The authors suggested that greater cuticular disrupt ion of the dried grass occurr ed at feeding and this may have
44 enhanced ruminal microbial colonization and therefore increas ed feed degradation and particle size reduction. Krueger et al. (2008b) applied a fibr olytic enzyme to bermudagrass hay immediately after it was cut, just before it was baled (after wilting) or just before it was fed and compared these to untreated hay in di ets of growing steer s. Steers fed hay treated with the enzyme after cutting had greater intakes of total DM, hay DM and total NDF and greater digestibilities of hay NDF, total CP and CP intake than those fed the untreated hay. The superiority of the enzyme at cutting treatment was attributed to greater forage moisture availability at t he time of enzyme application. Based on the results of several studies, Beauchemin et al (1999) suggested that fibrolytic enzymes are most effective when added to the dry com ponents of the feed as this allows greater opportunities for the enzyme to adhere to th e feed and therefore enhance digestion. However, the ideal moisture concentration that optimizes the activity of enzymes without jeopardizing their adherence to feeds has not been determined. Bowman et al. (2002) studied the effect of applying a fibrolytic enzyme (cellulose and xylanase; Promote N.E.T. Agribrands International, St. Louis, MO) product to the following components of the TMR: 1) nothing (control); 2) the concentrate (45% of TMR); 3) the supplement (4% of TMR), or the premix (0.2% of TMR). Enzyme supplementation did not affect milk yield or DMI, but digestibility of DM, OM, NDF, and ADF were greater in cows fed the concent rate-treated diet than the control diet. Because the concentrate accounted for the gr eatest proportion of the ration, the authors suggested that exogenous fibrolytic enzymes s hould be applied to a substantial portion of the diet to ensure their effectiveness. R ode et al. (1999) also reported that DMI was
45 unaffected but DM, NDF, and ADF digestibility and milk production increased when a xylanase and cellulase enzyme was applied to the concentra te portion of a TMR. Furthermore, Yang et al. (2000) reported that OM digestibi lity and milk yield increased when a xylanase-cellulase enzyme was applied to the concentrate portion of a TMR. These studies support enzyme addition to the concentrates but contradictory studies exist. No differences in milk yield or DM I occurred when enzymes were applied to a TMR versus a concentrate or forage (D ean et al., 2005b), to a TMR versus a concentrate (Phipps et al., 2000) or to alfalfa cubes alone versus a mixture of alfalfa cubes and a concentrate (Yang et al., 1999). To our knowledge, only one study has examined enzyme addition to the forage inst ead of the TMR and no effects on DMI or milk production by dairy cows were reported in that study (Dean et al., 2005b). Therefore, the ideal dietary component to which fibrol ytic enzymes should be applied remains elusive. Effect of the site of enz yme delivery The idea that exogenous polysaccharide-de grading enzymes could not survive the ruminal proteolysis (Chesson, 1994) has been refuted by studies demonstrating that exogenous polysaccharide-degrading enzymes could resist proteolysis by ruminal microorganisms and remain active in the duodenum (H ristov et al., 1997). Hristov et al. (2000) reported that once daily intrar uminal dosing of exogenous polysaccharidedegrading enzymes into heifers increased xylanase and cellulase activity in the rumen. These studies suggest that ruminal dosing is a potentially useful strategy for administering enzymes but it has not improved the performanc e of dairy cattle. Sutton et al. (2003) applied a xylanasecellulase enzyme to either the concentrate or TMR or infused it into the rumen of dairy cows. Milk yield and DMI were not affected by
46 treatment but total tract DM digestibility was greater w hen the enzyme was applied to the TMR. Lewis et al. (1996) studied the effect of applying cellulases and xylanases via the diet of steers or by ru minal infusion. Compared to the control treatment, enzyme treatment decreased ruminal pH and increa sed total VFA concentration 16 h after feeding. However, ruminal infusion produced lower DM disappearance and lower total tract digestibility of DM, NDF, and ADF than di etary application. Similar results were also reported by Sutton et al. (2003). T hese studies demonstrate that dietary enzyme addition is more effective than ruminal infusion. Effect of time of enzyme appli cation to the diet Beauchemin et al. (2004) suggested that a close feed-enzyme association might enable some form of preingestive hydrolysis of plant fiber leading to creation of binding sites between the enzyme and feed. Adding certain cellulase xylanase enzyme mixtures to the diet just pr ior to feeding was as effectiv e as treating the forage 2 wk (Yang et al., 1999) or 1 to 3 d before feeding (Lewis et al., 1996; Nussio et al., 1997). Sutton et al. (2003) reported that adding a cellulase xylanase enzyme 1 h prior to feeding improved total tract DM, OM, and star ch digestibility compared to adding the enzyme 24 h before feeding but milk yield and in take of nutrients did not differ. Allowing adequate time for the enzyme-f eed interaction is important in order to optimize hydrolysis of cell wall components and guarant ee further interaction of exogenous enzymes and ruminal microbes. However, studi es on the effects of the time of enzyme application have produc ed equivocal results. Therefore, the ideal duration of the enzyme substrate interaction pe riod has not been established.
47 Effect of the enzyme application rate Theoretically, increasing the enzyme applic ation rate should result in greater cell wall digestion. To examine this theory, Beauchemin et al. (1995) studied the effect of applying a cellulase xylanase enzyme at differ ent rates to alfalfa hay, timothy hay, and barley silage in diets of steers. The enzym e was applied at six in cremental levels of xylanase (international units, IU) and cellulase (filter paper units FPU; 1) 0, 0; 2) 1000, 40; 3) 2000, 80; 4)3900, 156; 5) 7900, 316; 6) 15800, 632 IU, FPU kg-1 of forage DM. Enzyme application had no effect on the performance of steers fed barley silage. Low application rates (treatments 2 to 4) increased (24 to 30%) the ADG of steers fed alfalfa due to greater digestible DMI but higher rates (treatments 5 and 6) were not effective. In contrast, higher application rates increased ADG and ADF digestibility of steers fed timothy hay by 36 and 17%, respectively but lower rates did not. Therefore, the relationship between enzyme concentration and animal response was non-linear and it differed for legumes and grasses. Lewis et al. (1999) reported that cows fed alfalfa hay and silage treated with a medium dose (2.5 ml/kg of forage DM) of a cellulase and xy lanase enzyme produced 4 kg more milk than cows receiving the contro l, low, and high doses (0; 1.25; 5 ml/kg of forage DM; respectively) of t he enzyme. Kung et al. (2000b) investigated effects of adding increasing levels (0, 1, 2.5 ml/kg of TMR) of a cellulase xylanase enzyme to the forage (corn silage and alfalfa hay) in a TM R fed to lactating cows. Cows fed forage treated with the low dose tended to have greater milk yield (39.5 kg/d) than those fed the control diet (37.0 kg/d) or the high dose (36.2 kg/d) but in take or efficiency of milk production was not improved by any treatm ent. These studies demonstrate that
48 application rate is an import ant factor affecting enzyme action but that the most appropriate rate is dependent on the com position of the enzyme and diet. Effects of stage of lactati on and parity of dairy cattle Enzyme supplementation should be most effe ctive when ruminal fiber digestion is compromised due to factors like acidosis, or when dietary glucose supply is inadequate to meet the needs of the cow such as in early lactation (Adesogan, 2005). In support, Beauchemin et al. (2000) reported that when cows in positive energy balance were fed enzyme-supplemented diets, intake of digestibl e energy increased but milk yield did not. Schingoethe et al. (1999) studied the re sponse to adding a cellulase xylanase enzyme to diets of lactating dairy cows for 12 weeks. Data from early (64 DIM) and midlactation (178 DIM) cows were analyzed separately to determine when it was most effective to feed the enzyme. Milk produc tion was increased by 10.8% in early lactation, but no response occurred for cows in midlactation. Knowlton et al. (2002) reported that enzyme treatment of a dairy cow diet numer ically increased milk production by early lactation (30 10.6 DIM) cows but decreased m ilk production by cows in late lactation (194 9.7 DIM). Lewis et al. (1999) r eported that more milk was produced by midlactation (213 DIM) cows fed a cellulase xylanase enzyme trea ted diet than cows fed the control diet (27.2 vs. 25.9 kg/d). The discrepancy between this and other studies could be due to differences in enzyme activity, diet composition, and trial duration. To determine effects of the time of initiating enzyme dosing on efficacy, Zheng et al. (2000) applied a cellulase xylanase enzym e to the corn silage and alfalfa hay in a TMR beginning at parturition (wk 0 to 18 postpartum), or before (wk -4 to 18 postpartum) or after (wk 6 to 18 postpartu m) parturition. Cows fed enzyme-treated forage had greater 3.5% FCM than cows fed t he untreated forage. The time of initiating
49 enzyme dosing had no effect on milk producti on but numerically, the greatest values occurred when enzyme addition began at parturition. Most of these studies suggest that responses to dietary enzyme addition are greater in early lactation than at later stages of lactation. However, as outlined above, several other factors determine the outcome of enzyme application to feeds. More research is needed to determine conditions that optimize and guarantee efficacy when fibrolytic enzymes are added to diets of dairy cows. Figure 2-4. Schematic diagr am showing possible cova lent cross links between polysaccharides and lignin in walls. dehydrodiferulic acid. a, Direct ester-linkage; b, direct ether-linkage;c, hydroxycinnamic acid esterified to polysaccharides; d, hydroxyc innainic acid esterified to lignin; e, hydroxycinnamic acid etherified to lig nin; f, FA ester-ether bridge; g, dehydrodiferulic acid diester bridge; h, dehydrodiferulic acid diester-ether bridge (Iiyama et al., 1994).
50 Silage Fermentation The aim of silage fermentation is to produce sufficient lactic acid to inhibit both the growth of undesirable epiphytic microor ganis ms and the activity of endogenous plant catabolic enzymes, thus maximizing nutrient preservation (Bolsen et al., 1992). Plant respiration, plant proteolytic activity, clos tridial activity, and aerobic microbial growth adversely affect silage ferment ation and quality. T herefore, these processes need to be limited to ensure preservati on of crop quality (Muck, 1988) The following section describes influences of these and other fact ors on the fermentati on, quality, and aerobic stability of silages. Factors Affecting Silage Fermentation Several factors affect the rate of l act ate production and hence the fermentation of silages. The most important factors incl ude the epiphytic population of microorganisms on the plant and the WSC concen tration, buffering capacity, and moisture concentration of the forage. However, environmental conditions, agronomic practices, and harvest conditions may increase the number and/or change the distribution of epiphytic microbes on plants and therefore affe ct the fermentation process. Moisture Concentration and Maturity Drier silages have higher pH values than wetter silages because of the greater osmotic pressure at high DM and these factor s collectively inhibit the growth of LAB in drier silages (Woolford, 1984; McDonald et al., 1991). Excess mois ture at ensiling predisposes crops to effluent production, whereas inadequate moisture at ensiling predisposes to heating and spoilage (Mu ck and Kung, 2007). Excess moisture encourages the growth of undesir able bacteria such as clostridia, which can dominate the ensiling process in grasses and alfalfa ( Medicago sativa ), resulting in a low quality
51 product (Muck and Kung, 2007). Clostridia can ferment lactate to butyrate resulting in significant loss of DM and energy. This se condary fermentation can be associated with substantial decarboxylation and deamination of amino ac ids from plant protein (McDonald et al., 1991). Moisture concentration typically decreases with plant maturity an d different studies have shown that fermentation is decreased with advancing plant maturity. Bal et al. (1997) observed a decline in lactate, acet ate, and ethanol concentration of corn ( Zea mays) silage as maturity advanced. Similarly, Neylon and Kung (2003) reported an increase in pH and a decline in lactate and acetate concentrations as maturity of corn silage increased. Epiphytic Bacteria The total microflora population on grasses and herbages varies between 105 and 109 colony forming units (cfu ) / g of the crop to be ens iled (Langston and Bouma, 1960 a, b). The epiphytic lactic acid bacteri a (LAB) are the essential microflora for spontaneous silage fermentation (Pahlow et al., 2003). The number of LAB varies with the type of forage and populations (cfu/g of fresh matter) of 105 on alfalfa, 106 on perennial grasses, and 107 on corn and sorghum ( Sorghum bicolor ) were reported by Pahlow et al. (2003). Howeve r, the LAB population also va ries with stage of maturity, and prevailing weather and is typically greates t on second to third cuts of alfalfa and grasses and on early maturing varieties of co rn (Bolsen et al., 1988; Muck, 1989). Whereas, the lowest numbers of LAB usually occur during c ool weather (Pahlow et al., 2003) because the optimal growth temper ature range for LAB is between 25 and 40C (Pahlow et al., 2003).
52 Enterobacteria are the second largest group (103 106 cfu g-1) of epiphytic microflora (Pahlow et al., 2003). They are ac tive in the silo and are important due to their competition with LAB. They ferment su gars into acetic acid, hydrolyze proteins, and deaminate peptides and amino acids (Rooke and Hatfield, 2003). They also reduce NO3 resulting in the production of nitrites and nitrogen oxide gases (Pahlow et al., 2003). Yeasts are frequently also present on gr owing forages. Yeasts ferment sugars such as glucose, maltose, and sucrose mainly into ethanol and CO2 and small amounts of propanol, 2-butanediol, 2-methylpropanol, pentanol, 3-methylbutanol, acetate, propionate, butyrate, and lactate (Schlegel, 1987; McDonald et al., 1991). When air penetrates the silo during fermentation, la ctate-fermenting yeas ts of the genera Candida and Hansenula dominate, whereas when anaerobic conditions are achieved, the population of the latter yeasts is reduced to about 15% and Saccharomyces spp., which do not utilize lactate dominate the population (McDonald et al., 1991). Members of the genera clostridia and bacilli appear to be scarce on fresh forage (Pahlow et al., 2003). They principally aris e from contamination from soil and farmyard manure (Ostling and Lindgren, 1991; Rammer et al., 1994). W hen low pH silage is not achieved, clostridia ferment lactate into butyric acid and degrade amino acids to a variety of products, which are of poor nutri tional value (McDonald et al., 1991). Bacilli can also be important facilitators of aerobic deterioration after other aerobic microorganisms have raised silage pH (Pahlow et al., 2003). Bacilli may be implicated in initiating aerobic deterioration in some s ilages as they possess an electron transport chain and can therefore metabolize lactic acid aerobically (Rooke and Hatfield, 2003).
53 Water-Soluble Carbohydrates The main nonstructural carbohydrates in temperate grasses are glucose, fructose, sucrose, and fructans (McDonald et al., 1991) These carbohydrates are soluble in water and are known as WSC. Fructans are the most abundant WSC in temperate grasses, whereas subtropical and tropical grasses accumulate starch instead of fructans (N elson, 1995). Glucose is the most fermented WSC by various species of LAB found on plants and it is fermented into la ctic acid, VFA, and alcohols by such bacteria (Muck and Kung, 2007). Ensiling crops with high WSC concentrations accelerates the fermentation by enhancing substr ate availability for LAB. Minimal WSC concentrations required to optimize the fe rmentation vary with forage type and DM concentration and are 14, 7, and 5 % (DM basis ) for alfalfa, cool-season grasses, and corn forage harvested at 35% DM (Lei bensperger and Pitt, 1988). Excessively high forage WSC concentrations may provide a sour ce of readily available nutrients for aerobic spoilage organisms such as yeasts (Mahanna and Chase, 2003), thus predisposing the silage to spoilage. Buffering Capacity The buffering capacity of plants is their ab ility to resist a change in pH. It is expressed as milliequivalents (meq) of alkali required to change the pH of 1 kg of the substrate from 4 to 6 (McDonald et al., 1991). The anionic fraction of forage crops, the organic acids, sulfates, nitrates, and chlorides, represent 75 to 90% of the total buffering constituents, and plant proteins account fo r 10 to 20% (Woolford, 1984). Forages with high buffering capacity require more acid to reduce their pH. Woolford (1984) noted that forages with low buffering capacity (250 to 350 meq NaOH kg-1 DM) are relatively easy to ensile, whereas forages with hi gh values (400 to 600 meq NaOH kg-1 DM) are
54 harder to ensile. Corn forage has a low buffering capacity of approximately 200 meq NaOH kg-1 DM (McDonald et al., 1991), therefore it is relatively easy to ensile. Climatic Factors The environmental temper ature at and during ensili ng may affect silage fermentation by influencing bacterial activity Ensiling at high temperatures reduces lactic acid concentration and aerobic stabi lity, and increases pH and DM losses (Weinberg et al., 2001; Ashbell et al., 2002) High ensiling temperatures reduce numbers of LAB (Weinberg et al., 1998, 2001), enhance proteolysis (Muck and Dickerson, 1988, Weinberg et al., 2001), and make the fermentation less homolactic (McDonald et al., 1996). Kim and Adesogan (2006) reported that fermentation of corn silage was adversely affected by wet conditions at harvest and high ensiling temperatures which resulted in greater pH and concentrations of residual WSC and ammonia-N, greater pr oteolysis, lower lactic to acetic ratio, and increased secondary fermentation. Most homofermentative lactobacilli grow optimally at 30 to 35C but Pediococcus spp. and some heterofermentative bacteria t end to be more thermotolerant and prefer temperatures of 40 to 45C (Woolford, 1984) Mulrooney and Kung (2008) reported that inoculant LAB were relatively stable when ex posed to temperatures of 30 to 35C for 3 to 6 h; however, exposure to 40 and 45C re sulted in marked reductions in viable cell counts of several inoculant bacteria within 3 h. Therefore, the authors suggested that thermotolerant bacteria should be used in inoculants and care should be taken to avoid exposing these bacteria to tem peratures exceeding 35 to 40C.
55 Factors Affecting Aerobic Stability Aerobic stability is the length of time it takes for silage to begin to heat during feedout. When silage is exposed to air, ce rtai n opportunistic microorganisms become metabolically active, produce heat, and consume nutrients from the silage resulting in spoilage. Aerobic instability increases nut rient losses from feeds and reduces feed intake and productivity of dairy (Hoffman and Ocker, 1997) and beef cattle (Whitlock et al., 2000). Yeasts Yeasts are facultative, anaerobic, heterotrophic microorganisms and are considered undesirable in silages. Silage yeasts ferment sugars to ethanol and CO2 under anaerobic conditions (Schlegel, 1987; Mc Donald et al., 1991). This decreases the amount of sugar available for lactic acid production. Intake of ethanolic silage by dairy cows can taint the flavor of milk (Randby et al. 1999). Many yeasts species in silages degrade lactic acid to CO2 and H2O under aerobic conditions, thereby, causing a rise in s ilage pH, and promoting t he growth of other spoilage organisms (McDonald et al., 1991). Woolfo rd et al. (1982) r eported that yeasts are essentially responsible for the aerobic in stability of corn silage. Some authors reported that during the first weeks of ens iling, yeast populations can increase up to107 cfu/g, though prolong ed storage will lead to a gradual decrease in yeast numbers (Jonsson and Pahlow, 1984; Middelhoven and van Baalen, 1988). Lactate-assimilating yeast such as Candida Cryptococcus and Pichia have been implicated as the primary causes of aerobic deterioration in silages (Jonsson and Pahlow, 1984).The presence of oxygen enhances the growth of yeasts during storage, wher eas high concentrations of formic or acetic acid reduce their growth (Oude Elferink et al., 1999). Yeast counts
56 greater than 105 cfu/g are usually indicative of aerobic instability (Pahlow and Zimmer, 1985; OKiely et al., 1987). Molds and Mycotoxins Molds are eukaryotic microorganisms that develop in silage when oxygen is present. Silage infested with molds are usually easily identified by the large filamentous structures and colored spores that many molds produce though some molds are not visible to the naked eye. Mold species regul arly isolated from silage belon g to the genera Penicillium, Fusarium, Aspergillus, Mucor, Byssochlamys, Absidia, Arthrinium, and Trichoderma (Woolford, 1984; Jonsson et al., 1990; Nout et al., 1993). Molds cause a reduction in feed value and palatabil ity, and have a negative effect on human and animal health due to the my cotoxins they produce. Silages that are heavily infested with mold s do not necessarily contain high levels of mycotoxins (Nout et al., 1993) and mycoto xins could be present when molds are not visible. Therefore, the re lationship between visible molds and occurrence of mycotoxins is not clear. Scudamore and Livesey (1998) reported that mycotoxicoses caused by mold mycotoxins range from digestive ups ets, fertility problems, and reduced immune function to serious liver or kidney dam age and abortions, depending on the type and amount of toxin present in the silage. Some important myco toxin-producing mold species are Aspergillus fumigatus, Penicillum roqueforti and Byssochlamys nivea. Penicillium roqueforti is acid tolerant and it can grow under low oxygen, high CO2 conditions. It is the predominant mold specie s in different types of silages (Lacey, 1989; Nout et al., 1993; Auer bach et al., 1998). Two species of Aspergillus, A. flavus and A. fumigatus and their aflatoxins are often reported in silage s from warm areas. Aspergillus flavus is common in hot and dry
57 regions where it colonizes corn plants in the field and produces aflatoxins and cyclopiazonic acid (Munkvold, 2003). Cyclopiazonic acid (CPA), which is also produced by Penicillium molds, is a potent specif ic inhibitor of the endoplasmic reticulum Ca++ ATPase (Goeger et al., 1988) Aspergillus fumigatus is a thermotolerant fungus that produces several different mycotoxins in cluding fumitremogens B and C, and gliotoxin (Cole et al., 1977). Molds of the genus Fusarium produce seve ral classes of important mycotoxins including the fumonisins, thichothecenes and zearalenone (DMello et al., 1999). Fumonisins are produced by Fusarium proliferatum and F. verticillioides as well as numerous other related Fusaria. These two species are extremely common on corn plants and cause ear and stalk rot diseases (P ayne, 1999). In addition, these fungi can grow inside the corn plant without causi ng disease symptoms (Bacon and Hinton, 1996). Maintenance of an anaerobic en vironment in the silo during the fermentation and storage phases and maintenance of aerobic stability during the feedout phase are important in silage preservation (Bolsen et al., 1996). Failure to achieve such conditions may cause lower recovery of nutrients, and the produc tion of poor quality silage, which can reduce DMI and animal pe rformance (Chen et al., 1994). Oxygen is the ultimate enemy of the ensiling process because most molds and yeasts require oxygen for growth. Thus, any management practice that hel ps exclude oxygen from the silage mass is helpful. Such practice s include harvesting at proper moisture concentrations, rapid filling, adequate packing and covering with plastic. This exclusion of oxygen from the silage promotes r apid fermentation by anaerobic hetero and
58 homofermentative bacteria, thereby reducing t he growth of yeasts and molds during the initial stages of fermentation. Bacilli Bacilli belong to the family Bacillaceae and they grow aerobically, however, some are facultative anaerobes (Pahlow et al., 2003). They can ferment a wide range of carbohydrates to acetate, lactate, but yrate, ethanol, 2,3-but anediol, and glyc erol (Woolford, 1977). The main habitat for Bacillus spp. is the soil (Claus and Berkeley, 1986) and they are scarce in fresh for age (Lindgren et al., 1985a) except when contaminated with soil. The number of Bacillus spores in cattle manure can range from 104 up to 106 cfu g-1 FM (Ostling and Lindgren, 1991; Rammer et al., 1994). Therefore, fertilizing with manure c an increase the number of Bacillus spores on the crop and consequently increase the number of Bacillus spores in silage. Bacillus spp. may be able to initiate aerobic deterioration of silage but more commonly, they contribute later to deterioration after it is initiated by y easts (Lindgren et al., 1985b, Spoelstra et al., 1985). Moisture Moisture sti mulates the growth of LAB but excess moisture encourages the growth of undesirable bacteria like clostridia. High moisture concentrations are also undesirable because compaction in the s ilo may produce seepage (effluent) losses, which contains high levels of soluble nutrients (Muck and Kung, 2007). Moisture concentration of crops affects O2 supply to spoilage microorganisms and the temperature rise produced fr om the heat generated by aerobi c microbial growth (Muck et al., 2003). Moisture concent ration also affects the amount of heat needed to raise silage temperature (i.e. the spec ific heat of the silage). The s pecific heat of water is 4.19
59 J /g H2O C-1, whereas that of forage DM is only 1.89 J /g DM C-1 (McDonald et al., 1991). Consequently, the heat released by micr obial respiration of a given amount of DM in silage containing 50% water produces more than a threefol d higher increase in temperature than the same loss in silage containing 80% water (Muck et al., 2003). This partly explains why drier silages are more susceptible to heating and deterioration. Packing Density Achiev ing high density in a silo reduces DM and nutrient losses resulting from plant respiration during filling or aerobic microbial growth at filling, storage, or feed-out (Muck et al., 2003). Porosity, which is determi ne by density and moisture concentration, determines the rate at which air moves into the silo and subsequently the amount of spoilage which occurs during storage and feed-out (Holmes and Muck, 1999). Inadequately packed silos or bags often have pa ssageways for air to move back rapidly from the open face. This exposes more of the silage to oxygen soon after opening, increasing the opportunity for spoilage and h eating (Muck and Kung, 2007). In bunker silos, density is determined primarily by t he weight and number of passes used by the tractor packing the silage into the bunker. In tower silos, density is increased by gravitational self compaction and therefore depends on depth of silage and silo factors such as height, diameter, and wall material (Muck et al., 2003). Silage Additives Additives are added to silages to direct the fermentation and prevent adverse effects of plant respiration, plant proteolytic activity, clostridial activ ity, and aerobic microbial growth on silage quality. Various bi ological, chemical, and nutrient additives are used for these purposes.
60 Chemical Additives Organic acids Several European studies have reported that formic acid treatment reduces NPN formation in direct-cut grass silages and improves their nutritive value for ruminants (McDonald et al., 1991). Formic acid-tr eated alfalfa silage had lower pH and NH3 concentrations than untreated controls and gr eater water-insoluble N (Lancaster and Brunswick, 1977; Barry et al., 1978). Nagel and Broderick (1992) showed that formic acid treatment of wilted alfalfa silage was more effective at reducing pH and had the lowest concentrations of lactic and acetic acid compared to untreated silage and formaldehyde treated silage. In addition, formic acid treatment decreased NPN formation and substantially improved N utilizat ion when fed to lactating dairy cows. Rooke et al. (1988) r eported that formic ac id-treated ryegrass ( Lolium perenne) silage had lower concentrations of ammonia-N and acetic and lactic acids and higher concentrations of WSC and ethanol than the c ontrol silage. Furthermore, when the silage was fed to sheep, N retention and silage DMI were improved by formic acid treated-silage. Despite its effi cacy, application of formic acid to forage is challenging because of its corrosive nature. Therefore, formic acid is not a widely used additive for silage preservation. Propionic acid-based preservatives have also been used to improve the aerobic stability of corn silages (Leaver, 1975) because of the antifungal nature of the acid (Britt et al., 1975; Leaver, 1975). These have been largely replaced by buffered propionic acid additives (Kung et al., 2000a) due to the co rrosive nature of propionic acid. Kung et al. (1998) reported subst antial improvements (120 160 h) in the aerobic stability of corn silage treated with relatively low concentra tions (0.1 to 0.2% of fresh forage weight)
61 of several additives that contained buffered propionic acid as their primary active ingredient. Kung et al. (2000a) examined the effect of various levels (0.1, 0.2, and 0.3%) of ammonia-N or a buffered propionic acid preservative on the fermentation of whole-plant corn. They reported that high levels (0.3%) of ammonia-N or buffered propionic acid markedly improved aerobic st ability of corn silage compared with the control; however, the number of yeasts and concentrations of lactate and acetate were similar in all treatments. Ammoniated s ilages had greater pH t han buffered propionic acid and untreated silages, but propionic acid concentration was only increased in silages treated with buffered propionic acid. Ammonia Moderate concentrations of ammonia (0.1 to 0.3%) have increased concentrations of lactic and acetic acids (Muck and Kung, 1997), decreased proteolysis (Huber et al., 1979, 1980), improved DM recovery (G oering and Waldo, 1980), and improved the aerobic stability of corn silage (Britt and Huber, 1975). Many researchers have suggested that addition of ammonia to silage improves aerobic stability because of its fungicidal properties (Depasq uale and Montville, 1990). K ung et al. (2000a) studied the effect of ammonia hydroxide ap plication at the rate of 0. 3 % of fresh forage on corn silage. They reported that the number of enterobacteria were less than 2.00 log cfu/g after 4 d of ensiling in contro l silages but remained high (> 5 log cfu/g) in ammoniated silages through 6 d of ensiling. The per sistence of enterobacteria and subsequent growth of heterofermentative LAB contributed to higher concentrations of acetic acid in ammoniated silages during their study. Th e number of yeasts in control silages increased rapidly whereas that in ammoni ated silages remained low for 144 h after aeration. Alii et al. (1983) reported that numbers of yeas ts decreased immediately in
62 high moisture corn after treatment with ammonia (1% of fresh forage). However, ammoniation is not widely used because it is corrosive on machinery, toxic at high doses, and hazardous. Biological Additives Homolactic Bacterial Inoculants The earlies t known use of LAB cultures is by French workers on sugar beet pulp at the beginning of this century (Wilkinson and Phipps, 1979). Inoculants containing selected strains of LAB have been develop ed to reduce dependence of the ensiling process on epiphytic LAB and on chemical additives. The principal function of these homofermentative inoculants is to ensure a r apid and efficient fermentation of WSC into lactic acid, a rapid decrease in pH and im proved silage conservation with minimal fermentation losses (Weinberg et al., 1993a). Fermentation of glucose by homolactic LAB via the Embden-Meyerhof-Parnas pathway is desirable because it yields high recovery of energy (99.3%) and DM (100%) and c onverts all of the glucose into lactic acid, a strong acid (McDonald et al., 1991). Inoculants containing homolactic LAB (> 105) can improve the fermentation quality of silages and reduce DM losses if the herbage contains sufficient fermentable ca rbohydrates and the inoculant bacteria dominate the epiphytic populatio n of LAB (Spoelstra,1991). One of the most widely used LAB in inoculants is Lactobacillus plantarum This bacterium is a facultative heterofermentative organism, which usually ferments hexoses homofermentatively into lactic acid but, when WSC concentrations ar e low, heterofermentative metabolism into lactic acid, carbon dioxide and ethanol (or acetic acid) occurs (Holzer et al., 2003). Muck (1993) reviewed several studies from 1985 to 1992 and reported that inoculant application improved intake and body -weight gain in about 25% of studies,
63 milk production was increased in 40% of t he studies and feed efficiency was improved in 45% of the studies. Adesogan et al. (2009) reviewed 38 studies from 1989 to 2009 and reported that inoculation wi th homofermentative bacteria increased digestibility of DM, organic matter (OMD), neut ral detergent fiber (NDFD), and acid detergent fiber (ADFD) in 36, 47, 31, and 36% of studies, respectively. Lactobacillus plantarum was the most frequently used (82% ) bacteria in the inoculants in these studies. Ely et al. (1981) applied L. plantarum to alfalfa, corn, sorghum, and wheat ( Triticum aestivum ) silages and reported that treat ed silages had a lower pH, higher lactic acid concentration, and greater recovery of DM, and CP than untreated silages. Combinations of L. plantarum and other homolactic LAB have also generally improved the fermentation. Driehuis et al. (1997) applied L. plantarum and Enterococcus faecium to ryegrass silage ensiled in lab and farm scale silos. After 180 d ensilage, treated silages had lower pH, DM loss, and ammonia-N concentration, and higher lactic acid concentrations compared wit h control silages. A summary of 14 lactation studies conducted in North America and Europe revealed that applying L. plantarum MTD1 to a variety of crops (grass, corn, and alfalfa) across a wide spectrum of DM contents (15 to 46% DM) resulted in a 4.6% increase in milk yield (Moran and Owen, 1994). Similarly, Moran and Owen (1995) summarized 19 comparisons among untreated and L. plantarum MTD1 treated silages fed to beef cattle. Cattle fed the treated silage ate 7.5% more DM and gained 11. 1% more weight. Kung et al. (1993) examined the effect of applying microbial inoculants to corn silage on the performance of lactating dairy cows in two experiments. Cows were assigned to the following treatments: Control, L. plantarum or L. plantarum and
64 Streptococcus faecium. Pr oduction of 3.5% FCM was greatest by cows fed silage treated with L. plantarum in both experiments. Muck (1993) reviewed several studies from 1985 to 1992 and reported that inoculant application increased milk production in 40% of the studies and feed efficiency was improved in 45% of the studies. Dry matter digestibility and animal performance were measured in 31 trials and reported that animal performance was improved in 9 of the 16 trials where the inoculant improved dry matter digestibility. When digestibility was not affected by the inoculant, only 2 of 15 trials had positive animal performance effects. The author suggested that increases in digestibility might be the key factor in explaining why inoculants improve animal performance. These studies cited above show that in many cases, homofermentative LAB have improved the fermentation of forages and enhanced animal performance but their effect on aerobic stability is less consistent. Weinberg et al. (1993b) noted that addition of a LAB inoculant containing E. faecium to wheat silage impair ed the aerobic stability of the silage. Addition of L. acidophilus had no positive effect on the fermentation of corn, sorghum, and wheat silages (Burghardi et al., 1980) and contributed to aerobic deterioration of these silages after opening (Moon et al., 1980). Muck and Kung, (1997) reported that inoculants, which were mainly homolactic improved aerobic stability in only 30% of 39 studies conducted between 1990 and 1995. The poor aerobic stability response to inoculation with homolactic LAB is attributable to the fact that the lactate they produce has a relatively low antifungal effect and is a gr owth substrate for certain spoilage yeasts (Pahlow et al., 2003). C onsequently, inoculants containing other types of bacteria that inhibit s poilage have been developed.
65 Heterolactic Bacterial Inoculants Lactobacillus buchneri Heterolactic bacteria ferment glucose into a mixture of ethanol, CO2, and acetic, butyric and lactic acids via a less efficient pathway than homolactic bacteria. However, the antifungal nature of the acet ic and butyric acids they produce has led to their use for improving aerobic stability of silages. Severa l studies have demonstrated that treatment of forages and feeds at ensiling with L. buchneri improves aerobic stability (Taylor and Kung, 2002; Nishino et al., 2004; Kung et al. 2007). Muck (2004) indicated that inoculation with Lactobacillus buchneri 40788 at the rate of 4 x 105 cfu/g is one of the most consistent methods for improving aerobic stability of corn silage. Doubling the application rate has not been more effectiv e (Huisden et al., 2009), but increasing the rate from 1 x 105 to 1 x 106 cfu/g of fresh material has (Ranjit and Kung, 2000). When applied at the rate of 106 cfu/g of fresh material L. buchneri increased aerobic stability of high moisture corn, corn silage, alfalfa silage, and small-grain silages relative to untreated controls (Muck, 2001; Taylor and Kung, 2002; Kleinschmit et al., 2005). Although the precise mechanism has not yet been determined, the beneficial impact of L. buchneri appears to be related to the production of acetic acid, which inhibits the growth of yeasts (Driehuis, et al., 1999a). The acetic acid can be further metabolized into propionic acid, a strong fungicidal agent by epiphytic L. diolivorans (Krooneman et al., 2002). Further inhibition of aerobic s poilage may be due to bacteriocins produced by L. buchneri. Yildirim (2001) and Yild irim et al. (2002) repor ted that buchnericin, a bacteriocin produced by L. buchneri had wide ranging bacteriocidal activity against spoilage bacteria such as Listeria monocytogenes and Bacillus cereus
66 Yeast and mold counts of L. buchneri inoculated silages are generally lower at feedout and do not increase as rapidly as in untreated controls exposed to air (Kung and Ranjit, 2001). Driehuis et al. (1999a) show ed that yeasts are affected in two ways by L. buchneri Firstly, the survival of yeasts during the anaerobic ensilage phase is reduced, and secondly, growth of yeasts during ex posure of silage to the air is inhibited. As a result, the temperatures of silages inoculated with L. buchneri tend to remain similar to ambient temperature for several days, even in warm weather (Taylor et al., 2000). Inoculation with L. buchneri is most beneficial under circumstances where problems with aerobic instability are expected. Corn silage, small-grain silages, and high-moisture corn are more susceptible to spoilage once exposed to air than legume silages and therefore the fo rmer often respond more favorably to inoculation with L buchneri (Muck, 1996). Few studies have compared the s poilage inhibiting effects of L. buchneri to those of other heterolactic bacteria. Danner et al. (2003) evaluated the effect of homofermentative ( L. rhamnosus, Pediococcus pentosaceous and L. plantarum ) or heterofermentative ( L. brevis or L. buchneri ) bacteria on the aerobic stability of silages. The silage inoculated with L. buchneri had the greatest aerobic stability and acetic acid concentration. Few studies have examined effects of inoc ulating silage with heterolactic bacteria on animal performance. Taylor et al. (2002) reported that treating barley ( Hordeum vulgare) silage with L. buchneri 40788 increased aerobic stability and reduced counts of yeasts and molds. However, when cows were fed the treated versus untreated silage, DMI, milk production, and milk composition were not affected by treat ment. Driehuis et
67 al. (1999b) reported that treating corn silage with L. buchneri improved aerobic stability but DMI and milk production were similar to t he untreated silage. Sim ilarly, treating high moisture shelled corn with L. buchneri improved aerobic stability but milk production was decreased when cows were fed the TMR containing treated versus untreated high moisture shelled corn (Kenda ll et al., 2002). Therefore, L. buchneri application has improved aerobic stability of vari ous forages but has not incr eased milk yield except in the following study. Treating alfalfa silage with L. buchneri 40788 improved aerobic stability and milk production was increased in cows fed treated versus untreated silages even though DMI and milk composition were unaffected (Kung et al., 2003a). Why milk production was improved in the latter study but not the others is uncl ear. More research evaluating effects of i noculating forages with L. buchneri on the performance of dairy cows are warranted. Propionibacteria Propionic acid bacteria can ferment gluc ose and lactate to acetate and propionic acid, which are two of the three main antif ungal acids in silages (Moon et al., 1983). Inoculation with Propio nibacteria has im proved aerobic stability in some studies (Higginbotham et al., 1996; Bo lsen et al., 1996; Dawson et al., 1998; Filya et al., 2006) but not others (Weinberg et al., 1995; Higgin botham et al., 1998; Pedroso et al., 2010). Dawson et al. (1998) evaluated the effect of Propionibacterium acidipropionici inoculation on fermentation and aerobic stability of corn s ilage. Inoculation improved aerobic stability, increased acetic and propion ic acid concentrations, reduced pH, and reduced numbers of yeasts and molds. In contrast, Higginbotham et al. (1998) examined the effect of microbial inoculants containing Propionibacteria either alone or with Pediococcus cerevisiae and P. cerevisiae plus L. plantarum The inoculants did
68 not affect the fermentation of corn silages but inoculated silages tended to heat more slowly and took a slightly longer time to reach their peak temperature than control silages. The authors concluded that the mi crobial inoculants eval uated did not prevent detrimental changes in quality wh en corn silage was exposed to air. The inconsistent effect of propionibacteria on aerobic stability is largely attributable to the slow growth of the organism at acidic pH (Weinberg et al., 1995; Higginbotham et al., 1998). Nevertheless, Propionibacteria is the main alternative antifungal bacterium to L. buchneri in commercially available inoculants in the US and studies comparing both types of antifungal inoculants are needed. Inoculants containing Homolactic and Heterolactic Bacteria Homofermentative LA B inoculants sometimes impair the aerobic stability of mature cereal silages by increasing the number of yeasts and fungi (Kennedy, 1990; Weinberg et al., 1993a). Adding a heterofementative LAB that produces fungicidal VFA has improved the stability of such silages (W einberg et al. 1999). The combination potentially reduces DM losses that often o ccur when heterofermentative bacteria alone are applied to forages. Driehuis et al. (2001) reported that adding Lactobacillus buchneri alone or in combination with P. pentosaceus and L. plantarum improved aerobic stability of grass silage, reduced yeast and mold counts, and led to lower DM loss compared to the untreated silage. In addi tion, Weinberg et al. (1999) reported that wheat and sorghum silages were more stable when treated with L. buchneri alone or with L. buchneri and L. plantarum whereas treatment with L. plantarum alone resulted in unstable silages. Filya (2003) reported that combination of L. buchneri and L plantarum reduced ammonia-N concentrations and fermentation losses in silages compared with L. buchneri alone. Huisden et al. (2009) reported that applying two
69 mixtures of homofermentative and heterofermentative bac teria resulted in similar reductions in lactate to acetate ratio, fewe r yeasts and more stable silages than Control silages. Adesogan et al. (2008) reviewed 8 studies in which combination inoculants (mainly L. buchneri and either L. plantarum or P. pediococcus ) were added to 16 forages and reported that DM recovery, lact ate, acetate, and aerobic stability were improved 63, 25, 69, and 94% of the time, whereas pH and yeast counts were reduced 25 and 88 % of the time, respectively. Therefore, most of the stud ies demonstrate that combination inoculants have im proved the aerobic stability of several types of forages but a few exceptions exist. Adesogan et al. (2004) showed that treatment with P. pentosaceus and L. buchneri improved the fermentation of bermudagrass ( Cynodon dactylon) silage but did not improve aerobic stab ility because control silages had high concentrations of fungicidal butyric acid Kleinschmit and Kung (2006b) examined the effect of adding Lactobacillus buchneri 40788 (4 x 105 cfu/g) and P. pentosaceus R1094 (1 x 105 cfu/g) to corn forage ensiled for differ ent durations ranging from 14 to 361 d. Inoculation increased acetate and 1,2 propanediol concentrations in silages ensiled for 56 to 361 d, decreased yeast counts in sila ges ensiled for 42, 56, 70 and 282 days, and increased aerobic stability in silages ensiled for 14, 56, and 361 d. The reason for the inconsistent improvement of aerobic stabili ty was not known. Most of studies on combination inoculants have been done in mini silos; consequently, animal responses to treatment of forages with such inoculants are unknown. Enzymes During the last two decades, enzymes have been evaluated as possible silage additives particularly those containing cellulolytic, hemicelluloly tic and amylolytic activities. Their primary functions are to break down cell-wall constituents and grain
70 starch in the crop to improve silage fe rmentation and animal utilization (Muck and Bolsen, 1991). Enzyme addition has increased lactic acid producti on (Rauramaa et al., 1987; Jaakkola et al., 1991; Kung et al., 1991), reduced silage pH (Rauramaa et al., 1987; Kung et al., 1991; Stokes 1992) reduced concentrations of acetic acid (Jaakkola et al., 1991; Stokes, 1992) and either in creased (Dean et al., 2005a), reduced (Jaakkola et al., 1991) or did not affe ct (Stokes, 1992) aerobic stabi lity. Enzyme treatment at ensiling has reduced NDF and ADF concentrations and increased NDF digestibility (Kung et al., 1991; Sheperd and Kung, 1996; D ean et al., 2005a) but did not increase milk production when enzyme-treated and untreated forages were fed (Shepherd and Kung, 1996; Dean et al., 2005b). Using enzymes as silage additives have had positive effects on silage and ruminal fermentation in many studies but has not consistently improved aerobic stability or animal performance. Future research shoul d examine if combinations of enzymes and spoilage-inhibiting bacteria can improve aerobic stability and animal performance. Nutrient Additives Crops with high moisture concentrations ar e prone to poor clostridial fermentati on and loss of nutrients from excessive produc tion of effluent. Effluent seepage into waterways can also cause eutrophication an d death of marine spec ies. Therefore, various sources of carbohydrates like cereal straw and sugar-beet pulp have been ensiled with forages as absorbents to incr ease the DM concentration and reduce the production of effluent (Kung et al., 2003b). Hamel eers et al. (1999) studied the effect of ensiling mixtures of molass ed sugar-beet pulp and forage corn harvested at four stages of maturity (154 to 235 g kg -1 DM). Addition of molassed-sugar beet pulp did not improve the fermentation but decreased effluent production except in the least mature
71 silage. Ferris and Mayne (1994) reported that ensiling ryegrass with sugar-beet pulp improved silage fermentation, reduced DM losses, and reduced effluent losses from the silo. When the sugar-beet pulp-treated ryegra ss silage was fed to dairy cows at three levels, DMI and milk fat and protein yields increased with increasing level of beet pulp inclusion in the diet. Moseley and Ramanathan (1989) ensiled a mixed crop of perennial ryegrass and white clover with not hing (Control), molassed-sugar beet pulp, rolled barley or formic acid and fed them to sheep. Dry matte r intake was increased by barley and sugar beet pulp treatm ents compared to the contro l and digestibility of DM and OM were greater when treated silages were fed. Rodrigues et al. (2005) studied the effect of ensiling elephantgrass with differ ent levels of citrus pulp. Silage DM concentration, in vitro DMD, and WSC increased linearly with increasin g level of citrus pulp but ammonia N and NDF concentrations dec reased. These studies reveal that nutrient additives can reduce effluent producti on and improve nutritive value of silages and enhance animal performance. However, addi tion of nutrient additives at ensiling is a difficult practice because of the in creased labor required and the need for uniform distribution of the adsorbent throughout the silage mass (Kung et al., 2003b). Summary Most of the published responses to additive treatment of ensiled forages indicate that they can improve silage fermentation and quality. However, their effects on animal performance have been variable. The use of chemicals as additives for forage conservation and improvement has been reduced due to their corrosiveness. Bacterial inoculants and enzymes are more appealing because they are benign and natural. Some of these have improved silage conservati on, nutritive value, and aerobic stability
72 but others have not. Effects of such bi ological additives on animal performance have been inconsistent. The objectives of this series of experiments were as follows: To determine the effect of dietary additi on of a fibrolytic enzyme preparation on the performance of dairy cows fed low or high concentrate diets. To compare simulated preingestive and rumi nal effects of a fibrolytic enzyme on various dietary components or diets and to determine which dietary components were most affected by enzyme action. To examine the effect of applying bacterial inoculants containing heterofermentative bacteri a alone or homofermentative and heterofermentative bacteria on the fermentation, quality, and aerobic stability of corn silage. To examine the effect of applying bacterial inoculants containing heterofermentative bacteri a alone or homofermentative and heterofermentative bacteria on the performance of lactating dairy cows.
73 CHAPTER 3 EFFECT OF FIBROLYTIC ENZYME APPL ICATION TO DIETS DIFFERING IN CONCENTRATE PROPORTION ON THE PERFORMANCE OF LACTATING DAIRY CATTLE Introduction Fibrolytic enzymes have been added to fo rages and ruminant diets to improve forage quality and animal performance. Enzym e application to forages immediately before in vitro incubation improved diges tion of DM and NDF (Feng et al., 1996) suggesting that applying fibrolytic enzymes at or just prior to feeding may enhance digestion of forages by cattl e. However, results of fi brolytic enzyme applic ation to ruminant diets have been equivocal. Some studies showed that enzyme supplementation increased milk production by dairy cattle (Rode et al., 1999; Kung et al., 2000b; Yang et al., 2000) but others did not (Beauchemin et al., 2000; Kung et al. 2002; Sutton et al. 2003). Adding fibrolytic en zymes has increased digestibility in some studies (Rode et al., 1999, Yang et al., 2000) but not others (Lewis et al. 1999). These discrepancies may be due to differences in enzyme activity, application rate and composition, stage of lactation of dairy cows, m ode and time of enzyme delivery, ruminal activity and stability of direct fed enzymes, enz yme-feed specif icity and the portion of the diet to which enzymes ar e applied (Beauchemin et al., 2004; Adesogan, 2005). A further problem may lie with use of enzymes that are incapable of sufficiently hydrolyzing forage cell walls to give repeat able results across a range of forages or diets. Most previous studies evaluated enzymes containing xyla nase and cellulase enzymes, which cannot hydrolyze ester linkages between hydroxycinnamic acids and sugars in plant cell walls. Ferulic acid esterases can hydrolyze such linkages (Williamson et al., 1998; Krueger et al., 2008a) thus releasing ferulic acid and opening
74 the remainder of the polysaccharides to furt her hydrolytic attack by other enzymes (Yu et al., 2005). Various studies have demonstr ated that esterase enzymes play an important role in ruminal cell wall degradation (Nsereko et al., 2000b; Dhiman et al., 2002; Krueger and Adesogan, 2008) particularly fo llowing pretreatment or co incubation with xylanases (Yu et al., 2002; Bartolome et al., 1995, 1997a, b) Most of such studies have been done in vitro. Consequently, littl e is known about effects of enzymes containing cellulase, xylanase and esterase acti vities on the performance of dairy cows. The objective of this study was to dete rmine the effect of dietary addition of a fibrolytic enzyme preparation on the perform ance of dairy cows fed low or high concentrate diets. The esterase xylanase enzyme preparati on tested had increased the NDF digestibility of alfalfa hay and corn silage by over 25% and it was more effective than using either enzyme alone (Eun and Beauchemin, 2006). We hypothesized that 1) enzyme application to the high concentrate diet would improve milk production, whereas application to the low conc entrate diet would improve the efficiency of milk production; 2) enzyme application to the low concentrate diet would result in as much milk production as that from the high concentrate diet that was not treated with the enzyme. Materials and Methods Cows, Treatments and Design Care of animals used in this study follow ed pr otocols approved by the University of Florida Institutional Animal Care and Use Committee. Sixty lactating Holstein cows in early lactation (22 + 3 DIM) were grouped by milk production and parity (multiparous and primiparous) and randomly assigned to 1 of 4 treatments arranged in a 2 x 2 factorial design. Cows were fed either a high or low concentrate TMR that was treated
75 with or without a prototype fibr olytic enzyme from Dyadic Inte rnational Inc., Jupiter, FL. The following treatments were investigated: 1) low concentrate untreated diet (LC; 67:33 roughage to concentrate ratio); 2) LC pl us enzyme (LCE); 3) high concentrate untreated diet (HC; 52:48 r oughage to concentrate ratio); and 4) HC plus enzyme (HCE). The dietary proportions of conc entrate were designed to approximate those typical of diets fed to dairy cows in the US (HC) and Western Europe (LC). Prior to the daily a.m. and p.m. feedings, t he enzyme solution was diluted in water (1:3 ratio v/v) and sprayed at a rate of 3.4 mg of enzyme/g DM on the TMR during mixing for 5 min in a 250-kg capacity Calan data ranger (American Calan Inc., Northw ood, NH). Separate data rangers were used to mix the enzym e-treated and untreated TMR. The roughage portion of the diets contained approximately 20% alfalfa hay, 72% corn silage, and 8% cottonseed hulls (DM basis). The exper imental diets were formulated to be isonitrogenous (Table 3-1) and to meet NRC ( 2001) guidelines for a dairy cow in early lactation producing 40 kg of milk. The experiment was conduc ted at the University of Florida Dairy Unit and it lasted for 63 d per co w. Cows were adapted to diets for the first 14 d and the last 49 d of the tr ial were used for sample collection. Cows were housed in a free-stall, open-sided barn fitted with continuously oper ated fans and misters for cooling and with Calan gates (American Calan Inc., Northwood, NH) for individual feed intake. Free-stalls were bedded with sand and maintained daily. Sufficient free-stalls were available to provide at least 1 free-sta ll per cow. Water and diets were available in ad libitum amounts. Enzyme Activity Xylanas e activity (EC 184.108.40.206) measured us ing the assay of Bailey et al. (1992) was 3633 U/ml with oat spelt xylan as the substrate (Sigma Chemical Company, St.
76 Louis, MO, USA). Endoglucanase (EC 3.2. 1.4) and exoglucanase (EC 220.127.116.11) activities measured with the Wood and Bhat (1988) assays were 880 U/ml and 70 U/ml using 1% (wt/vol) carboxymethylcellulose or microcrystalline cellulose (Avicel) as substrates, respectively (Sigma; Chemical Company, St. Louis, MO, USA). One unit of activity of the respective enzymes was def ined as micromoles of xylose or glucose released per min per g Assay conditions were 39oC and pH 6.0 to reflect ruminal conditions. Aryl and carboxyl esterase acti vities measured using the methods described by Gonzalez et al. (2006) were 0.38 mol/ min mg-1 and 0.28 mol/min mg-1 using p Nitrophenyl and naphthyl esters as substrates. Sampling and Analysis Cows were fed individually ( 0700 and 1200 h) and milked (1100 and 2300 h) twice daily. Milk samples from a.m. and p.m. milkings were collected twice every week and analyzed by Southeast Dairy labs (Belleview FL) for fat, protein and SCC using a Bentley 2000 Near Infrared Reflectance Sp ectrophotometer (Bentley Instruments Inc., Chaska, MN). Somatic cell scores were generated as described by Norman et al. (2000) for statistical analysis of SCC. Body we ight was measured at the beginning and end of the trial. Body condition score was measured on a 1 to 5 scale (Wildman et al., 1982). Weight of TMR and orts were recorded da ily for each cow. Duplicate samples of corn silage, alfalfa hay, and concentrates were collected weekly and composited monthly. Subsamples from eac h month were dried at 60C fo r 48 h in a forced air oven, ground to pass the 1-mm screen of a Wiley m ill (A. H. Thomas, Philadelphia, PA), and analyzed for DM (105oC for 16 h) and ash (512oC for 8 h). Concentrations of NDF and ADF were measured using the method of Van Soest et al. (1991) in an Ankom 200
77 Fiber Analyzer (Ankom Technologi es, Macedon, NY). Heat-stable -amylase and sulfite were used in the NDF assay. Nitrogen was determined by rapid combustion using a Macro elemental N analyzer (Vario MAX CN, model ID 25.00-5003; Elementar, Hanau, Germany) and CP was calculated as N x 6.25. Blood samples were collected weekly by coccygeal venipuncture into va cutainer tubes containing sodium heparin anticoagulant (Fisher Sc ientific, Pittsburgh, PA). The bl ood was centrifuged at 2,500 g for 20 min at 4C and the plasma was fr ozen (-20C). A Technicon Autoanalyzer (Technicon Instruments Corp ., Chauncey, NY) was used to measure plasma glucose (Bran and Luebbe Industrial Method 339; Gochman and Schmitz, 1972) and BUN (Bran and Luebbe Industrial Method 339; Marsh et al., 1965). In vivo apparent digestibility was calcul ated using chromic oxide as a marker (Schneider and Flatt, 1975). Chromic oxide pow der (Fisher Scientific, Fairlawn, NJ) was weighed (10 g/dose) into gelatin capsules (Jorgensen Lab. Loveland, CO) and dosed twice daily (0700 and 1900h) with a bal ling gun for 10 consecutive d between d 45 and 60. Fecal samples were collected at 0630 and 1830 h during the last 5 d of dosing. Feces were dried to a constant we ight at 55C in a forced-air oven, ground to pass through a 1-mm screen in a Wiley mill and a composite sample was made from all 10 fecal samples per cow. Chromium conc entration in the feces was determined using a Perkin Elmer 5000 atomic absorption spectr ometer (Perkin Elmer, Wellesley, MA), according to the procedure described by Willia ms et al. (1962). Appar ent digestibility of CP, ADF and NDF were calculated. In Situ Ruminal Degradability Four ruminally-cannulated, nonlactating Holstein cows were used to determine dietary treatment effects on ruminal ferment ation and in situ ruminal DM degradation.
78 This aspect of the experiment had a 4 x 4 Latin square design with four, 18-d periods. The first 14 d of each period were used to adapt cows to a new di et. On d 15, ruminal fluid samples were taken at 0, 2, 4, 6, 8 and 10 h after cows were fed in the morning. Approximately 100 ml of ruminal fluid was immediately filtered through 2 layers of cheesecloth, and pH was measured with an electrode (Accumet pH meter, model HP71, Fisher Scientific, Pittsburgh, PA). Subs equently, the ruminal fluid was acidified with 2 ml of 50% H2SO4 per 100 ml of ruminal fluid and centrifuged at 11,500 g for 20 min. The supernatant was frozen (-20oC) and subsequently analyzed for concentrations of lactate and VFA using the method of Muck and Dickerson (1988) and a High Performance Liquid Chromat ograph system (Hitachi, FL 7485, Tokyo, Japan) coupled to a UV Detector (Spectroflo w 757, ABI Analytical Kratos Division, Ramsey, NJ) set at 210 nm. The column was a Bio-Rad Am inex 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 an adaptation of the Noel and Hambleton (1976) procedure that involved co lorimetric N quantification. The ruminal degradation kinetics of t he experimental diets was measured in situ on d 16 to18. The TMR samples were dried (60C for 48 h), ground to pass a 4-mm screen with a Wiley mill and weighed (5 g as is) into preweighed polyester bags (10 20 cm, pore size 50 m, Bar Diamond Inc., Parma, ID). Samples were incubated for 0, 4, 8, 16, 24, and 48 h in quadruplicate in each of 2 ruminally fistulated cows fed that same diet in each period. All bags were in serted in the rumen simultaneously. After each incubation period, bags were removed and rinsed with cool water and frozen (-
79 20oC). Subsequently, all bags were thawed, washed using a rinse cycle without soap in a Kenmore Series 70 washing machine, dr ied for 48 h at 60C and weighed. The exponential model of McDonald (1981) was fitted to the DM degradation data with the NONLIN procedure of SAS (Version 9.2 SAS In stitute Inc., Cary, NC) to generate DM degradation parameters. The model is of the following form: P = A + B (1 e -c (t-lag)) where P is the DM degradation (%) at time t, A is the Y axis intercept representing the wash fraction (%), B is the potentially deg radable fraction (%), and c is the rate of degradation of the B fr action (%/h). Statistical Analysis A completely randomiz ed design with a 2 (control vs. enzyme) x 2 (LC vs. HC) factorial arrangement of treatments was used to anal yze the data. The MIXED procedure of SAS (Version 9.2 SAS Institute Inc., Cary, NC) and a model containing treatment, week (repeated measur e), parity, all interactions of these terms, and cow nested in treatment x pairty as the random effect was used to analyze the data from measurements that were repeated weekly. Milk production during the first 21 d of lactation was used as a covariate for analyzi ng milk production data. A similar model excluding the week effect and its interactions was used to analyze digestibility coefficients. The model for analyzing rumi nal fermentation data included treatment, period, time (repeated measur e), treatment x time and co w (random effect). Least square means are reported and results ar e presented on a DM basis. Contrast statements were used to determine the effects of enzyme application (control vs. enzyme), concentrate level (LC vs. HC), the interaction (enzyme treatment vs. concentrate level), and to compare the LC E and HC diets. The slice command of SAS
80 was used to detect differences among means at specific time points and the PDIFF statement of SAS also was used to compar e enzyme effects within each concentrate level. Treatment significance was declared at P < 0.05 and tendencies were declared at P > 0.05 < 0.10. Results and Discussion Enzyme application did not affect ( P = 0.14) DMI but tended to reduce intake of CP (P = 0.06; 4.5 vs. 4.2 kg/d), NDF ( P = 0.07; 7.9 vs. 7.4 kg/d), and ADF ( P = 0.07; 5.3 vs. 4.9 kg/d). Enzym e application increased ( P < 0.02 ) digestibility of DM (69.8 vs. 72.6%), CP (69.2 vs. 73.3%), ADF (50.4 vs. 54.8%), and NDF (53. 7 vs. 55.4%) (Table 3-2). Therefore, adding the enzyme increased nutrient digestion and release. Krueger et al. (2008b) reported that fibrolytic enzyme application to bermudagrass hay increased DMI and DM and NDF digestibility by beef steers. Yang et al. (1999) also reported that supplementation with a xylanas e cellulase enzyme increas ed NDF digestibility of an alfalfa barley silage-based TMR by dairy cows. They attributed the response to increased nutrient release and solubility of DM and NDF, which likely increased glycocalyx production, and thereby enhanced adhesion of fibrolytic bacteria to substrates. The enzyme-mediated digestibi lity increases in this study may have occurred via increased microbial colonization of feed partic les or by direct cell wall hydrolysis (Cheng et al., 1995; Yang et al., 1999). Increasing the concentrate amount increased DMI (21.5 vs. 24.8 kg/d, P = 0.003; Figure 3-1) and CP intake (4.1 vs. 4.6 kg/d, P = 0.001) and tended to increase ADF intake (P = 0.09; 4.9 vs. 5.2 kg/d). Likewis e, increasing the concentrate amount increased digestibility of DM (69.9 vs. 72.6%; P = 0.02) and CP (70.0 vs. 72.6%, P = 0.01) and tended ( P = 0.06) to increase ADF digestibility (50.1 vs. 54.4%). Cows fed
81 HC instead of LCE had greater ( P < 0.01) DMI (25.7 vs. 20.8 kg/d), CP intake (4.8 vs. 3.9 kg/d), and ADF intake (5.4 vs. 4.7 kg/d), and tended ( P = 0.06) to have greater NDF intake (8.0 vs. 7.3) Increasing the amount of concent rate in diets has caused linear increases in DMI in other studies (Llamas-Lamas and Combs, 1991; Weiss and Shockey, 1991). Eun and Beauchemin (2005) also reported that cows receiving high concentrate diets had greater DMI and greater DM digestibility than those receiving low concentrate diets. Substitution of concent rates for forages generally increases intake and digestibility because concentrates cause less ruminal fill, have a lower lignified polysaccharide concentration, a faster passage rate, and require less rumination than forages. Enzyme application effects on plasma gl ucose concentration differed with the amount of concentrates fed (Enzym e x concentrate interaction, P = 0.006) but the differences were small. (The PUN concentration was not affected by enzyme application, but feeding more concentrate s increased plasma urea N (13.5 vs. 15.0 mg/dl, P = 0.001; Table 3-3), reflecti ng the increased CP digestibility caused by enzyme treatment. Enzyme application did not statistically increase milk yield though numerical increases occurred at both c oncentrate amounts (Table 3-4, Figure 3-2). In some studies, dietary addition of fibrolytic enzym es increased milk production (Lewis et al., 1999; Rode et al., 1999; Yang et al., 2000) but no milk response was reported in others (Beauchemin et al., 2000; Elwakeel et al., 2007). Enzyme application effects on milk fat concentration seemed to differ with the amount of concentrates (Enzyme x concentrate interaction, P = 0.04) but milk fat concent rations did not differ ( P > 0.05) at the same
82 concentrate feeding amount. Enzyme application increased the efficiency of milk production ( P = 0.04 ; 1.44 vs. 1.60 kg milk/kg DMI) because of increased nutrient supply for milk production. That enzyme application increased feed efficiency across concentrate amounts partly confirms our firs t hypothesis that enzyme application would increase and the efficiency of milk production by cows. Increasing the dietary concentrate amount increased milk yield ( P = 0.02; 32.2 vs. 34.7 kg/d) and milk protein yield ( P = 0.02; 0.89 vs. 0.99 kg/d). These responses are attributable to the increase in concentration of dietary NFC, particul arly starch as the dietary concentrate level increased. Starch digestion in the rumen increases propionic acid production, which is a major gluconeogenic precursor in ruminants (Chen et al., 1994). Starch digestion also increases uptak e of amino acids and other nutrients by the mammary gland, improving milk and milk pr otein yields (Theurer et al., 1999). Despite the lower DMI of cows fed the LC E diet relative to the HC diet, milk production from both diets did not differ ( P = 0.43). Consequently t he efficiency of milk production was greater ( P = 0.01; 1.69 vs. 1.42 kg milk/kg DMI) in cows fed the LCE diet than those fed the HC diet. These re sults confirm our second hypothesis by indicating that enzyme application to the low c oncentrate diet made it as effective as the untreated high concentrate diet at stimulating milk production. Similarly, Schingoethe et al. (1999) reported that cows fed low-concent rate diets (45% of TMR) treated with a fibrolytic enzyme increased milk production to the same extent as that achieved when cows were fed a high concentrate (55%) diet without the enzyme. Such results imply that by adding such enzymes, higher forage diets can be fed without jeopardizing milk production, and this could lower the cost of the diet and reduce the risk of acidosis.
83 One concern about fibrolytic enzyme applic ation to diets containing considerable amounts of concentrates is that fermentation of starch in such diets may indirectly depress ruminal pH and predispose cows to ruminal acidosis (Eun and Beauchemin, 2005). Hristov et al. (1996a) also noted that applying fibrolytic enzymes to feed may decrease both chewing time and saliva output and thereby increase the risk of acidosis. Enzyme application did not affect ( P = 0.92; Table 3-5) the ruminal pH of cows in this study, but caused lower values 4 h after f eeding (Figure 3-3). T herefore, cows fed enzyme supplemented, high concentrate diets should be monitored closely for signs of subclinical ruminal acidosis. Enzyme application effects on ruminal amm onia concentration differed with time ( P = 0.02; Figure 3-4). Enzym e application increased total VFA concentration ( P = 0.03; 114.5 vs. 125.7 mM ; Figure 3-5). In contrast, seve ral studies reported that enzyme application had no effect on total VFA concen tration (Kung et al., 2002; Sutton et al., 2003; Eun and Beauchemin, 2005). Enzyme application also tended ( P = 0.10) to reduce acetate molar proportion ( 58.9 vs. 56.0; Figure 3-6) but did not affect propionate molar proportion or ammonia-N ( P > 0.05; Figure 3-7 and 34; respectively). Consequently, enzyme application reduced the acetate to propionate ratio ( P = 0.04; 3.09 vs. 2.87; Figure 3-8), im plying improved efficiency of energy utilization in the rumen. Increasing the amount of concentrate supplementation produced the expected pH decrease ( P < 0.001; 6.31 vs. 6.06), as did feeding the HC diet in stead of the LCE diet ( P = 0.006; 6.36 vs. 6.10). None of the aver age pH values was low enough to indicate acute ruminal acidosis, which is characte rized by ruminal pH below 5 (Nagaraja and
84 Town, 1990). However, ruminal pH of co ws fed the HC diet reached the subclinical acidosis threshold (pH <5.8; Ghorbani et al., 2002) 10 h after feedi ng. Feeding more concentrates reduced ( P = 0.004) molar acetate propor tion (59.8 vs. 55.1), and increased ( P < 0.05) concentration of total VFA (114.3 vs. 125.9 mM ) and lactate (5.5 vs. 7.0 mM ) and molar proportion of propionate (18.8 vs. 19.9). C onsequently, acetate to propionate ratio was reduced ( P = 0.003) by feeding more concentrates, implying increased ruminal energetic efficiency. E un and Beauchemin (2005) also reported that feeding more concentrates had no effect on molar acetate proportion, increased total VFA concentration and molar propionate pro portion; and decreased acetate propionate ratio. Enzyme application did not affect the rumi nal degradation of the diets (Table 3-6). Similarly, Krueger et al. (2008b) reported no effect of enzyme application on the rate or extent of degradation of be rmudagrass hay. However, f eeding more concentrates increased the immediately soluble dietary fraction ( P = 0.03; 43.3 vs. 33.6 %), and tended to decrease the potent ially degradable fraction ( P = 0.06; 45.0 vs. 53.9 %) likely reflecting differences in particle size distribution and NFC and fiber concentration between low and high concentrate diets. Conclusions This study shows that application of t he cellulase xylanase esterase enzyme preparation did not affect DMI or milk produc tion but increased nutrient digestibility, total VFA concentration, and the efficiency of milk production. Furthermore, enzyme application to the low concentrate diet resulted in as much milk production and DM digestibility as from cows fed the untreat ed high concentrate diet. These beneficial
85 effects of enzyme application we re primarily attributable to improved nutrient digestion and improved ruminal energy utilization.
86 Table 3-1. Ingredient and chemical compos ition of the untreat ed experimental diets Low concentrate High concentrate Ingredient composition, % DM Corn silage 49.20 37.00 Alfalfa hay 13.50 10.00 Cottonseed hulls 4.63 5.00 Corn meal 7.38 17.89 Citrus pulp 2.00 5.01 Whole cottonseed 1.81 4.84 SoyPlus1 7.90 5.93 Soybean meal 2.49 6.01 Cottonseed meal 7.80 5.10 Mineral mix2 3.26 3.25 Roughage : concentrate ratio 67:33 52:48 Chemical composition DM, % 64.9 72.2 Crude protein, % DM 18.6 18.5 Neutral detergent fiber, % DM 34.3 31.0 Acid detergent fiber, % DM 22.4 20.9 Hemicellulose 3, % of DM 11.9 10.1 Non-fiber carbohydrates 4, % of DM 36.5 39.7 1 West Central Soy, Ralston, IA. 2 Mineral mix contained 26.4% CP, 10.2% Ca, 8.6% Na, 5.1% K, 3.1% Mg, 1.5% S, 0.9% P, 2231 mg/kg of Mn, 1698 mg/kg of Zn, 512 mg/kg of Cu, 339 mg/kg of Fe, 31 mg/kg of Co, 26 mg/kg of I, 7.9 mg/kg of Se, 147,756 IU of vitamin A/kg, 787 IU of vitamin E/kg (DM basis). 3 Calculated as NDF ADF; 4 Calculated as NFC = 100 (CP+ash+fat+NDF); NRC (2001) values used for fat concentrations of ingredients.
87 Table 3-2. Effect of adding a fibrolytic enzyme1 (Enz) to diets containing low (33%) or high (48%) amounts of concentrate (conc) on intake and digestibility by dairy cows. Low Conc High Conc SEM Contrast P values No Enz Enz No Enz Enz Enz Conc LCE vs. HC Enz x Conc Item (LC) (LCE) (HC) (HCE) Intakes, kg/d DM 22.1 20.8 25.7 23.8 1.0 0.14 0.003 0.001 0.79 CP 4.2 3.9 4.8 4.4 0.16 0.06 0.001 0.003 0.80 NDF 7.8 7.3 8.0 7.4 0.29 0.07 0.43 0.06 0.87 ADF 5.1 4.7 5.4 5.0 0.19 0.07 0.09 0.01 0.84 Digestibility, % DM 68.5 71.2 71.1 74.0 1.1 0.02 0.02 0.91 0.90 CP 68.4a 71.5b 70.0x 75.1y 1.0 0.002 0.01 0.29 0.31 NDF 52.1 55.2 53.3 57.5 1.7 0.03 0.30 0.43 0.75 ADF 48.6 52.9 52.1 56.6 1.8 0.02 0.06 0.75 0.97 a. b, x, y At the same level of concentrate supplementation, means in the same row with different superscripts differed (P < 0.05). 1 Produced by Dyadic International Inc., Jupiter, FL
88 Table 3-3. Effect of adding a fibrolytic enzyme1 (Enz) to diets containing low (33%) or high (48%) amounts of concentrate (conc) on body weight, average daily gain (ADG), body condition score (BCS), and plasma metabolites by dairy cows. Low Conc High Conc SEM Contrast P values No Enz Enz No Enz Enz Enz Conc LCE vs. HC Enz x Conc Item (LC) (LCE) (HC) (HCE) Growth performance Initial wt, kg 574 568 579 558 16.3 0.41 0.89 0.61 0.63 Final wt, kg 593 593 609 584 14.7 0.39 0.82 0.43 0.39 ADG, kg/d 0.32 0.42 0.57 0.38 0.14 0.89 0.44 0.63 0.51 Initial BCS 2.55 2.43 2.46 2.48 0.11 0.29 0.71 0.61 0.19 Final BCS 2.42 2.39 2.40 2.45 0.03 0.81 0.60 0.84 0.20 Plasma metabolites Glucose, mg/dl 69.3a 71.3b 71.9 70.6 0.6 0.53 0.10 0.46 0.006 Urea N, mg/dl 13.4 13.6 14.5 15.4 0.4 0.13 0.001 0.06 0.31 1 Produced by Dyadic International Inc., Jupiter, FL
89 Table 3-4. Effect of adding a fibrolytic enzyme1 (Enz) to diets containing low (33%) or high (48%) amounts of concentrate (conc) on milk production, composition, and e fficiency of feed utilization by dairy cows. Low Conc High Conc SEM Contrast P values No Enz Enz No Enz Enz Enz Conc LCE vs. HC Enz x Conc Item (LC) (LCE) (HC) (HCE) Milk yield, kg/d 31.9 32.5 33.6 35.8 1.1 0.21 0.02 0.43 0.47 FCM 3.5%, kg/d 32.5 33.9 35.9 36.2 1.2 0.49 0.03 0.26 0.64 FCM 4%, kg/d 29.9 31.3 33.1 33.4 1.1 0.49 0.03 0.26 0.64 Milk protein, % 2.79 2.85 2.91 2.89 0.05 0.71 0.15 0.43 0.43 Milk fat, % 3.60 3.80 3.90 3.56 0.13 0.62 0.80 0.59 0.04 Milk protein, kg/d 0.88 0.90 0.97 1.00 0.04 0.59 0.02 0.19 0.96 Milk fat, kg/d 1.14 1.20 1.32 1.25 0.08 0.93 0.13 0.24 0.41 SCC x 1000/ml 412 158 417 465 144 0.88 0.68 0.85 0.47 Feed efficiency ( k g milk/k g DMI ) 1.46a 1.69b 1.42 1.51 0.08 0.04 0.16 0.01 0.35 a, b At the same level of concentrate supplementation, means in t he same row with different superscripts differed (P < 0.05). 1 Produced by Dyadic International Inc., Jupiter, FL
90 Table 3-5. Effect of adding a fibrolytic enzyme1 (Enz) to diets containing low (33%) or high (48%) amounts of concentrate (conc) on ruminal fermentation characteristics by dairy cows. Low Conc High Conc SEM Contrast P values No Enz Enz No Enz Enz Enz Conc LCE vs. HC Enz x Conc Item (LC) (LCE) (HC) (HCE) Ruminal pH 6.26 6.36 6.10 6.01 0.08 0.92 <0.001 0.006 0.14 Ammonia-N, m g /dl 12.4 13.0 12.7 13.0 0.78 0.61 0.87 0.81 0.87 Lactate, mM 6.57 4.41 5.14 8.94 0.55 0.15 0.01 0.37 <0.001 Total VFA, mM 110.4 118.2 118.6 133.1 4.8 0.03 0.01 0.96 0.49 Individual, molar p ro p ortion Acetate, (A) 60.1 59.5 57.7 52.4 1.73 0.10 0.004 0.42 0.18 Propionate, (P) 18.6 18.9 19.6 20.2 0.34 0.20 0.005 0.16 0.79 Iso-butyrate 2.70 2.64 2.90 2.49 0.22 0.30 0.92 0.39 0.43 Butyrate 11.7 12.0 12.3 12.7 1.40 0.81 0.67 0.90 0.97 A:P ratio 3.23 3.15 2.94 2.59 0.07 0.04 0.003 0.29 0.37 1 Produced by Dyadic International Inc., Jupiter, FL
91 Table 3-6. Effect of adding a fibrolytic enzyme1 (Enz) to diets containing low (33%) or high (48%) amounts of concentrate (conc) on the in situ ruminal degradabi lity of dry matter by dairy cows. Low Conc High Conc SEM Contrast P values No Enz Enz No Enz Enz Enz Conc LCE vs. HC Enz x Conc Item (LC) (LCE) (HC) (HCE) A, % 31.6 35.5 44.9 41.7 3.38 0.929 0.03 0.11 0.34 B, % 56.3 51.5 43.3 46.6 3.82 0.85 0.06 0.20 0.33 A+B, % 87.9 87.0 88.2 88.3 1.17 0.77 0.54 0.55 0.66 C, per h 0.05 0.06 0.05 0.05 0.01 0.60 0.31 0.30 0.31 L, h 0.0 0.39 1.16 0.63 0.42 0.88 0.15 0.27 0.33 1 Produced by Dyadic International Inc., Jupiter, FL; A = washout fraction; B = potentially degradable fraction A+B = total degradable fraction; C = fractional degradation rate of the B fraction; L = lag phase
92 Figure 3-1. Effect of adding a fibrolytic enzym e application to dairy cow diets with low or high levels of concentrates on dry matte r intake. LC = Low concentrate, LCE = Low concentrate + enzyme, HC = High concentrate, HCE = High concentrate + enzyme. and x indicate differences (* = P < 0.05; x = P < 0.10) between treatments at t hat week. Treatment x week, P = 0.41, SEM = 1.23. *x x
93 Figure 3-2. Effect of estera se-xylanase enzyme application to dairy cow diets with low or high levels of concentrates on milk yield. LC = Low concentrate, LCE = Low concentrate + enzyme, HC = High concentrate, HCE = High concentrate + enzyme. and x indicate differences (* = P < 0.05; x = P < 0.10) between treatments at that week. Treatment x week, P = 0.57, SEM = 1.18. *xx x
94 Figure 3-3. Effect of adding a fibrolytic enzyme to dairy cow diets with low or high levels of concentrates on ruminal pH. LC = Low concentrate, LCE = Low concentrate + enzyme, HC = High concentrate, HCE = High concentrate + enzyme. x indicates a difference ( P < 0.10) between treatme nts at that time. Treatment x time, P = 1.00, SEM = 0.16. x
95 Figure 3-4. Effect of adding a fibrolytic enzyme to dairy cow diets with low or high levels of concentrates on ruminal ammonia ni trogen concentration. LC = Low concentrate, LCE = Low concentrate + enzyme, HC = High concentrate, HCE = High concentrate + enzyme. indicates a difference ( P < 0.05) between treatments at that ti me. Treatment x time, P = 0.002, SEM = 1.79.
96 Figure 3-5. Effect of adding a fibrolytic enzyme to dairy cow diets with low or high levels of concentrates on ruminal total VFA c oncentration. LC = Low concentrate, LCE = Low concentrate + enzyme, HC = High concentrate, HCE = High concentrate + enzyme. indicates a difference ( P < 0.05) between treatments at that ti me. Treatment x time, P = 0.18, SEM = 6.24.
97 Figure 3-6. Effect of adding a fibrolytic enzyme to dairy cow diets with low or high levels of concentrates on ruminal acetate proportion.LC = Low concentrate, LCE = Low concentrate + enzyme, HC = High concentrate, HCE = High concentrate + enzyme. indicates a difference (P < 0.05) between tr eatments at that time. Treatment x time, P = 0.52, SEM = 4.19.
98 Figure 3-7. Effect of adding a fibrolytic enzyme to dairy cow diets with low or high levels of concentrates on ruminal propionate proportion. LC = Low concentrate, LCE = Low concentrate + enzyme, HC = High concentrate, HCE = High concentrate + enzyme. x indicates a difference ( P < 0.10) between treatments at that ti me. Treatment x time, P = 0.30, SEM = 0.81.
99 Figure 3-8. Effect of adding a fibrolytic enzyme to dairy cow diets with low or high levels of concentrates on ruminal acetate to propionate ratio. LC = Low concentrate, LCE = Low concentrate + enzyme, HC = High concentrate, HCE = High concentrate + enzyme. indicates a difference ( P < 0.05) between treatments at that ti me. Treatment x time, P = 0.63, SEM = 0.17.
100 CHAPTER 4 EFFECTS OF FIBROLYTIC ENZYME APPL ICATION ON THE DIGESTIBILITY OF CORN SIL AGE, ALFALFA HAY, TWO CONCENTRATES, AND COMPLETE DIETS UNDER SIMULATED RUMINAL AND PRERUMINAL CONDITIONS Introduction Exogenous fibrolytic enzyme products usually are applied to the diet before feeding because a preingestive enzyme-feed inte raction is necessary for any significant beneficial effects on ruminal digestion to be rea lized (Lewis et al., 1996; McAllister et al., 1999). The close enzyme feed associati on may enable partial hydrolysis of NDF and ADF (Krause et al., 1998) that causes a re lease of reducing sugars (Hristov et al., 1996a; Krueger and Adesogan, 2008). The hydrol ysis also may modify plant cell wall structure (Feng et al., 1996) and thereby increase fiber digestion. Another reason for applying enzymes to feed prior to ingestion is to enhance binding of the enzyme to the feed, thereby increasing the re sistance of the enzymes to ru minal proteolysis (Fontes et al., 1995). Despite the evidence for preingestive enzyme action, most attention has focused on ruminal enzyme effects. For instance, B eauchemin et al. (2003) stated that most of the improvements in forage quality resu lting from exogenous fibrolytic enzyme application are attributable to ru minal effects. Yang et al. ( 1999) reported t hat most of the effect of exogenous enzymes in diets of la ctating dairy cows occurred in the rumen. Yet infusion of exogenous enzymes into th e rumen is not considered an effective method of enzyme applicat ion (Lewis et al., 1996; Treacher et al., 1997; Hristov et al., 2000; Sutton et al., 2003) because the proper binding of enzymes to feeds that optimizes efficacy occurs in the preinges tive phase. Enzyme application to forage several months prior to feeding increased in vivo digestibility of DM and NDF in beef
101 steers, whereas enzyme application at f eeding had no effect (Krueger et al., 2008b), suggesting that an important preingestive effect occurred in the former treatment. Studies directly comparing preingestive versus ruminal enzyme action are needed to quantify the relative importance of these sites of enzyme action and to inform guidelines about the timing of enzyme application to feeds. Eun and Beauchemin (2007) repo rted that application of a certain mixture of developmental fibrolytic enzymes (FF and FT, Dyadic International, Jupiter, FL) improved the in vitro NDF digestibility of both forages by over 20%. Arriola et al. (2007; Chapter 3) reported th at adding the same enzyme mixture to diets of lactating dairy cows did not affect DMI but tended to incr ease milk production and therefore increased the efficiency of milk production. These e ffects were attributed to improved nutrient digestion and ruminal energy utilization. Ho wever, the dietary co mponent most affected by the enzyme was not known. Studies aimed at determining the ideal dietary component to which enzymes should be added have not produced consistent results. Milk production by dairy cows has been improved by adding enzymes to the forage (Lewis et al., 1999; Kung et al., 2000b) or concentrate (Rode et al., 1999; Yang et al., 2000) portions of diets or to the TMR (Beauchem in et al., 1999) in some studies but not others (Phipps et al., 2000; Sutton et al., 2003; Elwakeel et al., 20 07). Most of such studies have not involved concurrent enzyme addition to different dietary fractions therefore little is known about the target dietary fraction to which enzymes should be added. The objectives of this study were to compare simulated preingestive and ruminal effects of a fibrolytic enzyme on various the forages, concentrates, and TMR used by
102 Arriola et al. (2007) and to determine whic h dietary components were most affected by enzyme action. The hypotheses were that t he effect of the enzyme in the simulated rumen would exceed the simulated preingestive effect and the enzyme would exert the greatest effect on the for age component of the diet. Materials and Methods Dietary Substrates Samples of the alfalfa hay (AH), corn silage (CS), low corn (20%; LC) and high corn (34%; HC) concentrates, and low (33%) and high -(48%) concentrate TMR (TMRL and TMRH) from the study of Arriola et al. (2007) were used as substrates in this study. The substrates were dried in a forced-air oven at 60C for 48 h, ground to pass through a 1-mm screen usin g a Wile y mill (Arthur H. Thomas Company, Philadelphia, PA) and stored in air-tight plastic bags. Enzyme Activity The enzyme mixture was the same developmental en zyme from Dyadic International, Inc. (Jupiter, FL) used by A rriola et al. (2007; Chapter 3). Xylanase activity (EC 18.104.22.168) measured us ing the assay of Bailey et al. (1992) was 3633 U/ml with oat spelt xylan as the substrate (Sigma Chemical Company, St. Louis, MO, USA). Endoglucanase (EC 22.214.171.124) and exoglucanase (EC 126.96.36.199) activities measured with the Wood and Bhat (1988) assays were 880 U/ml and 70 U/ml using 1 % (wt/vol) of carboxymethylcellulose or 1% (wt/vol) solu tion of microcrystalline cellulose (Avicel, Sigmacell 50; Sigma; Chem ical Company, St. Louis, MO, USA) as substrates, respectively. One unit of activity of the re spective enzymes is defined as micromoles of xylose or glucose released per min. per g. Assay conditions were 39C and pH 6.0. Aryl and carboxyl esterase activities measured using the Gonzalez et al. (2006) assay were
103 0.38 mol min-1 mg-1 and 0.28 mol min-1 mg-1 using -naphthyl and p-nitrophenyl acetate esters as substrates, respectively. In vitro Fermentation and Degradability Four experiments were conducted to te st the experimental hypotheses. In Experiment 1, the objective was to determine effects of enzyme addition on c hemical composition. Exactly 6.8 mg of the fibrol ytic enzyme was diluted in 4 ml of citrate phosphate buffer (pH 6.0) and applied to 2 g of each substrate in beakers in triplicate at 23C. In addition, beakers containing each s ubstrate and the buffer alone were also prepared and regarded as respective controls. After 8 h, beakers we re dried overnight at 60C in a forced draft oven. Conc entrations of NDF and ADF were analyzed sequentially using the met hod of Van Soest et al. (1991) in an ANKOM Fiber Analyzer (ANKOM Technologies, Macedon, NY, USA). Heat stable -amylase and sodium sulfite were used in the NDF analysis. Hemicellul ose was calculated by difference from NDF and ADF. Water-soluble carbohydrate concentration was determined after water extraction, acid hydrolysis, and a colorimetr ic reaction with potassium ferricyanide (Hall et al., 1999). In Experiment 2, the obj ective was to determine effects of enzyme addition on substrate disappearance in a buf fer. Exactly 3.4 mg of the fibrolytic enzyme was diluted in 2 ml of citrate phosphate buffer (pH 6.0) and applied to 1 g of each substrate within 250 ml culture bottles in triplic ate. After 24 h of incubation at 23C in the buffer alone or the buffer enzyme solution, contents of culture bottles were filtered through a Whatman N 541 filter paper (#09851D, Fisher Sc ientific, Pittsburgh, PA, USA), and residues were dried at 60C overnight to determine DM disappearance in the buffer in each of 2 runs.
104 In Experiment 3 the objective was to compare the DM and NDF digestion of enzyme-treated substrates under simulat ed preingestive and ruminal conditions. Substrates were incubated for 24 h in the bu ffered enzyme solution as in Experiment 2 in each of 3 runs. Subsequently, 40 ml of ei ther distilled water (W) or buffered-ruminal fluid (RF) was added to each culture bottle, and the suspensions were incubated at 39C for 24 h. Incubation in W was used to si mulate preingestive effects of the enzyme, whereas incubation in RF simulated ruminal effects. The RF was collected by aspiration from a non-lactating non-pr egnant Holstein cow, filtered through two layers of cheesecloth, immediately transported in a pr e-warmed thermos flask to the laboratory, and mixed (1:2 ratio) under a CO2 stream with an anaerobic culture medium of Tilley and Terry (1963). Ruminal fluid donor cows were fed bermudagrass hay ad libitum supplemented with 750 g of soybean meal daily. The culture medium had been warmed to 39C to avoid exposing micr oorganisms to cold shock. After the 24 h incubation in RF or W, the contents of the culture bottles were filtered through a Whatman N 541 filter paper and residues were dried at 60C overnight to determine DM digestibility. The DM and NDF concentrations of substrates and digestion residues were measured as described previously and DM (DMD) and NDF di gestibility (NDFD) were calculated. Even though substrate disappear ance in the buffer or W did not involve enzymic or microbial digestion, DMD and NDF D will be used to describe so lubility in these media for simplicity. In Experiment 4 the objective was to det ermine effects of enzyme addition on DM and NDF digestibility under simu lated preingestive and ruminal conditions. Substrates were prepared and incubated in the buffered enzyme solution or in the buffer alone for
105 24 h. Subsequently, 40 ml of either W or RF was added to each culture bottle, and the suspensions were incubated at 39C for 24 h. Dry matter and NDF digestibility were determined as in Experiment 3. A 5 ml aliquot of the RF filtrate was frozen (-20oC) for volatile fatty acid (VFA), pH and NH3-N analysis. Filtrate samples containing ruminal fluid were analyzed for VFA using a Ga s Chromatography system (Perkin Elmer Autosystem XL, Waltham, MA) containing a S upelco (Sigma Aldrich, St. Louis, MO) packed column with the following specific ations: 2 m x 2 mm Tightspec ID, 4% Carbowax 20M on 80/120 B-DA. The pH of t he filtrate was measured with a pH meter (Accumet, model HP-71, Fisher Scient ific, Pittsburgh, PA) and ammonia-N was determined using an adaptation for the Tec hnicon auto analyzer (Technicon, Tarrytown, NY) of the Noel and Hamble ton (1976) procedure. The adaptation involved colorimetric quantification of N concentration. Statistical Analysis Data from experiments 1 and 2 were analyz ed as completely randomized d esigns with 2 (control versus enzyme) x 6 (substrates) factorial arrangement of treatments. Data from experiments 3 and 4 were analyz ed with completely randomized designs with 2 (water versus ruminal fluid) x 6 (substrat es) and 2 (control versus enzyme) x 2 (water versus ruminal fluid) x 6 (substrates) factorial treatment arrangement s, respectively. The GLIMMIX procedure of SAS (Version 9.2 SAS Institute Inc., Cary, NC) was used to analyze the data. The model used to anal yze individual treatment effects included substrate, enzyme treatment, and the interaction (Experiments 1 and 2), substrate and medium, and the interaction (Experiment 3) and substrate, medi um, enzyme treatment and all interactions (Experiment 4). Replic ate was the random term in each experiment.
106 The Tukey test was used to compare least square means and significance was declared at P < 0.05. Results and Discussion Experiments 1 and 2 The ingredient composit ion of the concentrates and TMR and the chemical composition of each substrate are shown in Table 4-1. In general, concentrations of NDF, ADF, and hemicellulose were greater in forages than the TMR and least in concentrates, whereas, WSC concentration was greatest in AH followed by concentrates and least in CS (Table 4-2). Across all substrates, enzyme treatment increased WSC concentration by 9% and dec reased concentrations of NDF, ADF, and hemicellulose by 3.9, 4.1, and 4.3%, respectively. The release of reducing sugars from hydrolyzed cell wall polysacchar ides agrees with reported effe cts of fibrolytic enzymes on various feeds and forages (Hristov et al., 1996a; Krause et al., 1998; Krueger and Adesogan, 2008). Greater release of sugars fr om cell walls could stimulate bacterial glycocalyx production, and thereby increase adhes ion of bacteria to substrates (Yang et al., 1999). However, the lack of a substrate x enzyme interaction for most of the chemical measures was surprising giv en that enzyme-feed s pecificity is often considered an important determinant of enzyme action (Beauchemin et al., 2004). Application of the enzyme had substr ate-dependent effects on DM disappearance in the buffer (substrate x enzyme interaction, P < 0.001; Table 4-3). Enzyme application increased the DMD of HC by 17% but did not affect thos e of other substrates. Therefore, HC seemed more susceptible to preingestive enzymatic hydrolysis in the buffer than other substrates perhaps because it contained the least NDF concentration.
107 Bowman et al. (2002) and Yang et al. (2000) also reported that enzyme application to the concentrate portion of the diet of lactat ing dairy cows improved in vivo DMD. Experiment 3 Incubation of enzyme-treated substrates in RF resulted in greater ( P < 0.001) in vitro DMD than incubation in W except for AH, which had similar DMD in both media (substrate x medium interaction; P < 0.001; Table 4-4). On average, DMD in RF was 76% greater than DMD in W, confirming that for all feeds except AH, ruminal digestion was more extensive than preing estive disappearance. The similar DMD result for AH in RF and W indicates that preingestive solubili ty of AH was as extensive as ruminal digestion of the substrate and suggests that digestion of AH in RF was due primarily to solubility rather than enzymatic or microbial hydrolysis. This may be because of the high concentration of water-soluble fractions in alfalfa hay (up to 48.5% of DM; Stefanon et al., 1996). On average, enzyme-treated substrates incubated in RF had greater ( P < 0.001) NDFD than those incubated in W (Table 4-4) but the magnitude of the increase was least for AH and greatest for the concent rates (substrate x medium interaction, P < 0.001). This highlights the greater insolubility of the fiber fractions in the concentrates relative to those in other substrates and illus trates the importance of microbial digestion of such fractions. Since untreated controls were not included in this experiment, it is not clear if the greater DMD and NDFD in RF versus W refl ect the intrinsic digestibility of the substrates or synergy bet ween the exogenous enzyme and ruminal microbes in RF
108 (Morgavi et al., 2000). This aspect was fu rther explored in Experiment 4, which compared untreated and enzym e-treated substrates in both media. Experiment 4 Incubation in RF resulted in greater DMD and NDFD than incubation in W but the magnitude of the responses differed with substrate type (substrate x medium interaction; P < 0.001 and = 0.003, respectively; T able 4-5 and 4-6). Averaged across control and enzyme treatment, increases in DMD of 26, 222, 101, 126; 144, and 152% occurred when AH, CS, LC, HC, TMRL, and TMRH were incubated in RF instead of W, respectively. Therefore, incubation medium had the greatest effect on the DMD of CS and the least on AH. This agrees with the lack of an incubation medium effect on DMD of AH in Experiment 3 and confirms that pr eingestive hydrolysis had a greater impact on AH than on other substrates. Enzyme effects on DMD and NDFD depended on the incubation medium ( P = 0.002 and <0.001, respectively) suggesting that preingestive and ruminal effects of the enzyme differed. Averaged across substr ates, enzyme addition increased DMD in W (26.3 vs. 23.9%) by 10% but had no effect on DMD in RF (53.5 vs. 54.2%). Enzyme treatment increased NDFD in W (14.9 vs. 8. 1%) by about 84% but did not affect NDFD in RF (36.4 vs. 36.3%). These results suggest that applying the enzyme contributed more to preingestive hydrolysis of the substrat es than to their digestion in ruminal fluid and indicates that the greater DMD and NDFD in RF versus W in Experiment 3 reflects the intrinsic digestibility of the substrates rather t han synergy between the exogenous enzyme and ruminal microbes in RF (Morgavi et al., 2000). This preingestive enzyme effect on DMD and NDFD explains some of t he enzyme-mediated increases in in vivo
109 apparent DMD, NDFD, and efficien cy of milk production in the previous study (Arriola et al., 2007). That enzymic hydrolysis increas ed the DMD of only HC after 24 h of incubation in the buffer (Table 4-3) but increa sed those of all substrates after 24 h of incubation in the buffer followed by 24 h of incubation in W (Table 4-5), suggests that the initial 24 h incubation in the buffer was too short to demonstrate preingestive effects of the enzyme on all substrates. Reasons why enzyme effects on DMD and NDF D in RF were not apparent are unknown. Many other studies evaluating enzyme effects on DMD of dairy cattle feeds have used 24 h ruminal fluid incubation dur ations to demonstrate positive enzyme effects (Eun and Beauchemin, 2007; Eun et al., 2007; Krueger and Adesogan, 2008). Nevertheless, greater en zyme effects on DMD and NDFD may have occurred if substrates were incubated for 30 (Oba and Allen, 1999) or 48 h (NRC, 2001) instead of 24 h to reflect ruminal NDF retention times in dairy cattle. Factors other than the duration of ruminal digestion also may be in volved. For instance, fibrolytic enzyme application decreased ruminal NDFD of a TMR fed to dairy cows but increased postruminal NDFD for unknown reas ons (Sutton et al., 2003). Ruminal pH and concentrations of total VFA and ammonia-N differed ( P < 0.001) with substrate type (Table 4-7) Ruminal pH was greates t for AH and least for CS, possibly reflecting the differences in the starch concentrations of the substrates, as well as their ammonia-N concentration and buffe ring capacity. Total VFA concentrations were less in forages than TMR and butyrate molar proportion was greatest for CS and least for AH. Ammonia-N concentration was least for CS and greatest for LC, partly due to their different CP concentrations.
110 Enzyme addition increased ( P = 0.002) total VFA conc entration (194 vs. 176 Mm) suggesting that the enzyme in creased supply of energy yi elding substrates from fermentation of the dietary substrates. Enzyme treatment also decreased ( P < 0.01) ruminal ammonia-N concentration (56 vs. 54 mg /L), which may reflect greater utilization of ammonia-N for microbial growth due to in creased availability of WSC during ruminal fermentation. Effects of enzyme addition on the mola r proportions of acetate and propionate tended to differ with th e substrate. Compared to Control, enzyme-treated AH had greater molar proportion of acetate ( P = 0.08) and lower propo rtion of propionate ( P = 0.07) resulting in a greater (P = 0.05) acetate to propionate ratio, but such differences did not occur in other substrates. Eun and Beauchemin (2007) report ed that adding the same enzyme to alfalfa hay increased total VFA concentration, propionate proportion, and NDFD, but did not affect acetate to pr opionate ratio. Whereas adding the enzyme to corn silage decreased acetate and pr opionate proportions and the acetate to propionate ratio but did not affect total VF A concentration. Differences between their results and those in this study may be due to differences between the forages and ruminal fluid activity as well as procedural di fferences for estimating these parameters. Beneficial enzyme effects on concentrations of cell walls and reducing sugars, digestibility of DM or NDF in W or RF, or ruminal fluid VFA or ammonia-N concentrations did not ( P > 0.05) depend on the substrate. This indicates that none of the substrates was preferentially hydrolyzed by the enzyme in RF or W and contradicts the hypothesis that enzyme effects on the forages would be greater. Therefore, it is unlikely that beneficial enzyme effects on DM D, NDFD, and efficiency of milk production in the study of Arriola et al. (2007) were due to preferential hydrolysi s of the concentrate
111 or either of the forages in the diets. Rather, the en zyme probably affected the entire TMR. Effects of enzyme addition to specific portions of diets hav e been contradictory. Yang et al. (2000) compared treating either the TMR or concentrate with an enzyme and reported that improvement s in DMD but not NDFD t ended to be greater when the concentrate was treated and only concentrate treatment improved milk yield. In contrast, Phipps et al. (2000) reported no di fferences between milk yield of cows fed enzyme-treated concentrates or TMR or the untreated TMR. Sutton et al. (2003) also reported no differences in DMI or milk yiel d of cows fed diets in which an enzyme was infused ruminally or added to the TMR or co ncentrate. These contradictions and the results of this study do not refute the ex istence of enzyme-feed specificity (Beauchemin et al., 2004) and its importance in determini ng enzyme effects. Rather, because the substrates, diets, and ingredients evaluated in this and other studies are comprised of various types and proportions of cell walls and other chemical components, they are probably not homogenous enough to reflect enzym e-feed specificity. Therefore, continued research on the best portion of the diet to which enzymes should be added may not identify an ideal ingred ient target for all diets. Conclusion Enzyme treatment decreased ruminal a mmonia-N conc entration and increased hydrolysis of cell walls, release of reduci ng sugars, digestibility of DM and NDF, and total VFA concentration regardless of substrat e type. Therefore, this study provided no convincing evidence that t he enzyme preferentially hydrolyzed specific dietary substrates. Substrate digestion in RF wa s consistently greater than that in W. However, enzyme effects on DMD and NDFD were consistently greater in W than in RF, indicating that preingestive effects of the enzyme were greater than ruminal effects
112 under the conditions of the study Preingestive effects were also greater for AH than other substrates, likely reflecting the high concent ration of water-soluble fractions in AH. These experiments involved 24 h incubations in RF or W, therefor e future research should investigate whether similar results are obtained with longer incubation periods.
113 Table 4-1. Ingredient compos ition of concentrate and TM R substrates and chemical composition of all substrates Item LC HC TMRL TMRH Ingredient, % DM Corn silage 0 0 49.2 37.0 Alfalfa hay 0 0 13.5 10.0 Cottonseed hulls 12.4 9.4 4.6 5.0 Corn meal 19.8 33.7 7.4 17.9 Citrus pulp 5.4 9.4 2.0 5.0 Whole cottonseed 4.9 9.1 1.8 4.8 SoyPlus1 21.2 11.2 7.9 5.9 Soybean meal 6.7 11.3 2.5 6.0 Cottonseed meal 20.9 9.6 7.8 5.1 Mineral mix2 8.7 6.1 3.3 3.3 Chemical composition AH CS LC HC TMRL TMRH DM, % 94.1 35.0 93.9 94.1 64.4 72.2 Ash, % DM 10.7 3.5 10.4 8.1 7.1 6.7 CP, % DM 18.7 9.0 31.1 25.1 18.6 18.5 NDF, % DM 43.6 44.7 27.9 25.4 38.1 33.3 ADF, % DM 23.2 18.4 13.8 12.9 17.9 15.7 Hemicellulose, % DM 20.4 26.2 14.1 12.5 20.2 17.6 1 West Central Soy, Ralston, IA. 2 Mineral mix contained 26.4% CP, 10.2% Ca, 8.6% Na, 5.1% K, 3.1% Mg, 1.5% S, 0.9% P, 2231 mg/kg of Mn, 1698 mg/kg of Zn, 512 mg/kg of Cu, 339 mg/kg of Fe, 31 mg/kg of Co, 26 mg/kg of I, 7.9 mg/kg of Se, 147,756 IU of vitamin A/kg, 787 IU of vitamin E/kg (DM basis). LC = Low concentrate; HC = High concentrate;TMR L = Low concentrate Total Mixed Ration; TMRH = High concentrate Total Mixed Ration
114 Table 4-2. Effect of enzyme application to different substrates on concentrations of NDF, ADF, hemicellulose, and WSC (Experiment 1). Substrate Measure Substrate Control Enzyme Mean NDF % of DM Alfalfa hay 43.6 42.7 43.2a Corn silage 44.7 43.4 44.0a Low concentrate 27.9 25.6 26.8d High concentrate 25.4 23.4 24.4e TMRL 38.1 36.2 37.1b TMRH 33.3 33.0 33.1c Enzyme mean 35.5x 34.1y Effects P values SEM Substrate <0.001 0.31 Enzyme <0.001 0.18 Substrate x enzyme 0.22 0.44 ADF, % of DM Alfalfa hay 23.2 23.6 23.4a Corn silage 18.4 17.9 18.2b Low concentrate 13.8 12.6 13.2e High concentrate 12.9 11.9 12.4e TMRL 17.9 16.7 17.3c TMRH 15.7 15.3 15.5d Enzyme mean 17.0x 16.3y Effects P values SEM Substrate effect <0.001 0.20 Enzyme effect <0.001 0.11 Substrate x enzyme effect 0.05 0.28 TMRL = Low concentrate Total Mixed Ration; TMRH = High concentrate Total Mixed Ration a, b, c, d, e Means within a column with x, y Means within a row with different superscripts differ, P < 0.001
115 Table 4-2. Continued Substrate Measure Substrate Control Enzyme Mean Hemicellulose, % of DM Alfalfa hay 20.4 19.2 19.8b Corn silage 26.2 25.5 25.9a Low concentrate 14.1 13.1 13.6d High concentrate 12.5 11.4 12.0e TMRL 20.2 19.5 19.9b TMRH 17.6 17.7 17.6c Enzyme mean 18.5x 17.7y Effects P values SEM Substrate <0.001 0.20 Enzyme <0.001 0.12 Substrate x enzyme 0.30 0.29 WSC, % of DM Alfalfa hay 9.4 9.8 9.6a Corn silage 2.2 3.7 2.9d Low concentrate 7.7 8.8 8.3b High concentrate 8.4 8.0 8.2b TMRL 6.0 6.6 6.3c TMRH 6.1 7.1 6.6c Enzyme mean 6.7y 7.3x Effects P values SEM Substrate effect 0.003 0.25 Enzyme effect <0.001 0.15 Substrate x enzyme effect 0.16 0.36 TMRL = Low concentrate Total Mixed Ration; TMRH = High concentrate Total Mixed Ration a, b, c, d, e Means within a column with x, y Means within a row with different superscripts differ, P < 0.001
116 Table 4-3. Effect of enzyme application on disappearance of DM from substrates incubated in a buffer for 24 h (Experiment 2). Substrate DM disappearance % Control Enzyme Alfalfa hay 36.7a 37.1a Corn silage 20.8e f 19.4 f Low concentrate 26.5b 27.8b High concentrate 22.0de 25.9bc TMRL 23.6cd 23.7cd TMRH 22.9de 22.8de Enzyme mean 25.4y 26.1x Effects P values SEM Substrate <0.001 0.34 Enzyme 0.02 0.20 Substrate x Enzyme <0.001 0.49 TMRL = Low concentrate Total Mixed Ration; TMRH = High concentrate Total Mixed Ration a, b, c, d, e, Interaction means with different superscripts differ, P < 0.001 x, y Means within a row with different superscripts differ, P < 0.05
117 Table 4-4. Effect of incubation medium on the dry matter (DMD) and neutral detergent fiber (NDFD) digestibility of enzymetreated substrates (Experiment 3). Substrate DMD % NDFD % Water Ruminal fluid Water Ruminal fluid Alfalfa hay 43.4b 43.1b 10.9d 22.7b Corn silage 22.2 f 54.4a 12.0cd 31.2a Low concentrate 33.2c 55.4a 0.8e 19.2bc High concentrate 29.4cd 55.8a 2.1e 22.2b TMRL 26.7de 52.9a 4.6ed 33.0a TMRH 24.9e f 55.9a 5.1ed 34.3a Mean 30.0y 52.9x 5.9y 27.1x Effects P values SEM P values SEM Substrate <0.001 0.44 <0.001 0.73 Medium <0.001 0.26 <0.001 0.42 Substrate x medium <0.001 0.62 <0.001 1.03 TMRL = Low concentrate Total Mixed Ration; TMRH = High concentrate Total Mixed Ration a, b, c, d, e,f Means within a measure (DMD or NDFD) wi th different superscripts differ, P < 0.001 x, y Means within a row with different superscripts differ, P < 0.05
118 Table 4-5. Effect of enzyme applicati on and incubation medium on the dry matte r digestibility (DMD) of substrates incubated for 24 h in water or ru minal fluid after incubation for 24 h in a buffer (Experiment 4). Medium Water Ruminal fluid Substrate Control Enzyme Mean Control Enzyme Mean Alfalfa hay 34.2 38.4 36.3d 44.9 46.5 45.8c Corn silage 15.6 17.8 16.7g 54.0 53.6 53.8b Low concentrate 26.5 27.4 27.0e 53.3 55.3 54.3b High concentrate 23.3 26.5 25.2e 56.4 57.5 56.9ab TMRL 21.9 23.7 22.8 f 55.7 55.7 55.7ab TMRH 21.7 23.3 22.5 f 56.7 56.8 56.8a Mean 23.9y 26.3x 53.5 54.2 Effects P values SEM Substrate <0.001 0.30 Enzyme <0.001 0.17 Medium <0.001 0.17 Substrate x medium <0.001 0.43 Enzyme x medium 0.002 0.25 Substrate x enzyme 0.10 0.43 Substrate x enzyme x medium 0.28 0.60 TMRL = Low concentrate Total Mixed Ration; TMRH = High concentrate Total Mixed Ration a ,b, c, d, e, f, g Substrate x medium means with different superscripts differ, P < 0.001 x, y Enzyme x medium means with diffe rent superscripts differ, P < 0.01
119 Table 4-6. Effect of enzyme application and incubation m edium on neutral detergent fiber digestibility (NDFD) of substrates of substrates incubated for 24 h in water or ruminal fluid after incubation for 24 h in a buffer (Experiment 4) Substrate NDFD Water Ruminal fluid Control Enzyme Mean Control Enzyme Mean Alfalfa hay 7.2 12.1 9.6d 27.4 29.4 28.4bc Corn silage 4.4 12.8 8.6d 31.9 34.2 33.1b Low concentrate 0.0 0.0 0.0e 22.7 24.3 23.5c High concentrate 0.0 7.1 1.3e 29.8 30.3 30.0b TMRL 21.2 25.6 23.4c 50.6 46.8 48.7a TMRH 28.1 33.5 30.8b 55.8 52.6 54.2a Mean 8.1y 14.9x 36.4 36.3 Effects P values SEM Substrate <0.001 0.88 Enzyme <0.001 0.51 Medium <0.001 0.51 Substrate x medium 0.003 1.25 Enzyme x medium <0.001 0.73 Substrate x enzyme 0.16 1.25 Substrate x enzyme x medium 0.61 1.76 TMRL = Low concentrate Total Mixed Ration; TMRH = High concentrate Total Mixed Ration a ,b,c,d,e Substrate x medium means with different superscripts differ, P < 0.001 x, y Enzyme x medium means with diffe rent superscripts differ, P < 0.01
120 Table 4-7. Effect of enzyme (Enz) application on pH, VFA and ammonia-N concentrations of substrates incubated for 24 h in ruminal fluid after incubation fo r 24 h in a buffer (Experiment 4) Substrate pH Total VFA, mM Acetate (A), mol/100 mol Propionate (P), mol/100 mol Butyrate, mol/100 mol A:P Ammonia-N, mg/L AH Control 7.17 135 57.6 32.0 8.9 1.93 58.3 Enz 7.16 165 67.3 24.4 7.1 2.76 60.4 Mean 7.17a 150 d 62.4 28.2 8.0c 2.3 59.3 b CS Control 6.01 160 57.4 28.4 13.2 2.03 30.1 Enz 5.91 194 57.3 28.4 13.2 2.02 26.3 Mean 5.96 d 177c 57.3 28.4 13.2a 2.02 28.2 d LC Control 7.15 178 60.4 28.2 10.3 2.15 76.0 Enz 6.91 180 59.3 29.0 10.6 2.05 75.9 Mean 7.03a b 179 b c 59.8 28.6 10.5 b 2.10 75.9a HC Control 6.71 184 58.2 30.4 10.4 1.92 65.9 Enz 6.72 190 57.6 30.4 11.0 1.90 60.8 Mean 6.72 b c 187a b c 57.9 30.4 10.7 b 1.91 63.3 b TMRL Control 6.44 205 60.3 28.1 10.6 2.15 53.1 Enz 6.51 216 60.5 27.7 10.8 2.19 49.4 Mean 6.47c 211a 60.4 27.9 10.7 b 2.17 51.3c TMRH Control 6.43 192 59.4 28.4 11.2 2.10 52.6 Enz 6.42 221 59.8 28.5 10.8 2.10 48.3 Mean 6.43c 207a b 59.6 28.4 11.0 b 2.10 50.4c SEM 1 0.10 9.18 1.89 1.44 0.42 0.15 1.55 Effect P values Substrate <0.001 <0.001 0.13 0.58 <0.001 0.16 <0.001 Enzyme 0.45 0.002 2 0.20 0.16 0.45 0.18 0.01 2 Substrate x enzyme 0.73 0.23 0.08 0.07 0.11 0.05 0.18 AH = Alfalfa hay; CS = Corn silage; LC = Low energy concentrate; HC = High energy concentrate; TMRL = Low concentrate Total Mix ed Ration; TMRH = High concentrate Total Mixed Ration a, b, c, d Means in the same column with different superscripts differ, P < 0.001 1 Substrate x enzyme SEM. 2 Main enzyme effect means for total VFA were 176 versus 194 mM, SEM = 3.48 and were 56 versus 54 mg/L, SEM = 0.63 for ammonia-N.
121 CHAPTER 5 EFFECT OF APPLYING BACTERIAL INO CULANT S ON THE FERMENTATION AND QUALITY OF CORN SILAGE Introduction Inoculants containing selected strains of lactic acid bacteria (LAB) have been develo ped to reduce the influence of epiphytic LAB on the outcome of ensiling forages. The principal function of homof ermentative inoculants is to ensure a rapid and efficient fermentation of water-soluble carbohydrates (W SC) into lactic acid, a rapid decrease in pH, and improved silage conservation with minimal fermentation losses (Weinberg et al., 1993a). However, such inoculants have not increased aerobic stability in many studies (Muck and Kung, 2007) and some hav e decreased aerobic stability (Moon et al. 1980) by enhancing the growth of spoilage yeas ts. Inoculating forages at harvest with Lactobacillus buchneri improves the aerobic stability of silages (Muck, 1996; Taylor et al., 2002) most likely because this organism converts lactic acid to acetic acid under anaerobic conditions (Oude Elferink et al., 2001). Recently, L. buchneri has been combined with homolactic bacteria to improve the aerobic stability and fermentation of silages (Kung et al., 2003a). Dri ehuis et al. (2001) reported that L. buchneri alone or in combination with P. pentosaceus and L. plantarum improved aerobic stability of grass silage, reduced yeast and mold counts, and had lower DM loss compared to the untreated silage. Other studies reported improved aerobic stability when combinations of homolactic and heterolactic inoculants we re applied to sorghu m (Filya, 2003) and corn (Huisden et al., 2009). However, Adesogan et al. (2004) r eported that treating bermudagrass with P pentosaceus and L. buchneri improved the fermentation but did not improve aerobic stability because their control silage was more stable due to a butyric fermentation. Kleinschmit and Kung (2006b) reported that an inoculant
122 containing Pedioccocus pentosaceus and L. buchneri reduced yeast counts and improved aerobic stability in a m anner that varied with the ensil ing duration. Kang et al. (2009) demonstrated that effect s of inoculants containing Lactobacillus casei and L. buchneri on aerobic stability of corn silage va ried with the corn hybrid tested. Therefore, more research on effects of combinations of L. buchneri and homolactic bacteria is needed. Propionic acid bacteria can ferment gluc ose and lactate to acetate and propionic acid (Moon, 1983) and a few studies have shown that such inoculants improved aerobic stability of silage (Dawson et al., 1998; Filya et al., 2004). Effects of combinations of propionibacteria and homolactic bacteria on silage fermentation and aerobic stability are not well known. The objective of this st udy was to examine t he effect of applying bacterial inoculants containing heteroferm entative bacteria alone or homofermentative and heterofermentative bacteria on the fermentation, quality, and aerobic stability of corn silage. Material and Methods Forage and Treatments A corn hybrid Vigoro 61R36 (Roy ster Cla rk, Inc., Greeley, CO) was grown at the Dairy Research Unit, University of Fl orida and harvested at 35% DM with a Claas Jaguar 850 (Claas of America, Columbus, IN) forage harvester. Forages were chopped to a theoretical length of cut of 1.9 cm and treated with 1) deionized water (CON); 2) Biotal Plus II (B2), containing 1 x 105 cfu/g of Pediococcus pentosaceus and Propionibacteria freudenreichii applied at 21.9 mg/kg of fresh forage; 3) Buchneri 40788 (BUC), containing 4 x 105 cfu/g of Lactobacillus buchneri applied at 8 mg/kg of fresh forage; 4) a combination inoculant, Buchneri 500 (B500), containing 4 x 105 cfu/g
123 of P. pentosaceus 12455 and L. buchneri 40788 applied at 8 mg/kg of fresh forage. Inoculants were dissolved in 950 mL of deionized water and sprayed uniformly onto the forages under constant mixing. All inocul ants were supplied by Lallemand Animal Nutrition, Milwaukee, WI. Four replicates of each treatment were weighed (10 kg) into polyethylene bags, within 20-L mini silos, sealed, and stored for 575 d at ambient temperature (25C) in a covered barn. Dr y matter recovery was calculated from the initial and final weights and the DM concentrations of the fresh and ensiled forage. Chemical Analysis Subsamples of the untreat ed fresh forage and each treated silage were collec ted, dried in a forced-air oven at 60C for 48 h and ground to pass a 1-mm screen using a Wiley mill (A. H. Thomas, Philadelphia, PA) Concentrations of NDF and ADF were measured using the method of Van Soest et al. (1991) in an Ankom 200 Fiber Analyzer (Ankom Technologies, Macedon, NY). St arch was determined using the procedure of Holm et al. (1986). The anthr one reaction assay (Ministry of Agriculture Fisheries and Food, 1986) was used to quantify water-soluble carbohydrates (WSC). Corn silage samples were also analyzed for aerobic stabili ty by placing thermocouple wires at the center of a bag containing 1 kg of silage, within an open-top polystyrene box. The silages were covered with 2 layers of cheesec loth 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 14 d. Aerobic stability was denoted by the time (h) before a 2C rise in silage tem perature above ambient temperature (23C). Silage subsamples from each silo were submitted to Dairyland Laboratories, Inc. Arcadia, WI for analysis of yeast and mold counts using YM-11 agar (AOAC, 1995),
124 VFA by HPLC (Canale et al., 1984), pH usi ng a pH meter (Orion 710+, Thermo Fisher Scientific Inc., Waltham, MA), and amm onia-N by distillation (AOAC, 1995). Phenol-chloroform Extraction of Total DNA Silage samples from each treatment were diluted with distilled water (1:2) and macerated in a blender. T he DNA was extr acted from t he water extract using the phenol-chloroform method (Giraffa et al., 2000). A 1.5 ml a liquot of each water extract was centrifuged at 12,500 g for 5 min, the supernatant was discarded and this step was repeated. The pellets were washed tw ice with TE buffer (10mM Tris-HCl-0.1 mM EDTA, pH 8.0). Washed cell pellets were resuspended in 500 l of TES buffer (50 mM Tris-HCl-1 mM EDTA-6.7% saccharose, pH 8.0) followed by incubation at 37C for 30 min with 200 l of lysozyme (50 mg/ml). Th is was followed by a second incubation at 56C for 30 min with 15 l of Proteinase K, and a third incubation at 56C overnight with 125 l of SDS (sodium dodecyl sulfate, 20% wt/vol). DNA was extracted by adding 200 l of phenol and chlorofo rm, centrifuging at 7,000 g for 5 min at 4C, and transferred to a new tube. This step was r epeated three times, follo wed by precipitation with cold (-20C) isopropanol (600 l) and centrifugation at 12,500 g for 30 min at 4C. A 1 ml aliquot of ethanol (70%) was added to the pelle ted DNA and centrifuged at 12,500 g for 5 min at 4C. Purified DNA was dried and resuspended overnight at 4C in 150 l of TE buffer. Ribonuclease HII (1 l; 5000 U/ml; New England BioLabs, Ipswich, MA) was added to resuspended DNA and after incubation at 37C for 1 h, DNA samples were stored at -20C until use. Conventional PCR Conditions The Polym erase chain reaction was performed to amplify a region of the bacterial 16S rRNA gene of L. buchneri with the forward primer LBR2 that corresponds to the
125 region from base 186 to 215 (5-GAAACA GGTGCTAATACCGTATAACAACCA-3) and the reverse primer LBR1 that corresponds to the region from base 316 to 345 (5CGCCTTGG-TAGGCCGTTACCTTACCAACA-3) (GenoMechanix, LLC. Gainesville, FL). The expected product size was 159 base pairs. A touchdown PCR was done in a PTC-100 Programmable Thermal Controller (MJ Research, Inc., Waltham, MA) in order to reduce the incidence of formation of false products, enhance correct annealing, and increase the specificity of the amplification (Kidd and Ruano, 1995). The initial denaturation step was set at 94C for 15 mi n and the annealing temper ature was set at 61C for the first cycle and reduced by 0.5C per cycle for the next nine cycles to reach a final melting temperature ( Tm) of 56C. The previous melting temperature was used for the remaining 20 cycles. An extension ti me of 30 s at 72C was utilized, with final extension of 7 min and hold at 4C. Am plification was done in a standard reaction mixture containing 16.75 l of H2O, 2.5 l of 10x Taq buffer advanced (5 Prime), 1 l of each primer, 2.5 l of 2mM dNTP (Thermo Fisher Scientific, Inc), 0.25 l of Taq Polymerase (5 U/l; 5 Prime), and 2 l of the template. Electrophoresis was conducted on 2.0% agarose gel stained wit h ethidium bromide, and DNA was visualized under UV light and photographed (Sam brook et al., 1989). Statistical Analysis The data were analyz ed as a complete ly randomized design using the GLM procedure of SAS (Version 9.2 SAS Institute Inc., Cary, NC). The general model was. Yij = + Ti + eij, where T = effect of treatment i, and eij = e rror term. The F-protected least significant difference test was used to compare least square means and significance was declared at P < 0.05.
126 Results and Discussion The chemical composition of the corn fo rages was normal for corn silage produced in Florida (Table 5-1; Huisden et al., 2009; Kang et al., 2009). The control silage had the greatest DM concentration but other ch emical components did not differ among the forages. C orn forage treated with B2 had a lower DM concentration than B500 and Control silages, whereas CP concentration was greater in CON and B2 silages than in the others (Table 5-2). As expected, WSC concentrations of all silages were reduced during the fermentation. The B500 silage had the greatest ( P < 0.05) residual WSC concentration (1.49 vs. 1.18 % DM), indicati ng that plant sugars were less extensively fermented by the bacteria in this inoculant co mpared to those in ot hers. In contrast, Filya (2003) reported that Control and L. plantarum -inoculated corn silages had greater ( P < 0.05) residual WSC than silages inoculated with L. buchneri alone or L buchneri and L. plantarum High residual WSC concentrations in silages are desirable because they reflect a more efficient fermentation in the silo and indi cate greater availability of energy-yielding substrates for ruminal microbes, but can also predispose to the growth of spoilage yeasts. All pH values were within the range of 3.8 to 4.1 (Table 5-3), which reflects adequate fermentation for rest ricting the growth of undesirable microorganisms like clostridia. Dry matter loss was lower in BUC silages compared with Control and B2 silages (5.0 vs. 14.3 %). Control and B2 si lages had greater pH and lower lactic acid concentration than BUC and B500 silages. The greater lactate concentration and lower pH of BUC silages explain the lower DM loss from this silage. Propionic acid concentration was greater in Control and B2 silages than BUC and B500 silages. Acetic acid concentration was greatest in the B2 silage (6.46 vs. 4.23 % DM), lowest in
127 the B500 silage and intermediate in Control and BUC silages. The B500 silage had the greatest lactic: acetic acid ratio (1.54 vs. 0.41). The fermentation results of the Control, BUC, and B500 silages were unusual. Normally, untreated corn silage undergoes a homolactic fermentation resulting in relatively low pH values (< 4.0) due to high concentrations of lactate and low concentrations of acetate and propionate. In contrast, the Control silage had among the highest pH values, lowest lactate conc entrations, and highest acetate and propionate concentrations, suggesting that the fermentation was dominated by heterolactic bacteria. Furthermore, the homolactic bacte ria in B2 and B500 did not dominate the fermentation sufficiently to result in great er lactate concentrations and lower DM loss relative to those in the Control silage. Results of inoculation with BUC and B500 were atypical in that application of Lactobacillus buchneri to corn forage usually increases the concentration of acetate and reduces the lactate concentration (Ranjit and Kung, 2000; Filya, 2003; Hu et al., 2009). This could be due to the presence of high concentrations of epiphytic L. buchneri on the control silages. T he conventional PCR analysis was performed to examine this theory. It rev ealed the presence of si milar populations of L. buchneri in all silages irrespective of treatment (Figure 5-1) and explains why inoculants containing L. buchneri did not have normal effects on the silages. Factors predisposing to high epiphytic populations of L. buchneri on corn forages or silages are unknown and warrant further research. Propionic acid bacteria can ferment gluc ose and lactate to acetate and propionic acid (Moon, 1983), theref ore the presence of Propionibacteria in B2 partly explains the greater acetate concentration and numerically greater prop ionate concentration of B2
128 silages relative to Control silages. However, inoculation with Propionibacteria has produced equivocal results on pr opionic acid concentration of silages, with few studies showing positive effects (Dawson et al ., 1998) and many others showing little to no effect (Higginbotham et al., 1998, Kung and Ranjit, 2001; Pedroso et al. 2010). Consequently, such bacteria have not usua lly increased aerobic st ability (Weinberg et al., 1995; Higginbotham et al., 1998; Pedroso et al., 2010). Yeast and mold counts were less than the threshold (105) typically associated with silage spoilage (Pahlow and Zimmer, 1985; OKiely et al., 1987) and did not differ among treatments (Table 5-4). C onsequently, all silages were stable for long periods (> 250 h) even though B500 silages were less stabl e than the others. That application of L. buchneri inoculants did not increase the aerobic st ability or decrease the yeast counts relative to those of untreated silages contr adicts various reports (Kleinschmit and Kung, 2006b; Hu et al., 2009; Huisden et al., 2009; Pedroso et al., 2010). This is likely because of the high population of L. buchneri in all silages. The greater deterioration of the B500 silage, which had one of the highest lactate concentrations, reflects the relatively low antifungal property of lactate. In fact, lactat e serves as a substrate for several spoilage yeasts such as those of the genera Candida and Pichia spp. (Pahlow et al., 2003). Acetate, butyrate, and propionate ar e the main antifungal acids in silages (Moon et al., 1983) and the total concentration of these acids was lower in the B500 silage than in others. Weinber g et al. (1993a) reported that high levels of residual WSC, combined with high lactate concentrations and insufficient concentrations of antifungal VFA in silages inoculated with homoferment ative LAB were associated with aerobic spoilage. The greater residual WSC conc entration and lower total antifungal acid
129 concentration may have made the B500 silage mo re susceptible to deterioration than others in this study. Conclusions Application of BUC improved lactate concentration, which made the fermentation more homolactic and contributed to a reduction in DM loss relative to values for Control silages. Control and B2 silages had greater pH values and lower lactate concentrations than BUC and B500 s ilages. Acetate conc entration was greatest in B2 silage, intermediate in Control and BUC silages, and lowest in B500 silage. However, aerobic stability was generally high (> 250 h) and was not improved by inoculant application. A conventional PCR analysis confirmed t he presence of similar populations of L. buchneri in all treatments and this probably explains the prolonged aerobic stab ility of all silages and some of the atypical fermentation results.
130 Table 5-1. Chemical composition of corn forages treated with or without bacterial inoculants before ensiling Item CON B2 BUC B500 SEM P values DM, % 35.4a 32.9 b 31.5 b 33.1 b 1.46 0.02 NDF, % DM 42.8 43.0 43.0 41.7 1.07 0.30 ADF, % DM 25.1 25. 1 25.3 25.7 0.97 0.81 Starch, % DM 25.5 28. 4 22.9 24.8 2.72 0.08 WSC, % DM 16.9 18.5 18.3 17.7 1.35 0.39 Means in the same row with different superscripts differed ( P < 0.05) CON = Control, no inoculant; B2 = Pediococcus pentosaceus and Propionibacteria freudenreichii ; BUC = Lactobacillus buchneri ; B500 = P. pentosaceaus and L. buchneri Table 5-2. Effect of bacterial inoculants on the chemical composition of corn silages Item CON B2 BUC B500 SEM P values DM, % 31.4 29.9 31.2 31.7 0.84 0.09 CP, % DM 10.5a 10.3a 9.86 b 9.86 b 0.31 0.03 NDF, % DM 44.4a 43.2a b 42.3 b 42.0 b 1.06 0.04 ADF, % DM 27.2 27.4 26.2 26.6 0.99 0.42 Starch, % DM 26.1 27. 8 28.3 25.2 2.81 0.41 WSC, % DM 1.22 b 1.14 b 1.18 b 1.49a 0.12 0.01 Means in the same row with different superscripts differed ( P < 0.05) CON = Control, no inoculant; B2 = Pediococcus pentosaceus and Propionibacteria freudenreichii ; BUC = Lactobacillus buchneri ; B500 = P. pentosaceaus and L. buchneri
131 Table 5-3. Effect of bacterial inoculants on DM losses and fermentation indices of corn silages Item CON B2 BUC B500 SEM P values DM loss, % 14.9a 13.6ab 5.0c 8.2ab 3.92 0.016 pH 4.10a 4.06a 3.90b 3.80b 0.05 <.001 NH3-N, % CP 8.25 9. 68 8.92 8.01 1.15 0.28 Total acids, % DM 7.15b 8.88a 8.40a 7.30b 0.58 0.005 Lactic acid, % DM 1.56b 1.31b 3.34a 4.17a 0.72 0.001 Acetic acid, % DM 4.85b 6.46a 4.88b 2.97c 0.59 <0.001 Propionic acid, % DM 0.72 1.09 0.17b 0.08b 0.16 <0.001 Butyric acid, % DM 0.01 0.01 0.01 0.07 0.06 0.47 Isobutyric acid, % DM 0.01 0.01 0.01 0.01 0.00 Ethanol, % DM 0.01 0. 10 0.01 0.01 0.07 0.28 La : Ac ratio 0.33b 0.21b 0.69b 1.54 0.35 0.001 Means in the same row with different superscripts differed ( P < 0.05) CON = Control, no inoculant; B2 = Pediococcus pentosaceus and Propionibacteria freudenreichii ; BUC = Lactobacillus buchneri ; B500 = P. pentosaceaus and L. buchneri Table 5-4. Effect of bacterial inoculants on fungal counts and aerobic stability of corn silages Item CON B2 BUC B500 SEM P values Yeasts, log cfu/g <3.00 <3.00 <3.00 <3.00 0.00 Molds, log cfu/g 3.4 3.67 4.15 3.75 0.71 0.54 Aerobic stability, h 390a 390a 381a 276b 53 0.07 Means in the same row with different superscripts differed ( P < 0.05) CON = Control, no inoculant; B2 = Pediococcus pentosaceus and Propionibacteria freudenreichii ; BUC = Lactobacillus buchneri ; B500 = P. pentosaceaus and L. buchneri
132 Figure 5-1. Gel electrophoresis analysis after polymerase chain reaction amplification of DNA from silages treated with or without inoculan ts: lane 1 and 8, DNA ladder/ marker (Promega, Corp.) lane 2, Pure culture of L buchneri, lane 3: CON, untreated control, lane 4: B2, Pediococcus pentosaceus and Propionibacteria freudenreichii lane 5: BUC L. buchneri lane 6: B500, P. pentosaceus plus L buchneri, lane 7: negative control. The expected product size was 159 base pairs (bp). 1 2 3 4 5 6 7 8 200 bp 100 bp
133 CHAPTER 6 EFFECT OF APPLYING BACTERIAL INO CULANT S TO CORN SILAGE ON THE PERFORMANCE OF DAIRY CATTLE Introduction Ensiling is a preservation method for mo ist forage crops based on conversion of water-soluble carbohydrates (WSC) into organic acids by lactic acid bacteria (LAB) under anaerobic conditions (McDonald et al ., 1991). Homofermentative LAB are used to increase the rate of acid ification and minimize DM and nutrient losses during forage fermentation. Bacteria typica lly used for this purpose include Lactobacillus plantarum, Enterococcus faecium, and Pediococcus spp. However, such bacteria have not been very successful at enhancing aerobic stability of silage (Muck and Kung, 2007) because the lactic acid they produce may facilitate the growth of spoilage yeasts. Propionic acid bacteria convert lact ate and glucose to antifungal acids like propionic and acetic acids, but they have not improved consistently the aerobic stability of forages (Weinberg et al., 1995; Higginbotha m et al., 1996, 1998; Filya et al., 2006) because the growth of propionic acid bacte ria is affected adversely by acidic environments (Weinberg et al., 1995; Higgi nbotham et al., 1996). Application of Lactobacillus buchneri inoculants has improved the aerobic stability of corn silage (Kleinschmit et al., 2005; Huisden et al., 2009), alfalfa silage (Kung et al., 2003a), and sugar cane silage (Pedroso et al., 2002). However, L. buchneri is heterofermentative, consequently, small losses of DM can occur during the fermentati on of forages treated with L. buchneri inoculants (Ranjit and Kung, 2000). Th is led to the recent development of dual purpose inoculants containi ng a mixture of homofermentative and heterofermentative LAB that reduce DM losses by incr easing the acidification rate and increase the aerobic stability, respectively.
134 Studies on effects of inocul ant application to forages on the performance of dairy cows are few and those available have pr oduced equivocal result s. Muck (1993) reviewed studies published fr om 1985 to 1992 and reported that inoculant application increased DMI and weight gain in about 25% of the studies, whereas milk production and feed efficiency were increased in 40 and 45% of studies, respectively. Kung et al. (1993) showed that feeding co rn silage inoculated with L. plantarum to lactating dairy cows improved 3.5% FCM and DMI. Wohlt ( 1989) reported that dairy cows fed corn silage inoculated with L. plantarum produced 0.7 kg/d more FCM than cows fed the untreated silage. Feeding alfalfa silage inoculated with L. buchneri 40788 increased milk production by dairy cows (Kung et al., 2003a), but feeding barley silage inoculated with L. buchneri 40788 did not (Taylor et al., 2002). To our knowledge, no studies have evaluated effects of inoculating corn s ilage with the newer i noculants containing homofermentative and heteroferm entative bacteria on the perfo rmance of dairy cows. The objective of this study was to exami ne the effect of applyi ng bacterial inoculants containing heterofermentative bacteri a alone or homofermentative and heterofermentative bacteria on the per formance of lactating dairy cows. Material and Methods Forage and Treatments A corn hybrid Vigoro 61R36 (Roy ster Cla rk, Inc., Greeley, CO) was grown at the Dairy Research Unit, University of Florida, harvested at 35% DM using a Claas Jaguar 850 (Monroe, NC) forage harvester and chopped to achieve a theoretical length of cut of 1.9 cm. Thirty-five tons of forage were ensiled within separate 2.4-m wide bags for 363 d after application of the fo llowing treatments: 1) nothing (CON), 2) Biotal Plus II (B2) containing Pediococcus pentosaceus and Propionibacteria freudenreichii (applied
135 at 1 x 105 cfu/g of fresh forage), 3) Buchneri 40788 (BUC) containing Lactobacillus buchneri 40788 (applied at 4 x 105 cfu/g), and 4) Buc hneri 500 (B500) containing P. pentosaceus 12455 (1 x 105) and L. buchneri 40788 (4 x 105 cfu/g). Inoculants were dissolved in water (0.95 L/ton) and applied via a sprayer mounted on the chopper. All inoculants were supplied by Lallemand Animal Nutrition, Milwaukee, WI. Diets, Cows, and Management The study was conducted from July to Oc tober 2007 at the Dairy Research Unit, University of Florida. Each of the four silages was mixed into a TMR consisting of 44% corn silage, 50% concentrate and 6% alfalfa hay (DM basis; Table 6-1). All diets were balanced to meet the nutrient r equirement of dairy cows in early lactation (NRC, 2001). Fifty-two lactating Holstein cows were classi fied according to milk production and parity, and randomly assigned to the four dietary treatments at 22 + 3 DIM. Cows were housed in a free-stall, opensided barn fitted with cont inuously operated fans and misters and drinking water was constantly av ailable in ad libitum amounts. Free-stalls were bedded with sand and cleaned daily. Sufficient free-stalls were available to provide at least 1 free-stall per cow. Cows were fed at an ad libitum level through individual Calan gates (American Calan Inc., Northwood, NH) twice daily (0800 and 1300 h). The initial 14 d were used for adaptation to di ets, and the subsequent 35 d were used to measure DMI and milk production. Sampling and Analysis Duplicate samples of each corn silage, alfalfa hay, and concentrate were collected weekly and composited monthly. Subsamples from each month were dried at 60C for 48 h in a forced draft oven, ground to pass 1 mm screen in a Wile y mill (Arthur H. Thomas Company, Philadelphia, PA) and anal yzed for DM (105C for 8 h) and ash
136 (512C for 8 h). Concentrations of NDF and ADF were measured using the method of Van Soest et al. (1991) in an Ankom 200 Fiber Analyzer (Ankom Technologies, Macedon, NY). Heat stable -amylase and sodium sulfite were used in the NDF analysis. Nitrogen was determined by rapid combustion using a Macro elemental N analyzer (Vario MAX CN, model ID 25.00-50 03; Elementar, Hanau, Germany) and CP was calculated as N x 6.25. Subsamples of corn silage were submitted to Dairyland Laboratories, Inc. (Arcadia, WI ) for analysis of yeast and mo ld counts using YM-11 agar (AOAC, 1995), VFA by HPLC (Canale et al., 1984), pH using an Orion 710+ electrode (Thermo Fisher Scientific, Waltham, MA), and ammonia-N by distillation (AOAC, 1995). Corn silage also was analyzed for aerobic stabili ty by placing thermocouple wires at the center of a bag containing 1 kg of silage, within an open-top polystyrene box. The thermocouple wires were connected to data loggers (Campbell Scientific Inc., North Logan, UT) that recorded the temperature every 30 min for 12 d. The silages were covered with 2 layers of cheesecloth to prevent drying. Aerobic stability was denoted by the time that elapsed before a 2C rise in silage temperature above ambient temperature (23C). The quantity of spoiled (v isibly heating, moldy, or darker) silage from each bag was weighed daily, composited weekly, and analyzed for DM (48 h at 60C). Body weight and BCS were measured every week at the beginn ing and end of the trial. Body condition score was measured on a 1 to 5 scale (Wildman et al., 1982) and by the same trained observer. Blood samples were collected weekly by coccygeal venipuncture into vacutainer tubes cont aining sodium heparin anticoagulant (Fisher Scientific, Pittsburgh, PA). T he blood was centrifuged at 2,500 x g for 20 min at 4C
137 and the plasma was frozen (-20C). A Tech nicon Autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was used to measure plasma glucose (Bran and Luebbe Industrial Method 339; Gochman and Schm itz, 1972) and BUN (Bran and Luebbe Industrial Method 339; Marsh et al., 1965). Cows were milked twice daily (1000 and 2100 h) and milk composition (fat, protein, and SCC) was measured on samples co llected from a.m. and p.m. milkings on two consecutive days in each week. Milk samples were analyzed by Southeast Dairy labs (Belleview, FL) using a Bentley 2000 N ear Infrared Reflectance Spectrophotometer (Bentley Instruments Inc., Chaska, MN). Somatic cell scores were generated as described by Norman et al. (2000) fo r statistical analysis of SCC. In vivo apparent digestibility was estimat ed by using chromic oxide as a marker. Chromic oxide powder (Fisher Scientific, Fairlawn, NJ) was weighed into gelatin capsules (Jorgensen Lab. Loveland, CO) and dosed twice daily with a balling gun (10 g/dose at 0700 and 1900h) for 10 c onsecutive d. Fecal samples were collected at 0630 and 1830 h during the last 5 d of dosing. Feces were dried to a constant weight at 55C in a forced-air oven, ground to pass through a 1-mm screen in a Wiley mill (Arthur H. Thomas Company, Philadelphia, PA) and a composite sample was made from all 10 fecal samples per cow. Chromium concentra tion in the feces was determined by using a Perkin Elmer 5000 atomic absorption spectr ometer (Perkin Elmer, Wellesley, MA). Apparent digestibility of CP, ADF and NDF were calculated by the marker ratio technique (Schneider and Flatt, 1975). Statistical Analysis The experiment had a completely randomiz ed design. The data was analyzed with the MIXED procedure of SAS ( SAS Institute, Inc. Cary, NC). A model contain ing
138 treatment, week (repeated measur e), parity and all interactions of these terms was used to analyze all performance measurements except apparent in vivo digestibility, which was analyzed with a similar model excludi ng the effect of week. Cow nested in treatment x parity was used as the random term. Milk producti on during the first 21 d of lactation was used as a covariate for analyzing milk production data. Pretrial BW was used as a covariate for analyzing BW and BCS. The covariance structure used was AR(1). The F-protected least significant diffe rent test was used to compare least square means. Significance was declared at P < 0.05. Results and Discussion All silages had normal chemical compositi on except for relati vely low NDF and ADF concentrations in B2 silage (Table 6-2). Chemical composition of silages did not differ (P > 0.05) among treatments. The BUC silage had 51% greater aerobic stability (> 177 h) than ot her silages. A meta-analysis based on 23 published studies with 43 experiments indicated that applying L. buchneri to corn, grass and small-grain silages improved their aerobic stability (Kleinschmit and Kung, 2006a). The improved stability is attributable to inhibition of fungal growth by the acetate produced when L. buchneri ferments lactate into acetate and 1, 2 pr opanediol (Driehuis et al., 1999a) and the propionic acid sometimes formed from fermentation of the 1,2 propanediol by epiphytic L. diolivorans (Krooneman et al., 2002) This theory is supported by t he numerically greater acetate and propionate concentrations in BUC versus the Control silages. Such differences have been statistically significant in most (Kleinschmit and Kung, 2006a, b; Huisden et al., 2009; Filya et al., 2007) but not all (Mari et al., 2009) other studies.
139 The B2 inoculant containing homolactic Pentosaceus pedioccocus and heterolactic, Propionibacteria freudenrechii may have not improved aerobic stability because of the types of bacteri a it contained. Effects of homolactic bacteria on aerobic stability have been variable (Weinberg et al., 1993b; Driehuis et al., 2001) because the lactate they produce has weak antifungal properties and it can be used as a growth substrate by certain yeasts. Addition of propionibacteria to forages has resulted in inconsistent effects on concentrations of strong antifungal acids (propionate and acetate) and aerobic stability of silage (W einberg et al., 1995; Higginbotham et al., 1996, 1998; Filya et al., 2006; Pedroso et al., 2010) because of the slow growth of propionic acid bacteria at acidic pH (Higgi nbotham et al., 1996; Weinberg et al., 1995). Effects of inoculants containing homolactic bacteria and L. buchneri like B500 on aerobic stability have been positive in some studies (Filya, 2003; Huisden et al., 2009) and dependent on ensiling duration or cultivar in others (Kleinschmit and Kung, 2006b; Kang et al., 2009). Numerically ( P > 0.1), the quantity of spoiled s ilage was 22% greater when B2 was applied, and 18 and 13% lower when BUC or B500 were applied, respectively. The lower spoilage losses of the BUC silage agrees with the greater aerobi c stability of this silage and supports other studies showing that application of L. buchneri increased the aerobic stability of (Driehuis et al., 1999b; Mari et al., 2009) and decreased spoilage losses from (Queiroz et al., 2010) corn silage made in farm-scale silos. No treatment x time interaction was detec ted for any of the animal measurements. Inoculant treatment did not affect intake of DM, CP, NDF, or ADF (Table 6-3). One of the early concerns about L. buchneri inoculation was that the high acetic acid
140 concentrations of inoculated silages would re duce feed intake in ruminant livestock. In agreement with previous studies (Driehuis et al., 1999b; Kendall et al., 2002; Taylor et al., 2002; Kung et al., 2003a), this study shows that inoculation with L. buchneri resulted in a trend for greater acetate c oncentration in the silage but this did not decrease DMI. Inoculant treatment did not affect digesti bility of DM or CP, but cows fed B2 had lower NDF (45.3 vs. 52.3 % of DM) and ADF (45.6 vs. 53.8 % of D M) digestibility than cows fed the control diet. Consequently, cows fed B2 had lower digestible NDF (2.5 vs. 3.0 kg/d) and ADF (1.6 vs. 2.0 kg/d) intake than cows fed the control diet. This may have been due to the numerically greater spoilage in the B2 silage. Whitlock et al. (2000) reported that DMI and NDF digestibility markedly decreased as the rate of inclusion of spoiled corn silage increased in a steer ration. Queiro z et al. (2010) also reported spoilage of corn silage resulted in decreased amounts of gross energy, CP, ash, NDF, and ADF in the silage. Body weight, ADG, BCS, and plasma gluc ose concentration were not affected by treatment (Table 6-4), whereas PUN concentration was greater in cows fed inoculated silages instead of the Control silage (13.2 vs. 11.4 mg/dl). Inoculant application typically decreases the WSC concentration of silages (McDonald, 1991). Feeding such silages instead of the Control silage may have reduc ed ruminal ammonia utilization, leading to greater PUN concentrations. The lower NDF and ADF digestibility of the B2 diet did not adversely affect milk production or composition (Table 6-5), possibly due to differences in NFC concentrations between diets. Milk yield, milk composition, milk component yield, somatic cell counts, and feed efficiency (k g milk/kg DMI) were unaffected by any
141 treatment. Milk produc tion was increased by L. buchneri -treatment in only one (Kung et al., 2003a) of the previous studies that investigated effects of application of L. buchneri to silages on milk production by cows (Dri ehuis et al., 1999b; Kendall et al., 2002; Taylor et al., 2002; Kung et al., 2003a). T he improvement in milk production occured when L. buchneri was applied to alfalfa at ensiling but application to corn and smallgrain cereals in the other studies had no effect. Therefore, the milk production results in this study agree with those in most of the previous studies. Conclusions Inoculant applic ation did not affect the ferme ntation of the corn silage. Inoculation with L. buchneri 40788 increased the aerobic stability of the silage and resulted in numerically less spoilage losses than the other treatments. Inoculation with combinations of L. buchneri 40788 and L. plantarum or Pedioccocus pentosaceus and Propionibacteria freudenrichii had no effect on aerobic stability. Inoculant application did not affect the feed intake, milk yield or efficiency of feed utilization by dairy cows. These results show that treating corn silage with L. buchneri 40788 can improve the aerobic stability of corn silage but none of the inoculants improv ed the performance of dairy cows.
142 Table 6-1. Ingredient and chemical co mposition of the experimental diets. % of TMR (DM basis) Item Ingredient composition Corn silage 44.0 Alfalfa hay 6.0 Citrus pulp 4.9 Corn gluten feed 4.6 Soy plus 9.1 Corn meal 17.9 Soybean meal 8.8 Mineral mix 1 4.7 Chemical composition Treatments 2 CON B2 BUC B500 DM, % 65.9 65.7 66.2 65.4 Ash, % DM 7.7 7.6 7.7 7.6 CP, % DM 18.2 18.1 18.3 18.3 NDF, % DM 29.9 26.9 28.9 28.6 ADF, % DM 18.7 16.7 17.6 17.8 Hemicellulose, % D M 11.2 10.2 11.2 10.8 NFC, % DM 40.8 43.9 41.8 42.1 1Mineral Mix contained 24.0% of CP, 9.5% of Ca, 1.0% of P, 7.0% of K, 2.5% of Mg, 7.9% of Na, 0.5% of S, 1400 mg/kg of Mn, 430 mg/kg of Cu, 1500 mg/kg of Se, 0.05 mg/kg of Cl, 15 mg/kg of I 2 CON = Control, no inoculant; B2 = Biotal Plus II, containing Pediococcus pentosaceus and Propionibacteria freudenreichii ; BUC = Buchneri 40788, containing Lactobacillus buchneri ; B500 = Buchneri B500, containing P. pentosaceaus and L. buchneri
143 Table 6-2. Chemical composition and fe rmentation indices of corn silages. Item CON B2+ BUC 500 SEM DM, % 34.0 33.8 34.8 32.9 1.24 CP, % DM 8.8 8.7 9.2 9.1 0.15 NDF, % DM 45.6 38.7 43.0 42.6 0.557 ADF, % DM 28.9 24.3 26.3 26.8 0.43 pH 4.00 3.90 4.02 3.90 0.17 Ammonia-N, % of CP 7.33 8.39 7.61 7.32 0.95 Total acids, % DM 4.34 4.89 5.10 4.63 1.05 Lactic acid, La, % DM 2.27 2.75 2.52 2.47 0.62 Acetic acid, Ac, % DM 1.62 1.86 2.04 1.89 0.57 Propionic acid, % DM 0.43 0.27 0.47 0.25 0.14 Butyric acid, % DM 0.01 0.01 0.01 0.01 0.00 Isobutyric acid, % DM 0.01 0.01 0.07 0.01 0.06 Ethanol, % DM 0.01 0.01 0.01 0.01 0.00 La:Ac ratio 1.49 1.49 1.23 1.36 0.25 Molds, log cfu/g 3. 25 3.25 3.58 3.25 0.55 Yeasts, log cfu/g 3. 00 3.00 3.00 3.72 0.42 Aerobic stability, h 95.2a 85.1a 177.8 b 77.5a 23.6 Spoiled corn silage, kg DM/d 60.0 73.3 49.4 52.5 8.2 Spoiled corn silage, % DM 24.9 27.4 20.7 22.2 Means in the same row with unlike superscripts differed ( P < 0.05) 1 CON = Control, no inoculant; B2 = Biotal Plus II, containing Pediococcus pentosaceus and Propionibacteria freudenreichii ); BUC = Buchneri 40788, containing Lactobacillus buchneri ; B500 = Buchneri 500, containing P. pentosaceaus and L. buchneri
144 Table 6-3. Effect of appl ying different inoculants1 to corn silage at ensiling on feed intake and digestibility in dairy cows. Control B2 BUC B500 SEM Item Intake, kg/d DM 19.4 20.9 20.3 19.5 0.8 CP 3.5 3.8 3.7 3.6 0.2 NDF 5.8 5.6 5.8 5.6 0.2 ADF 3.6 3.5 3.6 3.5 0.2 Digestibility, % DM 73.5 73.6 74.8 73.5 0.8 CP 72.0 72.9 72.3 72.3 0.9 NDF 52.3a 45.3b 53.9a 52.8a 2.0 ADF 53.8a 45.6b 54.2a 53.8a 2.0 Digestible intake, kg/d DM 14.3 15.4 15.2 14.3 0.6 CP 2.5 2.8 2.7 2.6 0.1 NDF 3.0a 2.5b 3.2a 2.9a 0.1 ADF 2.0a 1.6b 1.9a 1.9a 0.1 Means in the same row with unlike superscripts differed ( P < 0.05) 1 CON = Control, no inoculant; B2 = Biotal Plus II, containing Pediococcus pentosaceus and Propionibacteria freudenreichii ); BUC = Buchneri 40788, containing Lactobacillus buchneri ; B500 = Buchneri 500, containing P. pentosaceaus and L. buchneri
145 Table 6-4. Effect of appl ying different inoculants1 to corn silage at ensiling on growth performance and plasma metabolites in dairy cows. Item CON B2+ BUC 500 SEM Growth performance Covariate adjusted BW, kg 602 631 599 582 16 ADG, kg/d 0.06 0.19 0.18 0.41 0.15 Covariate adjusted BCS 2.49 2.52 2.47 2.45 0.04 Plasma metabolites Glucose, mg/dl 67.2 67.5 67.3 66.9 1.1 Urea N, mg/dl 11.4a 13.3 b 13.2 b 13.0 b 0.6 Means in the same row with unlike superscripts differed ( P < 0.05) 1 CON = Control, no inoculant; B2 = Biotal Plus II, containing Pediococcus pentosaceus and Propionibacteria freudenreichii ); BUC = Buchneri 40788, containing Lactobacillus buchneri ; B500 = Buchneri 500, containing P. pentosaceaus and L. buchneri
146 Table 6-5. Effect of appl ying different inoculants1 to corn silage at ensiling on milk yield, milk composition, and effi ciency of feed utilization. Item CON B2 BUC B500 SEM Milk yield, kg/d 32.4 33.3 33.3 30.3 1.3 3.5% FCM, kg/d 29.9 31.8 31.8 29.8 1.6 Milk fat, % 3.26 3.11 3.06 3.28 0.15 Milk protein, % 2. 79 2.81 2.73 2.81 0.06 Milk fat, kg/d 1.01 1.06 1.05 1.01 0.07 Milk protein, kg/d 1.27 1.28 1.24 1.26 0.03 SCC, x 1000/ml 1247 589 1524 1005 463 Feed efficiency (kg milk/kg DMI) 1.56 1.54 1.57 1.55 0.07 Means in the same row with unlike superscripts differed ( P < 0.05) 1 CON = Control, no inoculant; B2 = Biotal Plus II, containing Pediococcus pentosaceus and Propionibacteria freudenreichii ); BUC = Buchneri 40788, containing Lactobacillus buchneri ; B500 = Buchneri 500, containing P. pentosaceaus and L. buchneri
147 CHAPTER 7 GENERAL SUMMARY AND CONCLUSIONS A series of experiments was conducted to determine effects of application of biological additives on the qua lity of dairy cattle feeds and the performance of dairy cows. The objective of Experiment 1 was to determine the effect of dietary addition of a fibrolytic enzyme preparation containing cellulase, xy lanase and esterase activities on the performance of dairy cows fed low or hi gh concentrate diet s. Sixty lactating Holstein cows in early lactation (22 + 3 days in milk) were assigned to the following treatments: 1) Low concentrate (33%) diet (LC); 2) LC plus enzyme (LCE); 3) High concentrate (48%) diet (HC); 4) HC plus enzyme (HCE). The enzyme was sprayed at a rate of 3.4 mg of enzyme/g of DM on the TMR daily for 63 d. The first 14 d were used for adaptation to diets and the la st 49 d for measurements. In addition, four ruminallyfistulated cows were used to determine dietary treatment effects on indices of ruminal fermentation and in situ DM degradation in the rumen. Enzyme application did not affect intake of DM but increased digestibility of DM, CP, ADF, and NDF and increased the efficiency of milk production. Increasi ng the concentrate level reduced ruminal pH but increased intakes of DM and CP, digestibili ty of DM and CP, and milk yield and milk protein yield. Cows fed LCE instead of HC had less DMI, similar milk yield and greater efficiency of milk production. Enzyme applicat ion did not affect ruminal pH or ruminal degradation of the diets. Howe ver, increasing the level of concentrate supplementation decreased the pH, increased t he immediately soluble dietary fraction, and tended to decrease the potentially degradable fraction. In conclu sion, application of the enzyme increased nutrient digestion and the effici ency of milk production by the cows.
148 Experiment 2 was designed to determine if the enzyme used in Experiment 1 primarily exerted its hy drolytic effect prior to ingesti on or within the rumen. A second objective was to determine if the enzyme was more effective on specific components of the diet. Substrates were incubated in a bu ffer or a buffer enzyme solution in triplicate for up to 24 h and chemical composition and DM disappearance were measured. In addition, DMD and NDFD were determined after untreated or enzyme-treated substrates were incubated in W or RF for a further 24 h after the in itial incubation in the buffer or buffer-enzyme solution. Applicat ion of the enzyme reduced concentrations of NDF and hemicellulose, and increased water-soluble carbohydrate WSC concentration and DM disappearance. Incubation of enzymetreated substrates in RF resulted in greater DMD than incubation in W except fo r AH, which had similar DMD in both media. Enzyme addition increased DMD and NDFD in W by 10 and 84% respectively, but had no effect on DMD and NDFD in RF; suggesting that preingestive effects of the enzyme were greater than ruminal effects. En zyme effects on NDF, WS C, and hemicellulose concentration or DMD and NDFD in W or RF did not depend on the substrate. Therefore, this study provided no evidence that the enzym e preferentially hydrolyzed specific substrates and it suggested that preingestive effects of the enzyme were greater than ruminal effects under the conditions of the study. Experiment 3 determined the effect of bac terial inoculants on the fermentation and quality of corn silages. A corn hybrid Vi goro 61R36 (Royster Clark Inc.) was grown and harvested at 35% DM. Chopped corn forage was treated with 1) deionized water (CON); 2) Biotal Plus II (B2) inoculant containing Pediococcus pentosaceus and Propionibacteria freudenreichii ); 3) Buchneri 40788 (BUC) inoculant containing
149 Lactobacillus buchneri ; and 4) Buchneri 500 (B500) inoculant containing P. pentosaceus and L. buchneri Four replicates of each treatment were weighed into polyethylene bags within 20-L mini silos, which were stored for 575 d at ambient temperature (25C) in a covered barn. After silos were opened, aerobic stability, chemical composition, and yeast and mold counts were determined. The DNA from treated and untreated silages wa s isolated using a lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform extraction method. The DNA was used as a template for a conventional PCR with primers designed on the 16S rRNA gene to detect the presence of L. buchneri in silage samples. The WSC concentra tions of all silages were reduced during the fermentation. However, B500 had the greatest residual WSC concentration, suggesting that plant sugars we re less extensively fermented by the bacteria in this inoculant compared to those in other treat ments. Dry matter lo ss was lower in BUC silages compared with Control and B2 silages. Control and B2 silages had higher pH and propionic acid concentration and lower lactic acid concentrations than other treatments. The greater lactat e concentration and lower pH of BUC silages explain the lower DM loss from this silage. Acetat e concentration was greatest in B2 silage, intermediate in Control and BUC silages, and lowest in B500 silage. However, aerobic stability was generally high (> 250 h) and was not improved by inoculant application. The PCR analysis confirmed the pr esence of similar populations of L. buchneri in all treatments, perhaps explaining why aerobic stability was high in all silages. The inoculants had differing effects on the ferm entation of the silages with BUC producing the most desirable fermentation and least DM losses. However, none of the inoculants
150 improved aerobic stability, pr obably because all treatments had high populations of L. buchneri. Experiment 4 determined the effect of appl ying three different bacterial inoculants to corn silage on the performance of lactati ng dairy cows. Corn plants were harvested, chopped, and ensiled in 2.4-m wide bag silos after application of the same treatments as in Experiment 1. Each of the 4 silages was mixed into separate TMR consisting of 44% corn silage, 50% concentrate and 6% alfa lfa hay (DM basis). Fifty-two lactating Holstein cows in early lactation (22 DIM) we re fed for 49 d. Chemical composition and yeast and mold counts of silages did not di ffer among treatments. Treatment with BUC improved silage aerobic stability by 200% and numerically resulted in the least losses compared with other treatm ents. Inoculant treatment did not affect DMI or digestibility of DM or CP. However, cows fed B2 had lower NDF and ADF digestibility than cows fed the control diet. Consequently, cows fed B2 had lower digestible NDF and ADF intake than cows fed the control diet. Neve rtheless, milk yield, milk composition, and feed efficiency were not affected by treatment. Therefore, the inoculants did not affect the performance of the cows, but application of L. buchneri improved the aerobic stability of corn silage. Experiment 1 demonstrated that fibrolytic enzymes containing cellulase, xylanase and esterase improved the nutrient digestion and efficiency of feed utilization by dairy cows in early lactation. That feeding the low concentrate enzyme-treated diet resulted in greater efficiency of milk production than the untreated hi gh concentrate diet implies that the enzyme could be strategically used to reduce the level of concentrates in the diets of dairy cows and thereby reduce the risk of acidosis and associated metabolic
151 diseases without jeopardizing milk production. Future studies should examine the efficacy of the enzyme at other stages of lactation and determine the relative importance of the esterase, xylanase, and ce llulase enzymes in th e mixture. Such studies are needed to ascertain why this enzyme was effective at improving milk production because enzymes used in many si milar studies have been ineffective. Experiment 2 demonstrated t hat application of the enzyme increased cell wall hydrolysis, release of reducing sugars, and DMD and NDFD regardless of substrate type. This implies that there was no benefit to adding the enzyme to specific components of the diet instead of the TMR. Application of the enzyme to the TMR is attractive from the viewpoint of maximizing the distribution of the enzyme in the diet but this method could pose logistical di fficulties for some producers. That enzyme application to substrates in cubated in water increased their DMD and NDFD, whereas application to substrates incubated in ruminal fluid only affected concentrations of fermentation products, sugges ts that the preingest ive effect of the enzyme was important. Consequently, a prei ngestive enzyme substrate interaction period should be ensured when fibrolytic en zymes are added to diets of dairy cows. However, the ideal duration of the period ne eds to be verified by research. Future research should also use longer incubati on periods (30 and 48 h) to compare the preingestive and ruminal effects of the enzyme on the substrates. Application of bacterial inoculants re sulted in contrasting effects on the fermentation and aerobic stability of corn sil age in the mini-silo (Experiment 3) and farmscale silo experiments (Experiment 4) lik ely because of differences in the epiphytic bacterial populations of the untreated corn s ilages. In Experiment 3, the presence of L.
152 buchneri in Control silages proba bly prevented typical effects of inoculation with L. buchneri or Propionibacteria on aerobic stability. In Experiment 4, L. buchneri application produced the normal improvement in aerobic stabililty and resulted in numerically less spoilage losses than other treatments but feeding inoculated silages did not affect the performance of dairy cows Factors that predispose to high epiphytic populations of L. buchneri on corn silages need to be determined because such silages may be inherently aerobically stable. Inoculants that are more potent and consistently effective at improving ani mal performance are needed. Therefore, in addition to screening inoculant bacteria for their ferment ation enhancing and fungicidal properties, they should be selected based on their pot ential to improve nutrient digestion and utilization by dairy cows.
153 APPENDIX A METHOD FOR MEASURING HEMICELLULASE ACTI VITY Xylanase Assay Procedure adapted from Bailey et al. (1992) Xylose Standard 0.1 g of xylose per 100 ml distille d water (stock solution) Make a serial dilution to obtain 0, 0.2, 0.4, 0.6, and 0.8 mg Xylose a) 0 = 0 ml stock solution with 2 ml of distilled water b) 0.2 = 0.2 ml stock solution with 1.8 ml of distilled water c) 0.4 = 0.4 ml stock solution with 1.6 ml of distilled water d) 0.6 = 0.6 ml stock solution with 1.4 ml of distilled water e) 0.8 = 0.8 ml stock solution with 1.2 ml of distilled water Reagents Citrate phosphate buffer 0.1 M (pH 6.0): 0.3761 g of Citric acid 0.9114 g of Na2HPO4 Bring to 100 ml final volume with distilled water. DNS reagent 1 L volume (store in a dark bottle for no more than 2 weeks) 10 g 3,5 dinitrosalacylic acid 10 g NaOH 200 g Sodium Potassium Tartrate 0.5 g Sodium Sulfite 2 g Phenol Bring to 1 L final volume with distilled water Substrate: 1% xylan from oat spelts. 1 g of xylan is homogenized in approxim ately 60 ml of distilled water at 60C Heat and boil using a heating stirrer. Allow to cool overnight with continuous stirring Next day, bring to 100 ml final volume with distilled water Blank preparation Substrate blank: 0.1ml of substrate + 0.9 ml of buffer + 0.1 ml of water
154 Enzyme blank: 0.9 ml of buffer + 0.1 ml of enzyme (diluted in buffer) + 1.0 ml of distilled water Assay ** using 15 ml tubes 1) Add 1.0 ml of substrate 2) Add 0.9 ml of buffer 3) Incubate at 39C for 10 min 4) Add 0.1 ml of enzyme 5) Incubate at 39C for 5 min 6) At this point, standard tubes and blanks should be included for the next steps 7) Add 3 ml of DNS reag ent to terminate reaction 8) Boil in water bath for 5 min 9) Allow to cool in water 10) Read at 540 nm 11) Plot xylose standard 12) Plot samples against standard The following equation is produced: Xylose equivalents (mg) = a + b x Where x is the absorbance obtained after co rrection for the enzyme and the substrate blanks. Xylose equivalents are plotted on the Y-axis and the absorbance values are plotted on the X-axis. The units of activity are then expressed as mol xylose equivalents min-1 ml-1 of enzyme product. **All assays should be carried out at least in triplicate.
155 APPENDIX B METHODS FOR MEASURING CELLULASE ACTIVITY Endoglucanase Assay Procedure adapted from Wood and Bhat (1988). Glucose Standard 0.1 g of glucose per 100 ml distilled water (stock solution) Make a serial dilution t o obtain 0, 0.2, 0.4, 0.6, and 0.8 mg Glucose a) 0 = 0 ml stock solution with 2 ml of distilled water b) 0.2 = 0.2 ml stock solution with 1.8 ml of distilled water c) 0.4 = 0.4 ml stock solution with 1.6 ml of distilled water d) 0.6 = 0.6 ml stock solution with 1.4 ml of distilled water e) 0.8 = 0.8 ml stock solution with 1.2 ml of distilled water Reagents Citrate phosphate buffer 0.1 M (pH 6.0) DNS reagent 1 L volume (store in a dark bottle for no more than 2 weeks) Substrate: 1% (wt/vol) car boxymethylcellulose (CMC). 1 g of CMC is suspended in 100 ml of distilled water Stir under gentle heat until completely dissolved. Add sodium azide (0.02% wt/vol ) to prevent microbial growth Stored at 4C Blank preparation Substrate blank: 0.1ml of substrate + 0.9 ml of buffer + 0.1 ml of water Enzyme blank: 0.9 ml of buffer + 0.1 ml of enzyme (diluted in buffer) + 1.0 ml of distilled water Assay ** using 15 ml tubes 1) Add 1.0 ml of substrate
156 2) Add 0.9 ml of buffer 3) Incubate at 39C for 10 min 4) Add 0.1 ml of enzyme 5) Incubate at 39C for 5 min 6) At this point, standard tubes and blanks should be included for the next steps 7) Add 3 ml of DNS reag ent to terminate reaction 8) Boil in water bath for 5 min 9) Allow to cool in water 10) Read at 540 nm 11) Plot glucose standard 12) Plot samples against standard The following equation is produced: Glucose equivalents (mg) = a + b x Where x is the absorbance obtained after correction for the enzyme and the substrate blanks, and glucose equivalents are placed on the Y-axis. The absorbance is plotted on the X-axis to allow a direct inclusion of its value into a spreadsheet. The units of activity are then expressed as mol glucose equivalents min-1 ml-1 of enzyme product. **All assays should be carried out at least in triplicate.
157 Exoglucanase Assay Procedure adapted from Wood and Bhat (1988). Glucose Standard 0.1 g of glucose per 100 ml distilled water (stock solution) Make a serial dilution t o obtain 0, 0.2, 0.4, 0.6, and 0.8 mg Glucose a) 0 = 0 ml stock solution with 2 ml of distilled water b) 0.2 = 0.2 ml stock solution with 1.8 ml of distilled water c) 0.4 = 0.4 ml stock solution with 1.6 ml of distilled water d) 0.6 = 0.6 ml stock solution with 1.4 ml of distilled water e) 0.8 = 0.8 ml stock solution with 1.2 ml of distilled water Reagents Citrate phosphate buffer 0.1 M (pH 6.0) DNS reagent 1 L volume (store in a dark bottle for no more than 2 weeks) Substrate: 1% (wt/vol) microc rystalline cellulose (Avicel) 1 g of Avicel is suspended in 100 ml of disti lled water Stir continuously while pipeting (subs trate is not soluble in water). Blank preparation Substrate blank: 0.1ml of substrate + 0.9 ml of buffer + 0.1 ml of water Enzyme blank: 0.9 ml of buffer + 0.1 ml of enzyme (diluted in buffer) + 1.0 ml of distilled water Assay ** using 15 ml tubes 1) Add 1.0 ml of substrate 2) Add 0.9 ml of buffer 3) Incubate at 39C for 10 min 4) Add 0.1 ml of enzyme 5) Incubate at 39C for 120 min 6) The tubes are then boiled for 10 min to terminate the reaction.
158 7) After cooling, the tubes are centrifuged at 1000 x g for 10 min 8) A 1.0 ml of sample is withdrawn from each tube and 1.0 ml of distilled water is added 9) At this point, standard tubes and blanks should be included for the next steps 10) Add 3 ml of DNS reagent 11) Boil in water bath for 5 min 12) Allow to cool in water 13) Read at 540 nm 14) Plot glucose standard 15) Plot samples against standard The following equation is produced: Glucose equivalents (mg) = a + b x Where x is the absorbance obtained after correction for the enzyme and the substrate blanks, and glucose equivalents are placed on the Y-axis. The absorbance is plotted on the X-axis to allow a direct inclusion of its value into a spreadsheet. The units of activity are then expressed as mol glucose equivalents min-1 ml-1 of enzyme product. **All assays should be carried out at least in triplicate.
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186 BIOGRAPHICAL SKETCH Kathy Gisela Arriola, the youngest of four children was born in San Francisco, California but she grew up in Lima, Peru. She earned her B.S. in A nimal Science and acquired pre-professional experience in the Investigation Pr ogram for swine, beef, and dairy cattle at the Universidad Nacional Agra ria La Molina, Peru in 1998. After she graduated, she worked for one year as a management consultant for dairy farmers and then returned to Universidad Nacional Agraria La Molina to write a thesis for which she earned the title of Engineer in Animal Scienc es in 2000. In 2004, Kathy was accepted into the M.S. program in the Department of Animal Sciences, University of Florida under the guidance of Dr. Adegbola Adesogan. She received her M.S. degree in ruminant nutrition 2006 and was accepted into the Ph.D program in the D epartment of Animal Sciences at the University of Florida under the guidance of Dr. Adegbola Adesogan. While pursuing her graduate degrees, Kathy taught different classes and participated in several research projects. She was also an active member of the Animal Science Graduate Student Association an d Gamma Sigma Delta. Kathy would like to be employed as a consultant in the dairy industry in the future.