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Effect of Nonfiber Carbohydrates on Product Yield and Fiber Digestion in Fermentations with Mixed Ruminal Microbes

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EFFECT OF NONFIBER CARBOHYDRATES ON PRO DUCT YIELD AND FIBER DIGESTION IN FERMENTATIONS WITH MIXED RUMINAL MICROBES By LUCIA HOLTSHAUSEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Lucia Holtshausen

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This dissertation is dedicated to Heidi Bissell for her friendship duri ng the last three and a half years. It was her support, endless patience and encouragement that carried me through my PhD program.

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iv ACKNOWLEDGMENTS I would like to express my appreciation to all the people who made valuable contributions throughout my PhD program. Firs t, I would like to thank Dr. Mary Beth Hall, chair of my supervisory committee, for he lping me further devel op as a scientist and for her encouragement. I would also like to thank the other members of my committee: Dr. Ramon Littell for his assistance with the statistical analysis, Dr. Adegbola Adesogan for acting as assistant chair on short notice and for always being available to answer even the most trivial of questions, Dr. Christian Cruywagen for continued academic and moral support and for always taking a keen interest in my academic future, and the late Dr. Bill Kunkle for providing valuable practical insight at the onset of my program. Next I would like to thank all the people who helped at va rious stages with the in vitro studies and laboratory analyses: Alexandra Amorocho, Heid i Bissell, Jocelyn Croci, Faith Cullens, Bruno Amaral, Ashley Hughes, Celeste K earney, Colleen Larson, Sergei Sennikov and Tina Sheedy. Without their he lp I would not have been able to do the studies on such a large scale. I would like to thank Dr. Paul Weimer and Chri stine Odt for the analysis of organic acids and their patience in answer ing my questions, Dr. Glen Broderick for amino acid and ammonia nitrogen analys es, and Hangxin Hou from US Sugar in Clewiston (Florida) for sugar analysis. I w ould also like to tha nk Sabrina Robinson for various administrative favors throughout my PhD program. Last but not least I would like to thank the Liquid Feed Committee of the American F eed Industry Association and the Florida Milk Check-Off Progr am for funding this research.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................xi LIST OF ABBREVIATIONS........................................................................................xxiv ABSTRACT.....................................................................................................................xx v CHAPTER 1 REVIEW OF LITERATURE: MI CROBIAL FERMENTATION OF NONNEUTRAL DETERGENT FIBER CARB OHYDRATES, AND HOW IT MAY RELATE TO ANIMAL PERFORMANCE.................................................................1 Introduction................................................................................................................... 1 Chemical Structure and de gradation of NFC Sources..................................................2 Sucrose..................................................................................................................2 Starch.....................................................................................................................3 Pectic Substances...................................................................................................5 NFCs and Microbial Fermentation Products................................................................5 Organic Acids........................................................................................................6 Total volatile fatty acids.................................................................................6 Acetic acid......................................................................................................7 Propionic acid.................................................................................................9 Butyric acid..................................................................................................11 Branched chain volatile fatty acids..............................................................12 Lactic acid....................................................................................................13 Microbial Mass....................................................................................................14 Microbial composition.................................................................................14 Microbial protein yield.................................................................................15 Microbial -glucan.......................................................................................17 Other Factors Which Influen ce Microbial Product Yield...........................................18 Nitrogen Source...................................................................................................18 Fermentation pH..................................................................................................21 NFCs and Ruminal pH, Fiber Dige stion and Animal Performance...........................22 Ruminal pH and Fiber Digestion.........................................................................22 Animal Performance............................................................................................24

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vi Dry Matter Intake................................................................................................25 Milk Production and Composition......................................................................25 2 EFFECT OF PH ON MICROBIAL YI ELD AND NEUTRAL DETERGENT FIBER DIGESTION FROM IN VITRO FE RMENTATIONS OF SUCROSE AND ISOLATED NEUTRAL DETERGENT RESIDUE...................................................34 Introduction.................................................................................................................34 Materials and Methods...............................................................................................35 Substrates.............................................................................................................35 Medium and reducing solution............................................................................35 Fermentation........................................................................................................36 Sample Handling and Subsequent Analyses.......................................................37 Statistical Analysis..............................................................................................39 Results and Discussion...............................................................................................41 Residual Sucrose.................................................................................................41 Microbial Glycogen.............................................................................................43 Fermentation pH..................................................................................................44 Organic Acids......................................................................................................45 Protein Degradation Products..............................................................................47 Neutral Detergent Fiber Digestion......................................................................48 Microbial Crude Protein Yield and Efficiency....................................................49 Conclusions.................................................................................................................50 3 EFFECT OF NITROGEN SOURCE ON MICROBIAL YIELD AND NEUTRAL DETERGENT FIBER DIGE STION FROM IN VITR O FERMENTATIONS OF SUCROSE AND ISOLATED NEUTRAL DETERGENT RESIDUE......................58 Introduction.................................................................................................................58 Materials and Methods...............................................................................................59 Substrates.............................................................................................................59 Medium................................................................................................................60 Fermentation........................................................................................................60 Sample Handling and Subsequent Analyses.......................................................61 Statistical Analysis..............................................................................................64 Results and Discussion...............................................................................................66 Residual Substrate...............................................................................................66 Microbial Glycogen.............................................................................................67 Fermentation pH..................................................................................................67 Organic Acids......................................................................................................68 Protein Degradation Products..............................................................................69 Neutral Detergent Fiber Digestion......................................................................70 Microbial Crude Protein Yield and Efficiency....................................................71 Conclusions.................................................................................................................72

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vii 4 MICROBIAL PRODUCT YIELD AND NEUTRAL DETERGENT FIBER DIGESTION FROM IN VITRO FERMEN TATIONS WITH SUCROSE, STARCH AND PECTIN IN COMBINATION WITH ISOLATED BERMUDAGRASS NEUTRAL DETERGENT RESIDUE.......................................................................83 Introduction.................................................................................................................83 Materials and Methods...............................................................................................84 Substrates and Treatments...................................................................................84 Fermentation........................................................................................................86 Sample Handling and Subsequent Analyses.......................................................88 Statistical Analysis..............................................................................................91 Treatment mean comparisons and temporal pattern descriptions................91 Comparisons of maxima, minima and 24 h data..........................................92 Comparisons of NFC mixtures.....................................................................94 Results and Discussion...............................................................................................95 Residual Sugars (Sucrose, Glucose, Fructose)....................................................95 Microbial Glycogen Yield...................................................................................96 Fermentation pH..................................................................................................97 Organic Acids......................................................................................................99 Neutral Detergent Fiber Digestion....................................................................104 Microbial Crude Protein Yield..........................................................................106 Conclusions...............................................................................................................107 5 CONCLUSIONS......................................................................................................121 APPENDIX A ADDITIONAL FIGURES FOR CHAPTER 2.........................................................124 B ADDITIONAL FIGURES FOR CHAPTER 3.........................................................126 C FIGURES FOR CHAPTER 4...................................................................................128 D ADDITIONAL TABLE FOR CHAPTER 4.............................................................166 E CHAPTER 2 RAW DATA.......................................................................................168 F CHAPTER 3 RAW DATA.......................................................................................179 G CHAPTER 4 RAW DATA.......................................................................................193 LIST OF REFERENCES.................................................................................................281 BIOGRAPHICAL SKETCH...........................................................................................293

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viii LIST OF TABLES Table page 1-1 The effects of NFC source on ruminal or fermentation pH and organic acid profile.28 1-2 Effects of NFC source on dry matter inta ke, milk production and milk composition.31 2-1 Type and number of fermentation tubes per medium for each sampling hour, indicating the substrat e and analysis for which tubes we re reserved in an 24 h in vitro fermentation of sucrose and iNDF...................................................................51 2-2 Residual glucose, fructose, unhydroly zed sucrose, monos accharide sucrose equivalent (glucose+fructose) and sucrose equivalent at 0, 4 and 8 h, and averaged for 24 h in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue with initial medium pH of 6.8 or 5.6...........................................51 3-1 Type and number of fermentation tubes per medium for one sampling hour, indicating the substrat e and analysis for which tubes were reserved in a 16 h in vitro fermentation of sucrose and is olated neutral detergent fiber...........................73 3-2 Residual glucose, fructose, sucrose, m onosaccharide sucrose equivalent (glucose + fructose) and sucrose equivalent at 0, 4 and 8 h, and averaged for 16 h in vitro fermentations of sucrose and isolated be rmudagrass neutral detergent residue with different sources of nitrogen in media......................................................................74 3-3 Organic acid concentrations (least squa res means) at 16 h (corrected for blank fermentations) for in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue with differe nt sources of nitrogen in media.....................75 3-4 Maximum microbial crude protein (MCP) yield (hour of maximum) and efficiency of MCP yield at the point of maximum MCP yield for in vitro fermentations of iNDF and of SuNDF with different source of nitrogen in media. Values are least squares means...........................................................................................................75 4-1 Layout of treatments and substrate amount s for a series of three 24 h in vitro fermentations (performed in duplicate) of a mixed batch culture..........................108 4-2 Residual glucose, fructose, unhydrolyzed su crose and sucrose equi valent (mg) at 0 and 4 h for 24 h in vitro fermentations of iNDF, NFC sources (s ucrose, starch and pectin), and combinations of NFCs. Values are least squares means...................109

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ix 4-3 Maximum microbial glycogen (GLY) yi eld (mg), hour of maximum yield and temporal patterns for 24 h in vitro ferm entations of iNDF, NFC sources (sucrose and pectin), and combinations of NFCs Values are least squares means............110 4-4 Main effects and regression coefficien ts for maximum microbial glycogen (GLY) yield (mg) for increasing he xose equivalent amounts of NF Cs (sucrose, starch and pectin) fermented, and regression coe fficients for fermentations of NFC combinations..........................................................................................................111 4-5 Fermentation pH (mean, minimum, hour of minimum and tempor al pattern) for 24 h in vitro fermentations of iNDF, NFC s ources (sucrose, starch and pectin), and combinations of NFCs. Valu es are least squares means.......................................112 4-6 Main effects and regression coeffici ents for minimum fermentation pH for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations...113 4-7 Volatile fatty acid concentrations at 24 h and maximum lactate concentrations (hour of maximum indicated) for 24 h in vi tro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinat ions of NFCs. Values are least squares means. ...............................................................................................................114 4-8 Main effects and regression coefficients for maximum organic acid concentrations for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations...115 4-9 Residual NDFOM at 24 h and temporal patterns for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means..............................................................................117 4-10 Main effects and regression coefficients for residual NDFOM at 24 h for increasing hexose equivalent amounts of NFCs (suc rose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations...........................118 4-11 Microbial crude protein (MCP) yiel d (mean, maximum, hour of maximum and temporal pattern) for 24 h in vitro ferm entations of iNDF, NFC sources (sucrose, starch and pectin), and comb inations of NFCs. Values are least squares means..119 4-12 Main effects and regression coefficien ts for maximum microbial crude protein (MCP) yield for increasing hexose equivale nt amounts of NFCs (sucrose, starch and pectin) fermented, and regression co efficients for fermentations of NFC combinations..........................................................................................................120 D-1 Temporal patterns of organic acid concen trations for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs......167

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x E-1 Data used for statistical analysis in ev aluating the effect of pH on microbial yield and neutral detergen fiber digestion from in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue....................................................168 F-1 Data used for statistical analysis in evaluating the effect of nitrogen source on microbial yield and neutral detergen fiber digestion from in vitro fermentations of sucrose and isolated bermudagra ss neutral detergent residue................................179 G-1 Data used for statistical analysis in evaluating the effect on microbial yield and neutral detergen fiber digestion from in vitro fermentations of different sources (sucrose, starch and pectin), amounts (0, 40, 80 and 120 mg nominal hexose equivalents) and combinations (sucrose+s tarch, starch+pectin and sucrose+pectin) of NFCs. ...............................................................................................................193

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xi LIST OF FIGURES Figure page 2-1 Microbial glycogen yield (LSmeans standard error) for 24 h in vitro fermentations of SuNDF with initial medium pH of 6.8 ( ) or 5.6 ( ). SuNDF = sucrose + isolated bermudagra ss neutral detergent residue.....................................52 2-2 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF ( ) and SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF...........................................................................................................52 2-3 Acetate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue......................................................................................................53 2-4 Propionate concentrations (LSmeans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue...................................................................53 2-5 Butyrate concentrations (LSmeans sta ndard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue......................................................................................................54 2-6 Total volatile fatty acid concentrations (L Smeans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue...................................................................54 2-7 Lactate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue......................................................................................................55

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xii 2-8 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue...................................................................55 2-9 Ammonia nitrogen concentr ation (LSmeans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue...................................................................56 2-10 Total free amino acid concentration (LSmeans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue...................................................................56 2-11 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of (iNDF; ) and SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose + iNDF.........................................................................................................57 2-12 Microbial crude protein yield (LSmea ns standard error) for 24 h in vitro fermentations of SuNDF with an initial medium pH of 6.8 ( ) or 5.6 ( ). SuNDF = sucrose + isolated bermudagra ss neutral detergent residue..................................57 3-1 Microbial glycogen yield (least squares means standard error) for 16 h in vitro fermentations of SuNDF with media containi ng nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermuda grass neutral detergent residue.....................76 3-2 Fermentation pH (least squares means standard error) for 16 h in vitro fermentations of iNDF ( , ) and SuNDF ( , ) with media containing nitrogen in the form of non-pr otein nitrogen + true protein ( or ), true protein only ( or ) or non-protein nitrogen only ( or ). iNDF = isolated bermudagrass neutral detergent re sidue; SuNDF = sucrose+iNDF.........................76 3-3 Total volatile fatty acid concentrations (L Smeans standard error) for 16 h in vitro fermentations of SuNDF with media containi ng nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermuda grass neutral detergent residue.....................77 3-4 Acetate concentrations (LSmeans standa rd error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagra ss neutral detergent residue.....................................77

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xiii 3-5 Propionate concentrations (LSmeans standard error) for 16 h in vitro fermentations of SuNDF with media containi ng nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermuda grass neutral detergent residue.....................78 3-6 Butyrate concentrations (LSmeans sta ndard error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagra ss neutral detergent residue.....................................78 3-7 Lactate concentrations (LSmeans standa rd error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagra ss neutral detergent residue.....................................79 3-8 Ammonia nitrogen concentr ation (LSmeans standard error) for 16 h in vitro fermentations with no substrate ( , ) or SuNDF as the substrate ( , ) and media containing nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose+isolated bermudagrass neutral detergent residue.......................................79 3-9 Total free amino acid concentration (LSmeans standard error) for 16 h in vitro fermentations with no substrate ( , ) or SuNDF as the substrate ( , ) and media containing nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose+isolated bermudagrass neutral detergent residue.......................................80 3-10 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 16 h in vitro fermentation with no substrate ( , ) or SuNDF as the substrate ( , ) and media containing nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose+isolated bermudagr ass neutral detergent residue.......................80 3-11 Residual NDFOM for 16 h in vitro fermentations of iNDF (A; , ) and SuNDF (B; , ) with media containing nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). iNDF = isolated bermudagrass neutral de tergent residue; SuNDF = sucrose+iNDF.81 3-12 Microbial crude protei n yield for 16 h in vitro fermentations of iNDF ( , ) and SuNDF ( , ) with media containing nitrogen in the form of non-protein nitrogen + true protein ( or ), true protein only ( or ) or non-protein nitrogen only ( or ). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF...........................................................................................................82 A-1 Residual glucose content (LSmeans standa rd error) for 24 h in vitro fermentations of SuNDF with initial medium pH, befo re addition of reducing solution or inoculum, of 6.8 ( ) or 5.6 ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue....................................................................................................124

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xiv A-2 Residual fructose content (LSmeans standard error) for 24 h in vitro fermentations of SuNDF with initial me dium pH, before addition of reducing solution or inoculum, of 6.8 ( ) or 5.6 ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.................................................................125 A-3 Residual sucrose content (LSmeans standa rd error) for 24 h in vitro fermentations of SuNDF with initial medium pH, befo re addition of reducing solution or inoculum, of 6.8 ( ) or 5.6 ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue....................................................................................................125 B-1 Residual glucose content (LSmeans standa rd error) for 16 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagra ss neutral detergent residue...................................126 B-2 Residual fructose content (LSmeans standard error) for 24 h in vitro fermentations of SuNDF with media containi ng nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermuda grass neutral detergent residue...................127 B-3 Residual sucrose content (LSmeans standa rd error) for 24 h in vitro fermentations of SuNDF with media containing nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagra ss neutral detergent residue...................................127 C-1 Residual glucose content (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......128 C-2 Residual glucose content (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......129 C-3 Residual glucose content (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......129 C-4 Residual glucose content (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................130

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xv C-5 Residual glucose content (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................130 C-6 Residual glucose content (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................131 C-7 Residual fructose content (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................131 C-8 Residual fructose content (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................132 C-9 Residual fructose content (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................132 C-10 Residual fructose content (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................133 C-11 Residual fructose content (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC comb inations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................133 C-12 Residual fructose content (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................134

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xvi C-13 Residual sucrose content (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......134 C-14 Residual sucrose content (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......135 C-15 Residual sucrose content (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......135 C-16 Residual sucrose content (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................136 C-17 Residual sucrose content (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................136 C-18 Residual sucrose content (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................137 C-19 Microbial glycogen yield (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................137 C-20 Microbial glycogen yield (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................138 C-21 Microbial glycogen yield (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................138

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xvii C-22 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......139 C-23 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......139 C-24 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......140 C-25 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated bermudagrass neutra l detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................................................140 C-26 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated bermudagrass neutra l detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................................................141 C-27 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................141 C-28 Acetate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......142 C-29 Acetate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......142 C-30 Acetate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......143 C-31 Acetate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................143

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xviii C-32 Acetate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................144 C-33 Acetate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................144 C-34 Propionate concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................145 C-35 Propionate concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................145 C-36 Propionate concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................146 C-37 Propionate concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................146 C-38 Propionate concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC comb inations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................147 C-39 Propionate concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................147

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xix C-40 Butyrate concentrations (LSmeans sta ndard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......148 C-41 Butyrate concentrations (LSmeans sta ndard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......148 C-42 Butyrate concentrations (LSmeans sta ndard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......149 C-43 Butyrate concentrations (LSmeans sta ndard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................149 C-44 Butyrate concentrations (LSmeans sta ndard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................150 C-45 Butyrate concentrations (LSmeans sta ndard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................150 C-46 Total volatile fatty acid concentrations (L Smeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................151 C-47 Total volatile fatty acid concentrations (L Smeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................151 C-48 Total volatile fatty acid concentrations (L Smeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................152

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xx C-49 Total volatile fatty acid concentrations (L Smeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................152 C-50 Total volatile fatty acid concentrations (L Smeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC comb inations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................153 C-51 Total volatile fatty acid concentrations (L Smeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................153 C-52 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....................................................................................................154 C-53 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....................................................................................................154 C-54 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....................................................................................................155 C-55 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isol ated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.....................................155 C-56 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolat ed bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.....................................156

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xxi C-57 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.....................................156 C-58 Lactate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......157 C-59 Lactate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......157 C-60 Lactate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......158 C-61 Lactate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................158 C-62 Lactate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................159 C-63 Lactate concentrations (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................159 C-64 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......160 C-65 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......160 C-66 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......161

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xxii C-67 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated bermudagrass neutra l detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................................................161 C-68 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated bermudagrass neutra l detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................................................162 C-69 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................162 C-70 Microbial crude protein yield (LSmea ns standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................163 C-71 Microbial crude protein yield (LSmea ns standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................163 C-72 Microbial crude protein yield (LSmea ns standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolat ed bermudagrass neutral detergent residue. ...............................................................................................................164 C-73 Microbial crude protein yield (LSmea ns standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................164 C-74 Microbial crude protein yield (LSmea ns standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC comb inations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated berm udagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...............................................................165

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xxiii C-75 Microbial crude protein yield (LSmea ns standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate...................................................165

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xxiv LIST OF ABBREVIATIONS ADF acid detergent fiber ApH acidic pH medium B non-protein nitrogen+ true protein medium BCVFA branched chain volatile fatty acid BW body weight C true protein only medium CP crude protein DM dry matter DMI dry matter intake GLY microbial glycogen iNDF isolated neutral detergent residue MCP microbial crude protein MCPeff microbial crude protein efficiency N Nitrogen NDF neutral detergent fiber NDFCP neutral detergent fiber crude protein NDFOM neutral detergent organic matter NFC non-neutral detergent fiber carbohydrate NH3-N ammonia nitrogen NpH neutral pH medium NPN non-protein nitrogen NRC National Research Council NSC non-structural carbohydrate OM organic matter OMD organic matter digested OMI organic matter intake Pe Pectin RDP rumen degradable protein St Starch Su Sucrose SuNDF sucrose + isolated neutral detergent residue TCA trichloroacetic acid U non-protein nitrogen only medium VFA volatile fatty acid

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xxv Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF NONFIBER CARBOHYDRATES ON PRO DUCT YIELD AND FIBER DIGESTION IN FERMENTATIONS WITH MIXED RUMINAL MICROBES By Lucia Holtshausen December 2004 Chair: Mary Beth Hall Major Department: Animal Sciences Effects of nonfiber carb ohydrate (NFC) supplementation on fiber digestion and fermentation product yields were examined in three in vitro fermentation studies. Studies 1 and 2 respectively examined the effects of medium pH (5.6 vs. 6.7) and nitrogen source (non-protein nitrogen (U) vs. tr ue protein (C) vs. mixture (B)) on fermentation of isolated neutral detergent residue (iNDF) with or without sucrose (Su). Study 3 examined the effect of supplementing iNDF with starch, sucrose, pectin or their combinations. Anaerobic fermentations of 24 h (Studies 1 a nd 3) and 16 h (Study 2) were performed in batch culture with Goering and Van Soest medium and rumen inoculum. Fermentation samples were analyzed for residual substr ate, pH, iNDF digestion, and microbial fermentation products. In Study 1, maximum microbial crude prot ein (MCP) and glycogen (GLY) yields for Su+iNDF were greater at pH 6.7 (MCP: 19.4 mg; GLY:6.0 mg) than those at pH 5.7 (MCP:11.1 mg; GLY:3.5mg). At pH 6.7, 24 h iNDF digestion was greater for Su+iNDF

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xxvi (42.4%) than for iNDF (26.4%) and the reve rse was true at pH 5.6 (Su+iNDF: 2.2%; iNDF: 7.8%). In Study 2, maximum MCP yields from fermentations of Su+NDF in media containing C (15.7 mg), and B ( 14.3 mg) were greater than t hose containing U (8.05 mg). At 16 h, iNDF digestion for Su+iNDF was lower for U vs. B and C, with no difference among treatments for iNDF. Maximum GLY wa s similar among nitrog en treatments. In Study 3, sucrose decreased pH more than NFC combinations (sucrose+starch, starch+pectin, sucrose+pec tin) followed by pectin, starch and iNDF (6.83, 6.87, 6.89, 7.03 and 7.13, respectively). Residual iNDF was increased by sucrose (60.6%), not affected by starch (61.5%), and decreased by pectin (65.7%) and NFC combinations (63.6 %) compared to iNDF fermented al one (61.8%). Maximum MCP yield was greatest for NFC combinations followed by p ectin, starch, sucrose and iNDF fermented alone (22.4, 18.6, 16.6, 14.8 and 5.12 mg, respectively). The various types of NFCs as well as pH and nitrogen source al tered the yield of microbial products and extent of fiber diges tion. Treatment of NFCs as a uniform entity in ruminant nutrition is not warranted.

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1 CHAPTER 1 REVIEW OF LITERATURE: MICROBIA L FERMENTATION OF NON-NEUTRAL DETERGENT FIBER CARBOHYDRATES, AND HOW IT MAY RELATE TO ANIMAL PERFORMANCE Introduction In order to meet the nutriti onal needs of high producing b eef and dairy cattle, it is necessary to supplement diets with energy a nd protein rich feeds. Grain and byproduct feeds, such as molasses, have been used to increase the energy dens ity of ruminant diets (Huntington, 1997; Lykos et al., 1997). Othe r byproduct feeds, such as soybean hulls (Royes et al., 2001), almond hulls (Grasser et al., 1995), sugar beet pulp (Mansfield et al., 1994) and citrus pulp (Ammerman et al., 1963; Van Horn et al., 1975; Fegeros et al., 1995; Leiva et al., 2000; Arthington et al., 2002), have been used as substitutes for grains. These byproduct feeds have lower starch cont ents and higher neutral detergent soluble fiber (NDSF) contents (compared to grain feeds), and may have high sugar contents (Hall, 1998). Starch, sugars (monoand o ligosaccharides) and NDSF (non-starch, nonneutral detergent fiber polysaccharides) ar e three of the majo r components of the carbohydrate fraction of feeds referred to as non-neutral detergent fiber carbohydrates (NFCs). The NFC fraction of feedstuffs is estimate d from the following calculation: NFC = 100 – crude protein – ether extr act – ash – neutral detergen t fiber + neutral detergent insoluble crude protein (National Resear ch Council [NRC], 2001). The terms nonstructural carbohydrates (NSCs) and NFCs have at times been used interchangeably for the fraction derived by this ca lculation. However, NSCs refer to cell contents, and

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2 include organic acids (which are not carbohydrat es), monoand oligosaccharides, starch and fructans, whilst NFCs also include soluble fiber: pectic substances, -glucans and galactans (Van Soest et al., 1991). Therefore, NFCs include structur al and non-structural carbohydrates, as well as fibrous and nonfibrous carbohydrates (Hall, 1998). From a nutritional standpoint, some of the carbohydrates in plants are also designated as dietary fiber. This term refe rs to the non-starch polysaccharides that are not digestible by mammalian enzymes. Dietary fiber encompasses both NDSF and neutral detergent fiber (NDF), thus, includ ing both cell wall carbohydr ates and some cell contents. Cellulose, hemicelluloses, lignin, pectic substances, -glucans, fructans and galactans are all dietary fiber. Pectic substances, -glucans, fructans and galactans are soluble in neutral detergen t solution, and thus are in cluded in the NFC fraction. The NRC (2001) applies a total digestible nutrient content of 98% to the NFC fraction and it can therefore play a major role in the nutrien t supply to the animal. The rest of this discussion will focus on sucros e, starch and pectic substances (more specifically, pectin), as three of the major components of NFCs. Chemical Structure and degradation of NFC Sources When trying to understand or predict anim al responses to supplementation of NFC sources it is necessary to k eep in mind that this carbohydrate fraction is by no means a homogenous entity, chemically or nutritionall y. Some of the fundamental differences of the individual components are found in their chemical structure. Sucrose Sucrose is the primary vehicle for energy tr ansport in plants and the majority of plants convert sucrose into polymeric forms for storage (Van Soest, 1994). Sucrose, and its constituent monosaccharides glucose and fr uctose, are the predom inant saccharides of

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3 the monoand oligosaccharide component of NFC and are found in byproduct feeds such as molasses (Kunkle et al., 2000), sugar beet pulp (Hall, 2002) and citrus pulp (BenGhedalia et al., 1989). Sucrose is a disacchar ide consisting of single glucose and fructose monomers linked through an -1 2 linkage. It is considered to be 100% degradable in the rumen (Sniffen et al., 1992) and is reported to be fermented at a rate as high as 300%/h (Sniffen et al., 1983). Sucrose is hydr olyzed to glucose and fructose by the enzyme sucrase (Van Soest, 1994). Rumina l bacteria that ferment sucrose include Streptococcus bovis, Lachnospira multiparus, Lactobacillus ruminis, Lactobacillus vitulinis, Clostridium longispor um, Eubacterium cellulosolvens, and some strains of Eubacterium ruminantium, Butyrivibrio fibri solvens, Ruminococcus albus, Ruminococcus flavefaciens, Megaspaera elsdenii, Prevotella spp. Selenomonas ruminantium and Succinivibrio dextrinosolvens (Stewart et al., 1997). Starch Starch is a polymer of glucose molecules and is the major storage carbohydrate in most cereal grains. It consists of amylose, a predominantly linear -(1 4) linked polymer, and amylopectin, an -(1 4) linked polymer with -(1 6) linked branches, which can be present in various ratios. Amyl opectin comprises 70 to 80% of most cereal starches and amylose 20 to 30% (Rooney and Pflugfelder, 1986). The proportions of these two polysaccharides appear to affect the digestion characteristics of starch. There are contradictions in the literature as to the ease of hydrolysis of amylose as compared to amylopectin. Rooney and Pflugfelder (1986) described starch granules as having crystalline and amorphous areas, with amyl opectin comprising the majority of the crystalline region and amylose that of the amorphous region. According to Rooney and Pflugfelder (1986) amylase attack starts in the amorphous region and hydrolysis of the

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4 crystalline areas occurs more slowly. However, the author also stated that the digestibility of starch is inversely proportiona l to the amylose content. According to Piva and Masoero (1996) amylose is slowly degrad ed in the rumen, whereas amylopectin is more rapidly degraded. The reason for th e conflicting statements may be because amylose and amylopectin both contain crysta lline areas and the crystalline area of amylose has greater crystal strength (Van So est, 1994). Therefore, hydrolysis may start in the amorphous region of amylose, exposi ng the amylopectin for hydrolysis, while the crystalline region of amylose hydrolyzes more slowly. Nonetheless, starch fermentation in the rumen is considered to be extensiv e, but can vary from 40 to 90% (NRC, 2001) depending on factors such as structure (amy lose/amylopectin ratio; (Piva and Masoero, 1996), plant source (Rooney and Pflugfelder, 1986) and pr ocessing or physical form (Baldwin and Allison, 1983). Starch can be degraded by ruminal microbial enzymes as well as enzymes in the small intestine of the animal. A series of enzymes are required to degrade amylose and amylopectin to glucose in both the rumen a nd small intestine. These include randomly acting endo-amylases releasing maltodextrins from amylose and -amylases removing maltose units from the non-reducing end of the chain. Approximately 50% of amylopectin can be degraded by -amylase to maltose. The residue is hydrolyzed by glucoamylase (cleaving -(1 4) linkages), and -dextrin-6-glucanohydrolase and isoamylase (cleaving at the -(1 6) linked branch points). Maltose and maltodextrins are degraded to glucose by -glucosidase (Chesson and Forsberg, 1997). There are numerous amylolytic rumina l bacteria, which include Ruminobacter amylophilus

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5 Prevotella ruminicola Succinimonas amylolytica S. bovis S. ruminantium B. fibrisolvens E. ruminantium and Clostridium spp (Cotta, 1988). Pectic Substances Pectic substances are found in the middle lamella and other cell wall layers (Van Soest, 1994), but are not covale ntly linked to the lignified portions. They are almost completely digested (90-100%) in the rume n (Nocek and Tamminga, 1991; NRC, 2001). Pectic substances are a family of complex molecules that contain a great variety of monomers and potential branch -points. The pectin backbone consists of galacturonic acid monomers linked via -(1 4) linkages, and rhamnose inse rts. With the addition of neutral sugar side ch ains, made up largely of arabinose and galactose, bound to the rhamnose inserts, these complex molecules are referred to as pectic substances (Jarvis, 1984). Various degrees of methoxylation (J arvis, 1984) and acetylation (Marounek and Duškov, 1999) of the galacturonic acid bac kbone are possible. The acid groups in the backbone can also be associated with calcium ions (Van Soest, 1994). Animals do not have the enzymes to dige st pectin, but microorganisms in the rumen do (McDonald et al., 1995). At l east two enzymes, a methylesterase and polygalacturonidase, are required for pectin hydrolysis (Baldwin and Allison, 1983). Pectin-utilizing bacteria incl ude some of the prominent ruminal populations such as Fibrobacter succinogenes, P. ru minicola, B. fibrisolvens S. bovis and Lachnospira multiparus (Czerkawski and Breckenridge, 1969; Gradel and Dehority, 1972; Baldwin and Allison, 1983). NFCs and Microbial Fermentation Products Microbial fermentation products play an impor tant part in the nut rient supply to the ruminant animal and thus also in animal performance. Major products of microbial

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6 fermentation of carbohydrates include organi c acids, which are absorbed through the ruminal wall and serve as a source of ener gy for the animal, and microbial mass, which provides potentially metabolizable nutrients in the form of protein, glycogen, and lipids to the animal when they pass to the small in testine. The NFC fraction is considered a good source of readily available energy for micr obial growth (Ariza et al., 2001). It has been suggested that microbial growth is di rectly proportional to the rate of carbohydrate degradation (Russell et al., 1992). As docum ented in the Cornell Net Carbohydrate and Protein System model, simple sugars are cons idered to have a fast degradation rate, and starch and pectin an intermediate rate (S niffen et al., 1992). This would imply that supplementation with sugars would yield more microbial mass and other microbial fermentation products compared to starch and p ectin. There is evidence, both in vitro and in vivo, of differences in fermentation char acteristics among indivi dual NFC components (Table 1-1). Sometimes these results have not been consistent with the notion that individual NFC components with faster degradation rates resu lt in greater microbial yield (Hall and Herejk, 2001). It is important to understand how individual NFC components differ in their effect on microbial product yield and efficiency of yield to help predict and explain some of the variati on seen in animal performance when supplementing with NFCs. Differences among NFC components re garding microbial fermentation may also imply that the complement of NFCs in a part icular feedstuff is important when predicting animal response. Organic Acids Total volatile fatty acids Total volatile fatty acid (VFA) producti on is generally similar among different NFC sources (Table 1-1) both in vitro (Mansf ield et al., 1994; Ari za et al., 2001) and in

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7 vivo (Ben-Ghedalia et al., 1989; Khalili and Huhtanen, 1991a; Chamberlain et al., 1993; Moloney et al., 1994; O'Mara et al., 1997a; Leiva et al ., 2000; Sannes et al., 2002; Voelker and Allen, 2003c). However, Bach et al. (1999) reported an increase ( P > 0.05) in total VFA concentration for cracked corn compared with beet pulp and molasses in a continuous culture study. In contrast total VFA concentration increased ( P = 0.01) or tended to increase ( P = 0.07) in lactating dairy cows fed a total mixed ration (TMR) containing dried citrus pulp and high moisture ear corn in a 50:50 ratio compared to cows receiving a TMR containing high moisture ear corn or cracked shelled corn, respectively (Broderick et al., 2002b). The va rying results in these studies may be due to the fact that the combinations of NFCs that were compar ed differed, which may imply that the effect on VFA production from supplementation with individual NFC s ources may not be additive. Acetate, propionate and butyrate are th e major VFAs included in the total VFA concentration. Despite giving relatively similar total VFA yields there may be differences in the relative proportions of i ndividual VFAs from di fferent NFC sources. Acetic acid Acetate (acetic acid) is a li pogenic nutrient, a precursor of fatty acid synthesis and ultimately of milk fat synthesis in the ma mmary gland (Van Soest, 1994). Starch and sucrose (Sutton, 1979; Khalili and Huhtane n, 1991a; Chamberlain et al., 1993; Moloney et al., 1994; Heldt et al., 1999) have been associated with relative decreases in ruminal acetate concentration, whereas pectin had either no effect (Van Vuuren et al., 1993; Leiva et al., 2000) or increased (Br oderick et al., 2002b; Voelker and Allen, 2003c) acetate in the rumen (Table 1-1). In vitro fermentati ons of different carbohydrates showed a greater

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8 acetate production from pectin compared to starch and sucrose ( P < 0.05), which did not differ from each other ( P > 0.05; [Strobel and Russell, 1986]). The effect of sugars on the ruminal mola r proportion of acetate in vivo may depend on the amount of sucrose or glucose incl uded in the diet (Table 1-1). Sucrose supplementation at 10% of silage DM inta ke did not affect ruminal acetate molar proportion for sheep compared to th ose on the silage control diet ( P > 0.05; [Charmely et al., 1991]). When sucrose was substituted for corn at 3.2% of diet DM in a diet for lactating dairy cows, acetate pr oduction was also not affected ( P = 0.15; [Sannes et al., 2002]). Dextrose (glucose) at 5.6% of diet DM did not aff ect ruminal acetate proportions in heifers compared to a me dium concentrate diet contai ning 39.7% ground barley, or the control diet containi ng 10% ground barley ( P > 0.05; [Piwonka et al., 1994]). However, when starch or sucrose was supplemented at 200g/d (approximately 5% of daily diet DM) to a grass silage diet, ruminal acetate proportions for sheep on the starch-supplemented diet did not differ compared to those on the control diet, whereas sheep on the sucrosesupplemented diet had decreased ruminal acetate proportions ( P < 0.05; [Chamberlain et al., 1993]). When cane molasses, a source of sugars, was fed to steers at 61% of DM intake, ruminal acetate proportions was decreased compared to steers fed a diet with the same amount of barle y, a starch source ( P < 0.01; [Moloney et al., 1994]). Pectin is reported to ferment primarily to acetate (Czerkawski and Breckenridge, 1969; Marounek et al., 1985). When citrus pectin was fermented in cultures of B. fibrisolvens 787 and P. ruminicola AR29, 73.7 and 57.3% of metabolite carbon was captured in acetate, respectively (Marounek a nd Duškov, 1999). Citrus pulp can contain 25.2 to 43.7% neutral detergentsoluble fiber (Hall, 2002), of which pectin is a major

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9 component. Several continuous culture studies reported an in crease in acetate proportion for fermentations of beet pulp and citrus pul p compared to corn (Table 1-1), whether it was included at the same concentration as corn ( P < 0.05; [Bach et al., 1999]) and P = 0.03; [Ariza et al., 2001]) or repl aced a portion of the corn ( P < 0.05; [Mansfield et al., 1994]). This effect was also seen in vivo wh en increasing concentra tions of citrus pulp substituted for high moisture corn in lactati ng dairy cow diets resulted in a linear increase ( P < 0.01) in the ruminal acetate proportion (V oelker and Allen, 2003c). Ben-Ghedalia et al. (1989) also reported increased ruminal acetate proportions for cannulated rams fed dried citrus pulp compared to those fed barley ( P < 0.05). It would appear that fermentation of pectin in general increases the molar proportion of acetate compared to fermentation of sugars and starch, while sugars often decrease the acetate proportion when compared to starch. Propionic acid Propionate (propionic acid) is a precursor for glucose synthesis in the liver and thus important for the glucogenic energy supply to the ruminant. In vitro fermentation with mixed ruminal bacteria yielded similar propionate c oncentrations from starch and sucrose ( P > 0.05; [Strobel and Russell, 1986]), Table 11). The effect of sugars compared to starch on ruminal propionate pr oportion varies among in vivo studies. In some in vivo studies ruminal molar proportions of propionate did not differ between sugars and starch, whether small amounts (5.6% dextrose; Piw onka et al., 1994) or larger amounts (61% molasses; Moloney et al., 1994) of sugar were added to the diet. In contrast, ruminal propionate molar proportions in sheep on a st arch-supplemented diet was similar to that of sheep fed the control ryegrass silage diet ( P > 0.05), whereas ruminal propionate proportions increased ( P < 0.05) for sheep fed a sucrose supplementation (Chamberlain et

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10 al., 1993). In another contrasting study, rumina l molar proportions of propionate tended to increase with starch supplementation ( P = 0.11) compared to supplementation of sugars (sucrose, glucose and fructose) wh en a low amount of ruminally degradable protein (RDP; 0.031% BW/d) was supplem ented to steers, and increased ( P < 0.01) ruminal propionate proportions when a higher amount (0.122% BW/d) of RDP was supplemented (Heldt et al., 1999). It may be that other com ponents of the diet such as protein alter the yield of propionate from NFCs. Pectin yielded less ( P < 0.05) propionate compared to starch and sucrose when fermented in vitro with mixed ruminal bact eria (Strobel and Russell, 1986, Table 1-1). Citrus pectin fermented by a P. ruminicola AR29 in vitro culture yielded a small amount of propionate (3.2 mmol/L), while no propiona te production was detected in the culture with B. fibrisolvens 787 (Marounek and Duškov, 1999). Corn additions increased propionate molar proportions in continuous culture fermentations when compared to similar amounts of citrus pulp ( P = 0.02; [Ariza et al., 2001] and beet pulp ( P < 0.05; [Bach et al., 1999]). Broderick et al. (2002b) also reporte d higher ruminal propionate proportions in lactating dairy cows fed high moisture ear corn ( P < 0.01) and cracked shelled corn ( P = 0.04) compared to cows fed a diet in which citrus pulp substituted for 50% of high moisture ear corn. However, wh en beet pulp was substituted for 50% of the corn in a continuous culture study no diffe rence was found for the molar proportion of propionate ( P > 0.05, [Mansfield et al., 1994]). Othe r researchers also reported no effect on ruminal propionate proportions in lactating dairy cows wh en replacing beet pulp for ground corn (O'Mara et al., 1997a) and replac ing dried citrus pul p for corn hominy (Leiva et al., 2000). The varied propionate response when feeding citrus and beet pulp

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11 could be a result of the variation in composition of these feedstuffs, especially in the ratio of sugars to neutral detergent soluble fiber. It would appear that pectin yields less propionate than sugars and starch, with no clear difference between the latter two NFCs. Butyric acid Butyrate (butyric acid) supplies energy to the animal, mainly to the heart and skeletal muscle, in the form of -hydroxybutyrate (a ketone body; McDonald et al., 1995). It is lipogenic and can be used for the production of fat. Ruminally-produced butyrate is converted to -hydroxybutyrate in the rumina l epithelial cells, and is considered more effective than propionate or acetate in enha ncing development of ruminal papillae (Van Soest, 1994). Overall, it would appear that sucrose yields more butyrate than other NFCs (Table 1-1). In vitro fermentations with mixed ruminal microorganisms yielded more butyrate fr om sucrose compared to starch ( P < 0.05), which in turn yielded more butyrate than pectin ( P < 0.05; [Strobel and Russell, 1986]). Several in vivo studies also reported in creased butyrate production from sucrose compared to starch. Ruminal butyrate propor tions in cannulated steers increased with sugar (sucrose, glucose and fructose) supplem entation compared to supplementation with starch (Heldt et al., 1999). Khalili and H uhtanen (1991a) reported greater ruminal molar proportions of butyrate for bulls consuming a sucrose-supplemented diet compared to a grass silage and barley-based diet. Steers fed a molasses-ba sed diet also had increased ruminal butyrate proportions compared to thos e fed a barley-based diet (Moloney et al., 1994). Studies that have evaluated the fermentation of pectin or feeds that are reported to contain substantial amounts of pectin have shown differences among microorganisms in the yield of butyrate. Pectin fermentation in a B. fibrisolvens 787 culture yielded a small

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12 amount of butyrate (2.6 mmol/L), while no buty rate production was detected in a culture with P. ruminicola AR29 (Marounek and Duškov, 1999). In vivo comparisons of the fermentation of feeds high in starch and thos e that typically cont ain a high proportion of pectin (citrus and beet pulps) have shown no difference (Ben-Gheda lia et al., 1989; Leiva et al., 2000) or an increase in ruminal butyr ate concentration (Br oderick et al., 2002b; Voelker and Allen, 2003c) for animals consumi ng diets containing pulps Citrus pulp can contain between 12.5 and 40.2% sugars, and sugar beet pulp between 12.8 and 24.7% (Hall, 2002). The increase in the proportion of butyrate in these st udies may be a result of the fermentation of sugar rath er than of the soluble fiber content. This emphasizes the need to know the NFC complement of a f eedstuff when evaluating the effect of supplementation on fermentation and animal performance. Branched chain volatile fatty acids The branched chain volatile fatty acids (BCVFAs), isobutyric, iso-valeric and 2methylbutyric acid result from the deamin ation of valine, leucine and iso-leucine, respectively (Van Soest, 1994). Branched chain VFAs serve as carbon skeletons to ruminal microorganisms for the synthesis of microbial crude protein (MCP) from ammonia. In fact, the value of amino acids to cellulolytic orga nisms that have an obligate need for BCVFAs appears to be mainly as a source of BCVFAs (Stern, 1986). Sheep fed diets supplemented with sucrose or starch showed decreased proportions of ruminal BCVFAs compared to those fed a silage control diet (Chamberlain et al., 1993, Table 1-1), and the propor tion of BCVFAs for sheep fed the starch-supplemented diet (2.8 mol/100 mol) was nume rically higher than for those fed the sucrose supplement (1.8 mol/100 mol; difference was no t statistically tested). The ruminal concentrations of BCVFAs for lactating dairy cows were greater for animals fed a corn control diet

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13 compared to those receiving a diet with sucrose ( P = 0.02) substituted for corn at 3.2 % of diet DM (Sannes et al., 2002). Fermentation of corn by mixed ruminal microorganisms in continuous culture studi es gave higher proportions of BCVFAs as compared to citrus pulp ( P = 0.03; [Ariza et al., 2001]) and sugar beet pulp ( P > 0.05; [Mansfield et al., 1994] and P < 0.05; [Bach et al., 1999]). The apparently consistent thread here is that BCVFA concentrations are less for diets with more sucrose relative to starch. Lactic acid Compared to acetate, butyrate and propionate (average p Ka = 4.8), lactate (lactic acid, p Ka = 3.1) is a 10-fold stronger acid (Dawson et al., 1997). An increase in lactate concentration therefore has a greater potential to decrease ruminal pH. The fermentation of sugars and starch can yield lactate (Strobel and Russell, 1986), whereas pectin fermentation is generally not associated with lactate production (Strobel and Russell, 1986; Hatfield and Weimer, 1995). In vitro fe rmentations of sucrose with mixed ruminal microorganisms gave a higher lactate concen tration compared to fermentations with starch ( P < 0.05; [Strobel and Russell, 1986], Table 1-1). Heldt et al. (1999) also reported higher ruminal proporti ons of lactate for steers fed sugar supplements (sucrose, glucose, fructose) compared to those fed st arch. However, in a study with cannulated steers, animals fed a barley-based diet tended to have higher ruminal concentrations of Llactate compared to those rece iving a molasses-based diet ( P = 0.09; [Moloney et al., 1994]). This difference in lactate producti on response may have been a result of a difference in the source of starch (corn star ch vs. barley) and suga r (sucrose, glucose and fructose vs. molasses) supplem ented in the two studies.

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14 Although pectin is generally no t associated with the produc tion of lactate, pectin fermentation has been shown to yield sm all amounts of lactate (Czerkawski and Breckenridge, 1969). Cultures of B. fibrisolvens 787 and P. ruminicola AR29 both produced small amounts of lactate (1.5mmo l/L and 0.4mmol/L, respectively) from the fermentation of citrus pectin (Marounek a nd Duškov, 1999). In vivo studies showed no effect on lactate production in animal s fed diets containing citrus pulp ( P = 0.34; [Leiva et al., 2000]) or sugar beet pulp ( P = 0.72; [Voelker and Allen, 2003c]) compared to those fed corn hominy and high moisture corn supplem ents, respectively. In general, pectin is not expected to yield lactate to the extent that sugars and starch may. Microbial Mass Microbial composition The microbial mass that flows from the rumen to the small intestine forms a major part of the metabolizable nutrient supply to the ruminant animal. The composition of the microorganisms determines the potential specific nutrient contribution to the small intestine. Bacteria typica lly contain 50% protein, 20% RNA, 3% DNA, 9% lipid and 18% carbohydrate, but this composition can ch ange dramatically (Nocek and Russell, 1988). The two main components of microbial mass that contribute to the metabolizable nutrient supply in the small intestine ar e MCP and microbial storage carbohydrate ( glucan). Bacterial amino nitr ogen as a percentage of tota l nitrogen has been considered as relatively constant, but it can range from 54.9 to 86.7%, with an average of 66.5% (Clark et al., 1992). Large changes may also be seen in microbial glycogen content, especially when cultures are starved for nut rients other than energy (McAllan and Smith, 1974; Nocek and Russell, 1988).

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15 There are a variety of factor s that can affect microbial growth, of which supply of carbohydrate and nitrogen (Stern, 1986; H oover and Stokes, 1991; Clark et al., 1992) appear to be the most important. Another factor that may affect the efficiency of microbial growth is pH (Russell et al., 1992). The rest of this discussion will focus on the synthesis of MCP and glycogen from fermenta tion of starch, sucrose and pectin. The effect of nitrogen source (ammonia nitr ogen, amino acids) and pH on microbial fermentation product yield from NFCs will also be considered. Microbial protein yield Microbial crude protein has b een reported to supply from 34 (Clark et al., 1992); summary of 31 articles) to 80 (Owens a nd Bergen, 1983; Stern, 1986) or even 89% (Clark et al., 1992) of the total amino aci d nitrogen entering th e small intestine of ruminants (Owens and Bergen, 1983; Stern, 1986) Microbial crude pr otein is considered to have a good amino acid balance relative to the animal’s requirements (Clark et al., 1992) with a mean true digestibility of 84.7% (Storm et al., 1983). Accordingly, MCP is an important source of true protein to the animal. Carbohydrate fermentation in the rumen provi des both energy in the form of ATP, and carbon skeletons for MCP synthesis. Th e amount of carbohydrat e and its rate of fermentation regulate microbial metabolism and are in turn regulated by the physical and chemical form of carbohydrates (Stern et al ., 1994). Hall and Herejk (2001) compared the MCP yield from sucrose, starch and pec tin in an in vitro fe rmentation with mixed ruminal microorganisms. Microbial growth was initiated most rapidly on sucrose, followed by pectin and starch. Maximal yiel d was greatest for starch compared to sucrose and pectin ( P < 0.01), which did not differ from each other ( P = 0.30). An explanation offered for the pr oportional difference between maximal yields of MCP for

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16 pectin and starch was the difference between the NFCs in the amount of hexose, and relative amounts of carbon available to the micr oorganisms. However, this approach still overestimated the theoretical maximal yield of MCP from sucrose. In an in vivo study, Cameron et al. (1 991) found no effect on microbial nitrogen flow to the small intestine and efficiency of microbial crude protei n synthesis (MCPeff) in response to starch and dextrose suppleme ntation to mid-lactation Holstein cows. Dijkstra et al. (1992) suggeste d that variation in the result s on MCP yield and efficiency might be due to the lack of correction for microbial -glucan reaching the small intestine. For lack of a convenient method to distingui sh between dietary starch that escapes degradation in the rumen and -glucan (glycogen) stored in ruminal microorganisms, starch measured as glucose hydrolyzed from -glucan in the duodenal digesta will include both fractions. Therefore undigeste d feed starch content of digesta in the duodenum will be overestimated, and ruminal di gestion of starch will be underestimated, and that of dextrose, overestimated. E xpressing MCP yield as a proportion of the carbohydrate digested in the rumen, with an und erestimation of starch digestion in the rumen, will lead to overestimation of the MCPeff for starch. The efficiency of microbial yield from dextrose would be underestimate d, because the unfermen ted dextrose in the form of glycogen would not be accounted for. In a continuous culture system, Mansfi eld et al. (1994) found an increase ( P < 0.05) in non-ammonia nitrogen flow when beet pulp was substituted for corn, but there was no effect on MCPeff ( P > 0.05) and bacterial cr ude protein production ( P > 0.05). If there was less degradation of crude protein from th e feed, this may have resulted in a lower ammonia nitrogen concentration and less avai lable nitrogen for MCP synthesis, which

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17 could explain why no difference in bacter ial crude protein pr oduction was reported between beet pulp and corn. In the study by Mansfield et al. (1994) there were indeed decreased ammonia nitrogen con centrations in fermentations of beet pulp compared to fermentations of corn ( P < 0.05). Microbial -glucan Many species of ruminal bacteria produce polysaccharide, and some can store large amounts as an intracellular reserve (Tho mas, 1960; John, 1984; Lou et al., 1997). However, studies with P. ruminicola B14 (Russell, 1992) and F. succinogenes S85 (Maglione and Russell, 1997) indicated that ruminal bacteria might have a limited capacity to store polysaccharides as shown by a decrease of the viable cell number when the polysaccharide:protein rati o of the cultures exceeded 1.0. Little information exists on microbial -glucan production from different carbohydrate sources. As mentioned earlier, it is difficult to distinguish microbial glucan from dietary starch that passed through the rumen undegraded. McAllan and Smith (1974) reported a higher microbial -glucan content for animals on a diet with more than 70% concentrates (barley and flaked corn). In the same study, time after feeding also had an effect, with an increased microbial -glucan content from feeding through four to six hours after feeding. McA llan and Smith (1974) established a ratio of individual carbohydrates to nucle ic acids in samples of rumi nal bacteria to use as an estimate of the contribution of microbial ca rbohydrate in the duodenum. This may not be a very accurate way to quantify microbial -glucan content. Craig et al. (1987) confirmed the change in microbial -glucan content with time after feeding, and added that particle-associated micr obial populations had higher -glucan content compared to the liquid-associated populations in the ru men. Therefore, the ratio of individual

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18 carbohydrates to nucleic acids will depend on the method of sampling and isolating ruminal bacteria, as well as time of sampling. Ruminal microorganisms may incor porate and store carbohydrate as -glucan under conditions of excess avai lable carbohydrate (shortly after feeding) and potentially limiting nitrogen supply. McAllan and Smith (1974) reported diurnal variations in the glucan content of ruminal bacteria. It is possible that when the supply of available dietary carbohydrate runs out, microorganisms utilize the storage carbohydrate as a source of energy. This stored carbohydrat e can also become available to other microorganisms upon cell lysis or it can pass to th e small intestine and become part of the glucose supply to the animal. Other Factors Which Influence Microbial Product Yield Nitrogen Source Most ruminal microorganisms can synthesize MCP with a non-protein nitrogen (NPN) source such as urea, as the sole source of nitrogen (Oltjen, 1969 ). In a review of several studies Wallace et al. (1997) conclude d that microbial nitrogen derived from ruminal ammonia nitrogen can range from 40 to 100%. Maximal in vitro microbial growth has been reported at ammonia nitrogen concentrations of 5 to 8 mg/100 ml (Satter and Slyter, 1974) in continuous culture studies with purified substrates (starch, cerelose, wood pulp), a concentrate based diet (cracked corn) or a forage-concentrate combination (cracked corn, cerelose, lucerne hay, timot hy hay). Hume (1970) reported maximum in vivo MCP synthesis at a ruminal ammonia n itrogen concentration of approximately 9 mg/100ml in sheep fed diets containing urea only or a 50:50 ratio of urea and protein (casein, gelatin or zein). Ru minal ammonia is a source of nitrogen for MCP synthesis for both structural and non-stru ctural carbohydrate fermenting microorganisms. In the

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19 presence of increased levels of NFCs potential competition between NFC-utilizing bacteria and cellulolytic bacteria for ammonia c ould lead to an increase in the theoretical optimum ammonia concentration required for maximum growth of cellulolytic bacteria. However, over the longer time of the feedi ng cycle this apparent negative impact of increasing NFCs might be alleviated by cro ss-feeding among different ruminal microbial populations (McAllister et al., 1994). An example of cro ss-feeding was shown by Miura et al. (1980) when Megasphaera elsdenii (lactate utilizer) deam inated protein resulting from the lysis of Ruminobacter amylophilus (starch utilizer) wh ich provided BCVFA for the growth of Ruminococcus albus (cellulose and hemicellulose utilizer). Several studies reported no difference in ruminal ammonia concentration when comparing different NFC sources (Chamberlain et al., 1993; O'Mara et al., 1997a; Sannes et al., 2002). When interpreting the respons e to ruminal ammonia concentration it is important to keep in mind that this concentr ation is the net result of protein degradation in the rumen, ammonia abso rption across the ruminal wall ammonia passage from the rumen and ammonia incorporation into MCP. Therefore decreased ruminal ammonia concentration may indicate in creased utilization by rumina l microorganisms and potential increased MCP synthesis. In this case a decrease in ammonia concentration might be viewed as a positive response. On the other hand a decrease in ammonia concentration may also indicate a decrease in protein degr adation, in which case nitrogen might become limited and MCP synthesis decreased. If a decrease in ammonia concentration is accompanied by increased MCP synthesis, it ma y provide needed nutrients to support higher milk protein production.

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20 Although MCP synthesis can occur with ur ea as the sole source of nitrogen, the efficiency of yield may be lower compared to when peptides or ami no acids are supplied. Maeng et al. (1976) reported an optimum ratio of urea nitrogen to amino acid nitrogen of 75:25 for ruminal microbial growth in a seri es of in vitro fermentations with mixed ruminal microorganisms in which glucose, ce llobiose and starch we re the carbohydrate sources. However, nitrogen used for micr obial growth may be derived from sources other than. Amino acids and peptides, whic h are breakdown products of dietary protein, may be directly incorporated into microbial crude protein. Alte rnatively, amino acids from dietary origin can be degraded in the rumen to BCVFAs and ammonia, which can then be used for MCP synthesis. Both peptides and amino acids have been shown to stimulate MCP synthesis when substituted for ammonia in vivo (Rooke a nd Amstrong, 1989) and in vitro (Russell and Strobel, 1993). The importance of amino acids and peptides fr om dietary protein degradation for increasing both MCP production and energetic efficiency has been shown in several studies with batch culture fe rmentations (Maeng et al., 1976; Maeng and Baldwin, 1976a, 1976b). Russell and Sniffen ( 1984) reported an increase of 18.7% in ruminal bacteria yield with the addition of amino acids to mixed cultures with theoretically adequate ammoni a concentrations. A yield in crease of 28% in vivo (sheep) has also been reported when true protein was added to urea-containing diets (Hume and Purser, 1974). The advantages of peptides and ami no acids over NPN for microbial protein growth may depend on the species of bacteria and energy source (Cruz Soto et al., 1994). There appears to be a higher requirement fo r amino acids and peptides by amylolytic

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21 organisms (Maeng and Baldwin, 1976a, 1976b) and sugar-utilizing organisms (Hungate, 1966). However, proteins have also been shown to be superior to urea for maintenance of fiber digestion despite the fact that cellulolytic organisms primarily use ammonia as a nitrogen source. This may indicate that ce llulolytic bacteria may have some requirement for amino acids or peptides (Hoover, 1986), which may be related to the supply of BCVFAs. Varga et al. (1988) showed th at decreased BCVFAs are responsible for depressed fiber digestion in continuous culture fermentatio ns of formaldehyde-treated soybean meal. Gorosito et al. (1985), however suggested that amino acids or peptides might increase cell wall digestion more than BCVFAs alone. Pectin-fermenting ruminal bacteria include species that ferment bot h structural and non-st ructural carbohydrates (e.g. F. succinogenes and P. ruminicola respectively) and would therefore utilize ammonia, amino acids or peptides. It appears to be beneficial to supply nitrogen in the form of amino acids and peptides, whether to be incorporated directly or as source of BCVFAs, in addition to ammonia to optimize microbial growth. Fermentation pH As mentioned previously, NFC supplemen tation has the potential to decrease ruminal pH. Low pH in turn may decrease MCPeff (Russell et al., 1992), which may be related to energy spilling st rategies of ruminal microorganisms to cope with excess available carbohydrate and low pH (Russell a nd Strobel, 1993). One example of an energy spilling strategy i nvolves the ability of S. bovis to ferment glucose to lactate which only yields 2 ATP molecu les per glucose molecule as opposed to acetate, formate and ethanol which yield approximately 3 AT P molecules per glucose molecule (Russell and Baldwin, 1979). At a low pH, S. bovis decreases its intrace llular pH, which favors lactate production (Russell and Hino, 1985). Low pH has also been shown to increase

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22 the maintenance energy cost of ruminal micr oorganisms and thus decrease microbial cell yield (Shi and Weimer, 1992). It appears that cellulolytic bacteria are especially sens itive to low ruminal pH. However, a moderate decrease in pH from 6.8 to 6.0 does not always affect cellulolytic numbers (Slyter et al., 1970; Mackie et al ., 1978; Leedle et al., 1982; Van der Linden et al., 1984) and isolated fibrolytic enzyme activ ity remains high in this range (Stanley and Kesler, 1959; Smith et al., 1973). On the other hand, a decrease in pH below 6.0 has been reported to result in lo ss of fibrolytic activity and d ecreased numbers of cellulolytic bacteria in vitro and in vivo (Slyter et al., 1970; Stewart, 1977; Crawford et al., 1980; Hoover et al., 1984; Mould and rskov, 1984; M ould et al., 1984). At a pH between 4.5 and 5.0 there is virtually complete inhibition of fiber digestion (S tewart, 1977; Hoover et al., 1984; Mould et al., 1984). Russell and Dombrowski (1980) observed washout of cellulolytic bacteria in continuous culture fermentations at a pH below 6.0. Huhtanen and Khalili (1992) reported a decrease in ce llulolytic and hemicellulolytic enzymes at decreased ruminal pH, when sucrose was s upplemented to cattle on grass-silage based diets. It is thus not surpri sing that one of the major result s of decreased ruminal pH has been a decrease in fiber digestion, reported bo th in vitro and in vivo (Terry et al., 1969; Mould and rskov, 1984; Mould et al., 1984). NFCs and Ruminal pH, Fiber Dige stion and Animal Performance Ruminal pH and Fiber Digestion Decreased fiber digestion in vivo is of ten associated with supplementation of forage diets with readily fermentable carbohydrate sources. Cameron et al. (1991) reported decreases in ruminal neutral dete rgent fiber (NDF) and acid detergent fiber (ADF) digestion for lactating dairy cows recei ving supplements of starch and dextrose

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23 (glucose). Heldt et al. (1999) reported a decr ease in total tract NDF digestion relative to control diet-fed animals in steers suppleme nted at 0.3% BW of DM/d with starch, sucrose, glucose or fructose with low-qualit y, tallgrass-prairie hay. The diets in this study were supplemented with degradable inta ke protein at 0.031% BW of DM/d, which may have been below the amount needed to meet ruminal microbe requirements for a degradable nitrogen source. In a second study, with the same NFC sources, but with supplemental degradable intake protein of 0.122% BW of DM/d, total tract NDF digestion increased with NFC supplementation (Heldt et al ., 1999). Also, ruminal pH decreased more in animals consuming the star ch diet, and these animals had a lower total tract NDF digestion compared to animals fe d the sugar (sucrose, glucose and fructose) diets. Some of the decreases noted for fiber digestion may be the result of competition between NFC and fiber utilizing microorganisms for the nitrogen supply. A decrease in ruminal fiber digestion is ofte n attributed to a decr ease in ruminal pH (Hoover, 1986), caused by rapid fermentati on of NFCs and production of VFAs by ruminal microorganisms. Several studies have contradicted this concept and reported no effect on pH and varying effects on fiber di gestion as a result of supplementation with starch or sucrose (Cameron et al., 1991; Aldr ich et al., 1993; Casper et al., 1999). In a two-part study by Khalili and Huhtanen (1991a, b), a decrease in both pH and NDF digestion was reported in animals fed a gr ass silage and barley-based diet with supplementation of sucrose at 1 kg DM/day. However, the negative effect on pH and NDF digestion was alleviated when s odium bicarbonate was supplemented in combination with sucrose.

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24 The effect of NFC supplementation (esp ecially sucrose and starch) on fiber digestion might involve more than just the decrease of ruminal pH. It was Mould and rskov (1984) who first coined the term "carbohydrate effect to describe the initial impaired fiber digestion at a pH of appr oximately 6.2. The authors suggested that a series of events takes place: 1) rumina l microorganisms exhibit a preference for carbohydrate sources that are more readily available, 2) ferm entation of readily available carbohydrates produce organic acids and rumi nal pH decreases and 3) ruminal pH decreases below 5.5 resulting in a decrease of cellulolytic microorganisms and potentially completely inhibits fiber digestion. Piwonka and Firkins (1996) also suggested that there might be a carbohydrate effect related to mi crobially produced inhibitors, which is independent from pH. Substitution of dried, pelleted beet pulp for high moisture corn did not affect ruminal pH and increased both extent and ra te of NDF digestion (Voelker and Allen, 2003b) when fed to Holstein cows on an alfalfa a nd corn silage diet in early lactation. In several other studies increased NDF digestion as a result of supplementing sugar beet pulp (Van Vuuren et al., 1993) or citrus pul p (Zinn and Owens, 1993; Miron et al., 2002) for barley or corn has been reported. Animal Performance There are a multitude of animal studies that compared feedstuffs that differed in types of NFCs that predominated. The challe nge to interpreting these studies is that the diets were rarely characterized for th eir carbohydrate fractions and proportional substitution of carbohydrates was often not equa l. Accordingly, one should view these studies in a general sense to try and unders tand the impact of various carbohydrates on ruminal and animal performance, but th e results are anything but clear-cut.

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25 Dry Matter Intake Supplementation with NFCs is generally thought to result in decreased dry matter intake. However, total dry matter intake was not affected by form of NFC supplementation in several studies (Table 12; Charmely et al., 1991; Nombekela and Murphy, 1995; O'Mara et al., 1997a; O'Mara et al., 1997b; Leiva et al., 2000; McCormick et al., 2001; Broderick et al., 2002b; Ordway et al., 2002; Sannes et al., 2002; Cherney et al., 2003; Delahoy et al., 2003). The effect of NFC supplementation on forage intake alone also gave varied responses (Charmel y et al., 1991; O'Mara et al., 1997b; Heldt et al., 1999; Delahoy et al., 2003). The only report of a decrease ( P < 0.05) in forage intake with lactating dairy cows was in a study by O'Mara et al. (1997b) where perennial ryegrass was supplemented with molassed beet pulp. Several studies reported no effect of NFC type on forage intake for a variet y of forages and animals, including hay for steers (Heldt et al., 1999), pasture for lact ating dairy cows (Del ahoy et al., 2003) and silage for sheep (Charmely et al., 1991). Th e varying responses in forage and dry matter intake among studies investig ating the effect of NFC s upplementation may be due to differences in the amount, source and comb ination of NFC supplemented. However, it would appear that in general the different NFC sources do not differ from each other in their effect on dry matter intake, a nd potentially also forage intake. Milk Production and Composition The effect of different NFC sources on milk yield is inconsistent (Table 1-2). In some studies, substituting sucrose for some portion of corn had no effect on milk yield (Nombekela and Murphy, 1995; Ordway et al ., 2002; Cherney et al., 2003), while Sannes et al. (2002) reported a decrease ( P = 0.02) in milk production. In three studies, substituting beet pulp for ground corn (Dela hoy et al., 2003) and citrus pulp for corn

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26 (Solomon et al., 2000) or corn hominy (Lei va et al., 2000) had no effect on milk production. In other studies, substituting ci trus pulp for high moisture ear corn or cracked shelled corn ( P = 0.01 and P = 0.02, respectively; [Broderick et al., 2002b]) or corn meal ( P < 0.01; [Leiva et al., 2000]) decreased milk production. Milk composition changes in response to di fferent NFC sources also varies (Table 1-2). Sucrose substituted for corn (starch) had no effect on milk fat concentration (Nombekela and Murphy, 1995; Ordway et al ., 2002; Sannes et al., 2002; Cherney et al., 2003), while it decreased ( P = 0.04; [Sannes et al., 2002]), tended to increase ( P = 0.07; [Nombekela and Murphy, 1995] and P = 0.06; [Ordway et al., 2002]), or did not affect (Cherney et al., 2003) milk fat yield. Cows fed beet pulp subs tituted for corn had similar milk fat yields, but increased ( P < 0.05) milk fat concentratio ns (Mansfield et al., 1994). In contrast cows fed citrus pulp substituted for corn (Solomon et al., 2000), and corn hominy or corn meal (Leiva et al., 2000) had similar milk fat yields and concentrations. Varied responses to NFC supplementation in th ese studies may be due to differences in carbohydrate composition of feedstuffs such as beet pulp and citrus pulp, and also variation in composition within a particular feedstuff. Sucrose supplementation has decreased bot h milk protein yield (Sannes et al., 2002) and milk protein percentage (Nombeke la and Murphy, 1995) compared to corn. Beet pulp (Mansfield et al., 1994) and citrus pulp (Solomon et al., 2000; Broderick et al., 2002b) substituted for corn also decreased milk protein yield and milk protein concentration. In at least so me of the studies (Mansfield et al., 1994; Broderick et al., 2002b) decreased milk protein yield might have been due to a lower total milk yield.

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27 Starch and sucrose appear to be similar in their effects on milk yield, and starch may increase milk yield compared to pectin. There does not seem to be a consistent effect on milk composition from different NFC sources. With the effects of NFC on animal a nd microbial responses reported in the literature, and the variation in these responses it is clear that fu rther knowledge of the microbial fermentation product yields from di fferent NFC could be a vital resource in understanding and predicting animal response. It is also necessary to consider factors that may, in combination with NFC suppl ementation, affect substrate utilization, microbial product yield and nutrient supply to the ruminant animal. In this light, three in vitr o fermentation studies with mixed ruminal cultures were conducted to determine the effect of fermentation pH, nitrogen source and NFC supplementation on NDF digestion and fermenta tion product yields. The first two studies examined the effects of pH and nitrogen source on the fermentation of sucrose and isolated neutral detergent re sidue (iNDF). The final study examined the fermentation of iNDF together with starch, sucrose, pectin and their combinations.

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28Table 1-1. The effects of NFC source on ruminal or fermentation pH and organic acid profile. pH Total VFA1 C2 C3 C4 BCVFA Val Lac Reference and treatment (% of diet DM or fermentation substrate) m M --------------------mol/100 mol -----------------(Ariza et al., 2001) continuous culture Citrus pulp (23.6%) --104.2 68.9 16.7 11.4 3.0 ----Hominy feed (25.3%) --101.2 62.6 22.7 11.0 3.7 ----(Bach et al., 1999) continuous culture Control (lush pasture) 6.10 126.4 71.4 17.8 9.6 0.0 ----Beet pulp with molasses (44.7%) 6.04 124.3 60.9 20.2 15.6 0.9 ----Cracked corn (44.7%) 6.02 141.9 54.3 22.1 18.8 1.2 ----(Ben-Ghedalia et al., 1989) 4 cannulated rams Barley (76.5%) 6.18 82.4 65.0 17.6 14.3 1.9 1.4 --Dried citrus pulp (84.4%) 6.42 74.4 69.1 14.4 14.2 1.5 1.0 --(Broderick et al., 2002b) 6 cannulated lactating cows High moisture ear corn (38.4%) 6.14 102.0 63.3 20.6 11.2 1.6 1.9 --Cracked shelled corn (38.7%) 6.21 104.1 63.7 19.6 11.7 1.7 1.8 --High moisture ear corn (19.1%) + Citrus pulp (19.1%) 6.14 107.5 64.0 18.7 12.7 1.4 1.8 --(Chamberlain et al., 1993) 6 sheep Grass silage control (4 kg/d) 6.43 56.8 62.7 24.5 7.8 3.5 1.5 --Sucrose (200 g/d) 6.34 57.2 57.3 27.8 11.6 1.8 1.5 --Starch (200g/d) 6.25 65.4 62.8 24.1 8.9 2.8 1.4 --(Charmely et al., 1991) 8 sheep Alfalfa silage 6.73 84.9 70.1 19.2 --------Alfalfa silage + Sucrose (10% of silage DM intake) 6.63 90.6 65.8 21.6 --------

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29Table 1-1 Continued. pH Total VFA C2 C3 C4 BCVFA Val Lac Reference and treatment (% of diet DM or fermentation substrate) m M --------------------mol/100 mol -----------------(Heldt et al., 1999) 20 cannulated steers 0.031% BW/d RDP supplement Control 6.40 --76.1 13.3 9.0 0.9 0.4 0.2 Starch (0.3% BW of DM/d) 6.36 --70.3 17.1 10.0 1.9 0.8 0.1 Glucose (0.3% BW of DM/d) 6.28 --59.3 15.5 18.6 1.0 1.0 4.5 Fructose (0.3% BW of DM/d) 6.36 --58.8 13.5 19.7 1.0 1.0 6.0 Sucrose (0.3% BW of DM/d) 6.23 56.3 15.5 20.9 1.3 1.8 4.3 0.122% BW/d RDP supplement Control 6.56 --73.5 14.0 10.6 1.2 0.5 0.3 Starch (0.3% BW of DM/d) 6.13 --69.5 16.4 10.3 2.0 1.4 0.5 Glucose (0.3% BW of DM/d) 6.16 --61.5 14.1 17.5 1.7 1.7 3.5 Fructose (0.3% BW of DM/d) 6.29 --59.8 14.2 18.9 1.6 1.7 3.8 Sucrose (0.3% BW of DM/d) 6.22 --59.7 14.4 19.5 1.5 1.8 3.0 (Khalili and Huhtanen, 1991a) 4 cannulated bulls Control (starch) 6.28 105.0 63.6 17.8 14.9 --14.8 4.4 Sucrose (1 kg/d) 6.03 104.0 58.9 16.5 19.7 --23.5 12.5 (Leiva et al., 2000) 11 lactating cows (3 cannulated) Corn hominy diet (25.3%) 6.24 106.1 67.4 21.4 11.2 ----1.5 Citrus pulp diet (23.6%) 6.19 116.4 67.7 20.8 11.5 ----0.6 (Mansfield et al., 1994) continuous culture Corn (30.2%) --112.7 58.7 21.7 15.3 2.3 2.0 0.9 Corn (15.5%) + Beet pulp (15.3%) --109.5 61.6 20.5 14.0 1.9 2.0 0.9 (Marounek et al., 1985) in vitro; inoculum from goats Starch (9 g/150 ml, 8 h incubation) --76.3 64.5 29.1 6.4 ------Pectin (2 g/150 ml, 6 h incubation) --61.5 81.8 14.8 3.4 ------

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30Table 1-1. Continued. pH Total VFA C2 C3 C4 BCVFA Val Lac Reference and treatment (% of diet DM or fermentation substrate) m M -----------------mol/100 mol ---------------mg/dl (Moloney et al., 1994) 6 cannulated steers Barley (61% of DM intake) 6.94 71.2 66.5 15.8 14.0 --3.3 42.3 Molasses (61% of DM intake) 6.86 71.7 58.4 16.6 23.0 --2.0 28.9 (Piwonka et al., 1994) 6 cannulated heifers ------------------------mol/100 mol -----------------Control --82.4 70.0 16.7 9.6 ------Dextrose (5.6%) --91.2 68.9 18.1 9.9 ------Barley (39.7%) --90.5 68.7 16.7 11.1 ------(Sannes et al., 2002) 16 lactating cows (4 cannulated) ---------------------------m M -------------------------Corn (20%) --131.0 85.34 26.3 16.2 1.9 ----Corn (13.5%) + Sucrose (3.2%) --123.0 77.81 27.1 15.5 1.3 ----(Strobel and Russell, 1986) in vitro pH 6.7 Sucrose 6.70 --4.7 2.1 1.1 ----3.7 Starch 6.70 --5.1 2.9 0.8 ----0.9 Pectin 6.70 --10.1 1.3 0.2 ----ND2 in vitro pH 6.0 Sucrose 5.50 --1.7 1.1 0.7 ----8.3 Starch 5.80 --2.7 1.1 0.7 ----4.1 Pectin 5.80 --5.0 0.7 0.3 ----ND (Voelker and Allen, 2003c) 8 cannulated lact. cows ----------------------mol/100 mol --------------------Hi moisture corn (35.6%) 5.93 138.0 56.9 27.0 11.3 2.3 --0.3 Hi moisture corn (29.5%) + Citrus pulp (6%) 5.97 141.0 59.1 24.9 11.5 2.3 --0.3 Hi moisture corn (23.5%) + Citrus pulp (12%) 6.02 142.0 60.2 23.0 12.2 2.3 --0.3 Hi moisture corn (11.4%) + Citrus pulp (24%) 5.94 142.0 61.6 22.4 12.3 1.9 --0.1 1 VFA = volatile fatty acids; C2 = acetate; C3 = propionate; C4 = butyrate; BCVFA = branched chain VFA; Val = valerate; Lac = la ctate 2 ND = not detected

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31Table 1-2. Effects of NFC source on dry matte r intake, milk production and milk composition. Reference and treatment DMI1 Milk Fat Protein Fat Protein (% of diet DM) kg/d % (Broderick et al., 2002b), 48 lactating cows High moisture ear corn (38.4%) 20.9 35.2 1.24 1.04 2.94 3.53 Cracked shelled corn (38.7%) 21.4 35.1 1.19 1.06 3.02 3.38 High moisture ear corn (19.1%) + Ci trus pulp (19.1%) 19.7 32.1 1.07 0.88 2.85 3.44 (Charmely et al., 1991), 8 sheep Alfalfa silage 1.25 ----------Alfalfa silage + Sucrose (10% of silage DMI) 1.22 ----------Silage intake only Alfalfa silage 1.25 ----------Alfalfa silage + Sucrose (10% of silage DMI) 1.07 ----------(Cherney et al., 2003), 20 lactating cows High moisture corn (35.7%) 21.7 39.8 1.28 1.02 2.58 3.24 High moisture corn (32.1%) + Sucrose (3.6%) 21.4 39.5 1.29 1.02 2.59 3.27 High moisture corn (19.2%) 20.1 37.8 1.30 0.95 2.52 3.44 High moisture corn (17.3%) + Sucrose (1.9%) 20.6 38.9 1.33 0.97 2.49 3.47 (Delahoy et al., 2003), 28 lactating cows Ground corn (70.2%) 20.3 27.6 1.05 0.96 3.23 3.53 Ground corn (34.8%), Beet pul p (18.0%), Wheat middlings (17.4%) 20.2 27.4 1.08 0.95 3.19 3.63 (Fegeros et al., 1995), 26 sheep Maize (28.0%) + Barley (30.0%) --0.82 ----5.36 7.04 Maize (20.0%) + (Barley 15.0%) + Dried citrus pulp (30.0%) --0.78 ----5.32 7.27 (Friggens et al., 1995), 18 lacating cows Molassed sugar beet pulp (74.5%) --14.3 0.60 0.48 3.51 4.19 Molassed sugar beet pul p (37.3%) + Grain (38.2%) --14.9 0.57 0.51 3.41 4.05 Grain (Barley and Corn 76.4%) --14.5 0.58 0.50 3.56 3.98

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32Table 1-2. Continued. Reference and treatment DMI Milk Fat Protein Fat Protein (% of diet DM) kg/d % (Leiva et al., 2000) 11 lactating cows (3 cannulated) Corn hominy diet (25.3%) 21.4 32.8 1.12 0.93 2.83 3.43 Citrus pulp diet (23.6%) 20.9 31.3 1.11 0.85 2.71 3.54 184 lactating cows Corn meal diet (19.5%) 19.5 31.8 1.02 0.96 3.08 3.27 Citrus pulp diet (20.5%) 18.9 27.9 0.97 0.88 3.13 3.45 (Mansfield et al., 1994), 46 lactating cows Corn (30.2%) 21.5 32.2 1.18 0.97 3.64 3.01 Corn (15.5%) + Beet pulp (15.3%) 20.3 31.9 1.21 0.92 3.82 2.90 (McCormick et al., 2001), 32 lactating cows Ground corn (75.0%) 22.8 39.6 1.28 1.14 2.99 3.32 Ground corn (68.9%) + Brown sugar (5.0%) 22.9 38.7 1.30 1.13 2.97 3.39 (Nombekela and Murphy, 1995), 24 lactating cows Ground corn (39.9%) 19.0 28.4 0.96 0.96 3.51 3.40 Ground corn (38.4%) + Sucrose (1.5%) 19.1 29.3 0.97 0.95 3.28 3.30 (O'Mara et al., 1997a), 36 lactating cows Beet pulp (30.0%) 15.2 19.8 0.71 0.62 3.16 3.66 Beet pulp (10.6%) + Ground corn (20.0%) 13.7 21.2 0.78 0.64 3.04 3.64 (O'Mara et al., 1997b), 8 cannulated lactating cows Perennial ryegrass 13.6 ----------Perennial ryegrass + Molassed beet pulp (3 kg/day) 14.2 ----------Grass intake only Perennial ryegrass 13.6 ----------Perennial ryegrass + Molassed beet pulp (3 kg/day) 11.5 ----------

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33Table 1-2. Continued. Reference and treatment DMI Milk Fat Protein Fat Protein (% of diet DM) kg/d % (Ordway et al., 2002). prepartum diets (carry over effect measured; postpartum diet 15.1% ground corn) Ground corn (11.5%) 22.1 45.8 1.66 1.26 2.68 3.54 Ground corn (8.8%) + Sucrose (2.7%) 21.7 45.6 1.72 1.25 2.72 3.76 (Sannes et al., 2002), 16 lactating cows (4 cannulated) Corn (20.0%) 25.7 34.3 1.33 1.07 3.14 3.88 Corn (13.5%) + Sucrose (3.2%) 25.5 33.2 1.27 1.03 3.12 3.83 (Solomon et al., 2000), 20 lactating cows Corn (23.7%) 21.5 36.9 1.22 1.07 2.94 3.33 Dried citrus pulp (23.9%) 20.6 36.4 1.21 1.04 2.88 3.34 (Valk et al., 1990) Study 1 (18 lactating cows) Beet pulp (82.5%) 19.5 25.8 1.07 0.89 3.43 4.15 Maize meal (47.5%) + Maize bran (50.0%) 20.1 28.4 1.11 0.98 3.48 3.92 Study 2 (27 lactating cows) Beet pulp (78.4%) 21.2 30.9 1.29 1.02 3.32 4.19 Maize meal (87.5%) 20.7 31.6 1.17 1.03 3.27 3.70 Beet pulp (44.0%) + Maize meal (44.0%) 20.7 30.8 1.24 1.02 3.31 4.05 (Voelker and Allen, 2003a), 8 cannulated lactating cows High moisture corn (35.6%) 24.8 36.4 1.34 1.13 3.21 3.72 High moisture corn (29.5%) + Drie d citrus pulp (6%) 25.0 36.6 1.40 1.15 3.21 3.84 High moisture corn (23.5%) + Drie d citrus pulp (12%) 25.1 35.9 1.39 1.15 3.22 3.90 High moisture corn (11.4%) + Drie d citrus pulp (24%) 22.9 35.4 1.33 1.09 3.10 3.81 1 DMI = Dry Matter Intake

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34 CHAPTER 2 EFFECT OF PH ON MICROBIAL YI ELD AND NEUTRAL DETERGENT FIBER DIGESTION FROM IN VITRO FERMENTA TIONS OF SUCROSE AND ISOLATED NEUTRAL DETERGENT RESIDUE Introduction Dairy cattle diets are often supplemented w ith grains and byproduct feeds that have a large concentration of non-ne utral detergent fiber carbohydr ates (NFCs) in order to increase the energy intake of high producing ruminants. Sucrose, and its constituent monosaccharides glucose and fructose, are the predominant saccharides of the monoand oligosaccharide component of NFCs and are found in byproduct feeds such as molasses (Kunkle et al., 2000), sugar beet pulp (Hall, 200 2) and citrus pulp (Ben-Ghedalia et al., 1989). Sucrose is readily fermentable in th e rumen (Sniffen et al., 1983) and has been associated with a decrease in ruminal pH (Sutton, 1979; Khalili and Huhtanen, 1991a). Ruminal pH is considered one of the majo r modifiers of rumen fermentation (Hoover and Stokes, 1991). A decrease in ruminal pH belo w 6.0 has been associated with decreases in fiber digestion (Khalili and Huhtanen, 1991a, b; Grant and Weidner, 1992), microbial cell yield (Shi and Weimer, 1992) a nd efficiency of microbial protein synthesis (MCPeff; [Russell et al., 1992]), as well as altered volat ile fatty acid (VFA) profiles (Sutton, 1979; Chamberlain et al., 1993; Arab a et al., 2002). However, ther e is evidence that there are effects of readily fermented carbohydrates that are not pH related (Mould and rskov, 1984; Piwonka and Firkins, 1993). The objectives of this study were to comp are the effects of me dia with neutral or acidic starting pHs on 1) the yield of micr obial fermentation products and on neutral

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35 detergent fiber (NDF) digestion, from the in vitro fermentation of sucrose and isolated NDF, and 2) fermentation pH and NDF diges tion of isolated NDF fermented with or without sucrose supplementation. Materials and Methods Substrates Substrates used were isolated bermudagrass ( Cynodon dactylon L.) neutral detergent residue (iNDF; 92.8% DM, 99.4% OM, 98.6% NDFOM, 5.5% NDFCP) and a 50:50 mixture of iNDF and sucrose (SuNDF). The iNDF was prepared as described by Hall and Herejk (2001). Sucrose (S5-500, Fi sher Scientific, Atlanta, GA; 99.98% DM, 100% OM) and iNDF were analyzed prior to the study for DM and OM (AOAC, 1980), and iNDF for NDF using heat-stable -amylase (Termamyl 120L, Novo Nordisk Biochem, Franklinton, NC; (Van Soest et al ., 1991) and CP (AOAC, 1980). A total of 240 mg 0.5 mg of substrate, with iNDF a nd sucrose weighed individually, were transferred into duplicate 50 ml Nalgene hi gh speed, low density, pol yethylene centrifuge tubes (05-562-13, Fisher Scie ntific, Atlanta, GA) or Na lgene high speed polypropylene centrifuge tubes (05-562-10K, Fi sher Scientific, Atlanta, GA) depending on the type of analysis to be performed on the fe rmentation residues (Table 2-1). Medium and reducing solution The pH treatments consisted of one of tw o iso-nitrogenous media with neutral or acidic pH, respectively. The Goering and Van Soest (1970) medium served as the neutral medium (NpH) with initial pH of 6.8, a nd was modified by adding 4.4 ml of a 1 M citric acid solution per 100 ml of Goering and Van So est medium to obtain the acidic medium (ApH), with initial pH of 5.6 (P. J. Weimer and D. R. Mertens, personal communication). The media provided 6.85 mg non-protein n itrogen and 3.52 mg amino nitrogen per

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36 fermentation tube. Casein acid hydrolysate (A-2427, Sigma Chemical Co., St. Louis, MO) provided the amino nitrogen source in the medium. The reducing solution was mixed according to a modification of the Goer ing and Van Soest (1970) procedure (P. J. Van Soest, personal communication). For a volume of 100 ml, 0.625 g of L-cysteine hydrochloride (C-7880, Sigma Chemical Co., St. Louis, MO) and approximately 10 pellets of KOH (P250-3, Fisher Scientific, Atla nta, GA) were dissolved with stirring in 50 ml of distilled water. In a separate 250 ml glass beaker 0.625 g sodium sulfide (S4766, Sigma Chemical Co., St. Louis, MO) was dissolved with stir ring in 50 ml of distilled water. The solutions were combined when the cont ents of both beakers were in solution, and just before additi on of the reducing solution to the fermentation tubes. Fermentation Duplicate 24 h in vitro fermentation r uns using destructiv e sampling of mixed batch cultures were performed according to th e method of Goering and Van Soest (1970). Ruminal inoculum was obtained approximately 3 h post-feeding from a ruminally cannulated, non-pregnant, non-l actating Holstein cow under protocols approved by the University of Florida Institutional Animal Care and Use Committee. The donor cow received a diet of bermudagrass hay (10 kg DM/day), 48% crude protein soybean meal (900 g/d) and free choice mine ral supplement (Ca 1720%, P 9%, NaCl 25%, Mg 0.25%, Cu 0.15, Co 0.01%, I 0.01%, Mn 0.2%, Se 0.004%, Zn 0.4%, Fl 0.09%). The inoculum was filtered through four layers of cheeseclo th and one layer of glass wool, and maintained under anaerobic cond itions at 39C. Twen ty milliliters of the appropriate medium, 1 ml of reducing solution and 5 ml of filtered rumen fluid were added to each fermentation tube. After each addition, tube headspace was purged with CO2. Fermentation tubes were capped with rubber stoppers fitted with gas release valves,

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37 incubated (Equatherm Incubator Model C 1487, Curtin Matheson Scientific, Inc., Houston, TX) under anaerobic conditions at 39 C and destructively sampled at 0, 4, 8, 12, 16, 20 and 24 hours. Tubes were sw irled individually every 4 hours. Sample Handling and Subsequent Analyses At each sampling hour the fermentation tube s for the specific hour were removed from the incubator and placed in an ice bath to terminate the fermentation process. Approximately 5 min after tubes were remove d from the incubator pH was recorded on tubes reserved for NDF analys is. The tubes used for NDF analysis were stored at 10 C and were analyzed for residual NDFOM (NDF on an ash-free basis) within two days of completion of the fermentation. For NDF analysis, samples were allowed to equilibrate to room temperature, the pH was adjust ed up or down with minimal amounts of a 0.2 N NaOH or 1 M citric acid solution, respectively, to obtain a pH of between 6.9 and 7.1. Samples were quantitatively transferred to 600 ml Berzellius beakers and refluxed with 50 ml of neutral detergen t solution and heat-stable -amylase (Termamyl 120L, Novo Nordisk Biochem, Franklinton, NC) for 1 h (Van Soest et al., 1991). To ensure hydrolysis of -glucan, three doses of 0.2 ml heat-stable -amylase were used: one with addition of detergent, one 10 min before re moving the sample from the burner and one added to the Gooch crucible duri ng rinsing with boiling water. Fermentation tubes reserved for microbi al glycogen (GLY), residual sucrose equivalents (sucrose, and its hydrolysis products: glucose an d fructose), organic acids, ammonia-nitrogen (NH3-N) and total free amino acid anal yses were centrifuged at 15,000 x g for 30 min at 5 C. The supernatant was transferred to scintillation vial s and stored at -20 C until analysis for residual sucrose and organic acids by HPLC, and NH3-N and

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38 total free amino acid analyses as described by Broderick et al. ( 2004). The HPLC for analyzing residual sucrose was equipped with an anion exchange analytical column (CarboPac™ PA1, Dionex, Sunnyvale, CA), the mobile phase used was 100 m M NaOH, the flow rate 1.0 ml/min a nd the injection volume 10 L. The HPLC for analyzing organic acids was equipped with an organic acid column (PHX-87H, Bio-Rad Laboratories, Richmond, CA). The solvent used was 0.015 N H2SO4 / 0.0034 M EDTA, the flow rate 0.7 ml/min, the column temperat ure 45C and the injection volume 50 L. The pellets from the high-speed centrifuga tion were quantitatively transferred to 50 ml glass beakers using no more than 20 ml of a 0.2 N NaOH solution to rinse out the fermentation tubes and stored at -20 C until analysis for GLY. Beakers were removed from the freezer and samples were allowed to equilibrate to room temperature. Microorganisms were lysed with a 0.2 N NaOH solution (brought to a volume of 20 ml in the 50 ml glass beakers) in a boiling water bath for 15 min. Samples were cooled to room temperature and then neutralized to pH 7.0 0.1 with 6 N HCl. Samples were quantitatively transferred from the glass beaker s to funnels fitted w ith glass wool plugs for filtration into 100 ml volumetric flasks. B eakers, glass wool and funnels were rinsed with distilled de-i onized water (ddH2O), and then samples were brought to volume with ddH2O. Four milliliters of a 0.1 M sodium acetate buffer (pH ~ 4.5) and 50 l of amyloglucosidase (EC 3.2.1.3, A-3514, Sigma Chem ical Co., St. Louis, MO) were added to 4 ml of sample, incubated at 60C for 45 min, and analyzed for -glucan content as released glucose corrected for free glucose (Karkalas, 1985). Microbial crude protein (MCP) was estim ated as trichloroacetic acid (TCA)precipitated crude protein. Fermentation tube s were individually removed from the ice

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39 bath and 5.2 ml of a 120% (w/v) TCA soluti on were added in two equal increments to achieve a final concentration of 20.0% TCA. Fermentation tubes were returned to the ice bath for 45 min after which tubes were centrifuged at 7700 x g for 20 min at 5 C. The contents of each fermentation tube were th en quantitatively tran sferred into Whatman 541 filter paper (09-851D, Fisher Scientific, Atla nta, GA) in veined funnels set in 125 ml Erlenmeyer flasks, using approximately 50 ml of chilled 10% TCA to rinse the tubes, filter and residue. Samples were allowed to filter under gravity. The filtrate for each tube was filtered through a Whatman GF/A glass fiber filter (09-874-16D, Fisher Scientific, Atlanta, GA), using 10% TCA to rinse the flask, filter and residue. Both Whatman 541 and GF/A filters containing the TCA-precipitated material from one fermentation tube were placed together in a beaker and dried for 24 h at 55 C, before analysis for crude protein content as Kj eldahl nitrogen content x 6.25 (AOAC, 1980). Kjeldahl analysis blanks cons isted of a Whatman 541 filter an d a GF/A filter digested and distilled together in one Kjel dahl flask. The MCP and GLY c ontents of each tube were corrected for fermentation blanks at each hour and MCP for its content by substrate at hour 0. Statistical Analysis The experimental design was a split-split plot in time with a 2 x 2 factorial arrangements of treatments (media and substr ates). The data were analyzed using the PROC MIXED procedure of SAS (1999) with fermentation run (R) as a random variable, and medium (M) and substrate (S) as fixed va riables. Fermentation hour (H) was used as a class variable. Linear or quadratic temporal patterns within media and substrate treatments were determined usi ng orthogonal contrast statements.

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40 The Kenward-Roger method was used to calculate the denominator degrees of freedom for testing fixed effects. The c ontrast, NpH vs. ApH, was used for medium comparisons within substrate (iNDF, SuNDF). All values presented are least squares means. The model statement used was: Yijkl = + Mi + Sj + MSij + Hk + MHik + SHjk + MSHijk + ijkl Where: Yijkl = the dependent variable = overall mean Mi = medium (i = NpH, ApH) Sj = substrate (j = iNDF, SuNDF) Hk = hour (k = 0, 4, 8, 12, 16, 20, 24) MSij = interaction term for medium and substrate MHik = interaction term for medium and hour SHjk = interaction term for substrate and hour MSHijk = interaction term for medium, substrate and hour ijkl = residual error A treatment term (T) consisting of the in teraction between su bstrate and medium was used in the random statement to obtain appropriate standard errors for the least squares means. The random statement included the following terms: Rl + RTlm + RHlk + RTHlmk Where: Rl = fermentation run (m = 1, 2) Tm = treatment (n = 0, 1, 2, 3); number assigned to each M by S combination

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41 RTlm = interaction term for fermentation run and treatment RHlk = interaction term for fermentation run and hour RTHlmk = interaction term for ferm entation run, treatment and hour The sampling hour of maximum MCP or GLY yield within medium and fermentation run was defined as the hour with the maximum least squares means for these measures. Efficiency of MCP yield was expressed as maximum MCP (mg)/organic matter digested (OMD, mg). Organic matter digested (mg) was calculated as total sucrose (mg) minus residual sucrose equivale nt (mg) plus NDFOM digested (mg), minus GLY (mg). The sucrose equivalent at a ny specific sampling hour was calculated as residual sucrose (mg) + 0.95 x (residual fructose (mg) + residual glucose (mg)). Since NDF digestion, residual sucrose, MCP a nd GLY were not measured on the same fermentation tube, the least squares means fo r these measurements, within fermentation run at the hour of maximum MCP, were used to calculate MCP efficiency for individual treatments. The "hour" term and its inte raction terms were omitted from the above mentioned model to compare minimum values, maximum values, and results at a single sampling hour. Results and Discussion Residual Sucrose Sucrose disappeared rapidly from the fe rmentation medium, w ith only 69 and 40% of the original 120 mg sucrose recovered at 0 h in NpH and ApH, respectively (Table 22). Even when residual glucose, fructose and the unhydrolyzed sucrose were expressed as sucrose equivalent, only 83% of the or iginal substrate was accounted for NpH and ApH at 0 h. Residual sucrose amount, averag ed over the 24 h fermentation, did not differ

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42 for ApH and NpH, whereas glucose and sucros e equivalent contents were greater and fructose content tended to be gr eater for ApH compared to NpH. Amount of residual sucrose, and sucrose e quivalent did not diffe r between media at 0 h. By 4 h, and through 24 h, no residual sucros e could be detected in either medium. The ApH fermentation tended to contain more glucose and fructose at 0 h compared to NpH. At 4 h fructose amount and residual monosaccharide sucrose equivalent (glucose and fructose) were greater for ApH comp ared to NpH, whereas glucose was only numerically higher for ApH. Glucose and fructo se were not detected in NpH at 4 h, or in ApH at 8 h, and in subsequent hours for both media. It appears that ruminal microorganisms, depending on the pH of th e fermentation, do not utilize glucose and fructose similarly. For NpH, glucose and fr uctose disappeared from the fermentation by the same sampling time (4 h). However, for ApH, fructose remained in the fermentation for a longer period of time compared to glucose (8 and 4 h, respectively). Early disappearance of sucrose from the ferm entations is consistent with reports of sugar degradation rates of up to 300%/h (Sniffen et al., 1983). In a study by Henning et al. (1991) glucose was detected through 8 a nd 12 hours of fermentation when added at 12.5 g /L and 25 g/L of culture medium, respec tively. In the current study the initial concentration of sucrose (4.62 g/L) was only one-sixth to one-third of the amounts used by Henning et al. (1991), which c ould explain why no residual sugars were detected as early as 4 h in NpH. The present study and that of Henning et al. (1991) would indicate that sucrose or glucose disappearance is ra pid, but not instantaneous in fermentations with ruminal microorganisms. Residual s ugar concentrations in the current study

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43 confirmed that sucrose is readily utilized by ruminal microorganisms at near neutral pH (6.8) but may be more slowly utilized at a more acidic pH (5.6). Microbial Glycogen Although it is considered part of the mi crobial cell mass, glycogen represents available substrate that has been stored, but not yet metabolized by the cells. Maximum GLY yields for both media were recorded at 4 h, with 6.0 mg and 3.5 mg for NpH and ApH, respectively (Figure 2-1). In the ApH fermentation, sucrose-utilizing microorganisms converted less sucrose to GLY ( P = 0.04 at 4 h). Though the temporal pattern for GLY amount tended to differ between media ( P = 0.06 for medium by hour interaction), GLY appeared to decrease through 8 h for both ApH and NpH, and then remained relatively constant through 24 h. Ruminal microorganisms can store microbi al glycogen under conditions of excess carbohydrate supply (Thomas, 1960; John, 1984; L ou et al., 1997). Decreased pH has been shown to increase the maintenance en ergy required by ruminal microorganisms (Shi and Weimer, 1992). If more energy were di verted towards maintenance requirements in the ApH fermentation, there might not have been a relative excess of available carbohydrates, hence less microbial glycoge n storage occurred at the lower pH. However, the ratio of GLY to MCP (at th e hour of maximum GLY yield) was 0.67 and 1.68 for NpH and ApH, respectively. Studies with Prevotella ruminicola B14 (Russell, 1992) and Fibrobacter succinogenes S85 (Maglione and Russell, 1997) indicated a decreased viable cell number when the pol ysaccharide:protein ratio of the cultures exceeded 1.0, which might offer partial explanation for the lower microbial protein yield (Figure 2-12) and decreased NDF digestion (Figure 2-11) for ApH compared to NpH.

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44 Fermentation pH Temporal patterns for fermentation pH were somewhat similar in both media for the two substrates (Figure 2-2). The pH of iNDF fermentations increased over the 24 h fermentation (linear; P = 0.03 and P < 0.01 for NpH and ApH, respectively), whereas that for SuNDF decreased in the early hours and then increa sed through 24 h (quadratic pattern; P = 0.02 and P < 0.01 for NpH and ApH, respectively). However, substrates did not follow parallel paths for the two media, as indicated by a medium by hour interaction within substrate ( P < 0.01 for both iNDF and SuNDF). Minimum fermentation pH for ApH, with Su NDF as the substrate, was recorded at 8 h and was lower ( P < 0.01) than that for NpH (5.25 and 6.71, respectively), which was recorded at 4 h. Also, minimum fermentation pH for both media with SuNDF as the substrate was achieved at the same sampli ng hour that lactate concentration peaked (Figure 2-7). Thereafter the pH for thes e fermentations increased through 24 h. Minimum fermentation pH for iNDF in both media was recorded at 0 h, and this was lower ( P < 0.01) for ApH compared to NpH (6.07 and 6.99, respectively). The magnitude of pH change appeared to differ fo r the two media as well as for the substrates within each medium. At their minima, th e difference in pH between iNDF and SuNDF was numerically greater for ApH, a differen ce of 1.16 pH units at 8 h, compared to the difference of 0.31 pH units at 4 h for NpH. Strobel and Russell (1986) also reported a decrease in pH after 10 h in vitro fermenta tion of sucrose when the initial fermentation pH was 6.0, but not when the initial pH was 6.7. One explanation for the larger numerical d ecrease in fermentation pH for SuNDF in the current study could be the increased lactic (Figure 2-7) and acetic acid (Figure 2-3) production at 8 h for ApH compared to NpH. Another explanation could be a decreased

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45 buffering capacity due to the addition of citr ic acid to obtain a pH of 5.6 for the ApH medium. However, Grant and Me rtens (1992) reported that a 1 M citric acid solution used to adjust a phosphate-bi carbonate buffer to a pH of 5.8 was effective in maintaining the fermentation pH through 24 h for in vitro fermentations of alfalfa silage and corn grain. It is possible that the alfalfa silage and 1:1 alfalfa si lage:corn grain substrates used in that study did not provide as much rapidl y fermenting carbohydrate as did substrates in the present study, and therefore did not yield as much VFA to decrease pH. Organic Acids Temporal patterns for all organic acid c oncentrations differed for fermentations containing no substrate (blank fermentati ons; only inoculum) compared to those containing SuNDF for ApH and NpH ( P < 0.01 for medium by substrate by hour interactions; Figures 2-3 to 2-7) Organic acid concentrations were not corrected at each sampling hour for concentration in the blank fermentations (tubes with only medium and inoculum, but no substrate) since higher ace tate (Figure 2-3) a nd BCVFA (Figure 2-8) concentrations in blank fermentations fo r ApH would have resulted in negative net concentrations of these analytes from 8 h through the end of the fermentations. These results raise questions about the appropriate use of fermentation blanks to adjust for treatment values, especially when the fermentatio n pH is more acidic. It is not clear why the ApH fermentation for iNDF and ferm entation blanks yielded higher acetate concentrations compared to the ApH fermen tation with SuNDF. A partial explanation could be that the fermentation of plant organi c acids such as citric acid primarily yields acetate (Russell and Van Soest, 1984). At the end of the 24 h fermentation, with SuNDF as the substrate, there was no difference between ApH and NpH ( P = 0.36) for butyrate concentrations (Figure 2-5),

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46 whereas acetate (Figure 2-3), propionate (Figure 2-4) a nd total VFA concentrations (Figure 2-6) were greater for ApH ( P < 0.01, P = 0.04 and P < 0.01, respectively). In contrast, Strobel and Russell (1986) reco rded decreased aceta te and butyrate concentrations at a more acidic pH and no difference in propionate concentration in sucrose fermentations. In a continuous culture fermentation of alfalfa hay and corn grain, Calsamiglia et al. (2002) repor ted no effect on butyrate and an increase in propionate proportion at a more acidic pH (5.7). Howeve r, in contrast to the current study, the authors reported decreased total VFA con centrations, and a decr ease in the acetate proportion at the more acidic pH. The diffe rences among studies in VFA production may be in part due to different fermentation methods (continuous vs. batch culture), and different sources of inocula. Lactate was not detected in fermentations with only inoculum. Maximum lactate concentration did not differ ( P = 0.64) between ApH and NpH with SuNDF as the substrate (28.9 and 27.9 m M respectively), and this occurred at 4 h for NpH and at 8 h for ApH (Figure 2-7). It would appear that the lower pH resulted in delayed lactate production. In contrast, St robel and Russell (1986) r ecorded increased lactate concentrations for sucrose fermented by mixed ruminal microorganisms at pH 6.0 compared to 6.7. In the study by Strobel and Russell (1986) the increased lactate concentration was recorded after a 10 h fermentation, wh ich would correspond with a point in the current study where the lactate concentration appears to be greater for the ApH than for the NpH fermentation. For NpH, with SuNDF as the substrate, lactate concentration decreased from 4 h through 12 h and remained at zero till the end of the fermentation. However, for the ApH

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47 fermentation lactate was detectable in ApH until 16 h, and it was only at 20 and 24 h that lactate could no longer be detected. It woul d appear that lactat e utilization might be delayed at a more acidic pH. Both D(+) a nd L(-) isomers of lactic acid are produced in the rumen. However, D-lactate becomes the predominant isomer under more acidic pH (pH < 6) conditions and is considered to be le ss degradable compared to L-lactate. Also, Megasphaera elsdenii one of the major lactate utilizers in the rumen, is inhibited when the pH decreases below 5 .5 (Dawson et al., 1997). Protein Degradation Products Temporal patterns for BCVFA concentrati ons differed for fermentations containing no substrate (blank fermentations) compared to those containing SuNDF for ApH and NpH ( P < 0.01 for medium by substrate by hour in teraction; Figure 28). Higher BCVFA concentrations in the blank fermentations for ApH from 12 through 24 h could possibly be due to increased cell lysis and de-aminati on of microbial protein, leading to higher (at least numerically) NH3-N (Figure 2-9) and BCVFA concen trations. At the end of the 24 h fermentation of SuNDF, BCVFA concentration tended ( P = 0.11) to be lower for ApH compared to NpH. In contrast to the curre nt study, Calsamiglia et al. (2002) reported decreased BCVFA concentrations at a more acidic pH (5.7) in a continuous culture fermentation of alfalfa hay and corn grain. Total free amino acid and NH3-N concentrations are th e net result of protein breakdown and nitrogen utilization by rumi nal microorganisms in vitro. Ammonia nitrogen concentration, averaged acro ss the 24 h fermentation, was higher ( P < 0.01) for NpH compared to ApH with SuNDF as the s ubstrate (Figure 2-9). In the continuous culture study by Calsam iglia et al. (2002) NH3-N concentration also decreased at a more acidic pH (5.7). In another c ontinuous culture fermentation, this time with soybean meal,

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48 barley and corn silage as substrates, Erfle et al. (1982) reported decreased ammonia concentrations at a pH below 6 and attribut ed this to decreased microbial deaminase activity. In the same study, pr otease activity decreas ed at a more acidic pH, and as a result free amino acid concentrations decrea sed. In the current study, total free amino acid concentration was greater ( P < 0.01) for ApH compared to NpH (Figure 2-10). The lower total free amino acid concentration for NpH in this study may indicate greater utilization of amino acids for MCP synthesis, which coincided with a higher MCP yield ( P < 0.01) compared to ApH (Figure 2-12). Neutral Detergent Fiber Digestion Temporal patterns for NDF digestion diffe red for substrates within ApH and NpH ( P < 0.01 for both), as well as for ApH and NpH within each substrate ( P < 0.01 for iNDF and SuNDF; Figure 2-11). At 24 h, NDF digestibility for NpH was greater for SuNDF at 42.4% than for iNDF at 26.4% ( P < 0.01). The reverse was true for ApH, where 24 h NDF digestibility for iNDF (7.8%) tended to be greater ( P = 0.15) compared to SuNDF (2.2%). This would suggest that su crose addition is more detrimental to fiber digestion at a more acidic pH, but may actually have a positive effect under more neutral conditions. Fermentation pH for ApH decreased to 5.25 at 8 h. Decreased fiber digestion has been associated with a more acidic pH (Khalili and Huhtanen, 1991b; Kennelly et al., 1999). Several studies (Stewart, 1977; Hoove r et al., 1984; Mould et al., 1984) have reported almost complete inhibition of fiber di gestion at pH 5.0. A decrease in pH or supplementation of readily fermentable car bohydrates may decrease extent and increase lag of fiber digestion (Grant, 1994). The cu rrent study did not include enough data points in the early hours of the fermentation to adequa tely describe the lag phase. Nonetheless,

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49 evaluation of the graphed data suggests that sucrose addition in the NpH fermentation appeared to increase lag over the first 4 h of fermentation compared to fermentation of iNDF as sole substrate (Figure 2-11). Decreased fiber digestion can be attributed to a decrea se in pH as a result of increased VFA production, or a "carbohydrate effect" (Mould and rskov, 1984). The carbohydrate effect refers to a preference by ruminal microorganisms for more readily available carbohydrates. An increased competition for nutrients such as nitrogen among microbial populations fermenting NFC or NDF might also contribute to reduced fiber digestion by fibrolytic micr oorganisms. Mould and rs kov (1984) suggested that the carbohydrate effect might be a factor in reducing fiber digestion at a pH as high as 6.2. However, the addition of sucrose to the NpH fermentation in the current study resulted in increased fiber digestion. Heldt et al. (1999) also reported an increase in NDF digestion when supplementing starch, sucrose, glucose or fructose to steers on a tallgrass-prairie hay diet. In the study by Held t et al. (1999) pH was only slig htly decreased to 6.2, and in the current study the pH for the neutral fermentation never decreased below 6.7. Microbial Crude Protein Yield and Efficiency Microbial crude protein yiel d was greater (P < 0.01) for NpH compared to ApH over the 24 h fermentation (Figure 2-12). Max imum MCP yield was reco rded at 12 h for NpH (19.4 mg) and this was almost double the maximum amount achieved by ApH (11.1 mg at 20 h). Synthesis of MCP tended ( P = 0.10) to be more efficient for NpH compared to ApH (0.14 mg and 0.09 mg MCP/ mg OMD, respectively). Other studies have also show n a decrease in bacterial grow th at a lower pH (Strobel and Russell, 1986; Shi and Weimer, 1992; De Veth and Kolver, 2001). Decreased pH has been associated with a decrease in the e fficiency of microbial growth (Russell et al.,

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50 1992; Russell and Wilson, 1996), which may be rela ted to a potential increase in ruminal microorganisms' energy requirements for main tenance at a lower pH (Shi and Weimer, 1992). If more energy is expended on maintena nce it will lead to lo wer efficiency of microbial protein synthesis. Conclusions The fermentation pH alters the yields of products and fermentation of NDF when sucrose is included in the fermentation. T hough sucrose appears to be readily fermented regardless of pH, the utilization of the monosaccharide constituents (glucose and fructose) changes depending on pH. Fructose ut ilization may be delaye d at an acidic pH, whereas glucose utilization does not appear to be affected. At a more neutral pH (6.7) sucrose supplementation may increase fiber digestion, whereas at an acidic pH (5.6) sucrose supplementation may decrease fiber di gestion. To avoid the potential negative effect of sucrose supplementation on ruminal pH and fiber digestion adequate effective fiber should be provided in ra tions. Microbial protein yield and composition of microbial mass (microbial protein:glycoge n) changed with pH. The pot ential effects of sucrose supplementation on ruminal fermentation as they change with pH could be important to take into consideration when supplementing su crose to ruminant diet s that predispose the animals to more acidic or more neutral ruminal pH. Results from this study suggest that cons ideration be given to the interaction of sucrose supplementation and ruminal pH fo r predicting fiber digestion and yield of potentially metabolizable nutrients to the an imal. Animal studies further evaluating the effects of ruminal pH on substrate uti lization and nutrient su pply are warranted.

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51 Table 2-1. Type and number of fermentation tubes per medium for each sampling hour, indicating the substrat e and analysis for which tubes were reserved in an 24 h in vitro fermentation of sucrose and iNDF. Analysis Substrate1 pH and residual NDF Microbial crude protein Microbial glycogen, residual sucrose, organic acids, NH3-N2 and amino acid nitrogen No substrate 2 x HSPP3 2 x LDPE4 iNDF 2 x HSPP SuNDF 2 x HSPP 2 x HSPP 2 x LDPE 1 iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose + iNDF 2 NH3-N = ammonia nitrogen 3 HSPP = Nalgene high speed, polypropylene 4 LDPE = Nalgene high speed, low density, polyethylene Table 2-2. Residual glucose, fructose, unhydr olyzed sucrose, m onosaccharide sucrose equivalent (glucose+fructose) and su crose equivalent at 0, 4 and 8 h, and averaged for 24 h in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue with initial medium pH of 6.8 or 5.6. Time Medium pH Glucose Fructose Unhydrolyzed Sucrose Glucose+ Fructose1 Sucrose eq.2 mg Hour 0 6.8 8.45 9.31 83.4 16.9 100 5.6 27.0 27.4 48.5 51.6 100 SE3 3.09 3.68 25.0 6.43 18.7 P -value (medium) 0.11 0.15 0.36 0.13 1.00 Hour 4 6.8 -0.37 -0.02 -0.29 -0.37 -0.66 5.6 6.11 41.1 -0.24 44.8 44.6 SE 5.47 4.40 0.09 1.08 1.06 P -value (medium) 0.54 0.02 0.75 < 0.01 < 0.01 Hour 8 6.8 0.08 -0.05 -0.16 0.03 -0.13 5.6 -0.15 0.13 -0.17 -0.02 -0.19 SE 0.16 0.09 0.05 0.23 0.26 P -value (medium) 0.41 0.27 0.82 0.89 0.89 Avg. for 24 h 6.8 1.20 1.31 11.8 2.39 14.2 5.6 5.07 9.81 6.90 14.1 21.0 SE 0.90 1.14 3.60 0.94 2.95 P -value (medium) 0.01 0.11 0.37 < 0.01 < 0.01 P -value (medium x hour) 0.03 < 0.01 0.15 < 0.01 0.22 1 Calculated as residual (glucose + fructose) x 0.95 to give residual monosacch aride sucrose equivalent 2 Sucrose equivalent = residual (glucose + fructose) x 0.95 + unhydrolyzed sucrose 3 SE = standard error of least squares means

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52 -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 04812162024 Fermentation hourMicrobial glycogen (mg) Figure 2-1. Microbial glycogen yield (LSm eans standard error) for 24 h in vitro fermentations of SuNDF with initial medium pH of 6.8 ( ) or 5.6 ( ). SuNDF = sucrose + isolated bermuda grass neutral detergent residue. 5.0 5.5 6.0 6.5 7.0 7.5 8.0 04812162024 Fermentation hourFermentation pH Figure 2-2. Fermentation pH (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF ( ) and SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF.

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53 0 10 20 30 40 50 60 70 80 90 100 04812162024 Fermentation hourAcetate (m M ) Figure 2-3. Acetate concentrations (LSm eans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. 0 5 10 15 20 25 30 35 40 04812162024 Fermentation hourPropionate (m M ) Figure 2-4. Propionate concentrations (LSm eans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

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54 0 2 4 6 8 10 12 14 04812162024 Fermentation hourButyrate (m M ) Figure 2-5. Butyrate concentrations (LSm eans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. 0 20 40 60 80 100 120 140 04812162024 Fermentation hourTotal VFA (m M ) Figure 2-6. Total volatile fatty acid concentrat ions (LSmeans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

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55 -5 0 5 10 15 20 25 30 35 04812162024 Fermentation hourLactate (m M ) Figure 2-7. Lactate concentr ations (LSmeans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 04812162024 Fermentation hourBCVFA (m M ) Figure 2-8. Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagra ss neutral detergent residue.

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56 5 10 15 20 2504812162024Fermentation hourAmmonia Nitrogen (m M ) Figure 2-9. Ammonia nitrogen co ncentration (LSmeans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. 0 2 4 6 8 10 12 04812162024 Fermentation hourTotal Free Amino Acids (m M ) Figure 2-10. Total free amino acid concentra tion (LSmeans standard error) for 24 h in vitro fermentations containing no substrate ( ) or SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). SuNDF = sucrose + isolated bermudagrass neut ral detergent residue.

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57 50 60 70 80 90 100 04812162024 Fermentation hourResidual NDF (%) Figure 2-11. Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of (iNDF; ) and SuNDF ( ) with an initial medium pH of 6.8 ( or ) or 5.6 ( or ). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose + iNDF. 0 5 10 15 20 25 04812162024 Fermentation hourMicrobial crude protein (mg) Figure 2-12. Microbial crude pr otein yield (LSmeans standa rd error) for 24 h in vitro fermentations of SuNDF with an initial medium pH of 6.8 ( ) or 5.6 ( ). SuNDF = sucrose + isolated bermuda grass neutral detergent residue.

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58 CHAPTER 3 EFFECT OF NITROGEN SOURCE ON MICROBIAL YIELD AND NEUTRAL DETERGENT FIBER DIGE STION FROM IN VITR O FERMENTATIONS OF SUCROSE AND ISOLATED NEUTRAL DETERGENT RESIDUE Introduction Many ruminal microorganisms can synthesize microbial protein with a non-protein nitrogen (NPN) source such as urea, as the sole source of nitrogen (Oltjen, 1969). The efficiency of microbial prot ein synthesis (MCPeff) with urea as the sole source of nitrogen may be lower compared to when peptides or amino acids are supplied. The importance of amino acids and peptides from dietary protein degrad ation for increasing microbial protein (MCP) producti on and energetic efficiency has been shown in several studies with batch culture fermentations (Maeng et al., 1976; Maeng and Baldwin, 1976a, 1976b). Russell and Sniffen (1984) reported an increase of 18.7% in ruminal bacteria yield with the addition of amino acids to mixed cultures with theoretically adequate ammonia concentrations. The advantages of peptides and amino acids may depend on the species of bacteria and energy source (Cruz Soto et al., 1994). There appears to be a higher requirement for amino acids or pept ides by amylolytic organisms (Maeng and Baldwin, 1976a, 1976b) and sugar-utilizing orga nisms (Hungate, 1966), relative to fiber utilizers. Although cellulolytic organisms pr imarily use ammonia as nitrogen source, amino acids and peptides have been shown to increase in situ fiber digestion compared to ammonia nitrogen (NH3-N) (Yang, 2002). When a rapidly fermented carbohydrate source, such as sucrose, is available, there could be competition between sugar-utilizing bacteria and other microbial populations for available nitrogen. This has potential to

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59 affect the microbial growth of the different populations and alter ne utral detergent fiber (NDF) digestion. The objectiv e of the present study was to evaluate the effects of different nitrogen sources on microbial ferm entation products and NDF digestion from the in vitro fermentation of isolated NDF and sucrose. A secondary objective was to determine the effect of sucrose supplementa tion in combination with different nitrogen sources on fermentation pH and NDF digestio n compared to fermentation of isolated NDF only. Materials and Methods Substrates Substrates used were isolated bermudagrass ( Cynodon dactylon L. ) neutral detergent residue (iNDF; 92.8% dry matte r: DM, 99.4% organic matter: OM, 98.6% neutral detergent fiber OM: NDFOM, 5.4% ne utral detergent fiber crude protein: NDFCP) and a 50:50 mixture of iNDF and sucros e (SuNDF). The iNDF was prepared as described by Hall and Herejk (2001). Sucr ose (S5-500, Fisher Scientific, Atlanta, GA; 99.98% DM, 100% OM) and iNDF were analyzed prior to the onset of the study for DM and OM (AOAC, 1980), and iNDF for NDF using heat-stable -amylase (Termamyl 120L, Novo Nordisk Biochem, Franklinton, NC (Van Soest et al., 1991) and crude protein (AOAC, 1980). A total of 240 mg 0.5 mg of substrate, with iNDF and sucrose weighed individually, were transferred into duplicate 50 ml Nalgene high speed low density polyethylene (LDPE) centrifuge tubes (05-562-13, Fisher Scientific, Atlanta, GA) or Nalgene high speed polypropylene (HSPP) centrifuge tubes (05-562-10K, Fisher Scientific, Atlanta, GA) depending on the type of analysis to be performed on fermentation residues (Table 3-1).

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60 Medium Treatments consisted of one of three isonitrogenous media, containing different nitrogen sources. The Goering and Van So est (1970) medium contained NPN (3.52 mg N) and true protein (6.85 mg N)(B), and was modified to contain only NPN (U) by substituting urea (0.73 g urea/L of medium) for casein acid hydrolysat e, or to contain only true protein (C) by substituting cas ein acid hydrolysate (3.79 g casein acid hydrolysate/L of medium) + sodium bicar bonate (4.25 g sodium bicarbonate/L of medium) for ammoni um bicarbonate. Fermentation Duplicate 16 h in vitro fermentation r uns using destructive sampling of batch cultures were performed according to the method of Goering and Van Soest (1970). Casein acid hydrolysate (A-2427, Sigma Chemi cal Co., St. Louis, MO) was used as the amino nitrogen source in B and C media. The reducing solution was mixed according to a modification of the Goering and Van Soest (1 970) procedure (P. J. Van Soest, personal communication).The reducing solution was m odified as described by Van Soest and Robertson (1985). For a volume of 100 ml, 0.625 g of L-cysteine hydrochloride (C-7880, Sigma Chemical Co., St. Louis, MO) and approximately 10 pellets of KOH (P250-2, Fisher Scientific, Atlanta, GA) were dissolved wi th stirring in 50 ml of distilled water. In a separate glass beaker 0.625 g sodium sulf ide (S-4766, Sigma Chemical Co., St. Louis, MO) was dissolved with stirring in 50 ml of distilled water. The solutions were combined when the contents of both beakers were in solution, and just before addition of the reducing solution to the fermentation tubes. Rumen inoculum was obtained approximat ely 3 h post feeding from a ruminally cannulated, non-pregnant, non-l actating Holstein cow under approved protocols of the

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61 University of Florida Institutional Animal Care and Use Committee. The donor cow received a diet of bermudagrass hay (10 kg DM/day), 48% crude protein soybean meal (900 g/d) and free choice mine ral supplement (Ca 1720%, P 9%, NaCl 25%, Mg 0.25%, Cu 0.15, Co 0.01 %, I 0.01%, Mn 0.2%, Se 0.004%, Zn 0.4%, Fl 0.09%). The inoculum was filtered through four layers of cheeseclo th and one layer of glass wool and maintained under anaerobic c onditions at 39C. Twenty milliliters of the appropriate medium, 1 ml of reducing solution and 5 ml of inoculum were added to each fermentation tube. After each additio n, tube headspace was purged with CO2. Fermentation tubes were capped with rubber st oppers fitted with gas release valves, incubated (Equatherm Incubator Model C 1487, Curtin Matheson Scientific, Inc., Houston, TX) under anaerobic conditions at 39 C and destructively sampled at 0, 4, 8, 12 and 16 hours. Tubes were swirled in dividually to mix every 4 hours. Sample Handling and Subsequent Analyses At each sampling hour the fermentation tube s for the specific hour were removed from the incubator and placed in an ice bath to terminate the fermentation process. Approximately 5 min after tubes were remove d from the incubator pH was recorded on tubes reserved for NDF analys is. The tubes used for NDF analysis were stored at 10 C and were analyzed for residual NDF within tw o days of completion of the fermentation. For NDF analysis, samples were allowed to equilibrate to room temperature, were quantitatively transferred to 600 ml Berzellius beakers and refluxed with 50 ml of neutral detergent solution and heat-stable -amylase (Termamyl 120L, Novo Nordisk Biochem, Franklinton, NC) for 1 h (Van Soest et al., 1991). To ensure removal of -glucan, three doses of 0.2 ml heat-stable -amylase were used: one with addition of detergent, one 10

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62 min before removing the sample from the burner and one added to the Gooch crucible during rinsing with boiling water. Fermentation tubes reserved for microbi al glycogen (GLY), residual sucrose equivalents (sucrose, and its hydrolysis products: glucose an d fructose), organic acids, NH3-N and amino acid analyses were centrifuged at 15,000 x g for 30 min at 5 C. The supernatant was transfer red to scintillation vi als and stored at -20 C until analysis for residual sucrose and orga nic acids by HPLC, and NH3-N and amino acids by flowinjection analysis (Broderick et al., 2004). The HPLC for analyzing residual sucrose was equipped with an anion exchange anal ytical column (CarboPac™ PA1, Dionex, Sunnyvale, CA), the mobile phase used was 100 m M NaOH, the flow rate 1.0 ml/min and the injection volume 10 L. The HPLC for analyzing organic acids was equipped with an organic acid column (PHX-87H, Bio-Rad Laboratories, Richmond, CA). The solvent used was 0.015 N H2SO4 / 0.0034 M EDTA, the flow rate 0.7 ml/min, the column temperature 45C and the injection volume 50 L. The pellets from the high-speed centrifuga tion were quantitatively transferred to 50 ml glass beakers using no more than 20 ml of a 0.2 N NaOH solution to rinse out the fermentation tubes. Glass beakers were stored at -20 C until further analysis for GLY. Beakers were removed from the freezer and sa mples were allowed to equilibrate to room temperature. Microorganisms were lysed with a 0.2 N NaOH solution (brought to a volume of 20 ml in the 50 ml glass beakers) in a boiling water bath for 15 min. Samples were cooled to room temperature and then neutralized to pH 7.0 0.1 with 6 N HCl. Samples were quantitatively transferred from th e glass beakers to funnels fitted with glass wool plugs for filtration into 100 ml volumetric flasks. Beakers, glass wool and funnels

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63 were rinsed with distil led de-ionized water (ddH2O), and then samples were brought to volume with ddH2O. Four milliliters of a 0.1 M sodium acetate buffer (pH ~ 4.5) and 50 l of amyloglucosidase (EC 3.2.1.3, A-3514, Sigma Chemical Co., St. Louis, MO) were added to 4 ml of sample, incubated at 60 C for 45 min, and analyzed for -glucan content as released glucose corrected for free glucos e (Karkalas, 1985). Microbial crude protein was estimated as trichloroacetic acid (TCA)-precipitated crude protein. Samples were i ndividually removed from the i ce bath and a total of 5.2 ml of a 120% (w/v) TCA solution were added in two equal increments to achieve a final concentration of 20.0% TCA. Samples were re turned to the ice bath for 45 min after which tubes were centrifuged at 7700 x g for 20 min at 5 C. Each fermentation tube’s contents were then quantitatively transfe rred into Whatman 541 filter paper (09-851D, Fisher Scientific, Atlanta, GA) in veined f unnels set in 125 ml Erlenmeyer flasks, using approximately 50 ml of chilled 10% TCA to ri nse the tubes, filter and residue. Samples were allowed to filter under gravity. The filtrate was filtered through a Whatman GF/A glass fiber filter (09-874-16D, Fisher Scientific, Atlanta, GA), using 10% TCA to rinse the flask, filter and residue. Both What man 541 and GF/A filters containing the TCAprecipitated material from one fermentation t ube were placed togeth er in a beaker and dried for 24 h at 55 C, before analysis for crude prot ein content as Kjeldahl nitrogen content x 6.25 (AOAC, 1980). Kjeldahl anal ysis blanks consisted of a Whatman 541 filter and a GF/A filter digested and distille d together in one Kjeldahl flask. The MCP and GLY contents of each tube were correct ed for fermentation blanks at each hour, and MCP for its content by substrate at hour 0.

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64 Statistical Analysis The experimental design was a split-split plot in time with a 2 x 2 factorial arrangements of treatments (media and substr ates). The data were analyzed using the PROC MIXED procedure of SAS (1999) with fermentation run (R) as a random variable, and medium (M) and substrate (S) as fixed va riables. Fermentation hour (H) was used as a class variable. The Kenward-Roger met hod was used to calculate the denominator degrees of freedom for testing fixed effects. Orthogonal contrasts, B and C vs. U, and B vs. C, were used for medium comparisons ac ross substrates (iNDF, SuNDF) as well as within substrate. All values presented ar e least squares means. The model statement used was: Yijkl = + Mi + Sj + MSij + Hk + MHik + SHjk + MSHijk + ijkl Where: Yijkl = the dependent variable = overall mean Mi = medium (i = B, C, U) Sj = substrate (j = iNDF, SuNDF) Hk = hour (k = 0, 4, 8, 12, 16) MSij = interaction term for medium and substrate MHik = interaction term for medium and hour SHjk = interaction term for substrate and hour MSHijk = interaction term for medium, substrate and hour ijkl = residual error

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65 A treatment term (T) consisting of the interaction between substrate (S) and medium (M) were used in the random statemen t to obtain appropriate standard errors for the least squares means. The random statement included the following terms: Rl + RTlm + RHlk + RTHlmk Where: Rl = fermentation run (m = 1, 2) Tm = treatment (n = 0, 1, 2, 3, 4, 5); numb er assigned to M by S combinations RTlm = interaction term for fermentation run and treatment RHlk = interaction term for fermentation run and hour RTHklm = interaction term for ferm entation run, treatment and hour The sampling hour of maximum MCP or GLY yield within substrate, medium and fermentation run were defined as the hour with the maximum least squares means for these measures. The "hour" term and its interaction terms were omitted from the above model to compare maximum MC P yield and MCP efficiency, GLY at 4 h, and residual sucrose, fructose, glucose and sucrose equiva lent at 0, 4 and 8 h. The MCP efficiency was expressed as MCP (mg)/organic matte r digested (OMD, mg). Organic matter digested (mg) was calculated as the total sucrose (mg) minus residual sucrose equivalent (mg) plus iNDFOM digested (mg), minus GLY (mg). Since NDF digestion, residual sucrose, MCP and GLY were not measured on the same fermentation tube, the least squares means for these measurements, within fermentation run at the hour of maximum MCP, were used to calculate MCPeff for indi vidual treatments. Su crose equivalent was calculated as residual sucrose (mg) + 0.95 x (residual fructose (mg) + residual glucose (mg)). Total volatile fatty acids (VFA) is defined as the sum of acetate, propionate,

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66 butyrate and valerate. Organic acid concentr ations, but not protein degradation products (branched chain VFA (BCVFA), total free amino acids and NH3-N), were corrected for blank fermentations (no substrate, only inoc ulum). Orthogonal cont rasts were used to make comparisons among media across substr ates (iNDF + SuNDF) as well as within substrate. Results and Discussion Residual Substrate Residual substrate, defined as the amount (mg) of glucose, fructose and sucrose that could be detected in the supernatan t, did not differ among media over the 16 h fermentation ( P = 0.60, 0.37 and 0.24, respectively). However, there were some differences among media in the initial sampli ng hours (Table 3-2). At 0 h, U had more residual fructose and tended to have more re sidual glucose than th e other treatments. There was no difference among the media fo r sucrose content at 0 h, though U was numerically much smaller than B and C. The great variation and accordingly large standard errors may have prevented detection of differences. Sucros e equivalents did not differ among media at 0 h. The percentage of sucrose equivalent re maining at 0 h, as a proportion of the initial 120 mg sucrose, was 74.6%, 77.9% and 50.9% for B, C and U, respectively. Glucose was not detected in ei ther B or C at 4 h, and in U at 8 h, and at subsequent hours for all media. No fructose was detected for B at 4 h and for C and U at 8 h, and at subsequent hours for all media. Sucrose was re adily degraded regardless of the source of nitrogen, whereas ruminal microorganisms mo re readily utilized the monosaccharide constituents (glucose and fructose) when provided with true protein compared to NH3-N only. Fructose appeared to be utilized more slowly than glucose.

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67 Microbial Glycogen Maximum GLY accumulation for all treatments was achieved at 4 h, with a similar steady decline thereafter ( P = 0.46 for medium by hour interaction from 4 to 24 h; Figure 3-1). There was no detectable difference ( P = 0.54) in maximal glycogen accumulation among nitrogen sources (7.31, 7.17 and 6.84 mg for B, C and U, respectively). However, over the 16 h fermentation, U tended to have a lower ( P = 0.11) yield of GLY compared to B and C (3.09, 3.48 and 3.54 mg, respectivel y), whereas B and C did not differ ( P = 0.75). Microbial glycogen accumulation may have been reduced in U as compared to B and C as microorganisms had less preformed amino acids to incorporate into MCP and thus had to expend more energy for MCP synthe sis. Alternatively, a lack of sufficient BCVFAs rather than nitrogen may have reduced the efficiency of substrate use by the microorganisms provided only with NPN, a nd the limited amount of amino nitrogen supplied by the inoculum (Russell and Sniffen, 1984) Fermentation pH Fermentation pH was lower ( P < 0.01) with SuNDF as compared to iNDF (Figure 3-2). Among the media, mean fermentation pH for U was higher compared to B and C for both SuNDF ( P < 0.01) and iNDF ( P < 0.01), while B and C did not differ (SuNDF, P = 0.67; iNDF, P = 0.24). Aldrich et al (1993) reported a decrease in ruminal pH for cows fed a diet containing 65.7% compared to those fed a diet containing 52.4% rumen available protein in combination with a rapi dly degradable carbohydrat es source (starch). However, the comparison of different rume n degradable nitrogen sources (NPN and amino acids or peptides) on fermentation pH needs further investigation.

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68 The higher fermentation pH for U may be a result of the hydrol ysis of the added urea in the NPN fermentation to release ammoni a (Figure 3-8), which is alkaline, and the lower yield of organic acids for this medi um. The lower fermentation pH with SuNDF compared to iNDF as the substrate is likel y due to increased organic acid production in SuNDF fermentations which contained more readily fermented carbohydrate, however, organic acid concentrations were not measured on iNDF fermentations. Organic Acids In general, organic acid production incr eased in the presence of amino acid nitrogen compared to NH3-N only (Table 3-3). For ferm entations containing SuNDF as the substrate, concentrations of organic acids at 16 h, with the exception of lactate, were greater or tended to be greater (acetate) for B and C as compared to U. The organic acid concentrations of B and C medi a did not differ at 16 h. Orga nic acid concentrations did not follow similar temporal patterns (Figures 3-3 to 3-7) over the 16 h fermentation as indicated by medium by hour interactions ( P < 0.01 for total VFA, propionate, butyrate, valerate and lactate; P = 0.02 for acetate). Maximum lactate concentr ation did not differ ( P = 0.58) among media (27.6, 27.1 and 30.2 m M for B, C and U, respectively). Ho wever, maximum lactate concentration was detected at 4 h for B and at 8 h for C and U (Figure 3-7). At the end of the fermentation, lactate concentration was greater for U compared to B and C, and no lactate was detected in B and C at 16 h (Table 3-3). Growth rates for Megasphaera elsdenii a major ruminal lactate-utilizer, may be stimul ated in the presence of peptides and amino acids (Cruz Soto et al., 1994). The relati ve decrease in lactate disappearance in fermentations containing only NPN can be e xplained by impaired growth of lactate-

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69 utilizers due to the lack of amino acids or BCVFAs (needed together with ammonia for microbial protein synthesis). Protein Degradation Products Total free amino acid and NH3-N concentrations in fermentations are the net result of amount supplied in the medium and i noculum, as well as protein breakdown and nitrogen utilization by ruminal microorgani sms in vitro. At various sampling hours during the 16 h fermentation NH3-N (Figure 3-8) and tota l amino acid (Figure 3-9) concentrations appeared to be higher for blank fermen tations (no substrate, only inoculum) compared to fermentations contai ning SuNDF. This may have resulted from increased protein breakdown and decreased uti lization in blank ferm entations lacking in fermentable carbohydrate substrates, or incr eased utilization of protein breakdown products in fermentations with SuNDF as the substrate. For the entire fermentation, a nd at the 16 h endpoint, the NH3-N concentration for fermentations with SuNDF as the substrate was greater for U ( P < 0.01) compared to B and C, and B was greater than C ( P < 0.01). Total free amino acid concentration for the 16 h fermentation, with SuNDF as the substrate, was greater ( P < 0.01) for B and C compared to U, and C was greater than B ( P < 0.01). At 16 h, total free amino acid concentration for B and C was greater ( P = 0.02) than for U, and C tended to be greater than B ( P = 0.06). For the most part, relative di fferences in total amino acid and NH3-N concentrations among the three fermentations refl ected the differences in type of nitrogen source supplied at the onset: the greater the amount of NPN in the medium, the more NH3-N, and the more true protein, the more free amino acids. Branched chain VFAs are also products of protein degradation. The concentration of BCVFAs remained relatively low and si milar among media through the initial 12 h of

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70 fermentation (Figure 3-10). It was only at 16 h, with SuNDF as the substrate, that BCVFA concentration was greater ( P < 0.01) for B and C (2.09 and 3.07 m M respectively) compared to U (0.28 m M ), and C was greater than B ( P < 0.01). Increased microbial lysis and degradation of microb ial protein may occur when substrates supporting growth and maintenance becomes lim iting, which could explain the relative increase in protein degradation products ( BCVFA and ammonia) towards the end of the fermentation. Most research have been focused on comparing the effect of rumen degradable and rumen undegradable nitrogen sources on anim al performance and on protein breakdown product concentrations in the rumen. Information on the effect of different rumen degradable nitrogen sources (NPN vs. ami no acids and peptides) on measurements such as total free amino acid and ammonia nitrogen concentrations both in vitro and in the rumen needs further investigation. Neutral Detergent Fiber Digestion Fermentation pH did not decline below 6.56 for any treatment, and so it is not likely that pH had a negative effect on NDF digestion. At 16 h of fermentation, NDF digestion (100 – residual NDF) did not differ ( P = 0.68) among media with iNDF as substrate (18.5, 16.0 and 16.6% for B, C and U, respectively; Figure 3-11). At 16 h, digestion of NDF for SuNDF was greater ( P = 0.01) for B and C (21.0 and 19.5%, respectively) compared to U (14.4% ), and B and C did not differ ( P = 0.45). Digestion of NDF at 16 h, averaged across media, did not differ ( P = 0.28) between iNDF and SuNDF fermentations. Proteins may be superior to urea for main tenance of fiber digestion despite the fact that cellulolytic organisms primarily use ammoni a as nitrogen source. This may indicate

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71 that cellulolytic bacteria have some require ment for supplementation with amino acids or peptides (Hoover, 1986), which may be related to the supply of BCVFAs. Gorosito et al. (1985), however, suggested that amino acids or peptides might increase cell wall digestion over BCVFAs alone. There are not enough data points in the early hours to clearly define it, however, the patterns of the lines suggest that the additi on of sucrose increase d the lag time of NDF digestion in the early hours; iNDF declin ed below 95% residual NDF by 4 h, whereas, SuNDF did not reach that point until after 8 h. The apparent lag noted for NDF digestion may be the result of competition between NFC and fiber-utilizing microorganisms for the nitrogen supply. The addition of sucrose and true protein increased NDF digestion in the later hours of the fermentation. Proteolytic activity by bacteria that ferment readily available carbohydrates (e.g. sucrose; [Wallace et al., 1999]) could increase av ailable ammonia and BCVFAs, which are growth requirements fo r cellulolytic bacter ia (Hoover, 1986). Provision of limiting nutrients required by the microorganisms could enhance fiber digestion. Microbial Crude Protein Yield and Efficiency The yield of MCP over the 16 h fermentation was lower for U compared to B and C with SuNDF ( P = 0.01) as the substrate, and tended to be lower when iNDF ( P = 0.11) was fermented alone (Figure 3-12). Ther e was a greater MCP yield with SuNDF compared to iNDF ( P < 0.01) across all media. Maxi mum MCP yield was greater for B and C compared to U, and did not differ between B and C, for SuNDF fermentations (Table 3-4). For iNDF fermentations, how ever, there was no difference among media for

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72 maximum MCP yield. Therefore the benefit of amino acids or peptides for maximum MCP yield might only be apparent when sucrose is present in the fermentation. Microbes receiving NPN alone (U) were only 64% as efficient in their yield of MCP, with SuNDF as the substrate, as those receiving true protein (B and C), whereas B and C did not differ from each other (Table 3-4). The greater MCP and MCPeff with the addition of true protein is likel y due to direct inco rporation of amino acid or peptides into microbial protein (Cotta and Russell, 1982), or increased availability of carbon skeletons in the form of BCVFAs to support amino aci d synthesis (Russell and Sniffen, 1984). The importance of amino acids and peptides from dietary protein degrad ation for increasing both microbial protein producti on and energetic efficiency has been shown in several other studies with batch culture fermentati ons (Maeng et al., 1976; Maeng and Baldwin, 1976a, 1976b). Russell and Sniffen (1984) report ed an increase of 18.7% in ruminal bacteria yield in mixed culture fermentations with a mixture of carbohydrates (equal parts of glucose, maltose, sucrose, cellobiose a nd soluble starch) and the addition of amino acids with theoretically adequa te ammonia concentrations. Conclusions Addition of sucrose and source of nitrogen affected in vitro yield of fermentation products and NDF fermentation. Addition of true protein increased MCP yield and efficiency of yield from ruminal microorga nisms when sucrose was present and had a positive effect on MCP yield from NDF alone. True protein addition increased NDF digestion when sucrose was present, and increased total yield of organic acids. Maximum accumulation of GLY was not affect ed by nitrogen source when sucrose and NDF were fermented together. These resu lts imply that the sources of ruminally

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73 degradable nitrogen and the in clusion of sucrose may be im portant to consider in the prediction of fiber digestion and metabolizable nu trient supply in ruminant diets. Several animal studies have considered the effect of supplying ruminally degradable nitrogen compared to ruminally undegradable nitrogen on animal performance. Animal studies investigating the interaction of ruminally degradable nitrogen source and NFC source on ruminal measures and animal performance are warranted. Table 3-1. Type and number of fermentati on tubes per medium for one sampling hour, indicating the substrat e and analysis for which tubes were reserved in a 16 h in vitro fermentation of sucrose and is olated neutral detergent fiber. Analysis Substrate1 pH and residual NDF Microbial crude protein Microbial glycogen, residual sucrose, organic acids, NH3-N2 and amino acid nitrogen No substrate (fermentation blank) 2 x HSPP3 2 x LDPE4 iNDF 2 x HSPP 2 x HSPP SuNDF 2 x HSPP 2 x HSPP 2 x LDPE 1 iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose + iNDF 2 NH3-N = ammonia nitrogen 3 HSPP = Nalgene high speed, polypropylene 4 LDPE = Nalgene high speed, low density, polyethylene

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74 Table 3-2. Residual glucose, fructose, su crose, monosaccharide sucrose equivalent (glucose + fructose) and sucrose equiva lent at 0, 4 and 8 h, and averaged for 16 h in vitro fermentations of sucros e and isolated bermudagrass neutral detergent residue with different sources of nitrogen in media. Treatment1 Glucose Fructose Sucrose Glucose+Fructose2 Sucrose eq.3 Hour 0 Least squares means (mg) B 0.31 1.21 88.0 1.44 89.5 C 0.71 1.75 91.1 2.34 93.4 U 2.69 2.34 56.3 4.78 61.1 SE4 2.10 2.51 26.3 4.34 30.2 P -value Treatment 0.20 < 0.01 0.36 0.08 0.41 Contrasts B and C vs. U 0.11 < 0.01 0.20 0.05 0.24 B vs. C 0.70 0.06 0.89 0.35 0.87 Treatment1 Glucose Fructose Sucrose Glucose+Fructose2 Sucrose eq.3 Hour 4 Least squares means (mg) B -0.47 0.02 -0.34 -0.43 -0.77 C -0.72 5.92 -0.48 4.94 4.46 U 3.14 13.9 0.08 16.2 16.3 SE 1.88 8.62 0.22 9.94 10.0 P -value Treatment 0.32 0.51 0.07 0.48 0.46 Contrasts B and C vs. U 0.18 0.33 0.03 0.29 0.28 B vs. C 0.92 0.61 0.52 0.69 0.69 Hour 8 Least squares means (mg) B 0.13 0.06 -0.07 0.18 0.11 C 0.61 -0.03 -0.14 0.55 0.41 U 0.05 0.03 0.00 0.07 0.07 SE 0.11 0.05 0.04 0.06 0.05 P -value Treatment 0.06 0.55 0.21 0.02 0.02 Contrasts B and C vs. U 0.09 0.89 0.13 0.03 0.04 B vs. C 0.05 0.33 0.32 0.02 0.02 Average for 16 h Least squares means (mg) B 0.35 0.32 17.5 0.63 18.1 C 0.77 1.57 18.1 2.23 20.3 U 1.18 3.26 11.3 4.21 15.5 SE 0.73 2.14 5.28 2.68 7.95 P -value Treatment 0.60 0.37 0.24 0.48 0.79 Contrasts B and C vs. U 0.42 0.21 0.10 0.32 0.58 B vs. C 0.61 0.54 0.88 0.58 0.76 1 B = True protein + Non protein nitrogen; C = True protein only; U = Non-protein nitrogen only 2 Calculated as residual (glucose + fructose) x 0.95 to give residual monosacch aride sucrose equivalent 3 Sucrose equivalent = residual (glucose + fructose) x 0.95 + unhydrolyzed sucrose 4 SE = standard error of least squares means

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75 Table 3-3. Organic acid concen trations (least squares means) at 16 h (corrected for blank fermentations) for in vitro fermentations of sucrose and isolated bermudagrass neutral detergent residue with differe nt sources of nitrogen in media Treatment1 Total VFA2 Valerate C2 C3 C4 Lac m M B 46.0 2.53 20.0 17.8 5.61 0.00 C 43.9 2.54 17.5 18.4 5.44 -0.03 U 26.1 0.14 15.4 8.3 2.39 20.7 SE3 2.17 0.31 1.15 1.08 0.19 1.15 P -value Treatment 0.01 0.03 0.19 0.01 < 0.01 < 0.01 Contrasts B and C vs. U < 0.01 0.01 0.13 < 0.01 < 0.01 < 0.01 B vs. C 0.55 0.97 0.26 0.73 0.40 0.99 1 B = True protein + Non protein nitrogen; C = All true protein; U = Non protein nitrogen 2 VFA = volatile fatty acid; Lac = lactate; C2 = acetate; C3 = pr opionate; C4 = butyrate; BCVFA = branched chain VFA 3 SE = standard error of least squares means Table 3-4. Maximum microbial crude prot ein (MCP) yield (hour of maximum) and efficiency of MCP yield at the poin t of maximum MCP yield for in vitro fermentations of iNDF and of SuNDF with different source of nitrogen in media. Values are least squares means. Microbial crude protein Substrate1 Treatment2 Maximum, mg (h) Efficiency, mg/mg OMD4 SuNDF B 14.7 (12) 0.11 C 15.7 (16) 0.11 U 8.85 (12) 0.07 (SE3 = 1.30) (SE = 0.01) P -value Treatment effect < 0.01 0.06 Contrast B and C vs. U < 0.01 0.03 B vs. C 0.15 0.63 iNDF B 3.61 (16) 0.09 C 4.48 (12) 0.17 U 1.77 (16) 0.04 (SE = 1.30) (SE = 0.04) P -value Treatment effect 0.33 0.25 Contrast B and U vs. C 0.27 0.14 B vs. U 0.31 0.52 1 iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose + iNDF 2 B = Non-protein nitrogen + true protein, C = true protein only, U = non-protein nitrogen only 3 Standard error of least squares means 4 OMD = organic matter digested = {[Sucrose OM + NDFOM (mg) digested] – GLY (mg)}; for iNDF as substrate OM digested includes only NDF OM digested

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76 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0481216Fermentation hourMicrobial glycogen (mg) Figure 3-1. Microbial glycogen yield (least squares means standard error) for 16 h in vitro fermentations of SuNDF with medi a containing nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. 6.0 6.5 7.0 7.5 8.0 0481216 Fermentation hourFermentation pH Figure 3-2. Fermentation pH (l east squares means standard error) for 16 h in vitro fermentations of iNDF ( , ) and SuNDF ( , ) with media containing nitrogen in the form of non-pr otein nitrogen + true protein ( or ), true protein only ( or ) or non-protein nitrogen only ( or ). iNDF = isolated bermudagrass neutral detergent re sidue; SuNDF = sucrose+iNDF.

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77 -10 0 10 20 30 40 50 0481216 Fermentation hourTotal VFA (m M ) Figure 3-3. Total volatile fatty acid concentrat ions (LSmeans standard error) for 16 h in vitro fermentations of SuNDF with medi a containing nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. -5 0 5 10 15 20 25 0481216Fermentation hourAcetate (m M ) Figure 3-4. Acetate concentrations (LSm eans standard error) for 16 h in vitro fermentations of SuNDF with media cont aining nitrogen in the form of nonprotein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

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78 -5 0 5 10 15 20 0481216 Fermentation hourPropionate (m M ) Figure 3-5. Propionate concentrations (LSm eans standard error) for 16 h in vitro fermentations of SuNDF with media cont aining nitrogen in the form of nonprotein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0481216Fermentation hourButyrate (m M ) Figure 3-6. Butyrate concentrations (LSm eans standard error) for 16 h in vitro fermentations of SuNDF with media cont aining nitrogen in the form of nonprotein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

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79 -5 0 5 10 15 20 25 30 35 0481216 Fermentation hourLactate (m M ) Figure 3-7. Lactate concentr ations (LSmeans standard error) for 16 h in vitro fermentations of SuNDF with media cont aining nitrogen in the form of nonprotein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. -5 0 5 10 15 20 25 0481216 Fermentation hourAmmonia Nitrogen (m M ) Figure 3-8. Ammonia nitrogen co ncentration (LSmeans standard error) for 16 h in vitro fermentations with no substrate ( , ) or SuNDF as the substrate ( , ) and media containing nitroge n in the form of non-pr otein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose+isolated bermuda grass neutral detergent residue.

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80 -2 0 2 4 6 8 10 12 14 0481216 Fermentation hourTotal Amino Acids (m M) Figure 3-9. Total free amino acid concentra tion (LSmeans standard error) for 16 h in vitro fermentations with no substrate ( , ) or SuNDF as the substrate ( , ) and media containing nitrogen in the form of non-protein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose+isolated berm udagrass neutral detergent residue. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0481216 Fermentation hourBCVFA (m M ) Figure 3-10. Branched chain volatile fatty acid concentrations (LSmeans standard error) for 16 h in vitro fermentation with no substrate ( , ) or SuNDF as the substrate ( , ) and media containing nitrogen in the form of nonprotein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose+isolated bermudagrass neutral detergent residue.

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81 A 75 80 85 90 95 100 0481216 Fermentation hourResidual NDF (%) B 75 80 85 90 95 100 0481216 Fermentation hourResidual NDF (%) Figure 3-11. Residual NDFOM for 16 h in vitro fermentations of iNDF (A; , ) and SuNDF (B; , ) with media containing nitrogen in the form of nonprotein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). iNDF = isolated bermudagr ass neutral detergent residue; SuNDF = sucrose+iNDF.

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82 -5 0 5 10 15 20 0481216 Fermentation hourMicrobial crude protein (mg) Figure 3-12. Microbial crude pr otein yield for 16 h in vitr o fermentations of iNDF ( , ) and SuNDF ( , ) with media containing n itrogen in the form of non-protein nitrogen + true protein ( or ), true protein only ( or ) or non-protein nitrogen only ( or ). iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF.

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83 CHAPTER 4 MICROBIAL PRODUCT YIELD AND NEUT RAL DETERGENT FIBER DIGESTION FROM IN VITRO FERMENTATIONS WITH SUCROSE, STARCH AND PECTIN IN COMBINATION WITH ISOLATED BE RMUDAGRASS NEUT RAL DETERGENT RESIDUE Introduction Carbohydrates comprise between 70 and 80% of ruminant diets and are the major source of energy for both the ruminal microor ganisms and the ruminant animal. Nonneutral detergent fiber carbohydrates (NFCs) can make up 30 to 40% of diet dry matter in rations for high producing dairy cattle. The NFC fraction in feedstuffs includes sugars, organic acids, starch, -glucans, pectic substances a nd fructans and other carbohydrates soluble in neutral detergent (Hall et al., 1999). These have often been treated as a homogenous group regarding fermentation characte ristics. However, the fermentation of different NFCs produces different volatile fatty acid (VFA) pr ofiles (Strobel and Russell, 1986; Mansfield et al., 1994; Ariza et al., 2001) and has va ried effects on ruminal pH (Strobel and Russell, 1986; Khalili and Huht anen, 1991a), microbial product yield (Hall and Herejk, 2001; Sannes et al., 2002) and fiber digestion (Hel dt et al., 1999; Miron et al., 2002). The NFCs also vary in their eff ects on milk yield (O'Mara et al., 1997b; Leiva et al., 2000; Broderick et al., 2002b) and m ilk composition (Nombekela and Murphy, 1995; Broderick et al., 2002a). However, di fferences in animal performance among NFC sources have not been consistent. Understa nding of the fermentati on characteristics of individual NFCs and combinati ons of NFCs, could help to imp rove predictions of animal performance in response to NFC supplementation.

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84 The objective of this study was to evaluate the effects of different amounts and ratios of sucrose, starch and pectin in co mbination with isolated bermudagrass neutral detergent fiber (NDF) on microbial product yield and NDF digestion. Materials and Methods Substrates and Treatments Substrates used were isolated bermudagrass ( Cynodon dactylon L.) neutral detergent residue (iNDF; 96.4% DM, 99.5% OM, 99.0% NDFOM, 4.6% NDFCP), sucrose (S5-500, Fisher Scientific, Atlanta, GA; 99.98% DM, 100% OM), corn starch (S4126, Sigma Chemical Co., St. Louis, MO; 88.5% DM, 100% OM) and citrus pectin (P9135, Sigma Chemical Co., St. Louis, MO ; 95.2% DM, 96.0% OM). The iNDF was prepared from bermudagrass hay ground through the 1 mm screen of a Wiley mill (Arthur H. Thomas Co., Philadelphia, PA.). Fi fty grams of hay were transferred to a 3 L glass beaker and refluxed with 2 L of neutral detergent solution and 10 ml of heat stable -amylase (Termamyl 120L, Novo Nordisk Bi ochem, Franklinton, NC) for 1 h (Van Soest et al., 1991). The contents of the gl ass beaker was filtered through a nylon cloth (37 m pore size) and rinsed with boiling dist illed water until no foam was visible. The residue on the nylon cloth was transferred to a 2 L glass beaker and soaked overnight in 1 M ammonium sulfate solution (approximatel y 5 g of residue DM/200 ml) to remove residual detergent. The residue was then filtered under vacuum through nylon cloth and repeatedly rinsed with boiling distilled wate r until no more foaming from detergent was visible. The residue was then twice rinsed with acetone and filtered under vacuum until dry. After drying overnight at 55C in a fo rced-air oven, the residue was allowed to equilibrate with ambient humidity. The i NDF was included in all fermentation tubes containing substrate at 1 20 mg 0.5 mg (air dry).

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85 In each of the fermentations four amounts of two of the three NFC sources were examined individually and mixtures were also examined (Table 4-1). The weights of NFCs are expressed on a hexose equivalent we ight basis. Fermentation tubes with only iNDF were used to represent the treatments of 0 mg hexose equivalent NFCs. Hexose equivalent conversion coefficients used for sucrose, starch and pectin were 0.95, 0.90 and 1.14, respectively. These coefficients are used as multipliers to calculate the amount of substrate that yields one unit of simple sugars upon complete hydrolysis of the oligoor polysaccharides. The pectin value was based upon the manufacturer's analysis (multiplier to come up with needed mass of p ectin = 1/(% galacturaonic acid monomers [87.6%; as per specification sheet from Sigma Chemical Co. for Lot 108H0913 of P9135]). The sucrose multiplier is based upon th e molecular weight of sucrose divided by the weight of its constituent monomers: ( 342 MW sucrose / 360 MW glucose+fructose) = 0.95. For starch, the multiplier used in starch analyses to convert from released glucose to starch was used: 0.90. This value reflects the weight of a glucose molecule minus the weight of a water of hydrolysis by the molecula r weight of glucose: (180-18)/180 = 0.90). In this polysaccharide, the weight of one wa ter molecule is removed per glucose to form the glycosidic bond between glucose molecules. For use as a classi fication variable, the four equally spaced hexose equivalent treat ments are referred to as 0, 40, 80, and 120 mg for all NFC. Fermentation tubes with only iNDF were used to represent the treatments of 0 mg hexose equivalent NFCs. Substrates, with the excepti on of pectin, were weighed individually and transferred into duplicate 50 ml centrifuge tubes. Pectin did not readily go into solution or suspension upon addition of medium, but formed gel-like clumps. To avoid this probl em, three aqueous suspensions of pectin were

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86 formulated to deliver the desired amounts of pectin per fermentation tube. On the day before the start of the fermentation, three sepa rate pectin suspensions were prepared in individual 1 L glass beakers by adding the desired amount of p ectin to half of the amount of water to be used for solubilizing th e casein hydrolysate. These were stirred continuously with moderate heating for a pproximately 1 h and no more than 1.5 h, at which point all pectin was in suspension. P ectin suspensions were then covered with aluminum foil and kept at 4C overnight. Th e next morning, after dissolving the casein hydrolysate in the remainder of the water, the two solutions were combined and media preparation completed according to Goering and Van Soest (1970). Tubes destined for pH + NDF analyses or trichloroacetic acid (TCA) precipitation were made of Nalgene high-speed, polypr opylene (05-562-10K, Fi sher Scientific, Atlanta, GA), and those for residual sucr ose + organic acids + microbial glycogen analyses were made of Nalgene high-speed low-density polyethylene (05-562-13, Fisher Scientific, Atlanta, GA). Duplicate fermenta tion tubes were used for each of the three sets of analyses. This gave six fermenta tion tubes per treatment per sampling hour. Fermentation A series of six 24 h batch culture in vitr o fermentation runs were performed using mixed ruminal microbes as the inoculum acco rding to the method of Goering and Van Soest (1970). The macro-mineral solution was modified to include 2.22 g NaCl/L (Van Soest and Robertson, 1985). The reducing solution was mixed according to a modification of the Goering and Van Soest ( 1970) procedure (P. J. Van Soest, personal communication). For every 100 ml of reduci ng solution, 0.625 g of cysteine hydrochloric acid (C-7880, Sigma Chemical Co., St. Louis, MO) and 7 pellets of KOH (P250-2, Fisher Scientific, Atlanta, GA) were dissolved with stirring in 50 ml distilled water. In a

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87 separate beaker, 0.625 g of sodium sulfide (S -4766, Sigma Chemical Co., St. Louis, MO) was dissolved with stirring in 50 ml distilled water. The solutions were combined when the contents of both beakers were in soluti on, and just before a ddition of the reducing solution to the fermentation tubes. Trypt one (T-9410, Sigma Chemical Co., St. Louis, MO) was used as the source of am ino nitrogen in the medium. Rumen inoculum was obtained approximatel y 3 h post-feeding from two ruminally cannulated, non-pregnant, non-l actating Holstein cows unde r protocols approved by the University of Florida Institutional Animal Care and Use Committee. Both donor cows received bermudagrass hay (10 kg DM/day) and free choice mineral supplement (Ca 1720%, P 9%, NaCl 25%, Mg 0.25%, Cu 0.15, Co 0.01 %, I 0.01%, Mn 0.2%, Se 0.004%, Zn 0.4%, Fl 0.09%). In addition, 50 g of trace mineralized salt and vitamin mix (Ca 2.9%, P 0.18%, Mg 0.02%, K 0.2%, S 2.4%, Co 8250 ppm, Cu 19052 ppm, I 4762 ppm, Fe 7791, Mn 60041 ppm, Se 750 ppm, Zn 45063 ppm, vit. A 26 736 IU/kg, vit. D 8226 IU/kg) and 4 g of vit. E (50 000 UI/kg) was daily mixed in with the mineral supplement. One donor cow recei ved 48% crude protein soybean meal (900 g/d), while the other donor cow received a supplement containing 48% crude protein soybean meal (500 g/d), ground corn meal (500 g/d) and dried citrus pulp (500 g/d). Inoculum was collected into warmed, 3 L glass vessels via the rumen cannula using a 2.5 cm i.d. hose attached to a vacuum pum p. Equal amounts of inocula from the two donor cows were individually strained through two layers of cheesecloth into a CO2purged blender container (BlendMaster Model 50220, Type B15, Hamilton Beach/Proctor-Silex, Inc., Wash ington, NC). Inoculum was blended for 15 sec on low and 45 sec on high while continuously gassed with CO2. It was then strained through

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88 four layers of cheesecloth into an Erlenmeyer flask set in a 39C water bath, with the headspace continuously gassed with CO2. The inocula from both cows was combined in the Erlenmeyer flask. For inoculation of fermentation tubes, enough inoculum was poured out into a 600 ml glass beaker to fill tubes for one samp ling hour. The beaker was continuously gassed with CO2 to maintain anaerobic conditions, a nd set on a magnetic stir plate for continuous stirring during inocul ation of fermentation tubes. This was to ensure that the inoculum was consistent for all tubes of th e same hour. Twenty m illiliters of medium, 1 ml of reducing solution and 5 ml of inocul um were added to each fermentation tube, including duplicate fermentati on blanks (without any substrate) for each sampling hour. Fermentation tubes were capped with rubber st oppers fitted with gas release valves, incubated (Equatherm Incubator Model C 1487, Curtin Matheson Scientific, Inc., Houston, TX) under anaerobic conditions at 39 C and destructively sampled at 0, 4, 8, 12, 16, 20 and 24 hours. Tubes were individually swirled to mix contents every 4 hours. Sample Handling and Subsequent Analyses At each sampling hour the fermentation tube s for the specific hour were removed from the incubator and placed in an ice bath to terminate the fermentation process. Approximately 5 min after tubes were remove d from the incubator pH was recorded on tubes reserved for NDF analysis The tubes reserved for NDF analysis were stored at 10 C and were analyzed for residual NDF with in one week of the completion of the fermentation. For NDF analysis, samples were allowed to equilibrate to room temperature. Then they were quantitatively transferred to 600 ml Berzellius beakers and refluxed with 50 ml of neutral de tergent solution and heat-stable -amylase (Termamyl

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89 120L, Novo Nordisk Biochem, Franklinton, NC ) for 1 h (Van Soest et al., 1991). To ensure removal of -glucan, three doses of 0.2 ml heat-stable -amylase were used: one with addition of detergent, one 10 min befo re removing the sample from the burner and one added to the Gooch crucible during rinsing with boiling water. Fermentation tubes reserved for microbi al glycogen (GLY), residual sucrose equivalents (as sucrose, and its hydrolysis products: glucose and fructose) and organic acids, were centrifuged at 15,000 x g for 30 min at 5 C. The supernatant was transferred to serum vials and stored at -20 C until analysis for residual sucrose and organic acids by HPLC. The HPLC for analyzi ng residual sucrose was equipped with an anion exchange analytical column (CarboPac™ PA1, Dionex, Sunnyvale, CA), the mobile phase used was 100 m M NaOH, the flow rate 1.0 ml/min an d the injection volume 10 L. The HPLC for analyzing organic acids was equippe d with an organic ac id column (PHX-87H, Bio-Rad Laboratories, Richmond, CA ). The solvent used was 0.015 N H2SO4 / 0.0034 M EDTA, the flow rate 0.7 ml/min, the column temperature 45C and the injection volume 50 L. The pellets from the high-speed centrifuga tion were quantitatively transferred to 50 ml glass beakers using no more than 20 ml of a 0.2 N NaOH solution to rinse out the fermentation tubes. These samples were stored at -20 C until further analysis for GLY. For GLY analysis, beakers were removed from the freezer and samples were allowed to equilibrate to room temperature. Microorganisms were lysed with a 0.2 N NaOH solution (brought to a volume of 20 ml in the 50 ml glass beakers) in a boiling water bath for 15 min. Samples were cooled to room temperature and then neutralized to pH 7.0 0.1 with 6 N HCl. Samples were quantitatively transferred from the glass beakers to

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90 funnels fitted with glass wool plugs for filtration into 100 ml volumetric flasks. Beakers, glass wool and funnels were rinsed with distilled de-i onized water (ddH2O), and then samples were brought to volume with ddH2O. Two hundred micro-liters of sample and 800 l of ddH2O were dispensed into borosilicate glass tubes using a semi-automatic Hamilton Microlab 1000 dilutor (Hamilton Company, Reno, NV). One milliliter of a 0.1 M sodium acetate buffer (pH ~ 4.5) and 50 l of amyloglucosidase (EC 3.2.1.3, A-1602, Sigma Chemical Co., St. Louis, MO) were ad ded to the borosilicate glass tubes, which were then incubated at 60C for 45 min, and analyzed for -glucan content as released glucose corrected for free glucose (Karkalas, 1985). Microbial crude protein (MCP) was estimat ed as TCA-precipita ted crude protein. Fermentation tubes were indivi dually removed from the ice bath and 5.2 ml of a 120% (w/v) TCA solution were added to achieve a final concentration of 20% TCA. Samples were returned to the ice bath for 45 mi n after which tubes we re centrifuged at 7700 x g for 20 min at 5 C. Each fermentation tube's contents were then quantitatively transferred into Whatman 541 filter paper (09-851D, Fish er Scientific, Atlanta, GA) in veined funnels set in 125 ml Erlenmeyer flasks. A pproximately 50 ml of 10% TCA was used to rinse the tubes, filter and residue. Sample s were allowed to filter under gravity. The filtrate was filtered through a Whatman GF/A glass fiber filter (09-874-16D, Fisher Scientific, Atlanta, GA), using 10% TCA to rinse the flask, filter and residue. Both Whatman 541 and GF/A filters containing TCA-precipitated material from one fermentation tube were placed together in a beaker and dried for at least 24 h at 55 C, before analysis for crude protein content as Kjeldahl nitrogen content x 6.25 (AOAC, 1980). Kjeldahl analysis blanks consiste d of a Whatman 541 filte r and a GF/A filter

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91 digested and distilled togeth er in one Kjeldahl flask. The MCP and GLY contents of each tube were corrected for fermentation blanks at each hour, and MCP for its content by substrate at hour 0. Statistical Analysis Treatment mean comparisons and temporal pattern descriptions The experimental design was a split-split plot in time. Data were analyzed using the PROC MIXED procedure of SAS (1999) with fermentation run (R) as a random variable, and treatment (T) as a fixed variable. Fermentation hour (H) was used as a class variable. Equally spaced nominal amounts of hexose equivalents (0, 40, 80 and 120 mg) were used to designate treatments as class variables and polynomial contrasts were used for evaluating temporal patterns over the 24 h. All values presented are least squares means. The model statement used was: Yijkl = + Ti + Hj + THij + ijkl Where: Yijkl = the dependent variable = overall mean Ti = treatment (i = see Table 4-1) Hj = hour (j = 0, 4, 8, 12, 16, 20, 24) THij = interaction term for treatment and hour ijkl = residual error The random statement included the following terms: Rl + RTli + RHlj + RTHlij Where:

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92 Rl = fermentation run (l = 1, 2, 3, 4, 5, 6) Ti = treatment (i = see Table 4-1) RTli = interaction term for fermentation run and treatment RHlj = interaction term for fermentation run and hour RTHlij = interaction term for ferm entation run, treatment and hour The above model with the hour term om itted and using orthogonal contrasts was used to determine the linear or quadratic pa ttern of dependent va riable change with increasing nominal hexose equivalent in clusion of NFCs. The sampling hour of maximum MCP and GLY yield within treatme nt was defined as the hour with the maximum least squares means. The sucrose equivalent at any sp ecific sampling hour was calculated as residual sucrose (mg) + 0.95 x (residual fructose (mg) + residual glucose (mg)). Comparisons of maxima, minima and 24 h data Heterogeneity of regressi on is used to examine differences between or among treatments over time, doses, or other continuous variable. It is used to evaluate whether slopes of lines do or do not differ. Therefor e, heterogeneity of re gression analysis was used for comparisons of increasing hexose e quivalent amounts (mg DM) from different NFCs, on maxima, minima and 24 h values. Th e data were evaluate d for heterogeneity of regression using type one sums of squares and with the model statement: Yijkl = + Ti + Hj + HHjj + Ck + THij + THHijj + TCik + ijkl Where: Yijkl = the dependent variable = overall mean

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93 Ti = treatment (i = sucrose, starch, pectin) Hj = linear continuous term for hexose equivalents based on actual hexose equivalents (mg DM) weighe d into fermentation tubes HHjj = quadratic continuous term for hexos e equivalents based on actual hexose equivalents (mg DM) weighe d into fermentation tubes Ck = hexose equivalent as a cl ass variable (j = 0, 40, 80, 120) THij = interaction term for treatment and linear continuous hexose equivalents THHijj = interaction term for treatment and quadratic continuous hexose equivalents TCik = interaction term for treatment and linear continuous hexose equivalents ijkl = residual error The random statement included the following terms: Rl + RTli Where: Rl = fermentation run (l = 1, 2, 3, 4, 5, 6) RTli = interaction term for fermentation run and treatment The linear form of this model omitted quadratic terms for hexose equivalents (continuous). Significance of the linear or qu adratic term for hexose equivalent defined the order of the curve. The interaction term of treatment and linear or quadratic hexose equivalents (continuous) indica tes whether slopes differ among treatments. Significance of the treatment term in this model indicat es whether the intercep t values differ among treatments. Accordingly, if the slopes do not differ, but the intercep ts do, the analysis did not detect differences in response per hexose equivalent, however the mean values of the treatments could be said to differ. Th e interaction term of treatment and hexose

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94 equivalents as a class variable de scribes the lack of f it of the curves to the data points. If this term is significant, there is greater lack of fit. Orthogonal contrasts were performed by rec oding the treatments as new variable names to reflect the comparisons to be made (e.g. for contrast 1 = sucrose+starch vs. pectin, the new treatment codes were sucrose = 1, starch = 1, pectin = 2, and for contrast 2 = sucrose vs. starch, sucrose = 1 and starch = 2). The same models as above were used with contrast 1 or 2 substituting for treatment. The interaction of the new term for treatment and continuous hexose equivalents ( linear or quadratic) in dicates whether the slopes of the lines in th e contrast differ. Comparisons of NFC mixtures The question that needed to be addressed was whether the fermentation of mixtures of NFCs would give the value that would be expected if the products of the NFC were additive, or if there is some manner of inte raction that makes the yield of products from the mixture more or less than what would be expected of the individual contributions of NFC. Nominal hexose equivalents were used to describe a new con tinuous variable that converted the hexose equivalents to propor tions of the maximum value of hexose equivalent for each NFC source. For instan ce 0 mg nominal hexose equivalent = 0, 40 mg = 0.33, 80 mg = 0.67, and 120 mg = 1.0. The model statement used was: Yijkl = sui + stj + pek + su*stij + su*peik + st*pejk + ijkl Where: Yijkl = the dependent variable = overall mean sui = sucrose (i = hexose equivalents from sucrose)

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95 stj = starch (j = hexose equivalents from starch) pek = pectin (k = hexose equivalents from pectin) su*stij = interaction term for sucrose and starch su*peik = interaction term for sucrose and pectin st*pejk = interaction term for starch and pectin ijkl = residual error The random statement included the following term: Rl = fermentation run (l = 1, 2, 3, 4, 5, 6) The intercept and coefficients for each vari able can be used to calculate estimates for the dependent variable using 0, 0.33, 0.67, and 1.00 as the values inserted for su, st, or pe as a descriptor of the proportional amount of the maximum amount of hexose equivalent included for each NFC. In the tests of fixed effects, if the interaction terms are significant, that means that the yields of a product from that combination differs from the sum of what the individual NF C are predicted to contribute. Results and Discussion Residual Sugars (Sucrose, Glucose, Fructose) Despite detection of low quantities of gl ucose, fructose and unhydrolyzed sucrose in starch and pectin fermentations, it is the residual sugar contents in the fermentations containing sucrose, which would primarily be of interest as descriptors of remaining substrate (Table 4-2). At 0 h, varying amounts of sucrose were accounted for when amount of residual sucrose equivalent was e xpressed as a percenta ge of the original sucrose added to the fermentation (94.0, 96.6, 97.0, 86.0, 88.2, 93.1 and 85.9% for Su40, Su80, Su120, Su40St80, Su80St40, Su40Pe80 and S u80Pe40, respectively). No residual sucrose, glucose or fructose could be detected in any of the fermenta tions at 4 h (Table 4-

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96 2), or in subsequent hours. This agrees with degradation data from other in vitro studies with sucrose as substrate (Chapters 2 and 3). Also, simple sugars are documented in the Cornell Net Carbohydrate and Protein System model as having a fast degradation rate, and starch and pectin an intermediate rate (Sniffen et al., 1992). Residual starch and pectin were not analyzed fo r in the current study and ther efore no inference about the degradation rates for these two substrates can be made. At 0 h, fermentation of increasing amounts of sucrose gave a linear in crease (P < 0.01) in residual glucose and fructose content, and a quadratic increase (P < 0.01) in unhydrolyzed sucrose and sucrose equivalent content. Microbial Glycogen Yield For iNDF, sucrose and pectin fermentations, -glucan content in the fermentation residue reflects estimates of polysaccharide storage in rumen microorganisms, and can be referred to as GLY. However, for starch fermentations, -glucan content also includes any residual starch that ha s not been fermented. The method used to analyze for GLY only resulted in a 77.6% recovery of corn starch (data not repo rted). Therefore estimates of GLY will be inflated for starch fermen tations and NFC combinations that contain starch, but these estimates will not account for all the starch that has not been fermented. Accordingly, comparisons among NFC fermentations for GLY content will be limited to sucrose and pectin fermentations, and combin ations thereof, for lack of a way to distinguish between GLY and re sidual starch from fermen tations containing starch. Maximum GLY content differed among treatments ( P < 0.01), and it occurred at different sampling hours for sucrose and pect in fermentations (Table 4-3). Maximum GLY content increased linearly with increas ing amounts of NFC addition for sucrose and pectin fermentations. Though GLY content pe r hexose equivalent with increasing NFC

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97 inclusion did not differ for sucrose and p ectin fermentations, there was a tendency ( P = 0.07) for their intercepts to differ, with sucr ose having a value more than twice that of pectin (Table 4-4). There was no interaction between sucrose and pectin in fermentations that contained mixtures of these NFCs, whic h suggests that the effect of sucrose and pectin on GLY accumulation is additive. In general, GLY content for all treatmen ts decreased from the hour of maximum yield to 24 h, at which point it could no longer be detected. However, temporal patterns differed among treatments ( P < 0.01 for treatment by hour in teraction). Fermentation of iNDF alone yielded very small amounts of GLY and there was no detectable temporal pattern in GLY content (Table 4-4). Both p ectin and sucrose fermentations showed linear decreases in GLY content from the hour of maximum yield to 24 h for all amounts added. As documented in the Cornell Net Carbohydr ate and Protein System model, simple sugars (such as sucrose) are considered to have a fast degradation rate, and pectin an intermediate rate (Sniffen et al., 1992). Ruminal microorganisms may incorporate and store carbohydrate as -glucan under conditions of excess available carbohydrate (Thomas, 1960; John, 1984; Lou et al., 1997). It is also likely that when the supply of available dietary carbohydrate runs out, microorganisms utilize the storage carbohydrate as a source of energy (McAllan and Smit h, 1974). This could explain the greater accumulation of GLY for sucrose compared to pectin fermentations, and the decreased GLY content in both NFC fermentations from the point of maximum GLY yield through 24 h. Fermentation pH Fermentation pH never decreased below 6.60 for any of the treatments during the 24 h fermentations. However, temporal patterns ( P < 0.01) for the 24 h fermentation

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98 differed among the carbohydrate sour ces (Table 4-5). Fermentation of iNDF resulted in a linear increase in pH over the 24 h ferm entation, whereas tem poral patterns in fermentation pH for NFCs included linear quadratic, cubic and quartic patterns. Addition of individual NFCs ( P <0.01) or combinations of NFCs ( P < 0.01) to the fermentation decreased both mean and minimum pH compared to iNDF. Minimum pH differed among treatments ( P < 0.01) and was reco rded at different sampling hours (Table 4-5). The lowest ferm entation pH achieved was for Su120 at 4 h. Fermentation of increased amounts of NFCs gave quadratic decreases in minimum fermentation pH (Table 4-6). The decreas e in minimum fermentation pH per hexose equivalent with increasi ng NFC inclusion tended ( P = 0.11) to be greater for sucrose and starch fermentations compared to that of pectin fermentations, and the decrease in minimum fermentation pH per hexose equivale nt for sucrose and starch fermentations was similar. The intercepts, however, did differ ( P < 0.01) among the NFCs. Fermentation of combinations of NFCs gave a lower minimum pH ( P < 0.01) compared to iNDF fermentation. The sampling hour for which minimum pH of the combinations of NFCs was recorded varied depending on the component NFCs and their proportions (Table 4-5). The effect on minimum fermentation pH fr om the fermentation of NFC mixtures was not additive, which suggests th at the complement and not just the mass of NFCs in a ration may alter the ruminal pH. A decrease in ruminal pH is often associat ed with the rapid fermentation of NFCs. Several studies have contradict ed this concept and reported no effect on pH as a result of supplementation with starch or sucrose (Cameron et al., 1991; Aldrich et al., 1993; Casper et al., 1999). In a tw o-part study by Khalili and H uhtanen (1991a, b), a decrease

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99 in both pH and NDF digestion was reported in animals fed a grass silage and barleybased diet with supplementation of sucrose at 1 kg DM/day. However, the negative effect on pH and NDF digestion was a lleviated when sodium bicarbonate was supplemented in combination with sucrose. Therefore, NFC supplementation may not have a negative effect on ruminal pH when the buffering capacity is adequate due to supplemental buffers or including ade quate effective fiber in the ration. Organic Acids Maximum VFA concentrations were reco rded at 24 h for iNDF, all individual NFCs and NFC combinations. However, temporal patterns for individual VFA concentrations varied among treatments (Appendix D, Ta ble D-1). Organic acid concentrations at 24 h differed among treatments ( P < 0.01) and were greater for individual NFCs ( P < 0.01) and NFC combinations ( P < 0.01) compared to iNDF (Table 4-7). Fermentation of increased amounts of NF Cs gave a quadratic increase in total VFA concentrations at 24 h, with a similar millimolar VFA increase per additional mg of NFC hexose equivalent added and similar intercep t values for sucrose, starch and pectin fermentations (Table4-8). Organic acid concentrations are generally cons idered to relate to the rate and extent of carbohydrate fermentation. Similar to tal VFA production among different NFC sources have been reported both in vitro (Man sfield et al., 1994; Ar iza et al., 2001) and in vivo (Ben-Ghedalia et al., 1989; Khalili and Huhtanen, 1991a; Sannes et al., 2002; Voelker and Allen, 2003c), with some excepti ons (Bach et al., 1999; Broderick et al., 2002b). Bach et al. (1999) reported an increase ( P > 0.05) in total VFA concentration for cracked corn compared with beet pulp a nd molasses in a continuous culture study. In contrast, total VFA con centration increased ( P = 0.01) or tended to increase ( P = 0.07)

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100 for lactating dairy cows fed a total mixed ra tion (TMR) containing dried citrus pulp and high moisture ear corn in a 50:50 ratio comp ared to cows receiving a TMR containing high moisture ear corn or cracked shelled corn, respectively (Br oderick et al., 2002b). The varying results in these studies may be due to the fact that the combinations of NFCs that were compared differed, which may im ply that the effect on VFA production from supplementation with individual NFC sources of other nutrients may not be additive when supplemented in combinations. In the current study there was no interaction effect on total VFA concentration at 24 h between sucr ose and starch or sucrose and pectin in fermentations with these combinations of NFC (Table 4-8). However, there was an interaction effect between starch and pectin in ferm entations of starch-pectin combinations. This would suggest that the effect of starch and pectin on total VFA concentrations are additive when supplemente d together, whereas th e effect on total VFA concentration of sucrose in combination with either starch or pec tin is not additive. Fermentation of increased amounts of NFCs gave a linear increase in acetate concentrations at 24 h (Table 4-8). The increase in acetate concentration per hexose equivalent with increasing NFC inclusion was greater for pectin fermentations than that of starch and sucrose fermentation, whereas th at for starch and sucrose fermentations did not differ. Similar to the findings of this study, Strobel and Russell (1986) reported greater acetate production for p ectin compared to starch a nd sucrose, and no difference between sucrose and starch. Several in vivo studies have also associated starch and sucrose with relative decreases in rumina l acetate concentration (Sutton, 1979; Khalili and Huhtanen, 1991a; Chamberlain et al., 1993; Moloney et al., 1994; Heldt et al., 1999) and pectin with increased acetate in the rumen (Broderick et al., 2002b; Voelker and

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101 Allen, 2003c). There was no NFC interacti on effect on acetate concentrations when combinations of NFCs were fermented s uggesting that their effects are additive. Fermentation of increased amounts of NFCs gave quadratic increases in propionate concentration at 24 h (Table 4-8). The in crease in propionate c oncentration per hexose equivalent with increasing NF C inclusion tended to be greater for sucrose and starch fermentations than for pectin fermentations, whereas sucrose and starch did not differ. This is in agreement with similar ( P > 0.05) propionate concen trations for starch and sucrose fermentations with mixed ruminal b acteria, and less propi onate yielded from pectin fermentations ( P < 0.05; Strobel and Russell, 1986 ). The effect of sucrose compared to starch on ruminal propionate pr oportion varies among in vivo studies. In some in vivo studies ruminal molar proporti ons of propionate di d not differ between sugars and starch (Moloney et al., 1994; Piwonka et al., 1994), whereas others reported either an increase in the ruminal propionate proportion for sucrose compared to starch (Chamberlain et al., 1993) or an increase with starch supplementation compared to supplementation of sugars (sucrose, glucos e and fructose) when a higher amount (0.122% BW/d) of RDP was supplemented (Heldt et al ., 1999). Other components of the diet such as protein may have altered the yield of propi onate from NFCs. The effect of individual NFCs, when fermented in combinations, on pr opionate concentrati ons may be additive since no interaction between any two individual NFCs were recorded. Fermentation of increased amounts of NFCs gave quadratic increases in butyrate concentrations at 24 h (Table 4-8). The ferm entation of sucrose and starch gave a greater increase in butyrate concentrations per he xose equivalent with in creasing NFC inclusion compared to pectin fermentations, and that for sucrose and starch fermentations did not

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102 differ. This agrees with differences in but yrate production from fermentations of starch, sucrose and pectin noted by Strobel and Ru ssell (1986). Several in vivo studies also reported increased butyrate production when sucrose was fermented instead of starch (Khalili and Huhtanen, 1991a; Moloney et al., 1994), and feeds that typically contain a substantial proportion of sugar and pectin (cit rus and beet pulps) compared to those high in starch (Broderick et al., 2002b; Voelker and Allen, 2003c). Citrus pulp can contain between 12.5 and 40.2% sugars, and sugar beet pulp between 12.8 and 24.7% (Hall, 2002). The increase in the propor tion of butyrate in these studies may be a result of the fermentation of sugar rather than of the so luble fiber content. The effect on butyrate concentrations was additive for sucrose fermented in combination with either starch or pectin. However, the effect on butyrate con centrations for fermentation of starch in combination with pectin was not additive. Fermentation of increased amounts of NFCs gave quadratic increases in BCVFA concentrations at 24 h (Table 4-8), and indivi dual NFCs did not diffe r in their effect on BCVFA concentrations per hexose equiva lent with increasing NFC inclusion. Fermentation of sucrose and starch combinati ons, and starch and pectin combinations had an additive response in BCVFA concentrati ons, whereas combinations of sucrose and pectin further decreased BCVFA concentrations relative to fermentation of individual NFC. In continuous culture studies fermen tation of corn increased the proportion of BCVFAs compared to citrus pulp (Ariza et al ., 2001) and sugar beet pulp (Mansfield et al., 1994). Also in an in vivo study th e corn control diet increased BCVFA concentrations in lact ating dairy cows compared to t hose receiving a diet with sucrose substituted for corn at 3.2% of the diet DM (Sannes et al., 2002). It is not clear why

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103 responses in BCVFA concentra tions differed among these studies. However, it should be noted that it is a challenge to compare th e effect of NFCs on BCVFA concentrations among different studies. Branched chain VFAs are products of protei n degradation and is thus dependent on the source and amount of degradable protein in the ration or fermentation. Maximum lactate concentrations we re greater for individual NFCs ( P < 0.01) and NFC combinations ( P < 0.01) compared to iNDF (Tab le 4-7). The hour at which maximum lactate concentrations were recorded did not consistently coincide with the hour of minimum fermentation pH In general, the hour of minimum fermentation pH for sucrose fermentation often corresponde d with the hour of maximum lactate concentration, whereas the hour of minimum fermentation pH for starch and pectin fermentations appeared later in the ferm entation than the hour of maximum lactate concentration. Maximum lactate concentrations increased quadratically for increasing amounts of NFCs fermented (Table 4-8). Fermentations of sucrose, starch and pectin had similar increases in maximum lactate concentrations per hexose equivalent with increasing NFC inclusion, whereas that of sucrose ferm entations was greater than for starch fermentations. Greater lactate concentrations and proportions have b een reported both in vitro (Strobel and Russell, 1986) and in vivo (Heldt et al ., 1999) for fermentation of sugars compared to fermentation of starch. Compared to acetate, butyrate and propionate (average p Ka = 4.8), lactate (lactic acid, p Ka = 3.1) is a 10-fold stronger acid (Dawson et al., 1997). An increase in lact ate concentration therefore has a greater potential to

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104 decrease ruminal pH. In the current study, sucrose fermentation gave greater lactate concentrations and a lower fermentation pH compared to starch and pectin fermentations. Fermentations of sucrose in combinati on with starch gave reduced maximum lactate yields compared to sucrose or starch fermented separately, whereas the fermentation of sucrose in combination with pectin, and starch in combination with pectin, had additive responses in maximum lactate concentrations. In general, the fermentation of combinations of NFCs did not consistently give additive responses in individual VFA concentrations, which sugge sts that the complement of NFCs in a feedstuff or ration may alter the profile of organic acids produced. Neutral Detergent Fiber Digestion Digestion of NDF differed among NF C sources in temporal pattern ( P < 0.01) over the 24 h fermentation (Table 4-9). The majority of the treatments showed a cubic pattern in NDF digestion over the 24 h fermentation. Exceptions include a quartic pattern for Su40, a quadratic pattern for St40Pe80, and a linear increase fo r Su40St80 in NDF digestion. At 24 h, NDF digestion was lower ( P < 0.01) for fermentation of NFC combinations compared to that of iNDF fermented alone. Fermentation of increased amounts of NFCs linearly increased residual NDF at 24 h (Table 4-10). As the amount of NFC fermented increased, NDF digestion decrea sed in fermentations supplemented with starch and pectin, and increased in the sucrose-supplemented fermentation. The proportion of residual NDF per hexose equiva lent with increasing NFC inclusion was greater for pectin fermentations compared to sucrose and starch fermentations, whereas those for sucrose and starch fermentations di d not differ. Fermentations of sucrose in combination with starch or pectin had an additive response for NDF digestion, whereas

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105 fermenting starch and pectin in combination tended to decrease NDF digestion (increase proportion remaining) compared to NFC fermented individually. In contrast to the current study, Cameron et al. (1991) reported de creases in ruminal NDF and ADF digestion for lactating dairy co ws receiving supplements of starch and dextrose (glucose). Also in contrast, severa l studies reported increased NDF digestion for animals fed diets with beet pulp (Van Vuur en et al., 1993; Voelke r and Allen, 2003b) or citrus pulp (Zinn and Owens, 1993; Miron et al., 2002) subst ituted for high starch (corn or barley) diets. A decrease in ruminal fiber digestion is ofte n attributed to a decr ease in ruminal pH (Hoover, 1986), caused by rapid fermentation of NFCs and subsequent production of VFAs by rumen microorganisms. However, in the current study sucrose fermentations gave a lower minimum fermentation pH compared to starch and pectin fermentations, yet sucrose addition increased NDF digestion compared to NDF digestion for pectin fermentations. The results of the sucrose ferm entation agree with the results of Heldt et al. (1999) who reported an increase in NDF di gestion relative to the control diet-fed animals in steers supplemented at 0.3% BW of DM/d with sucrose, glucose or fructose with low-quality tallgrass-prairie hay and an apparently adequate supply of ruminally degradable protein (0.122% BW of DM/d). The results of the pectin fermentations may be explained in part by a "car bohydrate effect" that describes impaired fi ber digestion at a pH of approximately 6.2 (Mould and rskov, 1984). The authors suggested that it is related to a preference exhi bited by ruminal microorganisms for carbohydrate sources that are more readily available and competition for other nutrients (e.g. nitrogen) among ruminal microbial populations. Some of the decreases noted for fiber digestion may be

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106 the result of competition between NFC and fibe r utilizing microorganisms for a nitrogen supply. There might be a greater similar ity in the microbial populations fermenting pectin and NDF, which are both structural carbohydrates, than for microbial populations fermenting sucrose and NDF. This may result in a greater competition for nutrients when pectin and NDF are fermented together, and a potential negative e ffect on NDF digestion in such fermentations. Microbial Crude Protein Yield The yield of MCP was increased with fe rmentation of individual NFCs compared to iNDF fermentation ( P < 0.01). Carbohydrates sources also had different ( P < 0.01) temporal patterns of MCP yield over the 24 h fermentation (Table 4-11). Combinations of NFCs increased MCP yield compared to iNDF ( P < 0.01). Fermentation of increased amounts of NFCs gave linear increases in maximum MCP yields (Table 4-12). The increase per hexose equivalent with increas ing NFC inclusion was greater for pectin fermentations compared to sucrose and star ch fermentations, which did not differ from each other. In an in vitro fermentation with mixed ruminal microorganisms microbial protein yield was great er for starch fermentations co mpared to sucrose and pectin fermentations, with no difference between the latter two (Hall and Herejk, 2001). In the current study pectin was added to the fermentations in a suspension, which might have made it more readily available for fermen tation by ruminal microorganisms compare to pectin in a powder form used in the study by Hall and Herejk (2001). In general, fermentation of NFC comb inations increased maximum MCP yield compared to iNDF fermentation ( P < 0.01). Fermentation of combinations of NFCs showed no interaction effect on maximum MCP yields (Table 4-12). This would suggest

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107 that the effect of individual NFCs on maximum MCP yield is additive when fermented in combinations. Conclusions The various types and combinations of NFCs altered the yield of microbial products and extent of fiber digestion. Fe rmentation of sucrose increased GLY content compared to pectin fermentations, whereas pectin fermentations increased MCP compared to sucrose and starch. This w ould suggest that sucrose supplementation has potential to increase the -glucan supply and pectin suppl ementation may increase the protein supply to the small intestine of rumi nants. However the form of the sucrose and starch subtrates that were fe rmented differed from that for the pectin. Therefore more work is required to confirm that pectin s upplementation is more effective at increasing microbial protein yield than sucr ose or starch supplementation. Total VFA production was similar among the NFC sources. However, there was an effect of NFC source on organi c acid profile. Pectin fermentations increased acetate production, which may suggest an increase in precursors for fatty acid synthesis and ultimately for milk fat synthesis in the mammary gland. Sucrose and starch fermentations increased butyrate producti on, which is a precursor for energy supply mainly to the heart and skeletal muscle in the form of -hydroxybutyrate (a ketone body). Sucrose and starch also incr eased propionate production, which is a precursor for the glucogenic energy supply of the ruminant an imal. Sucrose fermentations increased lactate production and decrease d pH more than pectin and starch fermentations. This may imply that sucrose supplementation holds a greater risk for causing ruminal acidosis and also decreased fiber digestion in rumina nts. However, NDF digestion was increased

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108 for sucrose fermentations, which supports the contention that factors other than pH may play a role in the potential eff ect of NFCs on fiber digestion. The different effects of fermenting the indi vidual NFCs or mixtures of the NFCs on microbial fermentation product yield imply that the complement of NFCs in a particular feedstuff is important when predicting animal response. The treatment of all NFCs as a uniform entity in ruminant nutrition is not warra nted. Further in vitro and in vivo studies are needed to increase our ability to e xplain and predict animal response when supplementing diets with NFC sources. Table 4-1. Layout of treatments and substrate amounts for a series of three 24 h in vitro fermentations (performed in duplicate) of a mixed batch culture. Substrate Amount (mg)1 Fermentation A Fermentation B Fermentation C Treatment2 iNDF Su St iNDF St Pe iNDF Su Pe Blank 0 0 0 0 0 0 0 0 0 iNDF 120 0 0 120 0 0 120 0 0 Su40 120 40 0 120 40 0 Su80 120 80 0 120 80 0 Su120 120 120 0 120 120 0 St40 120 0 35 120 35 0 St80 120 0 71 120 71 0 St120 120 0 106 120 106 0 Pe40 120 0 38 120 0 38 Pe80 120 0 76 120 0 76 Pe120 120 0 114 120 0 114 Su40St80 120 40 71 Su80St40 120 80 35 St40Pe80 120 35 76 St80Pe40 120 71 38 Su40Pe80 120 40 76 Su80Pe40 120 80 38 1 iNDF amount on air dry basis; sucrose, starch and pectin amounts expressed as mg nominal hexose equivalent 2 iNDF = isolated bermudagrass neutral detergent residue; Su = sucrose; St = starch; Pe = pectin; numbers reflect nominal amount hexose equivalent of different NFCs

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109 Table 4-2. Residual glucose, fructose, unhydrol yzed sucrose and sucrose equivalent (mg) at 0 and 4 h for 24 h in vitro fermenta tions of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means. Item1 Amount2 Glucose Fructose Sucrose Sucrose. Equivalent4 0 h 4 h 0 h 4 h 0 h 4 h 0 h 4 h iNDF 0.05 -0.01 -0.01 -0.010.03 0.03 0.070.01 (SE4 = 0.32) (SE = 0.22) (SE = 0.59) (SE = 0.56) Individual NFCs Sucrose 40 7.67 0.19 5.09 -0.0123.8 -0.09 35.9 0.12 80 8.17 0.18 6.38 -0.0259.9 0.00 73.7 0.20 120 9.03 0.33 6.81 0.0595.7 0.01 110.8 0.41 Starch 40 -0.82 -0.01 -0.37 0.001.11 -0.06 -0.04-0.05 80 -0.91 0.05 -0.38 0.041.05 -0.01 -0.200.09 120 -0.92 -0.01 -0.39 0.040.96 -0.07 -0.30-0.04 Pectin 40 0.73 0.23 0.34 0.11-0.97 -0.03 0.050.26 80 0.89 0.24 0.57 0.03-0.70 -0.10 0.700.12 120 1.54 0.15 0.87 0.05-0.73 -0.05 1.570.11 (SE = 0.35) (SE = 0.25) (SE = 0.69) (SE = 0.68) NFC combinations Su40St80 10.5 0.01 8.13 0.0022.8 -0.07 40.4 0.01 Su80St40 10.6 -0.12 8.19 0.0049.6 -0.05 67.4 -0.09 St40Pe80 0.19 0.16 0.28 0.060.15 -0.02 0.570.12 St80Pe40 -0.12 0.11 0.02 0.080.10 -0.02 -0.020.09 Su40Pe80 7.73 0.15 5.27 0.1723.1 -0.21 35.5 0.09 Su80Pe40 8.79 -0.02 6.37 0.0850.7 0.09 65.1 0.14 (SE = 0.45) (SE = 0.31) (SE = 0.91) (SE = 0.95) 1 iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 2 Amount of NFC substrate in mg nominal hexose equivalent 3 Sucrose equivalent = residual (glucose + fructose) x 0.95 + unhydrolyzed sucrose 4 SE = standard error of least squares means

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110 Table 4-3. Maximum microbial glycogen (GLY ) yield (mg), hour of maximum yield and temporal patterns for 24 h in vitro fermentations of iNDF, NFC sources (sucrose and pectin), and combinations of NFCs. Values are least squares means. Item1 Amount2 Max. GLY, mg (h) Temporal pattern3 iNDF 0.51 (12) No detectable pattern (SE4 = 0.23) Individual NFCs Sucrose 40 3.08 (4) L ( P < 0.01) 80 3.59 (4) L ( P < 0.01) 120 4.27 (4) L ( P < 0.01) Pectin 40 1.34 (4) L ( P < 0.01) 80 1.72 (8) L ( P < 0.01) 120 2.43 (8) L ( P < 0.01) (SE = 0.26) NFC combinations Su40Pe80 3.45 (4) L ( P < 0.01) Su80Pe40 3.63 (4) L ( P < 0.01) (SE = 0.33) 1 iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; Pe = pectin 2 Amount of NFC substrate in mg nominal hexose equivalent 3 Pattern from hour of maximum GLY yield to 24 h: L = linear 4 SE = standard error of least squares means

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111 Table 4-4. Main effects and regression co efficients for maximum microbial glycogen (GLY) yield (mg) for increasing he xose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations. Main effects 1 Pvalues NFC2 0.07 He3 <0.01 NFC x He 0.47 NFC x HeCL4 0.77 Regression Coefficients Coefficients ( Pvalues) Individual NFCs Sucrose Pectin Intercept 2.59 (0.01) 0.99 (0.25) He 0.01 (<0.01) 0.10 (<0.01) NFC combination analysis 5 Coefficients ( Pvalues) Intercept 1.14 (0.01) Sucrose 3.41 (<0.01) Pectin 1.14 (<0.01) Sucrose x Pectin 0.69 (0.61) 1 Main effects ( P -values) and regression coefficients (estimates and P -values) for individual NFCs were obtained with a proc mixed heterogeneity of regression analysis 2 NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 3 He = hexose equivalent (continuous variable) 4 HeCL = hexose equivalent (class variable); NFC x HeCL = lack of fit term: if significant then regression line fits data poorly 5 Regression coefficients (estimates and P -values) for NFC combinations were obtained with a standard proc mixed regression analysis

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112 Table 4-5. Fermentation pH (mean, minimum, hour of minimum and temporal pattern) for 24 h in vitro fermentations of iN DF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Values are least squares means. pH Item1 Amount2 Mean Minimum (h) Temporal pattern3 iNDF 7.22 7.13 (4) L (P < 0.01) (SE4 = 0.01) (SE = 0.02) Individual NFCs Sucrose 40 7.13 6.99 (4) Qt (P = 0.04) 80 7.05 6.81 (4) Qt (P < 0.01) 120 6.93 6.68 (4) Qt (P < 0.01) Starch 40 7.16 7.12 (12) L (P = 0.04) 80 7.09 7.02 (12) Qt (P = 0.03) 120 7.03 6.94 (16) Qd (P = 0.01) Pectin 40 7.15 7.01 (4) C (P < 0.01) 80 7.07 6.90 (8) C (P < 0.01) 120 6.99 6.76 (8) Qt (P = 0.02) (SE = 0.01) (SE = 0.03) NFC combinations Su40St80 7.02 6.95 (8) Qd (P = 0.02) Su80St40 7.00 6.85 (4) C (P < 0.01) St40Pe80 7.01 6.86 (8) C (P = 0.05) St80Pe40 7.01 6.92 (12) Qt (P = 0.04) Su40Pe80 7.00 6.81 (8) Qt (P < 0.01) Su80Pe40 7.03 6.82 (4) Qt (P < 0.01) (SE = 0.02) (SE = 0.03) 1 iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 2 Amount of NFC substrate in mg nominal hexose equivalent 3 Pattern over 24 h fermentation: L = linear; Qd = quadratic; C = cubic; Qt = quartic 4 SE = standard error of least squares means

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113 Table 4-6. Main effects and regression coe fficients for minimum fermentation pH for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coeffici ents for fermentations of NFC combinations. Main effects 1 Pvalues NFC2 <0.01 He3 <0.01 He x He <0.01 NFC x He 0.10 NFC x He x He 0.14 NFC x HeCL4 0.99 Contrasts Intercept Slope Su and St vs. Pe 0.28 0.11 Su vs. St 0.06 0.43 Regression Coefficients5 Coefficients ( Pvalues) Individual NFCs Sucrose Starch Pectin Intercept 4.80E-08 (<0.01)6.70E-08 (<0.01) 9.60E-08 (<0.01) He 1.49E-06 (<0.01)4.65E-07 (<0.01) -3.17E-07 (<0.01) He x He -2.98E-10 (<0.01)1.33E-09 (<0.01) 8.46E-09 (<0.01) NFC combination analysis 6 Coefficients ( Pvalues) Intercept 6.80E-06 (<0.01) Sucrose 1.47E-07 (<0.01) Starch 6.00E-08 (<0.01) Pectin 9.50E-08 (<0.01) Sucrose x Starch -1.60E-04 (<0.01) Sucrose x Pectin -1.40E-04 (<0.01) Starch x Pectin -7.00E-05 (<0.01) 1 Main effects ( P -values) and regression coefficients (estimates and P -values) for individual NFCs were obtained with a proc mixed heterogeneity of regression analysis 2 NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 3 He = hexose equivalent (continuous variable) 4 HeCL = hexose equivalent (class variable); NFC x HeCL = lack of fit term: if significant then regression line fits data poorly 5 Coefficients are [H+] 6 Regression coefficients (estimates and P -values) for NFC combinations were obtained with a standard proc mixed regression analysis

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114 Table 4-7. Volatile fatty acid concentrations at 24 h and maximum l actate concentrations (hour of maximum indicated) for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), a nd combinations of NFCs. Values are least squares means. Item1 Amount2 Total VFA3 C2 C3 C4 Val Max. Lac (h) BCVFA m M Blank ------------3.68 iNDF 15.8 9.49 4.26 1.56 0.51 0.00 (4) 4.14 SE4 1.19 0.93 0.36 0.26 0.12 0.34 0.14 Individual NFCs Sucrose 40 28.4 15.0 6.98 4.91 1.55 8.02 (4) 3.39 80 *38.5 17.3 9.94 7.83 *3.44 19.5 (4) *2.90 120 53.5 23.8 18.9 7.37 3.59 31.0 (4) 3.19 Starch 40 **28.7 *16.7 7.82 *3.05 *0.78 0.62 (8) *3.92 80 38.9 21.6 11.8 4.47 1.06 0.72 (8) 3.93 120 47.0 24.9 15.0 5.92 1.19 0.77 (8) 3.80 Pectin 40 28.0 19.8 6.00 1.75 0.33 0.11 (0) 3.83 80 39.8 29.3 8.24 1.77 0.38 0.20 (0) 3.00 120 54.1 40.0 11.3 2.30 0.47 0.19 (0) *3.15 SE 1.35 *1.39 **1.46 1.03 *1.06 0.43 0.30 *0.31 0.14 0.37 0.16 *0.17 NFC combinations Su40St80 49.6 25.8 15.9 6.49 1.58 7.88 (4) 2.92 Su80St40 49.2 22.6 14.5 8.98 3.30 18.4 (4) 3.27 St40Pe80 47.8 32.9 11.5 2.93 0.40 0.14 (4) 2.62 St80Pe40 46.3 29.0 13.2 3.13 0.84 0.33 (8) 3.26 Su40Pe80 50.6 32.3 12.6 4.00 1.60 9.63 (4) 2.78 Su80Pe40 ***61.3 **33.4 15.3 **8.17 **4.34 21.4 (4) **2.98 SE 1.73 ***1.88 1.29 **1.40 0.58 *0.62 0.39 **0.42 0.17 **0.19 0.46 0.21 **0.23 1 Blank = no substrate; iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = su crose; St = starch; Pe = pectin 2 Amount of NFC substrate in mg nominal hexose equivalent 3 VFA = volatile fatty acid; C2 = acet ate; C3 = propionate; C4 = butyrate; Val = valerate; Lac = lactate; BCVFA = branched chain VFA 4 SE = standard error of least squares means *, **, *** Symbols indicate which SE corresponds with the least squares means in any given column

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115Table 4-8. Main effects and regression co efficients for maximum orga nic acid concentrations for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations. Total VFA1 C2 C3 C4 Val Max. Lac (h) BCVFA Main effects 2 Pvalues NFC3 0.49 <0.01 0.01 <0.01 <0.01 <0.01 0.09 He4 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.05 He x He 0.03 ----<0.01 <0.01 ----<0.01 ----NFC x He <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.17 NFC x He x He 0.25 ----0.07 <0.01 ----0.06 ----NFC x HeCL5 0.65 0.08 0.99 0.30 <0.01 <0.01 0.21 Contrasts Intercept; Slope Intercept; Slope Intercept; Slope Intercept; Slope Intercept; Slope Intercept; Slope Intercept; Slope Su and St vs. Pe 0.32 ; 0.83 <0.01 ; <0.01 0.01 ; 0.11 <0.01 ; <0.01 <0.01 ; <0.01 <0.01 ; 0.83 0.28 ; 0.17 Su vs. St 0.96 ; 0.14 0.25 ; 0.68 0.62 ; 0.27 0.11 ; 0.83 0.05 ; 0.96 0.05 ; 0.06 0.18 ; 0.16 Regression coefficients Coefficients ( Pvalues) Individual NFCs Sucrose: Intercept 25.8 (<0.01) 9.89 (<0.01) 10.1 (<0.01) -1.45 (0.20) 0.80 (0.05) -3.37 (0.05) 3.46 (<0.01) He -0.05 (0.68) 0.11 (<0.01) -0.15 (<0.01) 0.20 (<0.01) 0.03 (<0.01) 0.28 (<0.01) 0.00 (0.49) He x He 0.00 (<0.01) ----0.00 (<0.01) 0.00 (<0.01) ----0.00 (0.84) ----Starch: Intercept 15.2 (0.03) 12.9 (<0.01) 3.10 (0.15) 1.62 (0.17) 0.61 (0.12) 0.06 (0.96) 3.94 (<0.01) He 0.38 (<0.01) 0.12 (<0.01) 0.14 (0.01) 0.04 (0.19) 0.01 (0.05) 0.00 (0.97) 0.00 (0.74) He x He 0.00 (0.33) ----0.00 (0.45) 0.00 (0.98) ----0.00 (0.97) ----Pectin: Intercept 17.6 (0.01) 9.37 (<0.01) 4.52 (0.06) 2.21 (0.08) 0.22 (0.50) -0.12 (0.93) 3.97 (<0.01) He 0.22 (0.07) 0.27 (<0.01) 0.03 (0.6) -0.02 (0.49) 0.00 (0.49) 0.00 (0.91) 0.00 (0.02) He x He 0.00 (0.28) ----0.00 (0.38) 0.00 (0.34) ----0.00 (0.97) -----

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116Table 4-8. Continued Total VFA1 C2 C3 C4 Val Max. Lac (h) BCVFA NFC combination analysis 6 Coefficients ( Pvalues) Intercept 15.5 (<0.01) 10.1 (<0.01) 3.48 (<0.01) 1.97 (<0.01) 0.53 (0.06) -0.52 (0.36) 3.98 (<0.01) Sucrose 33.6 (<0.01) 13.3 (<0.01) 13.4 (<0.01) 6.72 (<0.01) 3.53 (<0.01) 30.9 (<0.01) -0.95 (<0.01) Starch 31.6 (<0.01) 16.0 (<0.01) 11.9 (<0.01) 3.79 (<0.01) 0.67 (<0.01) 1.45 (<0.01) -0.19 (0.41) Pectin 36.6 (<0.01) 29.0 (<0.01) 7.51 (<0.01) 0.04 (0.91) -0.23 (0.23) 0.06 (0.90) -1.10 (<0.01) Sucrose x Starch -2.09 (0.80) -1.00 (0.86) -3.71 (0.22) 2.45 (0.19) -0.75 (0.42) -10.9 (<0.01) -0.98 (0.38) Sucrose x Pectin 7.01 (0.41) 4.82 (0.43) -0.12 (0.97) 1.90 (0.33) 3.04 (<0.01) 3.11 (0.17) -0.21 (0.86) Starch x Pectin -17.6 (0.03) -8.96 (0.12) -4.61 (0.13) -3.44 (0.06) -0.67 (0.47) -0.84 (0.71) -2.36 (0.04) 1 VFA = volatile fatty acid; C2 = acetate; C3 = propionate; C4 = butyrate; Val = valerate; Lac = lactate; BCVFA = branched chain VFA 2 Main effects ( P -values) and regression coefficients (estimates and P -values) for individual NFCs were obtained with a proc mixed heterogeneity of regression analysis 3 NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 4 He = hexose equivalent (continuous variable) 5 HeCL = hexose equivalent (class variable); NFC x HeCL = lack of fit term: if significant then regression line fits data poorly 6 Regression coefficients (estimates and P -values) for NFC combinations were obtained with a standard proc mixed regression analysis

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117 Table 4-9. Residual NDFOM at 24 h and temporal patterns for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs. Valu es are least squares means. Item1 Amount2 Residual NDFOM3 (%) Temporal pattern4 iNDF 61.2 C (P < 0.01) (SE5 = 0.98) Individual NFCs Sucrose 40 60.8 Qt (P = 0.04) 80 60.7 C (P < 0.01) 120 60.1 C (P < 0.01) Starch 40 60.8 C (P < 0.01) 80 60.9 C (P < 0.01) 120 61.1 C (P < 0.01) Pectin 40 63.8 C (P < 0.01) 80 65.8 C (P < 0.01) 120 66.3 C (P < 0.01) (SE = 1.02) NFC combinations Su40St80 60.6 L (P < 0.01) Su80St40 61.6 C (P = 0.02) St40Pe80 65.7 Qd (P = 0.01) St80Pe40 64.0 C (P = 0.05) Su40Pe80 65.7 C (P = 0.04) Su80Pe40 63.1 C (P < 0.01) (SE = 1.12) 1 iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 2 Amount of NFC substrate in mg nominal hexose equivalent 3 NDFOM = neutral detergent fiber organic matter 4 Pattern over 24 h fermentation: L = linear; Qd = quadratic; C = cubic; Qt = quartic 5 SE = standard error of least squares means

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118 Table 4-10. Main effects and regression coe fficients for residual NDFOM at 24 h for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coeffici ents for fermentations of NFC combinations. Main effects 1 Pvalues NFC2 <0.01 He3 0.07 NFC x He <0.01 NFC x HeCL4 0.73 Contrasts Intercept Slope Su and St vs. Pe <0.01 <0.01 Su vs. St 0.98 0.28 Regression coefficients Coefficients ( Pvalues) Individual NFCs Sucrose Starch Pectin Intercept 61.3 (<0.01) 60.6 (<0.01) 62.9 (<0.01) He 0.00 (0.23) 0.00 (0.61) 0.03 (<0.01) NFC combination analysis 5 Coefficients ( Pvalues) Intercept 61.3 (<0.01) Sucrose -1.14 (0.04) Starch -0.48 (0.38) Pectin 5.58 (<0.01) Sucrose x Starch 2.77 (0.29) Sucrose x Pectin 3.50 (0.18) Starch x Pectin 4.17 (0.11) 1 Main effects ( P -values) and regression coefficients (estimates and P -values) for individual NFCs were obtained with a proc mixed heterogeneity of regression analysis 2 NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 3 He = hexose equivalent (continuous variable) 4 HeCL = hexose equivalent (class variable); NFC x HeCL = lack of fit term: if significant then regression line fits data poorly 5 Regression coefficients (estimates and P -values) for NFC combinations were obtained with a standard proc mixed regression analysis

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119 Table 4-11. Microbial crude protein (MCP) yield (mean, maximum, hour of maximum and temporal pattern) for 24 h in vitr o fermentations of iNDF, NFC sources (sucrose, starch and pectin), and comb inations of NFCs. Values are least squares means. Microbial protein yield Item1 Amount2 Mean, mg Max., mg (h) Temporal pattern3 iNDF 2.68 5.12 (16) C (P < 0.01) (SE4 = 0.81) (SE = 1.16) Individual NFCs Sucrose 40 7.15 10.8 (16) Qd (P < 0.01) 80 10.6 15.8 (12) Qd (P < 0.01) 120 12.9 17.9 (12) C (P = 0.04) Starch 40 5.97 12.0 (12) Qt (P < 0.01) 80 9.39 16.7 (16) Qt (P = 0.02) 120 12.0 21.0 (16) Qt (P = 0.02) Pectin 40 7.60 12.0 (12) Qt (P < 0.01) 80 11.3 18.7 (16) Qt (P < 0.01) 120 15.9 25.0 (8) Qt (P < 0.01) (SE = 0.96) (SE = 1.42) NFC combinations Su40St80 11.0 19.6 (12) Qd (P < 0.01) Su80St40 14.2 21.9 (16) Qd (P < 0.01) St40Pe80 15.4 23.8 (16) Qt (P = 0.01) St80Pe40 15.5 23.7 (16) Qt (P = 0.03) Su40Pe80 14.3 25.3 (16) Qt (P = 0.03) Su80Pe40 14.2 20.3 (16) Qd (P < 0.01) (SE = 1.29) (SE = 2.00) 1 iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 2 Amount of substrate in mg nominal hexose equivalent 3 Pattern over 24 h fermentation: Qd = quadratic; C = cubic; Qt = quartic 4 SE = standard error of least squares means

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120 Table 4-12. Main effects and regression co efficients for maximum microbial crude protein (MCP) yield for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and regression coefficients for fermentations of NFC combinations. Main effects 1 Pvalues NFC2 0.07 He3 <0.01 NFC x He <0.01 NFC x HeCL4 0.46 Contrasts Intercept Slope Su and St vs. Pe 0.12 <0.01 Su vs. St 0.47 0.07 Regression coefficients Coefficients ( Pvalues) Individual NFCs Sucrose Starch Pectin Intercept 7.33 (<0.01) 9.19 (<0.01) 5.81 (0.02) He 0.09 (<0.01) 0.11 (<0.01) 0.17 (<0.01) NFC combinations analysis 5 Coefficients ( Pvalues) Intercept 6.00 (<0.01) Sucrose 12.4 (<0.01) Starch 15.6 (<0.01) Pectin 19.3 (<0.01) Sucrose x Starch 1.67 (0.67) Sucrose x Pectin 2.87 (0.47) Starch x Pectin 5.10 (0.20) 1 Main effects ( P -values) and regression coefficients (estimates and P -values) for individual NFCs were obtained with a proc mixed heterogeneity of regression analysis 2 NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = starch; Pe = pectin 3 He = hexose equivalent (continuous variable) 4 HeCL = hexose equivalent (class variable); NFC x HeCL = lack of fit term: if significant then regression line fits data poorly 5 Regression coefficients (estimates and P -values) for NFC combinations were obtained with a standard proc mixed regression analysis

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121 CHAPTER 5 CONCLUSIONS Results from a series of in vitro fermen tations indicate that pH, nitrogen source, addition of non-neutral detergent fiber car bohydrates (NFCs) and NFC complement may all be important factors in determining produc t yield and effect on neutral detergent fiber (NDF) digestion by ruminal microorganisms. Since ruminal microorganisms and their fermentation products form a major part of th e nutrient supply to the ruminant animal these factors need to be considered when fo rmulating diets or predicting animal response. When feeding diets containing sucrose, th e current ruminal pH might affect the relative change in ruminal environment (e.g. pH, organi c acid and ammonia nitrogen concentrations), the degradation of substrates and the amounts of nut rients supplied to the animal. Sucrose appears to be readily fe rmented regardless of pH. However, the utilization of the monosaccharide constituents (glucose and fructose) differs depending on pH. At a more neutral pH sucrose s upplementation may increase fiber digestion, potentially increasing nutrie nt supply to the animal, but at acidic pH sucrose supplementation may decrease fiber digestion. Microbial protein synt hesis is higher at a more neutral pH, also adding to increased nutrient supply. Under acidic conditions, however, ruminal microorganisms might store more glycogen as a proportion of cell mass as compared to neutral fermentation c onditions, which could decrease microbial cell viability due to a large carbohydrate to prot ein ratio. The potential effects of a more acidic pH and sucrose on ruminal fermentation would be important to take into consideration when supplementing sucrose to a ruminant diet that already contains

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122 substantial amounts of readily fermentable car bohydrate. Therefore it is recommended that when supplementing sucrose to diets fo r ruminant animals, diets should include enough effective fiber or another form of buffe ring agent, such as sodium bicarbonate, to ensure that the ruminal pH remains near neut ral. Also, animal studies further evaluating the effects of ruminal pH on substrate ut ilization and nutrient supply are warranted. Carbohydrate substrate and n itrogen source may also a ffect in vitro yield of fermentation products and NDF fermentation. Addition of true protein increased microbial crude protein (MCP) yield and efficiency of yi eld (MCPeff) from ruminal microorganisms when sucrose was present a nd had a positive effect on MCP yield from NDF alone. True protein addition increased NDF digestion when sucrose was present, and increased total yield of organic ac ids. Maximum accumulation of microbial glycogen (GLY) was not affected by nitrogen source when sucrose and NDF were fermented together. These results imply that it would be advantageous to supply rumen degradable nitrogen in the form of amino aci ds or peptides in ruminant diets when a readily fermentable carbohydrate source such as sucrose is supplemented. Also, the sources of ruminal degradable nitrogen and th e inclusion of sucrose may be important to consider in the prediction of fiber dige stion and metabolizable nutrient supply in ruminant diets. Animal studies investigat ing the interaction of ruminally degradable nitrogen source and NFC source on ruminal measures and animal performance are warranted. Various types and combinations of NFCs altered the yield of microbial products and extent of fiber digestion. Supplementation of sucrose to ruminant diets may increase the supply of -glucan to the small intestine, which when hydrolyzed by enzymes in the

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123 small intestine and absorbed, becomes part of the metabolizable glucose supply to the ruminant animal. Supplementation of pectin on the other hand may increase the supply of metabolizable nitrogen to the small intestin e of the ruminant animal in the form of increased microbial protein s ynthesis. Among the three NFC sources evaluated sucrose had the greatest negative effect on pH and therefore may have more potential to cause ruminal acidosis. This may imply that sucrose also has the greatest potential to decrease fiber digestion in the rumen. However, sucr ose fermentations increased NDF digestion in the first in vitro study when the fermentation pH was near neutral and in the third in vitro study. This increase in NDF dige stion in the third in vitro study may also be attributed to the fact that fermentation pH in sucrose fermentations never decreased below 6, a pH considered critical for maintaining fiber-utilizing micr obial populations. Differences among NFC components regard ing microbial fermentation may imply that the complement of NFCs in a particular feedstuff is important when predicting animal response. The treatment of all NFCs as a uniform entity in ruminant nutrition is not warranted. Further in vitro and in vivo st udies are needed to increase our ability to explain and predict animal response wh en supplementing diets with NFC sources.

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124 APPENDIX A ADDITIONAL FIGURES FOR CHAPTER 2 -5 0 5 10 15 20 25 30 04812162024 Fermentation hourGlucose (mg) Figure A-1. Residual glucose content (LSm eans standard error) for 24 h in vitro fermentations of SuNDF with initial me dium pH, before addition of reducing solution or inoculum, of 6.8 ( ) or 5.6 ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

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125 -5 0 5 10 15 20 25 30 35 40 45 04812162024 Fermentation hourFructose (mg) Figure A-2. Residual fructose content (LSm eans standard error) for 24 h in vitro fermentations of SuNDF with initial me dium pH, before addition of reducing solution or inoculum, of 6.8 ( ) or 5.6 ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. -20 0 20 40 60 80 100 04812162024Fermentation hourSucrose (mg) Figure A-3. Residual sucrose content (LSm eans standard error) for 24 h in vitro fermentations of SuNDF with initial me dium pH, before addition of reducing solution or inoculum, of 6.8 ( ) or 5.6 ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

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126 APPENDIX B ADDITIONAL FIGURES FOR CHAPTER 3 -5 0 5 10 15 20 0481216 Fermentation hourGlucose (mg) Figure B-1. Residual glucose content (LSm eans standard error) for 16 h in vitro fermentations of SuNDF with media cont aining nitrogen in the form of nonprotein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

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127 -5 0 5 10 15 20 0481216 Fermentation hourFructose (mg) Figure B-2. Residual fructose content (LSm eans standard error) for 24 h in vitro fermentations of SuNDF with media cont aining nitrogen in the form of nonprotein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue. -20 0 20 40 60 80 100 120 0481216 Fermentation hourSucrose (mg) Figure B-3. Residual sucrose content (LSm eans standard error) for 24 h in vitro fermentations of SuNDF with media cont aining nitrogen in the form of nonprotein nitrogen + true protein ( ), true protein only ( ) or non-protein nitrogen only ( ). SuNDF = sucrose + isolated bermudagrass neutral detergent residue.

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128 APPENDIX C FIGURES FOR CHAPTER 4 -2.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 04812162024 Fermentation hourGlucose (mg) Figure C-1. Residual glucose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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129 -2.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 04812162024 Fermentation hourGlucose (mg) Figure C-2. Residual glucose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -2.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 04812162024 Fermentation hourGlucose (mg) Figure C-3. Residual glucose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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130 -2.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 04812162024 Fermentation hourGlucose (mg) Figure C-4. Residual glucose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isol ated bermudagrass neutral detergent residue; NFC = non-neutral deterg ent fiber carbohydrate. -2.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 04812162024 Fermentation hourGlucose (mg) Figure C-5. Residual glucose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolat ed bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.

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131 -2.0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 04812162024 Fermentation hourGlucose (mg) Figure C-6. Residual glucose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate. -2.0 0.0 2.0 4.0 6.0 8.0 10.0 04812162024 Fermentation hourFructose (mg) Figure C-7. Residual fructose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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132 -2.0 0.0 2.0 4.0 6.0 8.0 10.0 04812162024 Fermentation hourFructose (mg) Figure C-8. Residual fructose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -2.0 0.0 2.0 4.0 6.0 8.0 10.0 04812162024 Fermentation hourFructose (mg) Figure C-9. Residual fructose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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133 -2.0 0.0 2.0 4.0 6.0 8.0 10.0 04812162024 Fermentation hourFructose (mg) Figure C-10. Residual fructose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isol ated bermudagrass neutral detergent residue; NFC = non-neutral deterg ent fiber carbohydrate. -2.0 0.0 2.0 4.0 6.0 8.0 10.0 04812162024 Fermentation hourFructose (mg) Figure C-11. Residual fructose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolat ed bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.

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134 -2.0 0.0 2.0 4.0 6.0 8.0 10.0 04812162024 Fermentation hourFructose (mg) Figure C-12. Residual fructose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate. -20 0 20 40 60 80 100 04812162024 Fermentation hourSucrose (mg) Figure C-13. Residual sucrose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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135 -20 0 20 40 60 80 100 04812162024 Fermentation hourSucrose (mg) Figure C-14. Residual sucrose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -20 0 20 40 60 80 100 04812162024 Fermentation hourSucrose (mg) Figure C-15. Residual sucrose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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136 -20 0 20 40 60 80 100 04812162024 Fermentation hourSucrose (mg) Figure C-16. Residual sucrose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isol ated bermudagrass neutral detergent residue; NFC = non-neutral deterg ent fiber carbohydrate. -20 0 20 40 60 80 100 04812162024 Fermentation hourSucrose (mg) Figure C-17. Residual sucrose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolat ed bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.

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137 -20 0 20 40 60 80 100 04812162024 Fermentation hourSucrose (mg) Figure C-18. Residual sucrose content (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate. -1.0 0.0 1.0 2.0 3.0 4.0 5.0 04812162024 Fermentation hourMicrobial glycogen (mg) Figure C-19. Microbial glycogen yield (LSmean s standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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138 -1.0 0.0 1.0 2.0 3.0 4.0 5.0 04812162024 Fermentation hourMicrobial glycogen (mg) Figure C-20. Microbial glycogen yield (LSmean s standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -1.0 0.0 1.0 2.0 3.0 4.0 5.0 04812162024 Fermentation hourMicrobial glycogen (mg) Figure C-21. Microbial glycogen yield (LSmean s standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.

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139 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 04812162024 Fermentation hourFermentation pH Figure C-22. Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 04812162024 Fermentation hourFermentation pH Figure C-23. Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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140 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 04812162024 Fermentation hourFermentation pH Figure C-24. Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 04812162024 Fermentation hourFermentation pH Figure C-25. Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isol ated bermudagrass neutral detergent residue; NFC = non-neutral deterg ent fiber carbohydrate.

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141 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 04812162024 Fermentation hourFermentation pH Figure C-26. Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolat ed bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate. 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 04812162024 Fermentation hourFermentation pH Figure C-27. Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.

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142 0 5 10 15 20 25 30 35 40 45 04812162024Fermentation hourAcetate (m M ) Figure C-28. Acetate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. 0 5 10 15 20 25 30 35 40 45 04812162024Fermentation hourAcetate (m M ) Figure C-29. Acetate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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143 0 5 10 15 20 25 30 35 40 45 04812162024Fermentation hourAcetate (m M ) Figure C-30. Acetate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. 0 5 10 15 20 25 30 35 40 45 04812162024Fermentation hourAcetate (m M ) Figure C-31. Acetate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isol ated bermudagrass neutral detergent residue; NFC = non-neutral deterg ent fiber carbohydrate.

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144 0 5 10 15 20 25 30 35 40 45 04812162024Fermentation hourAcetate (m M ) Figure C-32. Acetate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolat ed bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate. 0 5 10 15 20 25 30 35 40 45 04812162024Fermentation hourAcetate (m M ) Figure C-33. Acetate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.

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145 -5 0 5 10 15 20 2504812162024Fermentation hourPropionate (m M ) Figure C-34. Propionate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -5 0 5 10 15 20 2504812162024Fermentation hourPropionate (m M ) Figure C-35. Propionate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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146 -5 0 5 10 15 20 2504812162024Fermentation hourPropionate (m M ) Figure C-36. Propionate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -5 0 5 10 15 20 2504812162024Fermentation hourPropionate (m M ) Figure C-37. Propionate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isol ated bermudagrass neutral detergent residue; NFC = non-neutral deterg ent fiber carbohydrate.

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147 -5 0 5 10 15 20 2504812162024Fermentation hourPropionate (m M ) Figure C-38. Propionate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolat ed bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate. -5 0 5 10 15 20 2504812162024Fermentation hourPropionate (m M ) Figure C-39. Propionate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.

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148 -2.0 0.0 2.0 4.0 6.0 8.0 10.004812162024Fermentation hourButyrate (m M ) Figure C-40. Butyrate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -2.0 0.0 2.0 4.0 6.0 8.0 10.004812162024Fermentation hourButyrate (m M ) Figure C-41. Butyrate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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149 -2.0 0.0 2.0 4.0 6.0 8.0 10.004812162024Fermentation hourButyrate (m M ) Figure C-42. Butyrate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -2.0 0.0 2.0 4.0 6.0 8.0 10.004812162024Fermentation hourButyrate (m M ) Figure C-43. Butyrate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isol ated bermudagrass neutral detergent residue; NFC = non-neutral deterg ent fiber carbohydrate.

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150 -2.0 0.0 2.0 4.0 6.0 8.0 10.004812162024Fermentation hourButyrate (m M ) Figure C-44. Butyrate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolat ed bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate. -2.0 0.0 2.0 4.0 6.0 8.0 10.004812162024Fermentation hourButyrate (m M ) Figure C-45. Butyrate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.

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151 -10 0 10 20 30 40 50 60 7004812162024Fermentation hourTotal VFA (m M ) Figure C-46. Total volatile fatty acid concentr ations (LSmeans sta ndard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -10 0 10 20 30 40 50 60 7004812162024Fermentation hourTotal VFA (m M ) Figure C-47. Total volatile fatty acid concentr ations (LSmeans sta ndard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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152 -10 0 10 20 30 40 50 60 7004812162024Fermentation hourTotal VFA (m M ) Figure C-48. Total volatile fatty acid concentr ations (LSmeans sta ndard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -10 0 10 20 30 40 50 60 7004812162024Fermentation hourTotal VFA (m M ) Figure C-49. Total volatile fatty acid concentr ations (LSmeans sta ndard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neut ral detergent fiber carbohydrate.

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153 -10 0 10 20 30 40 50 60 7004812162024Fermentation hourTotal VFA (m M ) Figure C-50. Total volatile fatty acid concentr ations (LSmeans sta ndard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NF C combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutra l detergent fiber carbohydrate. -10 0 10 20 30 40 50 60 7004812162024Fermentation hourTotal VFA (m M ) Figure C-51. Total volatile fatty acid concentr ations (LSmeans sta ndard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-ne utral detergent fiber carbohydrate.

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154 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.504812162024Fermentation hourBCVFA (m M ) Figure C-52. Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermenta tions of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.504812162024Fermentation hourBCVFA (m M ) Figure C-53. Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermenta tions of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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155 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.504812162024Fermentation hourBCVFA (m M ) Figure C-54. Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermenta tions of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.504812162024Fermentation hourBCVFA (m M ) Figure C-55. Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residu e; NFC = non-neutral detergent fiber carbohydrate.

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156 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.504812162024Fermentation hourBCVFA (m M ) Figure C-56. Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, a nd iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolated bermudagrass neutral detergent residu e; NFC = non-neutral detergent fiber carbohydrate. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.504812162024Fermentation hourBCVFA (m M ) Figure C-57. Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated

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157 bermudagrass neutral detergent residu e; NFC = non-neutral detergent fiber carbohydrate. -5 0 5 10 15 20 25 30 3504812162024Fermentation hourLactate (m M ) Figure C-58. Lactate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -5 0 5 10 15 20 25 30 3504812162024Fermentation hourLactate (m M ) Figure C-59. Lactate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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158 -5 0 5 10 15 20 25 30 3504812162024Fermentation hourLactate (m M ) Figure C-60. Lactate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -5 0 5 10 15 20 25 30 3504812162024Fermentation hourLactate (m M ) Figure C-61. Lactate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isol ated bermudagrass neutral detergent residue; NFC = non-neutral deterg ent fiber carbohydrate.

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159 -5 0 5 10 15 20 25 30 3504812162024Fermentation hourLactate (m M ) Figure C-62. Lactate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolat ed bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate. -5 0 5 10 15 20 25 30 3504812162024Fermentation hourLactate (m M ) Figure C-63. Lactate concentrations (LSm eans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.

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160 50 60 70 80 90 100 04812162024Fermentation hourResidual NDF (%) Figure C-64. Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. 50 60 70 80 90 100 04812162024 Fermentation hourResidual NDF (%) Figure C-65. Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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161 50 60 70 80 90 100 04812162024 Fermentation hourResidual NDF (%) Figure C-66. Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. 50 60 70 80 90 100 04812162024 Fermentation hourResidual NDF (%) Figure C-67. Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isol ated bermudagrass neutral detergent residue; NFC = non-neutral deterg ent fiber carbohydrate.

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162 50 60 70 80 90 100 04812162024 Fermentation hourResidual NDF (%) Figure C-68. Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolat ed bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate. 50 60 70 80 90 100 04812162024 Fermentation hourResidual NDF (%) Figure C-69. Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.

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163 -5 0 5 10 15 20 25 30 04812162024 Fermentation hourMicrobial protein (mg) Figure C-70. Microbial crude pr otein yield (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + sucrose at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -5 0 5 10 15 20 25 30 04812162024 Fermentation hourMicrobial protein (mg) Figure C-71. Microbial crude pr otein yield (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + starch at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.

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164 -5 0 5 10 15 20 25 30 04812162024 Fermentation hourMicrobial protein (mg) Figure C-72. Microbial crude pr otein yield (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry; ), and iNDF + pectin at 40 ( ), 80 ( ) or 120 mg ( ) hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue. -5 0 5 10 15 20 25 30 04812162024 Fermentation hourMicrobial protein (mg) Figure C-73. Microbial crude pr otein yield (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:starch. iNDF = isol ated bermudagrass neutral detergent residue; NFC = non-neutral deterg ent fiber carbohydrate.

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165 -5 0 5 10 15 20 25 30 04812162024 Fermentation hourMicrobial protein (mg) Figure C-74. Microbial crude pr otein yield (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + pectin ( ) or starch ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for pectin:starch. iNDF = isolat ed bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate. -5 0 5 10 15 20 25 30 04812162024 Fermentation hourMicrobial protein (mg) Figure C-75. Microbial crude pr otein yield (LSmeans standa rd error) for 24 h in vitro fermentations of iNDF (120 mg air dry) + sucrose ( ) or pectin ( ) at 120 mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 ( ) and 80:40 ( ) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral dete rgent fiber carbohydrate.

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APPENDIX D ADDITIONAL TABLE FOR CHAPTER 4

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167 Table D-1. Temporal patterns of organic ac id concentrations for 24 h in vitro fermen tations of iNDF, NFC sources (sucrose, star ch and pectin), and combinations of NFCs. Item1 Amount2 Total VFA3 C2 C3 C4 Val Lac BCVFA Temporal pattern4 ( P -value) iNDF (120 mg air dry) C (0.01) C (0.01) C (< 0.01) Qd (0.03) L (< 0.01) NDP C (< 0.01) NFC individual Sucrose 40 L (< 0.01) L (0.01) Qd (< 0.01) Qt (0.05) L (< 0.01) Qt (< 0.01) Qt (0.04) 80 L (< 0.01) L (< 0.01) Qd (< 0.01) C (< 0.01) C (< 0.01) Qt (< 0.01) C (< 0.01) 120 Qt (< 0.01) Qt (< 0.01) Qt (< 0.01) L (< 0.01) C (< 0.01) Qt (< 0.01) C (< 0.01) Starch 40 C (0.02) C (0.05) C (< 0.01 ) Qd (0.01) L (< 0.01) Qt (< 0.01) Qt (0.03) 80 Qt (0.05) C (0.03) Qt (< 0.01) L (< 0.01) L (< 0.01) C (< 0.01) Qt (< 0.01) 120 Qt (0.02) Qt (< 0.01) Qt (0.02) L (< 0.01) L (< 0.01) C (0.04) C (< 0.01) Pectin 40 Qt (0.02) Qt (0.04) Qd (0.03) Qd (0.03) L (0.01) C (< 0.01) C (< 0.01) 80 Qt (0.03) Qt (0.01) Qt (< 0.01) C (0.01) Qd (< 0.01) C (0.05) C (< 0.01) 120 Qt (< 0.01) Qt (0.04) Qt (< 0.01) L (< 0.01) Qd (< 0.01) Qt (< 0.01) C (0.02) NFC combinations Su40St80 Qd (0.05) L (< 0.01) Qd (0.01) L (< 0.01) C (0.02) Qt (< 0.01) C (< 0.01) Su80St40 Qd (< 0.01) L (< 0.01) Qt (0.03) L (< 0.01) C (0.03) Qt (< 0.01) C (0.02) St40Pe80 Qt (< 0.01) Qt (< 0.01) Qt (< 0.01) C (< 0.01) L (< 0.01) L (< 0.01) L (< 0.01) St80Pe40 Qt (0.02) Qd (< 0.01) Qt (< 0.01) C (0.02) L (< 0.01) C (< 0.01) C (0.04) Su40Pe80 Qt (< 0.01) Qt (< 0.01) Qt (< 0.01) C (< 0.01) C (0.05) Qt (< 0.01) C (0.04) Su80Pe40 L (< 0.01) L (< 0.01) L (< 0.01) L (< 0.01) L (< 0.01) Qt (< 0.01) L (< 0.01) 1 iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral detergent fiber carbohydrate; Su = sucrose; St = sta rch; Pe = pectin 2 Amount of NFC substrate in mg hexose equivalent 3 VFA = volatile fatty acid; C2 = acetate; C3 = propionate; C4 = butyrate; Val = valerate; Lac = lactate; BCVFA = branched chain VFA 4 L = linear; Qd = quadratic; C = cubic; Qt = quartic; NDP = no detectable pattern

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168APPENDIX E CHAPTER 2 RAW DATA Table E-1. Data used for statistical analys is in evaluating the effect of pH on microbial yield and neutral detergen fiber dige stion from in vitro fermentations of sucrose and isol ated bermudagrass neutral detergent residue. Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 3 NpH SuNDF 0 0.1114 0.1201 — — 0.1228 — — — — — — — — — — — — — 1 4 NpH SuNDF 0 0.1115 0.1199 — — -0.1227— — — — — — — — — — — — — 1 7 ApH SuNDF 0 0.1110 0.1200 — — 0.3774 — — — — — — — — — — — — — 1 8 ApH SuNDF 0 0.1118 0.1200 — — -0.3786— — — — — — — — — — — — — 1 9 NpH BL 0 — — — — — — — — — 0.0000 14.04384.1158 2.3390 0.23090.42660.5722 8.2307 10.6721 1 10 NpH BL 0 — — — — — — — — — 0.0000 13.95504.2600 1.9250 0.25000.33500.4050 8.4490 10.4990 1 11 NpH SuNDF 0 0.1117 0.1200 — — — 0.370767.68409.2148 9.9029 0.4350 14.33004.5450 1.9700 0.22500.31000.3350 8.1001 11.0689 1 12 NpH SuNDF 0 0.1111 0.1201 — — — -0.016576.28489.7920 10.13170.4500 14.24004.6200 1.9350 0.23000.29000.4000 8.9296 10.4949 1 13 ApH BL 0 — — — — — — — — — 0.0000 14.13974.1394 1.9570 0.26130.30230.4150 7.2441 10.6176 1 14 ApH BL 0 — — — — — — — — — 0.0000 14.11134.2247 2.0232 0.01530.31600.2905 8.6018 12.3786 1 15 ApH SuNDF 0 — — — — — -0.411917.164731.136832.85960.3609 14.18874.3412 1.9643 0.20110.27840.4022 8.0978 11.5255 1 16 ApH SuNDF 0 — — — — — -0.444912.835031.275632.20760.3752 14.51134.6520 1.9842 0.22100.27240.2981 8.1144 11.9552 1 17 NpH iNDF 0 0.2231 0.0000 0.2104 7.07— — — — — — — — — — — — — — 1 18 NpH iNDF 0 0.2230 0.0000 0.2138 7.03— — — — — — — — — — — — — — 1 19 NpH SuNDF 0 0.1116 0.1202 0.1071 7.05— — — — — — — — — — — — — — 1 20 NpH SuNDF 0 0.1114 0.1199 0.1070 7.02— — — — — — — — — — — — — — 1 21 ApH iNDF 0 0.2230 0.0000 0.2142 6.13— — — — — — — — — — — — — — 1 22 ApH iNDF 0 0.2230 0.0000 0.2150 6.13— — — — — — — — — — — — — —

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169Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 23 ApH SuNDF 0 0.1112 0.1203 0.1079 6.13— — — — — — — — — — — — — — 1 24 ApH SuNDF 0 0.1116 0.1199 0.1086 6.12— — — — — — — — — — — — — — 1 27 NpH SuNDF 4 0.1118 0.1199 — — 5.8304 — — — — — — — — — — — — — 1 28 NpH SuNDF 4 0.1114 — — 7.2047 — — — — — — — — — — — — — 1 31 ApH SuNDF 4 0.1116 0.1199 — — 2.1743 — — — — — — — — — — — — — 1 32 ApH SuNDF 4 0.1114 0.1200 — — 1.1084 — — — — — — — — — — — — — 1 33 NpH BL 4 — — — — — — — — — 0.0000 17.47005.5950 2.3150 0.30500.38500.5000 7.4663 14.5975 1 34 NpH BL 4 — — — — — — — — — 0.0000 17.72005.6150 2.3050 0.30500.40500.5400 9.2608 15.8433 1 35 NpH SuNDF 4 0.1114 0.1202 — — — 5.4948-0.3377-0.6881-0.163831.0773 22.73089.5896 3.7345 0.38130.33950.4962 5.4841 9.6161 1 36 NpH SuNDF 4 0.1112 0.1198 — — — 7.1012-0.3693-0.6764-0.237030.5183 22.58838.8958 3.8309 0.32190.39700.3917 7.1125 12.6730 1 37 ApH BL 4 . — — — — — — — 0.0157 20.61795.1884 2.4192 0.24560.40760.4807 8.5287 13.7264 1 38 ApH BL 4 . — — — — — — — 0.0000 18.85014.7426 1.9743 0.26640.22980.4387 7.3615 11.6482 1 39 ApH SuNDF 4 0.1115 0.1202 — — — 2.3190-0.1010-1.620148.94657.3136 21.56696.9717 2.7513 0.29920.36330.4755 9.2936 13.6376 1 40 ApH SuNDF 4 0.1115 0.1199 — — — 3.1510-0.1490-1.620145.62257.9548 22.79407.0779 2.8186 0.26760.37580.4669 8.5886 12.6489 1 41 NpH iNDF 4 0.2230 0.0000 0.2097 7.11— — — — — — — — — — — — — — 1 42 NpH iNDF 4 0.2228 0.0000 0.2105 7.09— — — — — — — — — — — — — — 1 43 NpH SuNDF 4 0.1115 0.1202 0.1069 6.74— — — — — — — — — — — — — — 1 44 NpH SuNDF 4 0.1116 0.1200 0.1070 6.73— — — — — — — — — — — — — — 1 45 ApH iNDF 4 0.2231 0.0000 0.2125 6.29— — — — — — — — — — — — — — 1 46 ApH iNDF 4 0.2231 0.0000 0.2142 6.22— — — — — — — — — — — — — — 1 47 ApH SuNDF 4 0.1118 0.1201 0.1087 6.01— — — — — — — — — — — — — — 1 48 ApH SuNDF 4 0.1117 0.1200 0.1084 6.01— — — — — — — — — — — — — — 1 51 NpH SuNDF 8 0.1118 0.1201 — — 20.2040— — — — — — — — — — — — — 1 52 NpH SuNDF 8 0.1112 0.1203 — — 19.6321— — — — — — — — — — — — — 1 55 ApH SuNDF 8 0.1114 0.1200 — — 7.9675 — — — — — — — — — — — — — 1 56 ApH SuNDF 8 0.1113 0.1200 — — 6.9017 — — — — — — — — — — — — — 1 57 NpH BL 8 — — — — — — — — — 0.0000 19.57006.1000 4.7700 0.40500.45500.6500 6.7059 14.6006 1 58 NpH BL 8 — — — — — — — — — 0.0000 20.01505.8800 3.1250 0.18000.41000.5900 6.8028 16.2393

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170Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 59 NpH SuNDF 8 0.1111 0.1202 — — — 1.6970-0.19760.1583 -0.004014.7600 35.780020.12505.7300 0.72500.39000.7700 4.7853 12.9973 1 60 NpH SuNDF 8 0.1117 0.1201 — — — 1.4828-0.21540.1519 -0.021415.4622 34.256519.32905.5070 0.37370.55540.6592 4.7905 12.6039 1 61 ApH BL 8 . — — — — — — — 0.0000 52.29336.2754 2.6938 0.35000.41090.5377 8.3546 14.5210 1 62 ApH BL 8 . — — — — — — — 0.0716 55.04626.1543 2.4535 0.24020.38850.4549 7.6446 13.7469 1 63 ApH SuNDF 8 0.1112 0.1201 — — — -0.2307-0.1750-0.41030.0057 28.4625 35.003713.47573.5863 0.21740.29500.5434 6.8616 10.9209 1 64 ApH SuNDF 8 0.1115 0.1201 — — — 1.4252-0.2529-0.32080.0279 26.2400 34.120013.22003.1550 0.10500.15500.4200 6.8102 10.8064 1 65 NpH iNDF 8 0.2226 0.0000 0.2087 7.17— — — — — — — — — — — — — — 1 66 NpH iNDF 8 0.2230 0.0000 0.2094 7.18— — — — — — — — — — — — — — 1 67 NpH SuNDF 8 0.1115 0.1197 0.1035 6.80— — — — — — — — — — — — — — 1 68 NpH SuNDF 8 0.1116 0.1200 0.1032 6.85— — — — — — — — — — — — — — 1 69 ApH iNDF 8 0.2229 0.0000 0.2109 6.49— — — — — — — — — — — — — — 1 70 ApH iNDF 8 0.2231 0.0000 0.2120 6.48— — — — — — — — — — — — — — 1 71 ApH SuNDF 8 0.1115 0.1198 0.1080 5.37— — — — — — — — — — — — — — 1 72 ApH SuNDF 8 0.1115 0.1199 0.1077 5.38— — — — — — — — — — — — — — 1 75 NpH SuNDF 12 0.1116 0.1200 — — 21.7672— — — — — — — — — — — — — 1 76 NpH SuNDF 12 0.1117 0.1203 — — 21.0245— — — — — — — — — — — — — 1 79 ApH SuNDF 12 0.1114 0.1202 — — 10.6327— — — — — — — — — — — — — 1 80 ApH SuNDF 12 0.1114 0.1203 — — 11.0411— — — — — — — — — — — — — 1 81 NpH BL 12 — — — — — — — — — 0.0000 22.78225.9063 3.7628 0.67340.60660.9356 5.5906 17.4949 1 82 NpH BL 12 — — — — — — — — — 0.0000 22.34505.9350 3.3600 0.75000.55500.9750 7.5302 17.8815 1 83 NpH SuNDF 12 0.1112 0.1201 — — — 3.5369-0.24440.5016 -0.04870.0000 39.220021.13509.2900 3.40501.05501.5200 2.7160 17.3216 1 84 NpH SuNDF 12 0.1115 0.1204 — — — 2.1444-0.14200.1729 -0.05190.0000 40.137722.68029.4351 3.38221.08271.3096 2.8851 17.3773 1 85 ApH BL 12 — — — — — — — — — 0.0300 78.96507.1250 2.9350 0.32000.39500.7300 8.8617 17.0561 1 86 ApH BL 12 — — — — — — — — — 0.0000 79.37506.8000 2.7550 0.31000.44500.7500 8.1236 16.3443 1 87 ApH SuNDF 12 0.1114 0.1203 — — — 0.8593-0.0709-0.15410.0197 19.8386 50.060921.52073.6253 0.22600.28620.4268 10.059315.0383 1 88 ApH SuNDF 12 0.1113 0.1202 — — — 0.4215-0.10400.0689 0.0241 19.0800 53.290022.55503.6200 0.18500.29000.3950 7.0308 11.2936 1 89 NpH iNDF 12 0.2226 0.0000 0.2074 7.22— — — — — — — — — — — — — — 1 90 NpH iNDF 12 0.2228 0.0000 0.2032 7.22— — — — — — — — — — — — — —

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171Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 91 NpH SuNDF 12 0.1112 0.1200 0.0960 6.88— — — — — — — — — — — — — — 1 92 NpH SuNDF 12 0.1112 0.1202 0.0930 6.86— — — — — — — — — — — — — — 1 93 ApH iNDF 12 0.2226 0.0000 0.2095 6.62— — — — — — — — — — — — — — 1 94 ApH iNDF 12 0.2228 0.0000 0.2086 6.60— — — — — — — — — — — — — — 1 95 ApH SuNDF 12 0.1113 0.1202 0.1098 5.43— — — — — — — — — — — — — — 1 96 ApH SuNDF 12 0.1118 0.1200 0.1072 5.46— — — — — — — — — — — — — — 1 99 NpH SuNDF 16 0.1114 0.1202 — — 18.4402— — — — — — — — — — — — — 1 100 NpH SuNDF 16 0.1112 0.1202 — — 18.5240— — — — — — — — — — — — — 1 103 ApH SuNDF 16 0.1114 0.1202 — — 12.3577— — — — — — — — — — — — — 1 104 ApH SuNDF 16 0.1113 0.1201 — — 12.1978— — — — — — — — — — — — — 1 105 NpH BL 16 — — — — — — — — — 0.0000 22.49105.4559 3.3622 0.97810.84051.5996 4.3989 21.2751 1 106 NpH BL 16 — — — — — — — — — 0.0000 22.37005.5900 3.3400 1.06500.93501.6250 3.5960 19.6991 1 107 NpH SuNDF 16 0.1113 0.1199 — — — 0.5649-0.05100.2483 0.0068 0.0000 45.170023.28009.3350 4.35001.12501.5350 — — 1 108 NpH SuNDF 16 0.1115 0.1200 — — — 1.1714-0.12930.2968 -0.02030.0000 45.415023.37009.3550 4.32501.08001.5400 1.4056 18.4075 1 109 ApH BL 16 — — — — — — — — — 0.0000 81.46007.5750 3.3800 0.39500.57501.0900 7.7167 17.7083 1 110 ApH BL 16 — — — — — — — — — 0.0000 80.61637.5995 3.2316 0.28920.50221.0653 6.7104 16.7367 1 111 ApH SuNDF 16 0.1118 0.1200 — — — 1.71840.0451 0.3686 0.1381 11.3850 67.410028.84503.9450 0.09000.31000.5150 8.2726 13.209 2 1 112 ApH SuNDF 16 0.1116 0.1201 — — — 1.84810.0184 0.5717 0.0625 10.7150 71.220030.08504.0200 0.19000.33500.4800 9.0806 14.137 7 1 113 NpH iNDF 16 0.2226 0.0000 0.1974 7.24— — — — — — — — — — — — — — 1 114 NpH iNDF 16 0.2228 0.0000 0.1937 7.24— — — — — — — — — — — — — — 1 115 NpH SuNDF 16 0.1113 0.1201 0.0840 6.87— — — — — — — — — — — — — — 1 116 NpH SuNDF 16 0.1114 0.1204 0.0809 6.83— — — — — — — — — — — — — — 1 117 ApH iNDF 16 0.2226 0.0000 0.2075 6.53— — — — — — — — — — — — — — 1 118 ApH iNDF 16 0.2229 0.0000 0.2052 6.70— — — — — — — — — — — — — — 1 119 ApH SuNDF 16 0.1117 0.1200 0.1094 5.55— — — — — — — — — — — — — — 1 120 ApH SuNDF 16 0.1113 0.1199 — 5.58— — — — — — — — — — — — — — 1 123 NpH SuNDF 20 0.1116 0.1200 — — 17.7011— — — — — — — — — — — — — 1 124 NpH SuNDF 20 0.1112 0.1202 — — 18.1954— — — — — — — — — — — — —

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172Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 127 ApH SuNDF 20 0.1116 0.1204 — — 10.7883— — — — — — — — — — — — — 1 128 ApH SuNDF 20 0.1116 0.1200 — — 10.3044— — — — — — — — — — — — — 1 129 NpH BL 20 — — — — — — — — — 0.0000 21.90664.8030 2.9677 1.49151.35491.9970 1.8837 18.9895 1 130 NpH BL 20 — — — — — — — — — 0.0000 23.01204.9836 3.1028 1.61931.34271.9864 2.0661 22.2596 1 131 NpH SuNDF 20 0.1114 0.1200 — — — 0.4940-0.1027-0.0066-0.02510.0000 49.270023.615010.50505.03001.21001.2200 1.0588 19.5832 1 132 NpH SuNDF 20 0.1114 0.1203 — — — 0.8980-0.1027-0.0080-0.02510.0000 50.390023.760010.63505.05501.29001.4300 1.0409 19.2715 1 133 ApH BL 20 — — — — — — — — — 0.0000 81.46007.7800 4.5750 0.46501.31001.9100 4.8662 20.1194 1 134 ApH BL 20 — — — — — — — — — 0.0000 81.05507.9500 4.6300 0.46501.32501.9850 4.7384 18.4607 1 135 ApH SuNDF 20 0.1117 0.1204 — — — 0.53060.1954 0.7880 0.0622 1.6880 89.286338.07575.1347 0.30320.38410.4700 6.3289 11.2347 1 136 ApH SuNDF 20 0.1114 0.1201 — — — -0.8196-0.1438-0.0518-0.13801.3221 89.049538.12874.5365 0.28260.32300.6207 6.0477 10.948 7 1 137 NpH iNDF 20 0.2227 0.0000 0.1804 7.32— — — — — — — — — — — — — — 1 138 NpH iNDF 20 0.2228 0.0000 0.1787 7.32— — — — — — — — — — — — — — 1 139 NpH SuNDF 20 0.1118 0.1202 0.0728 6.91— — — — — — — — — — — — — — 1 140 NpH SuNDF 20 0.1116 0.1202 0.0706 6.93— — — — — — — — — — — — — — 1 141 ApH iNDF 20 0.2229 0.0000 0.2031 6.79— — — — — — — — — — — — — — 1 142 ApH iNDF 20 0.2232 0.0000 0.2046 6.80— — — — — — — — — — — — — — 1 143 ApH SuNDF 20 0.1115 0.1201 0.1066 5.78— — — — — — — — — — — — — — 1 144 ApH SuNDF 20 0.1116 0.1199 0.1073 5.76— — — — — — — — — — — — — — 1 147 NpH SuNDF 24 0.1115 0.1200 — — 18.8930— — — — — — — — — — — — — 1 148 NpH SuNDF 24 0.1116 0.1203 — — 17.7395— — — — — — — — — — — — — 1 151 ApH SuNDF 24 0.1114 0.1201 — — 11.5385— — — — — — — — — — — — — 1 152 ApH SuNDF 24 0.1111 0.1204 — — 10.2237— — — — — — — — — — — — — 1 153 NpH BL 24 — — — — — — — — — 0.0000 21.69004.4750 2.3800 1.59001.35501.9400 1.3661 21.2830 1 154 NpH BL 24 — — — — — — — — — 0.0000 21.48004.2050 2.6950 1.54501.35001.8500 1.7411 24.1988 1 155 NpH SuNDF 24 0.1112 0.1201 — — — 0.3705-0.05620.0034 -0.00970.0000 51.305024.44009.9100 5.11501.21501.7100 0.9474 19.1206 1 156 NpH SuNDF 24 0.1115 0.1201 — — — 0.4696-0.0562-0.0016-0.00970.0000 51.445024.34509.8750 5.22001.15001.6400 0.9513 19.7636 1 157 ApH BL 24 — — — — — — — — — 0.0000 80.79538.1372 5.0507 0.52411.39772.0012 3.3667 16.8181 1 158 ApH BL 24 — — — — — — — — — 0.0000 79.46888.0652 5.0063 0.78511.40711.8047 3.6369 19.4424

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173Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 159 ApH SuNDF 24 0.1119 0.1200 — — — 1.3091-0.10590.8374 -0.06430.0502 86.309836.87816.4347 1.18360.42630.9579 6.0291 12.1153 1 160 ApH SuNDF 24 0.1116 0.1200 — — — 1.12050.1313 0.8242 0.0960 0.0300 86.810037.38505.0600 0.55500.33501.0250 6.2091 11.7841 1 161 NpH iNDF 24 0.2231 0.0000 0.1845 7.26— — — — — — — — — — — — — — 1 162 NpH iNDF 24 0.2228 0.0000 0.1583 6.96— — — — — — — — — — — — — — 1 163 NpH SuNDF 24 0.1116 0.1201 0.0649 6.90— — — — — — — — — — — — — — 1 165 ApH iNDF 24 0.2230 0.0000 — 6.78— — — — — — — — — — — — — — 1 166 ApH iNDF 24 0.2231 0.0000 0.2034 6.86— — — — — — — — — — — — — — 1 167 ApH SuNDF 24 0.1113 0.1203 0.1062 5.79— — — — — — — — — — — — — — 1 168 ApH SuNDF 24 0.1113 0.1201 0.1073 5.78— — — — — — — — — — — — — — 1 171 NpH SuNDF 24 0.1113 0.1202 0.0633 6.95— — — — — — — — — — — — — — 2 1 NpH iNDF 0 0.2228 0.0000 0.2168 6.95— — — — — — — — — — — — — — 2 2 NpH iNDF 0 0.2226 0.0000 0.2148 6.91— — — — — — — — — — — — — — 2 3 NpH SuNDF 0 0.1115 0.1199 0.1115 6.87— — — — — — — — — — — — — — 2 4 NpH SuNDF 0 0.1113 0.1201 0.1101 6.84— — — — — — — — — — — — — — 2 5 NpH BL 0 — — — — — — — — — 0.0000 15.86504.8250 2.1300 0.23000.14000.3050 7.9292 10.3062 2 6 NpH BL 0 — — — — — — — — — 0.0000 15.62504.8150 2.1450 0.22000.18000.2600 7.6134 10.9732 2 7 NpH SuNDF 0 0.1118 0.1200 — — — 0.383194.36367.6349 8.3624 0.3600 16.11005.0250 2.2700 0.20500.23500.2600 8.1881 11.2483 2 8 NpH SuNDF 0 0.1113 0.1200 — — — 0.078395.05957.1625 8.8500 0.3011 16.72533.7054 1.3985 0.14800.16330.2348 8.0268 11.7330 2 11 NpH SuNDF 0 0.1113 0.1200 — — 0.0821 — — — — — — — — — — — — — 2 12 NpH SuNDF 0 0.1113 0.1200 — — -0.0821— — — — — — — — — — — — — 2 13 ApH iNDF 0 0.2228 0.0000 0.2182 6.00— — — — — — — — — — — — — — 2 14 ApH iNDF 0 0.2227 0.0000 0.2180 6.00— — — — — — — — — — — — — — 2 15 ApH SuNDF 0 0.1116 0.1201 0.1107 6.06— — — — — — — — — — — — — — 2 16 ApH SuNDF 0 0.1114 0.1201 0.1103 6.09— — — — — — — — — — — — — — 2 17 ApH BL 0 — — — — — — — — — 0.0000 17.34122.9310 1.4186 0.02090.19820.0209 9.2548 12.9968 2 18 ApH BL 0 — — — — — — — — — 0.0000 17.22505.3500 2.3150 0.23000.24000.2800 8.3045 11.8049 2 19 ApH SuNDF 0 0.1116 0.1201 — — — -0.115385.505921.767221.10330.4150 17.95505.5350 2.4000 0.21000.23500.2900 8.0497 11.9899 2 20 ApH SuNDF 0 0.1116 0.1202 — — — -0.197778.475523.665223.31850.5150 18.24505.6500 2.4350 0.26000.24000.2950 7.8465 11.6758

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174Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 2 23 ApH SuNDF 0 0.1115 0.1200 — — 0.2499 — — — — — — — — — — — — — 2 24 ApH SuNDF 0 0.1118 0.1201 — — -0.2503— — — — — — — — — — — — — 2 25 NpH iNDF 4 0.2229 0.0000 0.2117 6.90— — — — — — — — — — — — — — 2 26 NpH iNDF 4 0.2229 0.0000 0.2123 6.95— — — — — — — — — — — — — — 2 27 NpH SuNDF 4 0.1113 0.1204 0.1103 6.65— — — — — — — — — — — — — — 2 28 NpH SuNDF 4 0.1116 0.1201 0.1095 6.71— — — — — — — — — — — — — — 2 29 NpH BL 4 — — — — — — — — — 0.0000 19.26486.1093 2.5485 0.41300.29720.4483 5.5130 9.6867 2 30 NpH BL 4 — — — — — — — — — 0.0000 18.84005.8600 2.8950 0.41000.32000.5300 6.0710 10.3297 2 31 NpH SuNDF 4 0.1116 0.1202 — — — 7.0433-0.2189-0.60620.0582 24.8501 26.456410.80864.2261 0.45610.26860.3851 7.4138 13.0591 2 32 NpH SuNDF 4 0.1114 0.1200 — — — 4.3002-0.23170.4910 0.2517 25.2000 25.935010.71004.2200 0.46500.27000.4550 5.9460 10.9710 2 35 NpH SuNDF 4 0.1117 0.1203 — — 10.1294— — — — — — — — — — — — — 2 36 NpH SuNDF 4 0.1115 0.1202 — — 9.5610 — — — — — — — — — — — — — 2 37 ApH iNDF 4 0.2227 0.0000 — — — — — — — — — — — — — — — — 2 38 ApH iNDF 4 0.2230 0.0000 0.2179 6.14— — — — — — — — — — — — — — 2 39 ApH SuNDF 4 0.1115 0.1202 0.1118 5.65— — — — — — — — — — — — — — 2 40 ApH SuNDF 4 0.1118 0.1202 0.1120 5.52— — — — — — — — — — — — — — 2 41 ApH BL 4 — — — — — — — — — 0.0000 26.67005.9800 2.6450 0.31500.23500.4500 9.0443 15.6055 2 42 ApH BL 4 — — — — — — — — — 0.0465 27.50586.2065 2.6540 0.29950.28920.4079 6.2693 12.0313 2 43 ApH SuNDF 4 0.1117 0.1202 — — — 4.5721-0.198314.083735.775019.3912 26.88168.6739 3.3128 0.35910.26810.5058 6.8313 11.1753 2 44 ApH SuNDF 4 0.1113 0.1201 — — — 3.7895-0.517813.612933.926519.2000 26.74008.7000 3.1800 0.28500.21000.4500 10.794416.7407 2 47 ApH SuNDF 4 0.1114 0.1202 — — 2.7532 — — — — — — — — — — — — — 2 48 ApH SuNDF 4 0.1117 0.1201 — — 2.2569 — — — — — — — — — — — — — 2 49 NpH iNDF 8 0.2230 0.0000 0.2075 7.00— — — — — — — — — — — — — — 2 50 NpH iNDF 8 0.2227 0.0000 0.2071 7.01— — — — — — — — — — — — — — 2 51 NpH SuNDF 8 0.1117 0.1202 0.1031 6.66— — — — — — — — — — — — — — 2 52 NpH SuNDF 8 0.1118 0.1200 0.1020 6.62— — — — — — — — — — — — — — 2 53 NpH BL 8 — — — — — — — — — 0.0000 22.13156.2768 3.0316 0.61550.35610.6358 6.8637 16.3386 2 54 NpH BL 8 — — — — — — — — — 0.0000 22.01506.1050 2.9450 0.66500.35500.6250 6.9025 18.8614

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175Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 2 55 NpH SuNDF 8 0.1114 0.1201 — — — 5.2929-0.14050.0102 -0.03896.5700 38.635019.27506.9800 1.24000.30000.6800 4.4838 15.2901 2 56 NpH SuNDF 8 0.1114 0.1199 — — — 4.3291-0.09460.0136 -0.13946.6900 38.205018.90507.0250 1.44500.29500.6800 4.3393 14.4568 2 59 NpH SuNDF 8 0.1115 0.1202 — — 9.6842 — — — — — — — — — — — — — 2 60 NpH SuNDF 8 0.1116 0.1200 — — 10.0153— — — — — — — — — — — — — 2 61 ApH iNDF 8 0.2227 0.0000 0.2158 6.33— — — — — — — — — — — — — — 2 62 ApH iNDF 8 0.2227 0.0000 0.2161 6.33— — — — — — — — — — — — — — 2 63 ApH SuNDF 8 0.1118 0.1201 0.1127 5.13— — — — — — — — — — — — — — 2 64 ApH SuNDF 8 0.1116 0.1202 0.1121 5.12— — — — — — — — — — — — — — 2 65 ApH BL 8 — — — — — — — — — 0.0550 56.04005.6500 3.1550 0.40000.32000.6400 6.1963 12.6230 2 66 ApH BL 8 — — — — — — — — — 0.0207 57.82986.1249 3.1866 0.37250.27930.5380 6.4818 12.8710 2 67 ApH SuNDF 8 0.1112 0.1202 — — — 3.1964-0.11020.0686 0.2453 31.1264 40.099713.88423.4248 0.28800.21080.4268 6.4481 10.7564 2 68 ApH SuNDF 8 0.1116 0.1203 — — — 2.8174-0.13080.0540 0.2458 29.9500 39.250013.51003.3500 0.29500.21000.4550 5.9917 9.8918 2 71 ApH SuNDF 8 0.1116 0.1202 — — 6.9391 — — — — — — — — — — — — — 2 72 ApH SuNDF 8 0.1118 0.1202 — — 6.3605 — — — — — — — — — — — — — 2 73 NpH iNDF 12 0.2226 0.0000 0.1998 6.91— — — — — — — — — — — — — — 2 74 NpH iNDF 12 0.2229 0.0000 0.1971 7.09— — — — — — — — — — — — — — 2 75 NpH SuNDF 12 0.1116 0.1201 0.0912 6.69— — — — — — — — — — — — — — 2 76 NpH SuNDF 12 0.1113 0.1201 0.0907 6.72— — — — — — — — — — — — — — 2 77 NpH BL 12 — — — — — — — — — 0.0000 23.75006.1850 3.5800 1.05000.56501.1750 4.0947 18.9294 2 78 NpH BL 12 — — — — — — — — — 0.0000 24.33906.2666 3.4971 1.06870.56191.1991 5.0477 20.5911 2 79 NpH SuNDF 12 0.1116 0.1202 — — — 3.1304-0.11240.1143 0.0060 0.0000 43.160020.41009.6400 3.49000.87501.3800 1.8108 16.9746 2 80 NpH SuNDF 12 0.1114 0.1201 — — — 2.5044-0.1168-0.10150.0082 0.0000 42.298920.080910.30253.21070.91231.4516 1.9397 16.8393 2 83 NpH SuNDF 12 0.1116 0.1202 — — 17.3626— — — — — — — — — — — — — 2 84 NpH SuNDF 12 0.1118 0.1202 — — 17.5226— — — — — — — — — — — — — 2 85 ApH iNDF 12 0.2228 0.0000 0.2168 6.59— — — — — — — — — — — — — — 2 86 ApH iNDF 12 0.2228 0.0000 0.2159 6.61— — — — — — — — — — — — — — 2 87 ApH SuNDF 12 0.1116 0.1201 0.1117 5.24— — — — — — — — — — — — — — 2 88 ApH SuNDF 12 0.1115 0.1202 0.1119 5.22— — — — — — — — — — — — — —

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176Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 2 89 ApH BL 12 — — — — — — — — — 0.0000 75.23506.5650 3.5400 0.43500.35000.7750 5.3788 14.0159 2 90 ApH BL 12 — — — — — — — — — 0.0000 77.09876.6758 3.6595 0.40320.36750.7809 5.8431 15.6357 2 91 ApH SuNDF 12 0.1115 0.1204 — — — 2.6485-0.01670.2642 -0.137325.2006 52.291319.45433.6378 0.30990.23370.5081 4.5547 8.3301 2 92 ApH SuNDF 12 0.1116 0.1202 — — — 2.2284-0.24420.2919 -0.135623.5850 52.155020.89503.6100 0.30500.21000.4750 6.6872 11.0473 2 95 ApH SuNDF 12 0.1114 0.1201 — — 6.6981 — — — — — — — — — — — — — 2 96 ApH SuNDF 12 0.1115 0.1201 — — 6.9428 — — — — — — — — — — — — — 2 97 NpH iNDF 16 0.2226 0.0000 0.1836 7.11— — — — — — — — — — — — — — 2 98 NpH iNDF 16 0.2228 0.0000 0.1792 7.17— — — — — — — — — — — — — — 2 99 NpH SuNDF 16 0.1115 0.1200 0.0800 6.73— — — — — — — — — — — — — — 2 100 NpH SuNDF 16 0.1114 0.1201 0.0811 6.71— — — — — — — — — — — — — — 2 103 NpH SuNDF 16 0.1113 0.1202 — — — — — — — — — — — — — — — — 2 104 NpH SuNDF 16 0.1114 0.1202 — — — — -0.0637-0.0177-0.0229— — — — — — — — — 2 107 NpH SuNDF 16 0.1111 0.1203 — — 18.7264— — — — — — — — — — — — — 2 108 NpH SuNDF 16 0.1114 0.1204 — — 18.7998— — — — — — — — — — — — — 2 109 ApH iNDF 16 0.2229 0.0000 0.2121 6.77— — — — — — — — — — — — — — 2 110 ApH iNDF 16 0.2228 0.0000 0.2136 6.75— — — — — — — — — — — — — — 2 111 ApH SuNDF 16 0.1117 0.1202 0.1123 5.42— — — — — — — — — — — — — — 2 112 ApH SuNDF 16 0.1116 0.1202 0.1123 5.39— — — — — — — — — — — — — — 2 115 ApH SuNDF 16 0.1115 0.1202 — — — — — — — — — — — — — — — — 2 116 ApH SuNDF 16 0.1113 0.1201 — — — — 0.2804 1.2927 0.1411 — — — — — — — — — 2 119 ApH SuNDF 16 0.1115 0.1200 — — 7.3143 — — — — — — — — — — — — — 2 120 ApH SuNDF 16 0.1116 0.1202 — — 7.6373 — — — — — — — — — — — — — 2 121 NpH iNDF 20 0.2227 0.0000 0.1651 7.11— — — — — — — — — — — — — — 2 122 NpH iNDF 20 0.2227 0.0000 0.1681 7.14— — — — — — — — — — — — — — 2 123 NpH SuNDF 20 0.1114 0.1202 0.0707 6.79— — — — — — — — — — — — — — 2 124 NpH SuNDF 20 0.1117 0.1204 0.0720 6.85— — — — — — — — — — — — — — 2 125 NpH BL 20 — — — — — — — — — 0.0000 27.64315.6559 3.8925 2.28941.22742.0189 1.6588 22.1759 2 126 NpH BL 20 — — — — — — — — — 0.0000 24.73504.8150 2.9200 1.94501.18501.8950 0.9026 15.4703

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177Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 2 127 NpH SuNDF 20 0.1118 0.1202 — — — 1.9689-0.1334-0.0238-0.01200.0000 52.585022.480011.53005.06001.00001.6300 0.7863 17.0543 2 128 NpH SuNDF 20 0.1116 0.1201 — — — 1.7629-0.1334-0.0049-0.01230.0000 53.659622.189411.25094.88231.02781.5871 1.4379 24.1084 2 131 NpH SuNDF 20 0.1118 0.1201 — — 19.7839— — — — — — — — — — — — — 2 132 NpH SuNDF 20 0.1115 0.1203 — — 19.7036— — — — — — — — — — — — — 2 133 ApH iNDF 20 0.2231 0.0000 0.2082 6.84— — — — — — — — — — — — — — 2 134 ApH iNDF 20 0.2228 0.0000 0.2118 6.85— — — — — — — — — — — — — — 2 135 ApH SuNDF 20 0.1113 0.1200 0.1114 5.66— — — — — — — — — — — — — — 2 136 ApH SuNDF 20 0.1116 0.1202 0.1133 5.64— — — — — — — — — — — — — — 2 137 ApH BL 20 — — — — — — — — — 0.0000 87.02647.5576 5.5663 0.69911.12811.8431 1.7051 13.1603 2 138 ApH BL 20 — — — — — — — — — 0.0000 86.81007.5100 5.5200 0.67001.13001.8850 3.5556 20.8428 2 139 ApH SuNDF 20 0.1116 0.1202 — — — 4.13140.0825 1.0300 -0.01970.0000 73.835030.58098.9743 2.37240.28570.8214 5.7512 11.7849 2 140 ApH SuNDF 20 0.1116 0.1199 — — — 3.55470.1178 0.9878 -0.03680.0000 74.235031.53508.6700 2.23500.29500.8050 7.2162 13.7544 2 143 ApH SuNDF 20 0.1117 0.1203 — — 11.8641— — — — — — — — — — — — — 2 144 ApH SuNDF 20 0.1117 0.1202 — — 11.4553— — — — — — — — — — — — — 2 145 NpH iNDF 24 0.2227 0.0000 0.1545 7.43— — — — — — — — — — — — — — 2 146 NpH iNDF 24 0.2230 0.0000 0.1592 7.43— — — — — — — — — — — — — — 2 147 NpH SuNDF 24 0.1115 0.1204 0.0658 7.19— — — — — — — — — — — — — — 2 148 NpH SuNDF 24 0.1115 0.1204 0.0629 7.16— — — — — — — — — — — — — — 2 149 NpH BL 24 — — — — — — — — — 0.0000 21.77005.0250 2.1350 1.89001.07501.8150 0.8949 20.7350 2 150 NpH BL 24 — — — — — — — — — 0.0000 24.21905.2247 2.4749 1.98601.21712.0267 0.8958 20.8142 2 151 NpH SuNDF 24 0.1114 0.1203 — — — 1.2316-0.0616-0.0995-0.00900.0000 52.117822.103710.64125.08381.01781.6526 0.8881 19.3491 2 152 NpH SuNDF 24 0.1115 0.1202 — — — 1.0174-0.0575-0.0995-0.00900.0000 55.315023.735011.11004.96501.06501.7000 0.9239 19.9850 2 155 NpH SuNDF 24 0.1115 0.1200 — — 13.9604— — — — — — — — — — — — — 2 156 NpH SuNDF 24 0.1116 0.1201 — — 13.7916— — — — — — — — — — — — — 2 157 ApH iNDF 24 0.2229 0.0000 0.2066 7.15— — — — — — — — — — — — — — 2 158 ApH iNDF 24 0.2231 0.0000 0.2090 7.18— — — — — — — — — — — — — — 2 159 ApH SuNDF 24 0.1113 0.1199 0.1112 6.16— — — — — — — — — — — — — — 2 160 ApH SuNDF 24 0.1115 0.1201 0.1108 6.15— — — — — — — — — — — — — —

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178Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 2 161 ApH BL 24 — — — — — — — — — 0.0000 88.80637.8058 6.1241 0.76991.21061.9856 2.3308 18.8831 2 162 ApH BL 24 — — — — — — — — — 0.0000 84.70417.4894 5.6510 0.68311.13521.8987 2.8673 20.6115 2 163 ApH SuNDF 24 0.1116 0.1202 — — — 3.67420.1027 0.8761 0.1782 0.0000 81.175025.855011.63503.90000.79501.2850 4.2137 14.8633 2 164 ApH SuNDF 24 0.1117 0.1203 — — — 3.47640.0609 1.0014 0.2185 0.0000 79.640032.605010.14003.19000.82501.4200 4.4632 14.1153 2 167 ApH SuNDF 24 0.1114 0.1203 — — 9.0765 — — — — — — — — — — — — — 2 168 ApH SuNDF 24 0.1112 0.1202 — — 8.9177 — — — — — — — — — — — — — 1 Ferm = fermentation; Med = medium: ApH = acidic medium, NpH = neutral medium; Sub = substrate: BL = no substrate; iNDF = isola ted bermudagrass neutral detergent residue, SuNDF = sucrose + iNDF; rNDF = residual NDF; MCP = microbial crude protein; GLY = microbial glycogen ; rSuc = residual sucrose; rGlc = residual glucose; rFruc = residual fr uctose; Lac = lactate; C2 = acetate; C3 = propionate; C4 = butyrate; Val = valerate ; Isobut = isobutyrate; Isoval+2MB = isovalerate + 2-Methylbutyrate; AA = free amino acids; NH3-N = ammonia nitrogen

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179APPENDIX F CHAPTER 3 RAW DATA Table F-1. Data used for statistical anal ysis in evaluating the effect of nitrogen source on microbi al yield and neutral deterg en fiber digestion from in vitro fermentations of sucrose a nd isolated bermudagrass neutral detergent residue. Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 1 B BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1658 0.2764 7.6161 10.3615 1 2 B BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1600 0.2450 7.4296 10.5887 1 3 B SuNDF 0 0.1114 0.1203 — — — 1.0889 96.10972.28954.0700 0.2000 0.52460.6162-0.0219-0.05530.1900 0.2300 8.0956 10.8926 1 4 B SuNDF 0 0.1115 0.1201 — — — 0.4244 103.89902.33263.6539 0.2234 0.52650.6298-0.0510-0.01870.1675 0.2132 4.9905 7.2073 1 7 B SuNDF 0 0.1116 0.1201 — — 0.0801 — — — — — — — — — — — — — 1 8 B SuNDF 0 0.1114 0.1201 — — -0.0800 — — — — — — — — — — — — — 1 9 B iNDF 0 0.2227 0.0000 — — 0.0864 — — — — — — — — — — — — — 1 10 B iNDF 0 0.2229 0.0000 — — -0.0865 — — — — — — — — — — — — — 1 11 B SuNDF 0 0.1116 0.1200 0.10956.98— — — — — — — — — — — — — — 1 12 B SuNDF 0 0.1116 0.1203 0.11037.02— — — — — — — — — — — — — — 1 13 B iNDF 0 0.2226 0.0000 0.21277.03— — — — — — — — — — — — — — 1 14 B iNDF 0 0.2228 0.0000 0.21547.07— — — — — — — — — — — — — — 1 15 C BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.2000 0.3800 10.0151 1.3816 1 16 C BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1800 0.2500 6.8941 0.9627 1 17 C SuNDF 0 0.1114 0.1201 — — — 0.9328 108.99881.98544.0989 0.2000 0.43250.0625-0.07000.05250.1600 0.2400 11.0758 1.8644 1 18 C SuNDF 0 0.1114 0.1201 — — — 0.5805 110.29562.26414.4591 0.1727 -0.2971-0.2161-0.2398-0.02130.0914 0.1270 9.0062 1.4813 1 21 C SuNDF 0 0.1116 0.1202 — — -0.1677 — — — — — — — — — — — — — 1 22 C SuNDF 0 0.1114 0.1200 — — 0.1674 — — — — — — — — — — — — —

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180Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 23 C iNDF 0 0.2226 0.0000 — — -0.1532 — — — — — — — — — — — — — 1 24 C iNDF 0 0.2231 0.0000 — — 0.1535 — — — — — — — — — — — — — 1 25 C SuNDF 0 0.1116 0.1203 0.10857.01— — — — — — — — — — — — — — 1 26 C SuNDF 0 0.1116 0.1203 0.10957.02— — — — — — — — — — — — — — 1 27 C iNDF 0 0.2229 0.0000 0.21317.03— — — — — — — — — — — — — — 1 28 C iNDF 0 0.2228 0.0000 0.21397.04— — — — — — — — — — — — — — 1 29 U BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.0632 0.1947 0.2515 7.0074 1 30 U BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.2048 0.3431 0.2061 5.6065 1 31 U SuNDF 0 0.1117 0.1205 — — — 0.8527 100.95284.99204.1184 0.1550 0.30920.24280.05670.02610.1450 0.2200 0.2541 7.1400 1 32 U SuNDF 0 0.1114 0.1200 — — — 0.6846 91.25485.77205.2260 0.1600 2.12920.15780.0717-0.03890.1350 0.2500 0.3000 8.1732 1 35 U SuNDF 0 0.1113 0.1201 — — -1.3532 — — — — — — — — — — — — — 1 36 U SuNDF 0 0.1115 0.1201 — — 1.3542 — — — — — — — — — — — — — 1 37 U iNDF 0 0.2226 0.0000 — — -1.2637 — — — — — — — — — — — — — 1 38 U iNDF 0 0.2230 0.0000 — — 1.2658 — — — — — — — — — — — — — 1 39 U SuNDF 0 0.1115 0.1201 0.10557.01— — — — — — — — — — — — — — 1 40 U SuNDF 0 0.1118 0.1203 0.10947.07— — — — — — — — — — — — — — 1 41 U iNDF 0 0.2231 0.0000 0.21567.04— — — — — — — — — — — — — — 1 42 U iNDF 0 0.2228 0.0000 0.21387.07— — — — — — — — — — — — — — 1 43 B BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.2214 0.4831 5.4986 9.9438 1 44 B BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.2900 0.4900 4.6405 8.4942 1 45 B SuNDF 4 0.1118 0.1201 — — — 7.8060 -0.16300.0246-0.0719 28.2150 0.13003.39981.2613-0.00590.2450 0.3800 3.7854 4.8287 1 46 B SuNDF 4 0.1114 0.1200 — — — 6.8693 -0.1945-0.1537-0.0795 27.8985 0.27603.47501.27250.04670.2823 0.3528 5.1141 6.7393 1 49 B SuNDF 4 0.1113 0.1203 — — 11.2125 — — — — — — — — — — — — — 1 50 B SuNDF 4 0.1113 0.1201 — — 11.4634 — — — — — — — — — — — — — 1 51 B iNDF 4 0.2227 0.0000 — — 1.1132 — — — — — — — — — — — — — 1 52 B iNDF 4 0.2231 0.0000 — — 2.4104 — — — — — — — — — — — — — 1 53 B SuNDF 4 0.1116 0.1202 0.10986.52— — — — — — — — — — — — — — 1 54 B SuNDF 4 0.1115 0.1204 0.10806.50— — — — — — — — — — — — — —

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181Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 55 B iNDF 4 0.2229 0.0000 0.21107.13— — — — — — — — — — — — — — 1 56 B iNDF 4 0.2230 0.0000 0.20987.18— — — — — — — — — — — — — — 1 57 C BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.2650 0.4350 . 1 58 C BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.2392 0.3715 11.1907 3.4697 1 59 C SuNDF 4 0.1117 0.1203 — — — 7.9746 -0.1569-0.021511.7511 25.1750 4.28993.64401.6385-0.01750.1900 0.3650 6.5981 0.6904 1 60 C SuNDF 4 0.1116 0.1201 — — — 6.3573 -0.18420.041210.7450 25.0687 4.33983.73851.1551-0.00760.2038 0.3617 5.5557 0.6044 1 63 C SuNDF 4 0.1114 0.1200 — — 8.8748 — — — — — — — — — — — — — 1 64 C SuNDF 4 0.1116 0.1200 — — 7.9680 — — — — — — — — — — — — — 1 65 C iNDF 4 0.2228 0.0000 — — -4.5156 — — — — — — — — — — — — — 1 66 C iNDF 4 0.2228 0.0000 — — -3.6942 — — — — — — — — — — — — — 1 67 C SuNDF 4 0.1117 0.1204 0.10786.58— — — — — — — — — — — — — — 1 68 C SuNDF 4 0.1115 0.1200 0.10816.45— — — — — — — — — — — — — — 1 69 C iNDF 4 0.2230 0.0000 0.21036.99— — — — — — — — — — — — — — 1 70 C iNDF 4 0.2227 0.0000 0.20956.88— — — — — — — — — — — — — — 1 71 U BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.1800 0.3150 0.3712 10.9493 1 72 U BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.1950 0.3100 0.4138 11.8845 1 73 U SuNDF 4 0.1118 0.1202 — — — 7.2300 0.3044 6.380927.4587 16.5323 3.08091.50810.4947-0.02690.1709 0.1859 0.3419 8.6223 1 74 U SuNDF 4 0.1119 0.1204 — — — 5.9489 0.0000 6.177628.3049 16.9300 3.40251.71500.8875-0.00750.3400 0.1950 0.5250 13.3896 1 77 U SuNDF 4 0.1115 0.1202 — — 4.0632 — — — — — — — — — — — — — 1 78 U SuNDF 4 0.1117 0.1202 — — 5.5384 — — — — — — — — — — — — — 1 79 U iNDF 4 0.2228 0.0000 — — 0.2064 — — — — — — — — — — — — — 1 80 U iNDF 4 0.2229 0.0000 — — 1.8451 — — — — — — — — — — — — — 1 81 U SuNDF 4 0.1116 0.1203 0.10916.62— — — — — — — — — — — — — — 1 82 U SuNDF 4 0.1116 0.1200 0.10926.51— — — — — — — — — — — — — — 1 83 U iNDF 4 0.2229 0.0000 0.21216.81— — — — — — — — — — — — — — 1 84 U iNDF 4 0.2231 0.0000 0.21076.98— — — — — — — — — — — — — — 1 85 B BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.1700 0.4000 5.2491 11.8492 1 86 B BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.2850 0.5300 — —

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182Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 87 B SuNDF 8 0.1114 0.1204 — — — 5.4005 -0.11630.19870.0593 24.0374 8.56045.78021.5188-0.05130.2651 0.4078 6.3469 9.9714 1 88 B SuNDF 8 0.1116 0.1202 — — — 6.0570 -0.14180.12630.0440 23.7450 9.91256.25501.5650-0.06000.1950 0.4650 4.3481 6.8676 1 91 B SuNDF 8 0.1114 0.1203 — — 13.2230 — — — — — — — — — — — — — 1 92 B SuNDF 8 0.1115 0.1203 — — 12.3994 — — — — — — — — — — — — — 1 93 B iNDF 8 0.2231 0.0000 — — 1.8764 — — — — — — — — — — — — — 1 94 B iNDF 8 0.2231 0.0000 — — 2.6936 — — — — — — — — — — — — — 1 95 B SuNDF 8 0.1117 0.1205 0.10776.77— — — — — — — — — — — — — — 1 96 B SuNDF 8 0.1118 0.1203 0.10616.75— — — — — — — — — — — — — — 1 97 B iNDF 8 0.2231 0.0000 0.20547.19— — — — — — — — — — — — — — 1 98 B iNDF 8 0.2230 0.0000 0.20677.22— — — — — — — — — — — — — — 1 99 C BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.2280 0.4713 12.1128 6.4069 1 100 C BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.3150 0.5550 9.2628 4.6730 1 101 C SuNDF 8 0.1115 0.1201 — — — 5.9930 -0.11340.43400.0584 24.6459 8.25377.17761.74060.04350.2241 0.4840 10.9952 2.1030 1 102 C SuNDF 8 0.1113 0.1201 — — — 5.3284 -0.08660.42640.0624 26.1350 7.29884.98141.2769-0.04020.2300 0.5000 7.6298 1.4557 1 105 C SuNDF 8 0.1118 0.1201 — — 16.2599 — — — — — — — — — — — — — 1 106 C SuNDF 8 0.1117 0.1204 — — 14.5313 — — — — — — — — — — — — — 1 107 C iNDF 8 0.2231 0.0000 — — 1.0528 — — — — — — — — — — — — — 1 108 C iNDF 8 0.2227 0.0000 — — 1.8139 — — — — — — — — — — — — — 1 109 C SuNDF 8 0.1118 0.1202 0.10766.76— — — — — — — — — — — — — — 1 110 C SuNDF 8 0.1114 0.1204 0.10416.78— — — — — — — — — — — — — — 1 111 C iNDF 8 0.2230 0.0000 0.20527.14— — — — — — — — — — — — — — 1 112 C iNDF 8 0.2228 0.0000 0.20677.18— — — — — — — — — — — — — — 1 113 U BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.1650 0.2450 1.0830 21.8470 1 114 U BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.1844 0.2715 0.7072 16.3340 1 115 U SuNDF 8 0.1115 0.1202 — — — 4.6759 0.0000 0.15430.1040 26.9550 6.74303.31231.35380.15320.1650 0.2600 0.9145 17.5515 1 116 U SuNDF 8 0.1115 0.1200 — — — 4.3556 0.0000 0.00000.0000 27.5300 6.28803.49231.27880.00820.1500 0.1950 1.0946 19.2755 1 119 U SuNDF 8 0.1116 0.1201 — — 10.8814 — — — — — — — — — — — — — 1 120 U SuNDF 8 0.1115 0.1200 — — 9.5706 — — — — — — — — — — — — —

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183Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 121 U iNDF 8 0.2231 0.0000 — — 0.4402 — — — — — — — — — — — — — 1 122 U iNDF 8 0.2231 0.0000 — — — — — — — — — — — — — — — — 1 123 U SuNDF 8 0.1116 0.1205 0.10596.92— — — — — — — — — — — — — — 1 124 U SuNDF 8 0.1114 0.1204 0.10606.88— — — — — — — — — — — — — — 1 125 U iNDF 8 0.2229 0.0000 0.20607.45— — — — — — — — — — — — — — 1 126 U iNDF 8 0.2228 0.0000 0.20687.46— — — — — — — — — — — — — — 1 127 B BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.0000 0.0000 7.5122 4.7885 1 128 B BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.0000 0.0000 11.8436 9.7923 1 129 B SuNDF 12 0.1118 0.1200 — — — — — — — — — — — — 0.0000 0.0000 12.0368 8.0988 1 130 B SuNDF 12 0.1116 0.1201 — — — — — — — — — — — — 0.0000 0.0000 11.0489 9.7332 1 133 B SuNDF 12 0.1118 0.1200 — — — — — — — — — — — — — — — — 1 134 B SuNDF 12 0.1117 0.1201 — — — — — — — — — — — — — — — — 1 135 B iNDF 12 0.2229 0.0000 — — — — — — — — — — — — — — — — 1 136 B iNDF 12 0.2228 0.0000 — — — — — — — — — — — — — — — — 1 137 B SuNDF 12 0.1114 0.1200 0.1062— — — — — — — — — — — — — — — 1 138 B SuNDF 12 0.1116 0.1203 0.1068— — — — — — — — — — — — — — — 1 139 B iNDF 12 0.2229 0.0000 0.2119— — — — — — — — — — — — — — — 1 140 B iNDF 12 0.2231 0.0000 0.2108— — — — — — — — — — — — — — — 1 141 C BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.3050 0.6850 11.2495 10.1219 1 142 C BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.3641 0.7751 10.8827 10.3974 1 143 C SuNDF 12 0.1114 0.1201 — — — 4.0313 0.1150 1.54710.1356 11.0900 15.14589.44532.73440.13530.2550 0.6900 8.4321 3.7682 1 144 C SuNDF 12 0.1118 0.1201 — — — 3.7751 0.0017 1.49820.1507 10.2700 16.030815.47032.76940.13030.2500 0.6950 8.9749 4.4018 1 147 C SuNDF 12 0.1117 0.1200 — — — — — — — — — — — — — — — — 1 148 C SuNDF 12 0.1118 0.1203 — — 14.1618 — — — — — — — — — — — — — 1 149 C iNDF 12 0.2229 0.0000 — — 3.8999 — — — — — — — — — — — — — 1 150 C iNDF 12 0.2229 0.0000 — — 2.2570 — — — — — — — — — — — — — 1 151 C SuNDF 12 0.1118 0.1204 0.09896.68— — — — — — — — — — — — — — 1 152 C SuNDF 12 0.1117 0.1200 0.09466.67— — — — — — — — — — — — — —

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184Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 153 C iNDF 12 0.2230 0.0000 0.18747.14— — — — — — — — — — — — — — 1 154 C iNDF 12 0.2229 0.0000 0.19747.19— — — — — — — — — — — — — — 1 155 U BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.1871 0.4046 1.2769 22.8742 1 156 U BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.2150 0.3300 1.0929 21.0903 1 157 U SuNDF 12 0.1116 0.1204 — — — 2.8944 0.0000 -0.0023-0.0027 24.1550 9.49445.35981.6400-0.05090.1550 0.2150 1.2279 20.1872 1 158 U SuNDF 12 0.1116 0.1203 — — — 2.4300 0.0000 -0.0023-0.0027 24.8923 8.77135.26801.5314-0.05190.1582 0.2245 1.2128 20.0421 1 161 U SuNDF 12 0.1116 0.1204 — — 11.0402 — — — — — — — — — — — — — 1 162 U SuNDF 12 0.1117 0.1205 — — 9.8866 — — — — — — — — — — — — — 1 163 U iNDF 12 0.2229 0.0000 — — 0.9415 — — — — — — — — — — — — — 1 164 U iNDF 12 0.2231 0.0000 — — -0.8741 — — — — — — — — — — — — — 1 165 U SuNDF 12 0.1118 0.1203 0.10036.92— — — — — — — — — — — — — — 1 166 U SuNDF 12 0.1113 0.1202 0.10386.84— — — — — — — — — — — — — — 1 167 U iNDF 12 0.2227 0.0000 0.19637.40— — — — — — — — — — — — — — 1 168 U iNDF 12 0.2230 0.0000 0.19977.41— — — — — — — — — — — — — — 1 169 B BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.5300 1.1900 4.8012 19.8484 1 170 B BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.6050 1.2600 3.8819 18.9232 1 171 B SuNDF 16 0.1118 0.1203 — — — 1.3011 0.0019 0.60600.0958 0.0000 19.519017.78785.51222.07690.7703 1.2335 2.1426 12.6875 1 172 B SuNDF 16 0.1115 0.1202 — — — 1.9256 0.0135 0.60110.1361 0.0000 20.062516.51505.86752.13500.7550 1.1750 2.3582 14.2038 1 175 B SuNDF 16 0.1115 0.1201 — — 16.4290 — — — — — — — — — — — — — 1 176 B SuNDF 16 0.1114 0.1204 — — 15.6029 — — — — — — — — — — — — — 1 177 B iNDF 16 0.2231 0.0000 — — 5.1579 — — — — — — — — — — — — — 1 178 B iNDF 16 0.2230 0.0000 — — 3.5236 — — — — — — — — — — — — — 1 179 B SuNDF 16 0.1116 0.1204 0.08446.78— — — — — — — — — — — — — — 1 180 B SuNDF 16 0.1118 0.1201 0.08296.82— — — — — — — — — — — — — — 1 181 B iNDF 16 0.2231 0.0000 0.17547.11— — — — — — — — — — — — — — 1 182 B iNDF 16 0.2231 0.0000 0.17957.11— — — — — — — — — — — — — — 1 183 C BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.6852 1.6678 — — 1 184 C BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.8405 1.6558 6.1693 13.3502

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185Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 1 185 C SuNDF 16 0.1117 0.1200 — — — 2.7022 -0.00730.75940.1552 0.0000 19.376019.68175.44122.16821.2650 2.1200 3.5002 6.5415 1 186 C SuNDF 16 0.1118 0.1202 — — — 1.4532 -0.04300.74590.1316 0.0000 19.586019.95675.75122.26821.3100 2.0400 4.7521 9.2913 1 189 C SuNDF 16 0.1117 0.1204 — — 16.9135 — — — — — — — — — — — — — 1 190 C SuNDF 16 0.1115 0.1204 — — 16.9167 — — — — — — — — — — — — — 1 191 C iNDF 16 0.2232 0.0000 — — 5.1557 — — — — — — — — — — — — — 1 192 C iNDF 16 0.2231 0.0000 — — 3.9278 — — — — — — — — — — — — — 1 193 C SuNDF 16 0.1114 0.1201 0.08546.82— — — — — — — — — — — — — — 1 194 C SuNDF 16 0.1115 0.1201 0.08296.79— — — — — — — — — — — — — — 1 195 C iNDF 16 0.2231 0.0000 0.18317.09— — — — — — — — — — — — — — 1 196 C iNDF 16 0.2231 0.0000 0.18287.05— — — — — — — — — — — — — — 1 197 U BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.3046 0.2945 1.2034 21.8217 1 198 U BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.1950 0.2500 1.0264 19.9790 1 199 U SuNDF 16 0.1114 0.1201 — — — 1.4612 0.0000 -0.0045-0.0051 18.7631 16.45709.35582.76200.12200.1822 0.1822 1.1479 19.1339 1 200 U SuNDF 16 0.1115 0.1203 — — — 1.7014 0.0000 -0.0045-0.0051 18.7300 14.84559.24192.46010.16910.1500 0.1350 1.2135 19.8234 1 203 U SuNDF 16 0.1119 0.1205 — — 10.0886 — — — — — — — — — — — — — 1 204 U SuNDF 16 0.1114 0.1201 — — 10.1044 — — — — — — — — — — — — — 1 205 U iNDF 16 0.2228 0.0000 — — 3.6154 — — — — — — — — — — — — — 1 206 U iNDF 16 0.2232 0.0000 — — 3.8407 — — — — — — — — — — — — — 1 207 U SuNDF 16 0.1118 0.1202 0.09426.93— — — — — — — — — — — — — — 1 208 U SuNDF 16 0.1116 0.1203 0.08996.88— — — — — — — — — — — — — — 1 209 U iNDF 16 0.2231 0.0000 0.17607.30— — — — — — — — — — — — — — 1 210 U iNDF 16 0.2231 0.0000 0.18687.32— — — — — — — — — — — — — — 2 1 B BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1518 0.1822 8.9943 9.0308 2 2 B BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.0900 0.2050 7.8397 7.9636 2 3 B SuNDF 0 0.1115 0.1201 — — — 0.0841 59.3853-1.7158-1.4389 0.2450 1.24380.22460.12990.02420.2050 0.1900 7.5064 7.5691 2 4 B SuNDF 0 0.1116 0.1205 — — — 0.1962 92.7013-1.6710-1.4389 0.1682 1.00710.18570.10940.03160.1122 0.1835 — — 2 7 B SuNDF 0 0.1113 0.1200 — — 2.0956 — — — — — — — — — — — — — 2 8 B SuNDF 0 0.1114 0.1200 — — -2.0965 — — — — — — — — — — — — —

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186Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 2 9 B iNDF 0 0.2227 0.0000 — — 1.5665 — — — — — — — — — — — — — 2 10 B iNDF 0 0.2230 0.0000 — — -1.5684 — — — — — — — — — — — — — 2 11 B SuNDF 0 0.1116 0.1202 0.10937.06— — — — — — — — — — — — — — 2 12 B SuNDF 0 0.1116 0.1200 0.10947.04— — — — — — — — — — — — — — 2 13 B iNDF 0 0.2228 0.0000 0.21367.04— — — — — — — — — — — — — — 2 14 B iNDF 0 0.2229 0.0000 0.21397.00— — — — — — — — — — — — — — 2 15 C BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1409 0.2114 11.8680 0.8926 2 16 C BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1800 0.1500 8.9579 0.6736 2 17 C SuNDF 0 0.1117 0.1198 — — — 0.1962 76.3971-0.4398-0.6956 0.1850 0.6578-0.0363-0.07070.00720.0750 0.1900 11.9896 0.9974 2 18 C SuNDF 0 0.1115 0.1201 — — — 0.1241 68.7214-0.9733-0.8582 0.2079 0.6529-0.0292-0.03640.04890.1673 0.2231 14.6845 1.2873 2 21 C SuNDF 0 0.1115 0.1198 — — 1.3966 — — — — — — — — — — — — — 2 22 C SuNDF 0 0.1112 0.1201 — — -1.3968 — — — — — — — — — — — — — 2 23 C iNDF 0 0.2228 0.0000 — — -0.6139 — — — — — — — — — — — — — 2 24 C iNDF 0 0.2229 0.0000 — — 0.6141 — — — — — — — — — — — — — 2 25 C SuNDF 0 0.1115 0.1202 0.10887.06— — — — — — — — — — — — — — 2 26 C SuNDF 0 0.1116 0.1199 0.10786.93— — — — — — — — — — — — — — 2 27 C iNDF 0 0.2228 0.0000 0.21336.99— — — — — — — — — — — — — — 2 28 C iNDF 0 0.2228 0.0000 0.21296.98— — — — — — — — — — — — — — 2 29 U BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1365 0.1517 0.4707 8.7029 2 30 U BL 0 0.0000 0.0000 — — — — — — — — — — — — 0.1150 0.2050 0.3328 5.7084 2 31 U SuNDF 0 0.1117 0.1199 — — — 0.2562 25.12120.00000.0000 0.1650 0.3230-0.0156-0.00480.00440.1500 0.2050 0.4424 8.2335 2 32 U SuNDF 0 0.1115 0.1200 — — — 0.1681 7.9664 0.00000.0000 0.1700 0.32800.2244-0.0898-0.05060.0550 0.0950 0.3313 5.8361 2 35 U SuNDF 0 0.1115 0.1200 — — -0.8892 — — — — — — — — — — — — — 2 36 U SuNDF 0 0.1118 0.1204 — — 0.8918 — — — — — — — — — — — — — 2 37 U iNDF 0 0.2231 0.0000 — — 1.0268 — — — — — — — — — — — — — 2 38 U iNDF 0 0.2231 0.0000 — — -1.0268 — — — — — — — — — — — — — 2 39 U SuNDF 0 0.1116 0.1201 0.10987.05— — — — — — — — — — — — — — 2 40 U SuNDF 0 0.1115 0.1201 0.10846.99— — — — — — — — — — — — — —

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187Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 2 41 U iNDF 0 0.2231 0.0000 0.21427.01— — — — — — — — — — — — — — 2 42 U iNDF 0 0.2231 0.0000 0.21287.05— — — — — — — — — — — — — — 2 43 B BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.1300 0.2700 6.9974 8.9578 2 44 B BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.1853 0.2654 3.8949 5.1360 2 45 B SuNDF 4 0.1117 0.1204 — — — 7.6183 -0.7654-1.25650.4098 26.5988 5.12062.07890.81810.00010.1333 0.2409 6.7273 6.7012 2 46 B SuNDF 4 0.1115 0.1204 — — — 6.9377 -0.2506-0.4931-0.1900 24.6650 3.89891.78620.9660-0.00020.1550 0.3050 8.4593 8.3316 2 49 B SuNDF 4 0.1116 0.1200 — — 7.8787 — — — — — — — — — — — — — 2 50 B SuNDF 4 0.1116 0.1198 — — 5.7488 — — — — — — — — — — — — — 2 51 B iNDF 4 0.2230 0.0000 — — 0.2798 — — — — — — — — — — — — — 2 52 B iNDF 4 0.2228 0.0000 — — -0.9434 — — — — — — — — — — — — — 2 53 B SuNDF 4 0.1115 0.1197 0.11026.73— — — — — — — — — — — — — — 2 54 B SuNDF 4 0.1117 0.1204 0.10996.56— — — — — — — — — — — — — — 2 55 B iNDF 4 0.2228 0.0000 0.21007.17— — — — — — — — — — — — — — 2 56 B iNDF 4 0.2230 0.0000 0.20957.09— — — — — — — — — — — — — — 2 57 C BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.1750 0.2850 7.8430 1.6254 2 58 C BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.0662 0.3259 13.0860 2.4937 2 59 C SuNDF 4 0.1116 0.1202 — — — 7.2540 -1.2484-2.1980-0.7270 22.5850 4.65343.37620.7530-0.00680.1400 0.2350 . 2 60 C SuNDF 4 0.1116 0.1200 — — — 7.0939 -0.3451-0.71101.9282 27.4742 14.60515.30601.96240.54301.2988 0.3804 9.0842 0.5141 2 63 C SuNDF 4 0.1117 0.1197 — — 7.8431 — — — — — — — — — — — — — 2 64 C SuNDF 4 0.1117 0.1205 — — 8.1535 — — — — — — — — — — — — — 2 65 C iNDF 4 0.2231 0.0000 — — 0.2361 — — — — — — — — — — — — — 2 66 C iNDF 4 0.2231 0.0000 — — 0.6468 — — — — — — — — — — — — — 2 67 C SuNDF 4 0.1116 0.1201 0.10926.60— — — — — — — — — — — — — — 2 68 C SuNDF 4 0.1118 0.1200 0.10916.60— — — — — — — — — — — — — — 2 69 C iNDF 4 0.2229 0.0000 0.21057.13— — — — — — — — — — — — — — 2 70 C iNDF 4 0.2230 0.0000 0.20886.96— — — — — — — — — — — — — — 2 71 U BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.0806 0.2418 0.7180 14.4564 2 72 U BL 4 0.0000 0.0000 — — — — — — — — — — — — 0.7347 0.2503 0.6476 12.7626

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188Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 2 73 U SuNDF 4 0.1113 0.1204 — — — 6.8297 0.0000 0.00000.0000 18.0891 2.44820.76020.38180.04300.1566 0.1768 1.0867 18.6572 2 74 U SuNDF 4 0.1118 0.1200 — — — 7.3421 0.0000 0.00000.0000 26.7540 9.57302.93430.6037-0.09330.0213 0.0414 0.6877 12.6292 2 77 U SuNDF 4 0.1114 0.1201 — — 0.0963 — — — — — — — — — — — — — 2 78 U SuNDF 4 0.1117 0.1197 — — 0.4295 — — — — — — — — — — — — — 2 79 U iNDF 4 0.2228 0.0000 — — -4.2130 — — — — — — — — — — — — — 2 80 U iNDF 4 0.2232 0.0000 — — -4.0778 — — — — — — — — — — — — — 2 81 U SuNDF 4 0.1115 0.1205 0.10816.62— — — — — — — — — — — — — — 2 82 U SuNDF 4 0.1116 0.1200 0.10956.80— — — — — — — — — — — — — — 2 83 U iNDF 4 0.2231 0.0000 0.21187.32— — — — — — — — — — — — — — 2 84 U iNDF 4 0.2231 0.0000 0.20987.22— — — — — — — — — — — — — — 2 85 B BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.1654 0.3558 4.5496 7.1464 2 86 B BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.9418 0.3384 5.8547 9.0172 2 87 B SuNDF 8 0.1117 0.1203 — — — — -0.01720.09910.1044 26.8058 4.35082.88750.6461-0.31110.1012 0.1467 5.9177 9.6471 2 88 B SuNDF 8 0.1118 0.1204 — — — — 0.0017 0.08640.0385 27.3500 4.24032.73080.5581-0.24740.1050 0.1850 5.5600 9.2909 2 91 B SuNDF 8 0.1117 0.1201 — — 6.9283 — . — — — — — — — — — 2 92 B SuNDF 8 0.1117 0.1202 — — 8.4861 — . — — — — — — — — — 2 93 B iNDF 8 0.0000 0.0000 — — 0.4718 — . — — — — — — — — — 2 94 B iNDF 8 0.0000 0.0000 — — 0.1432 — . — — — — — — — — — 2 95 B SuNDF 8 0.1117 0.1202 0.10696.74— — . — — — — — — — — — 2 96 B SuNDF 8 0.1115 0.1200 0.10716.76— — . — — — — — — — — — 2 97 B iNDF 8 0.2231 0.0000 0.20897.18— — . — — — — — — — — — 2 98 B iNDF 8 0.2228 0.0000 0.20757.17— — . — — — — — — — — — 2 99 C BL 8 0.0000 0.0000 — — — — . — — — — — 0.1430 0.2605 9.7354 2.9712 2 100 C BL 8 0.0000 0.0000 — — — — . — — — — — 0.1500 0.2600 — — 2 101 C SuNDF 8 0.1115 0.1198 — — — 5.3684 -0.18910.7839-0.1310 29.6757 9.62375.30191.61470.22930.1930 0.4469 10.7154 1.1109 2 102 C SuNDF 8 0.1116 0.1206 — — — 5.2003 -0.15700.7802-0.1021 — — — — — — — 6.5473 0.7817 2 105 C SuNDF 8 0.1115 0.1199 — — 10.9643 — — — — — — — — — — — — — 2 106 C SuNDF 8 0.1114 0.1201 — — 9.5654 — — — — — — — — — — — — —

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189Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 2 107 C iNDF 8 0.2229 0.0000 — — 1.8052 — — — — — — — — — — — — — 2 108 C iNDF 8 0.2228 0.0000 — — 2.3023 — — — — — — — — — — — — — 2 109 C SuNDF 8 0.1118 0.1202 0.10816.75— — — — — — — — — — — — — — 2 110 C SuNDF 8 0.1114 0.1201 0.10776.74— — — — — — — — — — — — — — 2 111 C iNDF 8 0.2229 0.0000 0.20837.19— — — — — — — — — — — — — — 2 112 C iNDF 8 0.2228 0.0000 0.19957.23— — — — — — — — — — — — — — 2 113 U BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.1700 0.2250 1.1214 20.5233 2 114 U BL 8 0.0000 0.0000 — — — — — — — — — — — — 0.9427 0.4204 — — 2 115 U SuNDF 8 0.1114 0.1203 — — — 4.5758 0.0000 0.01680.0000 27.9800 5.33232.21300.7039-0.39770.1000 0.1700 0.7808 13.2187 2 116 U SuNDF 8 0.1116 0.1199 — — — 4.8720 0.0000 0.01940.0000 38.3669 16.63796.45272.45940.17831.3277 0.6591 0.6853 11.5208 2 119 U SuNDF 8 0.1114 0.1202 — — 5.8836 — — — — — — — — — — — — — 2 120 U SuNDF 8 0.1115 0.1201 — — 5.3089 — — — — — — — — — — — — — 2 121 U iNDF 8 0.2231 0.0000 — — -0.4107 — — — — — — — — — — — — — 2 122 U iNDF 8 0.2229 0.0000 — — -0.8919 — — — — — — — — — — — — — 2 123 U SuNDF 8 0.1117 0.1204 0.10796.89— — — — — — — — — — — — — — 2 124 U SuNDF 8 0.1117 0.1202 0.10706.88— — — — — — — — — — — — — — 2 125 U iNDF 8 0.2230 0.0000 — 7.46— — — — — — — — — — — — — — 2 126 U iNDF 8 0.2230 0.0000 0.20847.40— — — — — — — — — — — — — — 2 127 B BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.2041 0.3826 — — 2 128 B BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.7605 0.2569 7.8718 13.5572 2 129 B SuNDF 12 0.1116 0.1201 — — — 2.1738 -0.08331.02540.0933 14.7223 17.768413.61653.17980.53321.3204 0.4627 6.1341 8.7851 2 130 B SuNDF 12 0.1114 0.1202 — — — 1.7895 -0.08431.03840.1131 14.6489 15.771812.99723.12480.45401.2944 0.4261 5.9623 8.4822 2 133 B SuNDF 12 0.1116 0.1203 — — 14.3182 — — — — — — — — — — — — — 2 134 B SuNDF 12 0.1116 0.1203 — — 12.5932 — — — — — — — — — — — — — 2 135 B iNDF 12 0.2230 0.0000 — — 2.4567 — — — — — — — — — — — — — 2 136 B iNDF 12 0.2227 0.0000 — — 2.4701 — — — — — — — — — — — — — 2 137 B SuNDF 12 0.1116 0.1198 0.10346.79— — — — — — — — — — — — — — 2 138 B SuNDF 12 0.1118 0.1205 0.10136.78— — — — — — — — — — — — — —

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190Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 2 139 B iNDF 12 0.2230 0.0000 0.19877.17— — — — — — — — — — — — — — 2 140 B iNDF 12 0.2228 0.0000 0.20047.20— — — — — — — — — — — — — — 2 141 C BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.2300 0.5000 11.8329 6.1511 2 142 C BL 12 0.0000 0.0000 — — — — — — — — — — — — 1.2291 0.3754 — — 2 143 C SuNDF 12 0.1116 0.1202 — — — 2.0137 0.1108 2.75540.0887 17.7450 13.177312.46081.1735-0.04950.2150 0.4500 9.8320 1.6927 2 144 C SuNDF 12 0.1116 0.1202 — — — 2.2539 0.1129 2.64510.0300 18.6508 16.710112.37632.58940.13350.0000 0.3948 11.3770 2.3943 2 147 C SuNDF 12 0.1114 0.1203 — — 11.8198 — — — — — — — — — — — — — 2 148 C SuNDF 12 0.1115 0.1204 — — 13.3762 — — — — — — — — — — — — — 2 149 C iNDF 12 0.2229 0.0000 — — 4.8857 — — — — — — — — — — — — — 2 150 C iNDF 12 0.2231 0.0000 — — 3.9695 — — — — — — — — — — — — — 2 151 C SuNDF 12 0.1115 0.1198 0.10166.81— — — — — — — — — — — — — — 2 152 C SuNDF 12 0.1115 0.1202 0.10286.75— — — — — — — — — — — — — — 2 153 C iNDF 12 0.2229 0.0000 0.19767.25— — — — — — — — — — — — — — 2 154 C iNDF 12 0.2229 0.0000 0.20987.25— — — — — — — — — — — — — — 2 155 U BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.1938 0.3621 — — 2 156 U BL 12 0.0000 0.0000 — — — — — — — — — — — — 0.1800 0.3700 0.8864 15.2510 2 157 U SuNDF 12 0.1115 0.1203 — — — 1.9096 0.0000 0.01490.0000 26.7600 7.31303.32771.2936-0.12060.1400 0.2050 1.1826 16.8542 2 158 U SuNDF 12 0.1116 0.1197 — — — 1.3411 0.0000 0.01070.0000 30.0198 12.22015.99922.11480.35751.1539 0.6028 — — 2 161 U SuNDF 12 0.1114 0.1200 — — 8.2325 — — — — — — — — — — — — — 2 162 U SuNDF 12 0.1114 0.1204 — — 6.2456 — — — — — — — — — — — — — 2 163 U iNDF 12 0.2231 0.0000 — — -3.7376 — — — — — — — — — — — — — 2 164 U iNDF 12 0.2230 0.0000 — — -3.6496 — — — — — — — — — — — — — 2 165 U SuNDF 12 0.1116 0.1201 0.10187.00— — — — — — — — — — — — — — 2 166 U SuNDF 12 0.1114 0.1202 0.10287.03— — — — — — — — — — — — — — 2 167 U iNDF 12 0.2232 0.0000 0.19887.53— — — — — — — — — — — — — — 2 168 U iNDF 12 0.2232 0.0000 0.19917.53— — — — — — — — — — — — — — 2 169 B BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.2450 0.8100 7.6387 16.7876 2 170 B BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.9816 0.3862 5.6819 13.2920

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191Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 2 171 B SuNDF 16 0.1115 0.1202 — — — 1.1930 -0.13200.97650.1480 0.0000 17.246318.16845.12002.63250.7350 1.1600 3.6623 11.3550 2 172 B SuNDF 16 0.1117 0.1200 — — — 1.2330 -0.11261.04590.0103 0.0000 23.266018.89195.94123.27471.8643 0.6614 3.3673 11.8837 2 175 B SuNDF 16 0.1117 0.1204 — — 13.0393 — — — — — — — — — — — — — 2 176 B SuNDF 16 0.1118 0.1204 — — 12.0508 — — — — — — — — — — — — — 2 177 B iNDF 16 0.2228 0.0000 — — 2.7531 — — — — — — — — — — — — — 2 178 B iNDF 16 0.2229 0.0000 — — 2.9951 — — — — — — — — — — — — — 2 179 B SuNDF 16 0.1113 0.1201 0.09236.89— — — — — — — — — — — — — — 2 180 B SuNDF 16 0.1117 0.1205 0.09316.88— — — — — — — — — — — — — — 2 181 B iNDF 16 0.2231 0.0000 0.18597.28— — — — — — — — — — — — — — 2 182 B iNDF 16 0.2230 0.0000 0.18657.24— — — — — — — — — — — — — — 2 183 C BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.2050 1.0200 — — 2 184 C BL 16 0.0000 0.0000 — — — — — — — — — — — — 1.1109 0.3125 9.9598 11.0693 2 185 C SuNDF 16 0.1116 0.1200 — — — 0.9328 -0.14781.53190.1024 -0.0599 15.886617.13465.37442.76670.8300 1.8150 5.4314 4.0210 2 186 C SuNDF 16 0.1116 0.1204 — — — 1.3331 -0.18801.50690.1135 -0.0599 15.256616.93465.18942.96671.0200 1.8900 6.7007 5.0380 2 189 C SuNDF 16 0.1116 0.1201 — — 15.2291 — — — — — — — — — — — — — 2 190 C SuNDF 16 0.1116 0.1204 — — 13.7437 — — — — — — — — — — — — — 2 191 C iNDF 16 0.2230 0.0000 — — 1.8832 — — — — — — — — — — — — — 2 192 C iNDF 16 0.2230 0.0000 — — 3.0332 — — — — — — — — — — — — — 2 193 C SuNDF 16 0.1115 0.1200 0.09516.85— — — — — — — — — — — — — — 2 194 C SuNDF 16 0.1116 0.1201 0.09576.86— — — — — — — — — — — — — — 2 195 C iNDF 16 0.2228 0.0000 0.19177.22— — — — — — — — — — — — — — 2 196 C iNDF 16 0.2228 0.0000 0.19127.17— — — — — — — — — — — — — — 2 197 U BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.1400 0.1300 1.3455 19.4901 2 198 U BL 16 0.0000 0.0000 — — — — — — — — — — — — 0.1300 0.2400 1.8095 23.0433 2 199 U SuNDF 16 0.1117 0.1205 — — — 1.1209 0.0000 0.0049-0.0019 21.9156 12.78817.37292.30510.15450.1159 0.1159 1.3812 17.9959 2 200 U SuNDF 16 0.1115 0.1199 — — — 1.2010 0.0000 0.0097-0.0019 23.5400 17.30257.06502.04500.12750.0900 0.1300 — — 2 203 U SuNDF 16 0.1116 0.1204 — — 5.5402 — — — — — — — — — — — — — 2 204 U SuNDF 16 0.1114 0.1203 — — 6.4548 — — — — — — — — — — — — —

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192Ferm1 Tube ID Med Sub Hr iNDF (g DM) Sucrose (g DM) rNDF (g OM) pH MCP (mg) GLY (mg) rSuc (mg) rGlc (mg) rFruc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) Total AA (m M ) NH3-N (m M ) 2 205 U iNDF 16 0.2229 0.0000 — — 0.5867 — — — — — — — — — — — — — 2 206 U iNDF 16 0.2230 0.0000 — — -0.9799 — — — — — — — — — — — — — 2 207 U SuNDF 16 0.1114 0.1202 0.09957.00— — — — — — — — — — — — — — 2 208 U SuNDF 16 0.1117 0.1203 0.09866.96— — — — — — — — — — — — — — 2 209 U iNDF 16 0.2228 0.0000 0.18757.47— — — — — — — — — — — — — — 2 210 U iNDF 16 0.2228 0.0000 0.19377.50— — — — — — — — — — — — — — 1 Ferm = fermentation; Med = medium: B = Non-protein + true protein nitrogen, C = True protein nitrogen only, U = Non-protein ni trogen only; Sub = substrate: BL = no substrate; iNDF = isolated bermudagrass neutral detergen t residue, SuNDF = sucrose + iNDF ; rNDF = residual NDF; MCP = m icrobial crude protein; GLY = microbial glycogen; rSuc = residual sucrose; rGlc = residu al glucose; rFruc = residual fr uctose; Lac = lactate; C2 = acet ate; C3 = propionate; C4 = butyrate; Val = valerate; Isobut = isobutyrate; Isoval+2MB = isovalerate + 2-Methylbutyrat e; AA = free amino acids; NH3-N = ammonia nitrogen

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193APPENDIX G CHAPTER 4 RAW DATA Table G-1. Data used for statistical anal ysis in evaluating the effect on microbial yield and neutral detergen fiber digestion from in vitro fermentations of different source s (sucrose, starch and p ectin), amounts (0, 40, 80 and 120 mg nominal hexose equivalents) and combinations (sucrose+starc h, starch+pectin and sucr ose+pectin) of NFCs. Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 1 BL 0 0.0000 0.0000 0.00000.0000 7.21— — — — — — — — — — — — — 1 2 BL 0 0.0000 0.0000 0.00000.0000 7.20— — — — — — — — — — — — — 1 3 NDF 0 0.1156 0.0000 0.00000.0000 7.2397.5345 — — — — — — — — — — — — 1 4 NDF 0 0.1159 0.0000 0.00000.0000 7.2198.6355 — — — — — — — — — — — — 1 5 Su40St0 0 0.1159 0.0000 0.03830.0000 7.1197.7392 — — — — — — — — — — — — 1 6 Su40St0 0 0.1160 0.0000 0.03810.0000 7.14— — — — — — — — — — — — — 1 7 Su80St0 0 0.1156 0.0000 0.07620.0000 7.2398.6127 — — — — — — — — — — — — 1 8 Su80St0 0 0.1159 0.0000 0.07640.0000 7.2498.0977 — — — — — — — — — — — — 1 9 Su120St0 0 0.1157 0.0000 0.11390.0000 7.2297.7226 — — — — — — — — — — — — 1 10 Su120St0 0 0.1159 0.0000 0.11440.0000 7.2198.3666 — — — — — — — — — — — — 1 11 Su0St40 0 0.1157 0.0317 0.00000.0000 7.1399.3386 — — — — — — — — — — — — 1 12 Su0St40 0 0.1160 0.0320 0.00000.0000 7.1199.0908 — — — — — — — — — — — — 1 13 Su0St80 0 0.1161 0.0641 0.00000.0000 7.1399.5454 — — — — — — — — — — — — 1 14 Su0St80 0 0.1162 0.0639 0.00000.0000 7.1098.6582 — — — — — — — — — — — — 1 15 Su0St120 0 0.1159 0.0957 0.00000.0000 7.2398.5459 — — — — — — — — — — — — 1 16 Su0St120 0 0.1161 0.0959 0.00000.0000 7.2198.0243 — — — — — — — — — — — — 1 17 Su40St80 0 0.1157 0.0637 0.03800.0000 7.19102.9297— — — — — — — — — — — —

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194Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 18 Su40St80 0 0.1159 0.0637 0.03850.0000 7.1298.4563 — — — — — — — — — — — — 1 19 Su80St40 0 0.1158 0.0320 0.07620.0000 7.1197.5515 — — — — — — — — — — — — 1 20 Su80St40 0 0.1157 0.0319 0.07590.0000 7.1397.7226 — — — — — — — — — — — — 1 23 NDF 0 0.1161 0.0000 0.00000.0000 — — -0.1575 — — — — — — — — — — — 1 24 NDF 0 0.1160 0.0000 0.00000.0000 — — 0.1574 — — — — — — — — — — — 1 25 Su40St0 0 0.1159 0.0000 0.03850.0000 — — -0.1018 — — — — — — — — — — — 1 26 Su40St0 0 0.1161 0.0000 0.03850.0000 — — 0.1019 — — — — — — — — — — — 1 27 Su80St0 0 0.1158 0.0000 0.07620.0000 — — 0.1562 — — — — — — — — — — — 1 28 Su80St0 0 0.1157 0.0000 0.07630.0000 — — -0.1563 — — — — — — — — — — — 1 29 Su120St0 0 0.1159 0.0000 0.11400.0000 — — -0.0520 — — — — — — — — — — — 1 30 Su120St0 0 0.1157 0.0000 0.11420.0000 — — 0.0520 — — — — — — — — — — — 1 31 Su0St40 0 0.1161 0.0319 0.00000.0000 — — 0.3662 — — — — — — — — — — — 1 32 Su0St40 0 0.1160 0.0320 0.00000.0000 — — -0.3664 — — — — — — — — — — — 1 33 Su0St80 0 0.1160 0.0639 0.00000.0000 — — 0.7238 — — — — — — — — — — — 1 34 Su0St80 0 0.1159 0.0636 0.00000.0000 — — -0.7223 — — — — — — — — — — — 1 35 Su0St120 0 0.1157 0.0957 0.00000.0000 — — 0.1077 — — — — — — — — — — — 1 36 Su0St120 0 0.1160 0.0957 0.00000.0000 — — -0.1079 — — — — — — — — — — — 1 37 Su40St80 0 0.1159 0.0639 0.03820.0000 — — 0.5208 — — — — — — — — — — — 1 38 Su40St80 0 0.1158 0.0639 0.03830.0000 — — -0.5208 — — — — — — — — — — — 1 39 Su80St40 0 0.1159 0.0322 0.07630.0000 — — -0.7795 — — — — — — — — — — — 1 40 Su80St40 0 0.1160 0.0321 0.07640.0000 — — 0.7799 — — — — — — — — — — — 1 41 BL 0 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.21540.2537 1 42 BL 0 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.14010.2007 1 43 NDF 0 0.1161 0.0000 0.00000.0000 — — — — 0.0709 0.0112 0.3015 -0.0178-0.17190.1992-0.06970.01850.00000.2915 1 44 NDF 0 0.1159 0.0000 0.00000.0000 — — — — -0.0134-0.0012 -0.2511-0.0178-0.3394-0.3481-0.0554-0.14080.14200.1882 1 45 Su40St0 0 0.1161 0.0000 0.03840.0000 — — — — 10.36849.2626 23.22170.41190.34270.18200.4238-0.10760.25960.1782 1 46 Su40St0 0 0.1157 0.0000 0.03790.0000 — — — — 8.0466 8.0042 21.36790.3506-0.4747-0.3438-0.12890.19420.15530.2445 1 47 Su80St0 0 0.1158 0.0000 0.07600.0000 — — — — 9.1230 7.4985 51.10670.42270.0378-0.8901-0.0610-0.21190.15680.0000

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195Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 48 Su80St0 0 0.1160 0.0000 0.07650.0000 — — — — 12.570611.3322 53.58190.2187-0.3988-0.1942-0.0356-0.01500.22420.2994 1 49 Su120St0 0 0.1157 0.0000 0.11390.0000 — — — — 12.65128.9090 72.01460.3659-0.2642-0.1027-0.1139-0.00600.17180.2998 1 50 Su120St0 0 0.1158 0.0000 0.11410.0000 — — — — 11.946611.1762 90.55390.2397-0.2726-0.2188-0.0853-0.00600.21010.2548 1 51 Su0St40 0 0.1160 0.0321 0.00000.0000 — — — — 0.0036 -0.0062 0.1838 -0.0178-1.5632-0.1670-0.1214-0.21190.06750.2435 1 52 Su0St40 0 0.1157 0.0321 0.00000.0000 — — — — -0.01080.0014 -0.2433-0.0178-0.1148-0.20050.01720.06340.16650.3161 1 53 Su0St80 0 0.1161 0.0638 0.00000.0000 — — — — 0.0214 0.0119 0.2010 -0.0178-0.5639-0.1092-0.07840.01720.22030.2382 1 54 Su0St80 0 0.1157 0.0639 0.00000.0000 — — — — -0.0108-0.0038 -0.2823-0.0178-0.1352-0.8377-0.23870.01770.19180.0000 1 55 Su0St120 0 0.1162 0.0955 0.00000.0000 — — — — 0.0003 -0.0374 -0.3421-0.0178-0.1260-0.0862-0.08420.01950.21280.2794 1 56 Su0St120 0 0.1162 0.0955 0.00000.0000 — — — — -0.0134-0.0090 -0.2719-0.0178-0.4120-0.32850.0546-0.21190.00000.1684 1 57 Su40St80 0 0.1159 0.0640 0.03830.0000 — — — — — — — 0.42190.37930.16990.0938-0.01370.17300.3827 1 58 Su40St80 0 0.1159 0.0638 0.03840.0000 — — — — 9.9186 9.2522 15.56990.34980.0702-0.0784-0.07680.00840.18000.2380 1 59 Su80St40 0 0.1158 0.0320 0.07630.0000 — — — — 11.23949.6409 50.46970.3226-0.19110.2491-2.1860-0.05270.00000.1954 1 60 Su80St40 0 0.1162 0.0322 0.07620.0000 — — — — 14.702614.0362 43.59790.5384-0.2872-0.1284-0.05890.02000.19510.2737 1 61 BL 4 0.0000 0.0000 0.00000.0000 7.14— — — — — — — — — — — — — 1 62 BL 4 0.0000 0.0000 0.00000.0000 7.09— — — — — — — — — — — — — 1 63 NDF 4 0.1159 0.0000 0.00000.0000 7.1695.3193 — — — — — — — — — — — — 1 64 NDF 4 0.1161 0.0000 0.00000.0000 7.1595.7873 — — — — — — — — — — — — 1 65 Su40St0 4 0.1160 0.0000 0.03800.0000 6.9992.3743 — — — — — — — — — — — — 1 66 Su40St0 4 0.1158 0.0000 0.03830.0000 6.9997.8206 — — — — — — — — — — — — 1 67 Su80St0 4 0.1157 0.0000 0.07600.0000 6.8695.3884 — — — — — — — — — — — — 1 68 Su80St0 4 0.1161 0.0000 0.07600.0000 6.8593.0134 — — — — — — — — — — — — 1 69 Su120St0 4 0.1160 0.0000 0.11450.0000 6.7395.7774 — — — — — — — — — — — — 1 70 Su120St0 4 0.1162 0.0000 0.11400.0000 6.6999.0158 — — — — — — — — — — — — 1 71 Su0St40 4 0.1160 0.0320 0.00000.0000 7.1492.5534 — — — — — — — — — — — — 1 72 Su0St40 4 0.1162 0.0318 0.00000.0000 7.1392.8469 — — — — — — — — — — — — 1 73 Su0St80 4 0.1159 0.0640 0.00000.0000 7.1190.8379 — — — — — — — — — — — — 1 74 Su0St80 4 0.1162 0.0637 0.00000.0000 7.1190.8799 — — — — — — — — — — — — 1 75 Su0St120 4 0.1157 0.0957 0.00000.0000 7.1492.5155 — — — — — — — — — — — —

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196Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 76 Su0St120 4 0.1158 0.0955 0.00000.0000 7.1591.9002 — — — — — — — — — — — — 1 77 Su40St80 4 0.1161 0.0640 0.03820.0000 7.0290.8659 — — — — — — — — — — — — 1 78 Su40St80 4 0.1160 0.0636 0.03820.0000 7.0192.1952 — — — — — — — — — — — — 1 79 Su80St40 4 0.1157 0.0319 0.07630.0000 6.8291.6177 — — — — — — — — — — — — 1 80 Su80St40 4 0.1161 0.0321 0.07600.0000 6.8692.9240 — — — — — — — — — — — — 1 83 NDF 4 0.1162 0.0000 0.00000.0000 — — 3.4324 — — — — — — — — — — — 1 84 NDF 4 0.1163 0.0000 0.00000.0000 — — 3.6381 — — — — — — — — — — — 1 85 Su40St0 4 0.1158 0.0000 0.03800.0000 — — 6.2121 — — — — — — — — — — — 1 86 Su40St0 4 0.1158 0.0000 0.03850.0000 — — 6.7206 — — — — — — — — — — — 1 87 Su80St0 4 0.1157 0.0000 0.07640.0000 — — 9.6818 — — — — — — — — — — — 1 88 Su80St0 4 0.1157 0.0000 0.07600.0000 — — 10.6259 — — — — — — — — — — — 1 89 Su120St0 4 0.1158 0.0000 0.11430.0000 — — 12.4921 — — — — — — — — — — — 1 90 Su120St0 4 0.1159 0.0000 0.11440.0000 — — 11.6563 — — — — — — — — — — — 1 91 Su0St40 4 0.1158 0.0323 0.00000.0000 — — 4.0550 — — — — — — — — — — — 1 92 Su0St40 4 0.1158 0.0319 0.00000.0000 — — 3.0313 — — — — — — — — — — — 1 93 Su0St80 4 0.1161 0.0641 0.00000.0000 — — 1.9125 — — — — — — — — — — — 1 94 Su0St80 4 0.1157 0.0640 0.00000.0000 — — 4.0092 — — — — — — — — — — — 1 95 Su0St120 4 0.1159 0.0956 0.00000.0000 — — 2.2400 — — — — — — — — — — — 1 96 Su0St120 4 0.1161 0.0959 0.00000.0000 — — 2.2248 — — — — — — — — — — — 1 97 Su40St80 4 0.1160 0.0638 0.03850.0000 — — 5.0382 — — — — — — — — — — — 1 98 Su40St80 4 0.1158 0.0637 0.03830.0000 — — 3.7042 — — — — — — — — — — — 1 99 Su80St40 4 0.1162 0.0322 0.07610.0000 — — 9.7871 — — — — — — — — — — — 1 100 Su80St40 4 0.1159 0.0321 0.07620.0000 — — 9.8985 — — — — — — — — — — — 1 101 BL 4 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.27060.4626 1 102 BL 4 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.25590.4521 1 103 NDF 4 0.1163 0.0000 0.00000.0000 — — — 0.28510.0247 -0.0602 0.0051 0.0434-1.27080.35630.19270.04490.33190.6201 1 104 NDF 4 0.1160 0.0000 0.00000.0000 — — — -0.1196-0.00750.0758 -0.04460.0000-1.5184-0.39260.17090.06630.26540.3843 1 105 Su40St0 4 0.1161 0.0000 0.03810.0000 — — — 2.16890.0058 -0.0941 -0.02546.62501.56502.07190.23540.02840.19430.7377

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197Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 106 Su40St0 4 0.1160 0.0000 0.03820.0000 — — — 1.76420.0731 0.0394 -0.03946.64091.34552.09200.06050.10050.15180.7464 1 107 Su80St0 4 0.1158 0.0000 0.07600.0000 — — — 2.99220.0204 -0.0974 -0.029617.84112.23243.23460.22840.08220.28470.8450 1 108 Su80St0 4 0.1157 0.0000 0.07630.0000 — — — 1.35560.1225 0.0212 -0.034217.02852.44932.5284— — 0.00000.0000 1 109 Su120St0 4 0.1162 0.0000 0.11430.0000 — — — 3.48460.0193 -0.0902 0.0182 29.07992.88034.27550.43970.06180.26650.7334 1 110 Su120St0 4 0.1160 0.0000 0.11440.0000 — — — 3.15370.2187 0.0472 -0.008229.19912.96504.29040.44320.09760.29830.5426 1 111 Su0St40 4 0.1159 0.0319 0.00000.0000 — — — 17.62050.0211 -0.0232 -0.03570.0298-0.9949— -0.23690.12100.27990.7702 1 112 Su0St40 4 0.1159 0.0323 0.00000.0000 — — — 13.1272-0.01010.0654 -0.04720.0000-0.9586-0.0235-0.24490.09470.25400.8186 1 113 Su0St80 4 0.1159 0.0636 0.00000.0000 — — — 41.00430.0253 -0.0213 -0.04030.0496-0.44890.3506-0.17510.08520.31520.6744 1 114 Su0St80 4 0.1158 0.0639 0.00000.0000 — — — 40.8488-0.01010.0498 -0.04460.0619-0.41670.3913-0.17350.08030.30940.8054 1 115 Su0St120 4 0.1162 0.0956 0.00000.0000 — — — 43.95070.0251 -0.0493 -0.01920.0000-0.86680.2565-0.23070.06360.25220.7176 1 116 Su0St120 4 0.1159 0.0957 0.00000.0000 — — — 52.5407-0.01010.0858 -0.03390.0512— -0.1259-0.38310.05270.23761.9426 1 117 Su40St80 4 0.1160 0.0641 0.03790.0000 — — — 41.82760.1745 -0.0757 -0.04366.95721.43732.43370.3484-0.00220.21970.6569 1 118 Su40St80 4 0.1159 0.0637 0.03840.0000 — — — 25.3155-0.01010.0680 -0.04206.75511.06452.12570.1304-0.01460.27380.5458 1 119 Su80St40 4 0.1160 0.0321 0.07620.0000 — — — 23.43960.0105 -0.0769 -0.021318.65493.02683.57010.34080.10010.27250.6619 1 120 Su80St40 4 0.1162 0.0323 0.07610.0000 — — — 16.65170.1771 0.0368 -0.044617.39022.10673.28670.3235-0.03400.2615— 1 121 BL 8 0.0000 0.0000 0.00000.0000 7.22— — — — — — — — — — — — — 1 122 BL 8 0.0000 0.0000 0.00000.0000 7.22— — — — — — — — — — — — — 1 123 NDF 8 0.1161 0.0000 0.00000.0000 7.1986.1236 — — — — — — — — — — — — 1 124 NDF 8 0.1157 0.0000 0.00000.0000 7.2085.6924 — — — — — — — — — — — — 1 125 Su40St0 8 0.1159 0.0000 0.03850.0000 7.0784.2950 — — — — — — — — — — — — 1 126 Su40St0 8 0.1162 0.0000 0.03800.0000 7.0683.9958 — — — — — — — — — — — — 1 127 Su80St0 8 0.1157 0.0000 0.07630.0000 6.9482.4604 — — — — — — — — — — — — 1 128 Su80St0 8 0.1160 0.0000 0.07620.0000 6.9685.4787 — — — — — — — — — — — — 1 129 Su120St0 8 0.1159 0.0000 0.11450.0000 6.8385.8187 — — — — — — — — — — — — 1 130 Su120St0 8 0.1161 0.0000 0.11440.0000 6.8086.1236 — — — — — — — — — — — — 1 131 Su0St40 8 0.1159 0.0319 0.00000.0000 7.1185.0121 — — — — — — — — — — — — 1 132 Su0St40 8 0.1161 0.0319 0.00000.0000 7.1286.5710 — — — — — — — — — — — — 1 133 Su0St80 8 0.1158 0.0642 0.00000.0000 7.0085.9799 — — — — — — — — — — — —

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198Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 134 Su0St80 8 0.1161 0.0640 0.00000.0000 7.0485.0498 — — — — — — — — — — — — 1 135 Su0St120 8 0.1162 0.0958 0.00000.0000 6.8886.9461 — — — — — — — — — — — — 1 136 Su0St120 8 0.1160 0.0958 0.00000.0000 7.0186.7325 — — — — — — — — — — — — 1 137 Su40St80 8 0.1160 0.0637 0.03790.0000 6.9684.5832 — — — — — — — — — — — — 1 138 Su40St80 8 0.1158 0.0639 0.03810.0000 6.9383.9167 — — — — — — — — — — — — 1 139 Su80St40 8 0.1157 0.0321 0.07620.0000 6.9384.0764 — — — — — — — — — — — — 1 140 Su80St40 8 0.1157 0.0320 0.07600.0000 6.8684.6151 — — — — — — — — — — — — 1 143 NDF 8 0.1157 0.0000 0.00000.0000 — — 3.0803 — — — — — — — — — — — 1 144 NDF 8 0.1162 0.0000 0.00000.0000 — — 4.0050 — — — — — — — — — — — 1 145 Su40St0 8 0.1158 0.0000 0.03850.0000 — — 9.2717 — — — — — — — — — — — 1 146 Su40St0 8 0.1157 0.0000 0.03850.0000 — — 10.5234 — — — — — — — — — — — 1 147 Su80St0 8 0.1160 0.0000 0.07620.0000 — — 12.6480 — — — — — — — — — — — 1 148 Su80St0 8 0.1160 0.0000 0.07610.0000 — — 12.0250 — — — — — — — — — — — 1 149 Su120St0 8 0.1159 0.0000 0.11430.0000 — — 17.0202 — — — — — — — — — — — 1 150 Su120St0 8 0.1158 0.0000 0.11440.0000 — — 14.9376 — — — — — — — — — — — 1 151 Su0St40 8 0.1161 0.0319 0.00000.0000 — — 4.8435 — — — — — — — — — — — 1 152 Su0St40 8 0.1158 0.0318 0.00000.0000 — — 5.0652 — — — — — — — — — — — 1 153 Su0St80 8 0.1162 0.0638 0.00000.0000 — — 11.1336 — — — — — — — — — — — 1 154 Su0St80 8 0.1160 0.0639 0.00000.0000 — — 7.8043 — — — — — — — — — — — 1 155 Su0St120 8 0.1160 0.0959 0.00000.0000 — — 14.5660 — — — — — — — — — — — 1 156 Su0St120 8 0.1159 0.0956 0.00000.0000 — — 15.6200 — — — — — — — — — — — 1 157 Su40St80 8 0.1160 0.0636 0.03800.0000 — — 11.5743 — — — — — — — — — — — 1 158 Su40St80 8 0.1160 0.0640 0.03830.0000 — — 12.1710 — — — — — — — — — — — 1 159 Su80St40 8 0.1158 0.0319 0.07620.0000 — — 15.6850 — — — — — — — — — — — 1 160 Su80St40 8 0.1157 0.0319 0.07620.0000 — — 16.3148 — — — — — — — — — — — 1 161 BL 8 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.36061.1007 1 162 BL 8 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.31271.1325 1 163 NDF 8 0.1157 0.0000 0.00000.0000 — — — -0.07970.0072 -0.0658 -0.00520.00002.32010.87070.63920.16410.33140.8144

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199Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 164 NDF 8 0.1157 0.0000 0.00000.0000 — — — 1.2300-0.00650.0508 -0.00960.00002.17230.91270.32540.20230.34691.1373 1 165 Su40St0 8 0.1161 0.0000 0.03840.0000 — — — 1.15020.0079 -0.0852 0.0095 3.66336.01154.54161.56590.28700.42800.9382 1 166 Su40St0 8 0.1157 0.0000 0.03800.0000 — — — 2.7849-0.00650.0485 0.0086 3.41406.24834.24091.68930.30670.34250.9656 1 167 Su80St0 8 0.1157 0.0000 0.07640.0000 — — — 1.96760.0023 -0.1083 0.0508 13.48678.3573— 1.78380.21060.31230.9498 1 168 Su80St0 8 0.1162 0.0000 0.07630.0000 — — — 2.87060.1111 0.0281 -0.009612.72797.84116.24651.81730.24260.30850.9616 1 169 Su120St0 8 0.1162 0.0000 0.11440.0000 — — — 2.29450.0072 -0.0974 0.0098 21.79399.96729.63322.00050.14020.28880.9656 1 170 Su120St0 8 0.1161 0.0000 0.11430.0000 — — — 3.27530.1392 0.0037 -0.009621.233310.44049.40292.21450.30930.30310.6840 1 171 Su0St40 8 0.1162 0.0323 0.00000.0000 — — — 5.8091-0.00570.0176 -0.01220.00005.15012.86870.60860.31600.23171.1728 1 172 Su0St40 8 0.1160 0.0319 0.00000.0000 — — — 8.3468-0.0065-0.0116 -0.00960.00005.67142.78830.61610.33510.33011.1559 1 173 Su0St80 8 0.1158 0.0640 0.00000.0000 — — — 21.4281-0.00530.0205 -0.00980.09897.12364.01710.78920.22900.32161.1766 1 174 Su0St80 8 0.1159 0.0637 0.00000.0000 — — — 16.9328-0.0065-0.0376 -0.01220.03978.20644.32111.23880.19380.34230.6092 1 175 Su0St120 8 0.1159 0.0960 0.00000.0000 — — — 36.71630.0306 0.1294 -0.01000.16037.11421.23900.84030.21400.27390.9736 1 176 Su0St120 8 0.1157 0.0959 0.00000.0000 — — — 21.5956-0.0065-0.0474 -0.00700.00009.47452.96170.90640.12780.00001.0353 1 177 Su40St80 8 0.1162 0.0640 0.03790.0000 — — — 12.76640.0012 -0.0705 0.0117 3.368412.38909.52522.59700.37870.30721.0271 1 178 Su40St80 8 0.1162 0.0636 0.03840.0000 — — — 18.1628-0.0065-0.1078 0.0008 3.788511.16758.75752.55920.44810.32720.5619 1 179 Su80St40 8 0.1161 0.0322 0.07630.0000 — — — 10.2247-0.0005-0.0830 0.0275 12.945011.02288.80423.10860.41410.30830.7054 1 180 Su80St40 8 0.1159 0.0321 0.07640.0000 — — — 9.0046-0.0039-0.0116 -0.007012.331011.79049.11422.94030.55330.31981.2016 1 181 BL 12 0.0000 0.0000 0.00000.0000 7.22— — — — — — — — — — — — — 1 182 BL 12 0.0000 0.0000 0.00000.0000 7.23— — — — — — — — — — — — — 1 183 NDF 12 0.1160 0.0000 0.00000.0000 7.2277.0607 — — — — — — — — — — — — 1 184 NDF 12 0.1162 0.0000 0.00000.0000 7.2176.0388 — — — — — — — — — — — — 1 185 Su40St0 12 0.1158 0.0000 0.03800.0000 7.1074.8568 — — — — — — — — — — — — 1 186 Su40St0 12 0.1157 0.0000 0.03800.0000 7.1275.7271 — — — — — — — — — — — — 1 187 Su80St0 12 0.1161 0.0000 0.07650.0000 6.9975.3861 — — — — — — — — — — — — 1 188 Su80St0 12 0.1159 0.0000 0.07610.0000 7.0175.7804 — — — — — — — — — — — — 1 189 Su120St0 12 0.1157 0.0000 0.11390.0000 6.8775.0987 — — — — — — — — — — — — 1 190 Su120St0 12 0.1157 0.0000 0.11420.0000 6.8475.9965 — — — — — — — — — — — — 1 191 Su0St40 12 0.1159 0.0322 0.00000.0000 7.1375.6011 — — — — — — — — — — — —

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200Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 192 Su0St40 12 0.1157 0.0318 0.00000.0000 7.1175.1885 — — — — — — — — — — — — 1 193 Su0St80 12 0.1162 0.0640 0.00000.0000 7.0275.5917 — — — — — — — — — — — — 1 194 Su0St80 12 0.1162 0.0639 0.00000.0000 6.9875.4129 — — — — — — — — — — — — 1 195 Su0St120 12 0.1157 0.0957 0.00000.0000 6.9775.2783 — — — — — — — — — — — — 1 196 Su0St120 12 0.1161 0.0957 0.00000.0000 6.9576.2809 — — — — — — — — — — — — 1 197 Su40St80 12 0.1162 0.0636 0.03840.0000 6.9773.6248 — — — — — — — — — — — — 1 198 Su40St80 12 0.1160 0.0637 0.03800.0000 6.9874.8219 — — — — — — — — — — — — 1 199 Su80St40 12 0.1157 0.0323 0.07610.0000 6.9572.8543 — — — — — — — — — — — — 1 200 Su80St40 12 0.1159 0.0322 0.07650.0000 6.9072.5538 — — — — — — — — — — — — 1 203 NDF 12 0.1162 0.0000 0.00000.0000 — — 7.2329 — — — — — — — — — — — 1 204 NDF 12 0.1158 0.0000 0.00000.0000 — — 5.8882 — — — — — — — — — — — 1 205 Su40St0 12 0.1162 0.0000 0.03820.0000 — — 11.5607 — — — — — — — — — — — 1 206 Su40St0 12 0.1162 0.0000 0.03840.0000 — — 12.1806 — — — — — — — — — — — 1 207 Su80St0 12 0.1158 0.0000 0.07620.0000 — — 16.9204 — — — — — — — — — — — 1 208 Su80St0 12 0.1161 0.0000 0.07620.0000 — — 18.6856 — — — — — — — — — — — 1 209 Su120St0 12 0.1157 0.0000 0.11450.0000 — — 20.3520 — — — — — — — — — — — 1 210 Su120St0 12 0.1158 0.0000 0.11420.0000 — — 21.6045 — — — — — — — — — — — 1 211 Su0St40 12 0.1159 0.0322 0.00000.0000 — — 16.2899 — — — — — — — — — — — 1 212 Su0St40 12 0.1161 0.0322 0.00000.0000 — — 12.3265 — — — — — — — — — — — 1 213 Su0St80 12 0.1161 0.0640 0.00000.0000 — — 14.4625 — — — — — — — — — — — 1 214 Su0St80 12 0.1161 0.0638 0.00000.0000 — — 14.2602 — — — — — — — — — — — 1 215 Su0St120 12 0.1159 0.0159 0.00000.0000 — — 19.2964 — — — — — — — — — — — 1 216 Su0St120 12 0.1157 0.0957 0.00000.0000 — — 19.0585 — — — — — — — — — — — 1 217 Su40St80 12 0.1161 0.0637 0.03790.0000 — — 18.2346 — — — — — — — — — — — 1 218 Su40St80 12 0.1160 0.0636 0.03800.0000 — — 19.6961 — — — — — — — — — — — 1 219 Su80St40 12 0.1159 0.0322 0.07630.0000 — — 20.1496 — — — — — — — — — — — 1 220 Su80St40 12 0.1160 0.0319 0.07620.0000 — — 19.9493 — — — — — — — — — — — 1 221 BL 12 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.41920.6509

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201Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 222 BL 12 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.31301.5951 1 223 NDF 12 0.1161 0.0000 0.00000.0000 — — — 0.08370.0000 -0.1018 -0.00370.00005.08092.85460.48510.36910.38851.9416 1 224 NDF 12 0.1161 0.0000 0.00000.0000 — — — 1.30970.0025 -0.0472 0.0001 0.00005.10492.86070.95210.34530.57571.3799 1 225 Su40St0 12 0.1160 0.0000 0.03810.0000 — — — 2.29050.0003 -0.1008 -0.00050.56399.28956.28422.82360.60860.51061.9082 1 226 Su40St0 12 0.1157 0.0000 0.03800.0000 — — — 2.13500.0077 -0.0502 0.0053 0.52688.53226.25742.87420.03590.62791.9821 1 227 Su80St0 12 0.1160 0.0000 0.07630.0000 — — — 2.53770.0000 -0.0434 0.0105 4.316810.39129.04474.61281.32250.38781.6012 1 228 Su80St0 12 0.1159 0.0000 0.07640.0000 — — — 1.3237-0.0001-0.0424 -0.00254.097710.06818.83434.97791.57650.44531.6722 1 229 Su120St0 12 0.1157 0.0000 0.11400.0000 — — — 2.95240.0001 -0.0406 -0.00410.047716.421316.94186.63692.49310.48891.8046 1 230 Su120St0 12 0.1157 0.0000 0.11420.0000 — — — 2.04930.0211 -0.0995 -0.00240.000014.877516.13307.68442.50560.45341.5287 1 231 Su0St40 12 0.1160 0.0323 0.00000.0000 — — — 4.6668-0.00010.0094 -0.00510.00008.83855.84541.23070.35930.54431.9359 1 232 Su0St40 12 0.1161 0.0323 0.00000.0000 — — — 4.58510.0415 -0.1022 -0.00250.0000— 5.71701.36510.40380.56231.9107 1 233 Su0St80 12 0.1162 0.0638 0.00000.0000 — — — 7.36200.0214 0.0332 -0.00510.000012.96348.24261.61590.36290.00001.8746 1 234 Su0St80 12 0.1160 0.0640 0.00000.0000 — — — 5.97650.0441 -0.0736 -0.00510.000015.03089.90671.86410.54800.61811.7873 1 235 Su0St120 12 0.1158 0.0957 0.00000.0000 — — — 16.52410.0281 0.0477 -0.00510.000016.059011.04371.94530.45390.50941.9470 1 236 Su0St120 12 0.1157 0.0957 0.00000.0000 — — — 17.02650.0103 -0.1022 -0.00250.105216.030012.40172.01640.52710.59801.9634 1 237 Su40St80 12 0.1158 0.0640 0.03840.0000 — — — 10.97020.0147 -0.0028 -0.00330.035216.266912.92174.44411.07990.61042.2720 1 238 Su40St80 12 0.1161 0.0637 0.03820.0000 — — — 11.79550.0233 -0.1022 0.0001 0.000015.323711.86444.50971.09100.64942.2002 1 239 Su80St40 12 0.1159 0.0320 0.07610.0000 — — — 7.45170.0153 -0.0061 -0.00471.138515.142212.21116.81572.23300.53001.9896 1 240 Su80St40 12 0.1159 0.0322 0.07630.0000 — — — 8.4405-0.0001-0.0969 -0.00251.171413.859511.55966.68402.13770.54991.8583 1 241 BL 16 0.0000 0.0000 0.00000.0000 7.33— — — — — — — — — — — — — 1 242 BL 16 0.0000 0.0000 0.00000.0000 7.33— — — — — — — — — — — — — 1 243 NDF 16 0.1159 0.0000 0.00000.0000 7.2969.2824 — — — — — — — — — — — — 1 244 NDF 16 0.1161 0.0000 0.00000.0000 7.2669.3462 — — — — — — — — — — — — 1 245 Su40St0 16 0.1158 0.0000 0.03820.0000 7.2069.4298 — — — — — — — — — — — — 1 246 Su40St0 16 0.1162 0.0000 0.03830.0000 7.2069.5569 — — — — — — — — — — — — 1 247 Su80St0 16 0.1161 0.0000 0.07620.0000 7.0869.4357 — — — — — — — — — — — — 1 248 Su80St0 16 0.1162 0.0000 0.07650.0000 7.0869.1099 — — — — — — — — — — — — 1 249 Su120St0 16 0.1161 0.0000 0.11450.0000 6.8869.1673 — — — — — — — — — — — —

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202Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 250 Su120St0 16 0.1157 0.0000 0.11390.0000 6.9268.1410 — — — — — — — — — — — — 1 251 Su0St40 16 0.1158 0.0319 0.00000.0000 7.0967.1872 — — — — — — — — — — — — 1 252 Su0St40 16 0.1158 0.0322 0.00000.0000 7.1868.3533 — — — — — — — — — — — — 1 253 Su0St80 16 0.1157 0.0639 0.00000.0000 7.0669.9365 — — — — — — — — — — — — 1 254 Su0St80 16 0.1157 0.0637 0.00000.0000 7.0768.8592 — — — — — — — — — — — — 1 255 Su0St120 16 0.1157 0.0957 0.00000.0000 6.9769.1285 — — — — — — — — — — — — 1 256 Su0St120 16 0.1160 0.0959 0.00000.0000 6.9869.8517 — — — — — — — — — — — — 1 257 Su40St80 16 0.1160 0.0638 0.03840.0000 6.9768.6875 — — — — — — — — — — — — 1 258 Su40St80 16 0.1161 0.0637 0.03820.0000 6.9967.2882 — — — — — — — — — — — — 1 259 Su80St40 16 0.1160 0.0319 0.07630.0000 6.9967.3442 — — — — — — — — — — — — 1 260 Su80St40 16 0.1161 0.0322 0.07610.0000 7.0267.8251 — — — — — — — — — — — — 1 263 NDF 16 0.1158 0.0000 0.00000.0000 — — 6.3057 — — — — — — — — — — — 1 264 NDF 16 0.1160 0.0000 0.00000.0000 — — 6.0925 — — — — — — — — — — — 1 265 Su40St0 16 0.1159 0.0000 0.03800.0000 — — 9.8022 — — — — — — — — — — — 1 266 Su40St0 16 0.1158 0.0000 0.03840.0000 — — 11.2525 — — — — — — — — — — — 1 267 Su80St0 16 0.1159 0.0000 0.07600.0000 — — 14.3191 — — — — — — — — — — — 1 268 Su80St0 16 0.1157 0.0000 0.07630.0000 — — 14.5254 — — — — — — — — — — — 1 269 Su120St0 16 0.1159 0.0000 0.11410.0000 — — 17.8561 — — — — — — — — — — — 1 270 Su120St0 16 0.1160 0.0000 0.11420.0000 — — 17.1244 — — — — — — — — — — — 1 271 Su0St40 16 0.1157 0.0323 0.00000.0000 — — 9.1084 — — — — — — — — — — — 1 272 Su0St40 16 0.1160 0.0318 0.00000.0000 — — 8.7030 — — — — — — — — — — — 1 273 Su0St80 16 0.1159 0.0641 0.00000.0000 — — 14.5693 — — — — — — — — — — — 1 274 Su0St80 16 0.1157 0.0639 0.00000.0000 — — 14.1643 — — — — — — — — — — — 1 275 Su0St120 16 0.1158 0.0958 0.00000.0000 — — 17.9055 — — — — — — — — — — — 1 276 Su0St120 16 0.1162 0.0957 0.00000.0000 — — 17.3777 — — — — — — — — — — — 1 277 Su40St80 16 0.1160 0.0640 0.03850.0000 — — 14.1412 — — — — — — — — — — — 1 278 Su40St80 16 0.1159 0.0641 0.03840.0000 — — 14.3533 — — — — — — — — — — — 1 279 Su80St40 16 0.1157 0.0322 0.07590.0000 — — 16.3124 — — — — — — — — — — —

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203Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 280 Su80St40 16 0.1162 0.0321 0.07610.0000 — — 16.5057 — — — — — — — — — — — 1 281 BL 16 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.66681.0817 1 282 BL 16 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.30982.3044 1 283 NDF 16 0.1159 0.0000 0.00000.0000 — — — -0.1110-0.03780.0434 -0.00040.00007.71273.02080.73920.42740.69472.5264 1 284 NDF 16 0.1162 0.0000 0.00000.0000 — — — -0.53380.0436 -0.0290 0.0110 0.00007.28812.90950.69920.44120.68212.4385 1 285 Su40St0 16 0.1157 0.0000 0.03810.0000 — — — 0.8579-0.04480.0426 -0.00120.000010.51005.86443.04860.99290.87102.7724 1 286 Su40St0 16 0.1158 0.0000 0.03810.0000 — — — 1.04880.0511 -0.0290 -0.00210.000010.64795.98613.08860.91920.87902.6145 1 287 Su80St0 16 0.1159 0.0000 0.07650.0000 — — — 1.2686-0.04660.0577 0.0018 0.000011.98828.95446.35482.68960.83702.3877 1 288 Su80St0 16 0.1160 0.0000 0.07610.0000 — — — 1.36720.0402 0.0022 -0.00210.000012.51439.17956.53772.68770.81042.3831 1 289 Su120St0 16 0.1157 0.0000 0.11440.0000 — — — 0.3954-0.0480-0.0286 0.0008 0.000019.110417.68726.47992.83860.79212.1943 1 290 Su120St0 16 0.1160 0.0000 0.11440.0000 — — — 1.2862-0.0482-0.0290 0.0031 0.000019.063917.90596.9151— 0.0000— 1 291 Su0St40 16 0.1158 0.0318 0.00000.0000 — — — 1.6812-0.0138-0.0199 0.3353 0.000012.23216.02151.56170.48770.77672.5390 1 292 Su0St40 16 0.1161 0.0322 0.00000.0000 — — — 2.02800.0013 -0.0264 0.0005 0.000012.52106.14551.38820.39940.69242.3944 1 293 Su0St80 16 0.1162 0.0639 0.00000.0000 — — — 10.0260-0.0197-0.0211 0.3320 0.000015.48648.75292.15700.73750.94712.7405 1 294 Su0St80 16 0.1161 0.0640 0.00000.0000 — — — 5.7984-0.0066-0.0290 0.0005 0.000016.11449.18552.28120.64930.85812.7542 1 295 Su0St120 16 0.1160 0.0958 0.00000.0000 — — — 7.5760-0.0368-0.0290 0.3990 0.000021.772813.57812.68580.57860.99311.5021 1 296 Su0St120 16 0.1158 0.0958 0.00000.0000 — — — 7.2227-0.0092-0.0264 -0.00210.076818.454611.48822.74190.71750.89982.7453 1 297 Su40St80 16 0.1157 0.0639 0.03850.0000 — — — 9.9582-0.0231-0.0287 0.2198 0.000018.043813.02125.08461.38410.94562.5463 1 298 Su40St80 16 0.1160 0.0638 0.03820.0000 — — — 9.2047-0.0082-0.0184 0.0006 0.000018.068112.70864.59751.22160.94202.4673 1 299 Su80St40 16 0.1161 0.0320 0.07600.0000 — — — 2.7517-0.0466-0.0023 0.1756 0.000017.696712.57077.05692.61000.87312.1534 1 300 Su80St40 16 0.1162 0.0322 0.07630.0000 — — — 3.0328-0.0482-0.0238 0.0005 0.000015.957512.15916.8102— 0.0000— 1 301 BL 20 0.0000 0.0000 0.00000.0000 7.37— — — — — — — — — — — — — 1 302 BL 20 0.0000 0.0000 0.00000.0000 7.38— — — — — — — — — — — — — 1 303 NDF 20 0.1160 0.0000 0.00000.0000 7.3264.2993 — — — — — — — — — — — — 1 304 NDF 20 0.1157 0.0000 0.00000.0000 7.3264.9090 — — — — — — — — — — — — 1 305 Su40St0 20 0.1157 0.0000 0.03830.0000 7.1764.7294 — — — — — — — — — — — — 1 306 Su40St0 20 0.1157 0.0000 0.03850.0000 7.2064.6396 — — — — — — — — — — — — 1 307 Su80St0 20 0.1160 0.0000 0.07640.0000 7.1163.1351 — — — — — — — — — — — —

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204Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 308 Su80St0 20 0.1158 0.0000 0.07630.0000 7.1263.1506 — — — — — — — — — — — — 1 309 Su120St0 20 0.1161 0.0000 0.11430.0000 6.9963.0827 — — — — — — — — — — — — 1 310 Su120St0 20 0.1157 0.0000 0.11450.0000 6.9863.2930 — — — — — — — — — — — — 1 311 Su0St40 20 0.1162 0.0319 0.00000.0000 7.1964.9079 — — — — — — — — — — — — 1 312 Su0St40 20 0.1162 0.0323 0.00000.0000 7.2264.1926 — — — — — — — — — — — — 1 313 Su0St80 20 0.1162 0.0637 0.00000.0000 7.1264.9973 — — — — — — — — — — — — 1 314 Su0St80 20 0.1159 0.0641 0.00000.0000 7.0964.7113 — — — — — — — — — — — — 1 315 Su0St120 20 0.1158 0.0955 0.00000.0000 6.9465.2137 — — — — — — — — — — — — 1 316 Su0St120 20 0.1161 0.0959 0.00000.0000 6.9666.0355 — — — — — — — — — — — — 1 317 Su40St80 20 0.1159 0.0636 0.03810.0000 7.0365.1595 — — — — — — — — — — — — 1 318 Su40St80 20 0.1160 0.0642 0.03820.0000 7.0562.5978 — — — — — — — — — — — — 1 319 Su80St40 20 0.1158 0.0321 0.07590.0000 7.0664.3167 — — — — — — — — — — — — 1 320 Su80St40 20 0.1160 0.0321 0.07640.0000 7.0663.6725 — — — — — — — — — — — — 1 323 NDF 20 0.1158 0.0000 0.00000.0000 — — 8.1799 — — — — — — — — — — — 1 324 NDF 20 0.1161 0.0000 0.00000.0000 — — 7.3395 — — — — — — — — — — — 1 325 Su40St0 20 0.1160 0.0000 0.03800.0000 — — 11.6742 — — — — — — — — — — — 1 326 Su40St0 20 0.1158 0.0000 0.03800.0000 — — 11.1581 — — — — — — — — — — — 1 327 Su80St0 20 0.1157 0.0000 0.07590.0000 — — 15.7819 — — — — — — — — — — — 1 328 Su80St0 20 0.1159 0.0000 0.07610.0000 — — 14.0050 — — — — — — — — — — — 1 329 Su120St0 20 0.1162 0.0000 0.11420.0000 — — 18.4753 — — — — — — — — — — — 1 330 Su120St0 20 0.1158 0.0000 0.11420.0000 — — 19.2096 — — — — — — — — — — — 1 331 Su0St40 20 0.1160 0.0321 0.00000.0000 — — 8.7931 — — — — — — — — — — — 1 332 Su0St40 20 0.1157 0.0318 0.00000.0000 — — 9.1294 — — — — — — — — — — — 1 333 Su0St80 20 0.1157 0.0640 0.00000.0000 — — 14.8903 — — — — — — — — — — — 1 334 Su0St80 20 0.1159 0.0641 0.00000.0000 — — 14.6734 — — — — — — — — — — — 1 335 Su0St120 20 0.1162 0.0959 0.00000.0000 — — 18.2055 — — — — — — — — — — — 1 336 Su0St120 20 0.1162 0.0957 0.00000.0000 — — 18.5256 — — — — — — — — — — — 1 337 Su40St80 20 0.1160 0.0639 0.03810.0000 — — 14.6821 — — — — — — — — — — —

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205Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 338 Su40St80 20 0.1160 0.0636 0.03820.0000 — — 13.5446 — — — — — — — — — — — 1 339 Su80St40 20 0.1157 0.0320 0.07590.0000 — — 15.6928 — — — — — — — — — — — 1 340 Su80St40 20 0.1160 0.0320 0.07640.0000 — — 14.3187 — — — — — — — — — — — 1 341 BL 20 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.45283.1574 1 342 BL 20 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.98494.0016 1 343 NDF 20 0.1160 0.0000 0.00000.0000 — — — -0.4068-0.02750.0051 0.1096 0.000011.62013.58751.27930.30660.96843.0134 1 344 NDF 20 0.1157 0.0000 0.00000.0000 — — — 1.03990.0191 -0.0049 -0.04520.000010.5200— — — — — 1 345 Su40St0 20 0.1162 0.0000 0.03790.0000 — — — 1.0664-0.02230.0002 0.1229 0.000016.12707.26463.78630.87420.99762.9302 1 346 Su40St0 20 0.1157 0.0000 0.03810.0000 — — — 1.6015-0.04210.0321 -0.04250.000015.26816.82083.54620.85570.87242.5819 1 347 Su80St0 20 0.1160 0.0000 0.07650.0000 — — — 0.8251-0.0237-0.0014 0.1379 0.000016.49619.42406.81032.38190.86052.5309 1 348 Su80St0 20 0.1161 0.0000 0.07610.0000 — — — 1.3641-0.04210.0133 -0.04520.000015.35008.46505.72772.23780.00002.2802 1 349 Su120St0 20 0.1158 0.0000 0.11410.0000 — — — 1.4710-0.02430.1017 -0.04340.000023.655318.61936.80472.49700.62572.4647 1 350 Su120St0 20 0.1159 0.0000 0.11420.0000 — — — 1.75590.0021 -0.0049 -0.04000.000022.528418.82267.24002.76560.83612.4283 1 351 Su0St40 20 0.1158 0.0320 0.00000.0000 — — — 1.1501-0.04100.1804 -0.03140.000016.98687.09032.35680.52021.08732.9327 1 352 Su0St40 20 0.1161 0.0319 0.00000.0000 — — — 2.1515-0.0135-0.0049 -0.04520.000016.99337.13792.40130.51201.05503.0359 1 353 Su0St80 20 0.1156 0.0640 0.00000.0000 — — — 2.1349-0.03830.1914 -0.04520.000020.893610.90453.4590— — — 1 354 Su0St80 20 0.1160 0.0641 0.00000.0000 — — — 3.1010-0.0212-0.0049 -0.04000.000022.146910.80173.46870.80201.08052.9506 1 355 Su0St120 20 0.1160 0.0957 0.00000.0000 — — — 6.5525-0.03650.2214 -0.04170.000023.573214.12184.29680.97671.15722.9681 1 356 Su0St120 20 0.1160 0.0958 0.00000.0000 — — — 8.6477-0.0421-0.0023 -0.03740.000024.543813.79144.06060.80421.08132.8712 1 357 Su40St80 20 0.1161 0.0639 0.03810.0000 — — — 3.7715-0.03880.1077 -0.04410.000024.676214.28066.10161.18890.97952.6225 1 358 Su40St80 20 0.1162 0.0636 0.03800.0000 — — — 4.2918-0.02120.0081 -0.04260.000024.399914.20115.98771.20410.96142.6774 1 359 Su80St40 20 0.1160 0.0321 0.07590.0000 — — — 2.5475-0.03980.0999 -0.04520.0000— 13.51608.11352.76940.86742.6728 1 360 Su80St40 20 0.1161 0.0321 0.07590.0000 — — — 2.6186-0.04210.0081 -0.04260.000023.710614.06977.95492.70500.94922.6455 1 361 BL 24 0.0000 0.0000 0.00000.0000 7.38— — — — — — — — — — — — — 1 362 BL 24 0.0000 0.0000 0.00000.0000 7.45— — — — — — — — — — — — — 1 363 NDF 24 0.1159 0.0000 0.00000.0000 7.3161.4399 — — — — — — — — — — — — 1 364 NDF 24 0.1158 0.0000 0.00000.0000 7.3260.9529 — — — — — — — — — — — — 1 365 Su40St0 24 0.1157 0.0000 0.03840.0000 7.2158.6695 — — — — — — — — — — — —

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206Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 366 Su40St0 24 0.1160 0.0000 0.03810.0000 7.2258.7023 — — — — — — — — — — — — 1 367 Su80St0 24 0.1156 0.0000 0.07600.0000 7.1460.2459 — — — — — — — — — — — — 1 368 Su80St0 24 0.1158 0.0000 0.07640.0000 7.0959.5176 — — — — — — — — — — — — 1 369 Su120St0 24 0.1161 0.0000 0.11440.0000 6.9960.8010 — — — — — — — — — — — — 1 370 Su120St0 24 0.1160 0.0000 0.11390.0000 7.0059.5978 — — — — — — — — — — — — 1 371 Su0St40 24 0.1159 0.0323 0.00000.0000 7.1863.9495 — — — — — — — — — — — — 1 372 Su0St40 24 0.1160 0.0320 0.00000.0000 7.2158.2545 — — — — — — — — — — — — 1 373 Su0St80 24 0.1161 0.0642 0.00000.0000 7.0661.2484 — — — — — — — — — — — — 1 374 Su0St80 24 0.1161 0.0638 0.00000.0000 7.0758.1166 — — — — — — — — — — — — 1 375 Su0St120 24 0.1161 0.0959 0.00000.0000 6.9560.9800 — — — — — — — — — — — — 1 376 Su0St120 24 0.1162 0.0955 0.00000.0000 6.9261.5552 — — — — — — — — — — — — 1 377 Su40St80 24 0.1158 0.0638 0.03800.0000 7.0158.8897 — — — — — — — — — — — — 1 378 Su40St80 24 0.1159 0.0636 0.03820.0000 6.9761.3503 — — — — — — — — — — — — 1 379 Su80St40 24 0.1157 0.0319 0.07650.0000 7.0359.4775 — — — — — — — — — — — — 1 380 Su80St40 24 0.1157 0.0318 0.07630.0000 7.0258.4899 — — — — — — — — — — — — 1 383 NDF 24 0.1158 0.0000 0.00000.0000 — — 7.2428 — — — — — — — — — — — 1 384 NDF 24 0.1158 0.0000 0.00000.0000 — — 5.9933 — — — — — — — — — — — 1 385 Su40St0 24 0.1160 0.0000 0.03810.0000 — — 10.2140 — — — — — — — — — — — 1 386 Su40St0 24 0.1161 0.0000 0.03820.0000 — — 9.5846 — — — — — — — — — — — 1 387 Su80St0 24 0.1157 0.0000 0.07610.0000 — — 14.0082 — — — — — — — — — — — 1 388 Su80St0 24 0.1158 0.0000 0.07630.0000 — — 12.9619 — — — — — — — — — — — 1 389 Su120St0 24 0.1157 0.0000 0.11420.0000 — — 16.7120 — — — — — — — — — — — 1 390 Su120St0 24 0.1158 0.0000 0.11390.0000 — — 15.4655 — — — — — — — — — — — 1 391 Su0St40 24 0.1161 0.0319 0.00000.0000 — — 7.6514 — — — — — — — — — — — 1 392 Su0St40 24 0.1161 0.0323 0.00000.0000 — — 8.0539 — — — — — — — — — — — 1 393 Su0St80 24 0.1159 0.0638 0.00000.0000 — — 12.0792 — — — — — — — — — — — 1 394 Su0St80 24 0.1162 0.0641 0.00000.0000 — — 13.2072 — — — — — — — — — — — 1 395 Su0St120 24 0.1161 0.0958 0.00000.0000 — — 17.6899 — — — — — — — — — — —

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207Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 1 396 Su0St120 24 0.1157 0.0958 0.00000.0000 — — 17.3873 — — — — — — — — — — — 1 397 Su40St80 24 0.1162 0.0637 0.03830.0000 — — 12.6960 — — — — — — — — — — — 1 398 Su40St80 24 0.1162 0.0639 0.03790.0000 — — 13.5373 — — — — — — — — — — — 1 399 Su80St40 24 0.1158 0.0322 0.07610.0000 — — 15.0552 — — — — — — — — — — — 1 400 Su80St40 24 0.1160 0.0318 0.07640.0000 — — 14.8470 — — — — — — — — — — — 1 401 BL 24 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.88473.2724 1 402 BL 24 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 1.19513.4312 1 403 NDF 24 0.1159 0.0000 0.00000.0000 — — — 0.3690-0.00490.0485 -0.00060.000012.53164.03141.94540.27001.20993.3865 1 404 NDF 24 0.1160 0.0000 0.00000.0000 — — — 0.2718-0.0041-0.0175 -0.00060.000011.98864.10341.9723-0.20691.22203.2652 1 405 Su40St0 24 0.1158 0.0000 0.03820.0000 — — — 0.6162-0.00780.0492 -0.00060.000016.00883.04464.33961.60021.08832.9396 1 406 Su40St0 24 0.1158 0.0000 0.03800.0000 — — — 1.0631-0.0092-0.0175 -0.00060.000017.11067.59304.30530.60351.02922.8651 1 407 Su80St0 24 0.1158 0.0000 0.07590.0000 — — — 0.3810-0.00660.0610 0.0024 0.000020.027612.09038.50032.12001.33392.3275 1 408 Su80St0 24 0.1157 0.0000 0.07620.0000 — — — 0.8257-0.0092-0.0175 -0.00060.000018.361610.94947.2208— — — 1 409 Su120St0 24 0.1158 0.0000 0.11410.0000 — — — 0.6999-0.00680.0687 0.0066 0.000025.952419.22107.69912.41610.99902.7593 1 410 Su120St0 24 0.1159 0.0000 0.11430.0000 — — — 0.9087-0.0092-0.0175 -0.00060.000025.763419.47697.91292.49290.49552.8415 1 411 Su0St40 24 0.1158 0.0318 0.00000.0000 — — — 0.9451-0.00820.0896 -0.00060.000016.26047.3424— — — — 1 412 Su0St40 24 0.1157 0.0319 0.00000.0000 — — — 1.2233-0.0092-0.0175 -0.00060.0000— 7.66673.16560.19491.20852.7344 1 413 Su0St80 24 0.1158 0.0637 0.00000.0000 — — — 2.3386-0.00920.0072 0.0022 0.000022.957210.96064.28080.14701.11082.9635 1 414 Su0St80 24 0.1158 0.0638 0.00000.0000 — — — 2.3349-0.0092-0.0175 -0.00060.000022.597711.00574.11860.22681.13383.0947 1 415 Su0St120 24 0.1161 0.0958 0.00000.0000 — — — 2.0954-0.00920.1451 -0.00060.000026.436914.99784.80320.34921.18453.0556 1 416 Su0St120 24 0.1158 0.0957 0.00000.0000 — — — 2.8039-0.0092-0.0038 -0.00060.000028.071815.15134.82550.45971.24293.2008 1 417 Su40St80 24 0.1160 0.0641 0.03840.0000 — — — 2.0954-0.00900.0716 0.0101 0.000026.989715.13386.62270.80101.07832.9261 1 418 Su40St80 24 0.1160 0.0637 0.03830.0000 — — — 3.5161-0.0092-0.0175 -0.00060.000026.681715.32366.93830.95381.08591.1448 1 419 Su80St40 24 0.1162 0.0321 0.07600.0000 — — — 2.6735-0.00920.0721 0.0006 0.000024.610614.23218.63822.21830.97472.7987 1 420 Su80St40 24 0.1162 0.0320 0.07620.0000 — — — — -0.0092-0.0061 -0.00060.000025.228014.96288.37882.03130.98922.6784 2 1 BL 0 0.0000 0.0000 0.00000.0000 7.05— — — — — — — — — — — — — 2 2 BL 0 0.0000 0.0000 0.00000.0000 6.99— — — — — — — — — — — — — 2 3 NDF 0 0.1158 0.0000 0.00000.0000 7.0595.6884 — — — — — — — — — — — —

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208Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 4 NDF 0 0.1159 0.0000 0.00000.0000 7.0694.7459 — — — — — — — — — — — — 2 5 Su40St0 0 0.1159 0.0000 0.03840.0000 7.0295.4362 — — — — — — — — — — — — 2 6 Su40St0 0 0.1157 0.0000 0.03840.0000 7.0494.9038 — — — — — — — — — — — — 2 7 Su80St0 0 0.1160 0.0000 0.07630.0000 7.0694.2360 — — — — — — — — — — — — 2 8 Su80St0 0 0.1156 0.0000 0.07590.0000 7.0494.4639 — — — — — — — — — — — — 2 9 Su120St0 0 0.1158 0.0000 0.11420.0000 7.0694.3066 — — — — — — — — — — — — 2 10 Su120St0 0 0.1160 0.0000 0.11410.0000 7.0494.5809 — — — — — — — — — — — — 2 11 Su0St40 0 0.1156 0.0320 0.00000.0000 7.0795.1560 — — — — — — — — — — — — 2 12 Su0St40 0 0.1158 0.0322 0.00000.0000 7.0394.7384 — — — — — — — — — — — — 2 13 Su0St80 0 0.1161 0.0638 0.00000.0000 7.0896.3976 — — — — — — — — — — — — 2 14 Su0St80 0 0.1160 0.0639 0.00000.0000 7.0794.9258 — — — — — — — — — — — — 2 15 Su0St120 0 0.1158 0.0958 0.00000.0000 7.0795.0839 — — — — — — — — — — — — 2 16 Su0St120 0 0.1161 0.0959 0.00000.0000 7.0595.1915 — — — — — — — — — — — — 2 17 Su40St80 0 0.1159 0.0638 0.03810.0000 7.0694.3144 — — — — — — — — — — — — 2 18 Su40St80 0 0.1159 0.0640 0.03830.0000 7.0794.8322 — — — — — — — — — — — — 2 19 Su80St40 0 0.1157 0.0318 0.07640.0000 7.0894.2123 — — — — — — — — — — — — 2 20 Su80St40 0 0.1158 0.0321 0.07630.0000 7.1394.9975 — — — — — — — — — — — — 2 23 NDF 0 0.1157 0.0000 0.00000.0000 — — -0.4292 — — — — — — — — — — — 2 24 NDF 0 0.1161 0.0000 0.00000.0000 — — 0.4306 — — — — — — — — — — — 2 25 Su40St0 0 0.1160 0.0000 0.03850.0000 — — 0.0836 — — — — — — — — — — — 2 26 Su40St0 0 0.1158 0.0000 0.03800.0000 — — -0.0832 — — — — — — — — — — — 2 27 Su80St0 0 0.1161 0.0000 0.07590.0000 — — 0.1969 — — — — — — — — — — — 2 28 Su80St0 0 0.1157 0.0000 0.07640.0000 — — -0.1970 — — — — — — — — — — — 2 29 Su120St0 0 0.1159 0.0000 0.11390.0000 — — -0.2883 — — — — — — — — — — — 2 30 Su120St0 0 0.1157 0.0000 0.11450.0000 — — 0.2888 — — — — — — — — — — — 2 31 Su0St40 0 0.1160 0.0323 0.00000.0000 — — 0.1954 — — — — — — — — — — — 2 32 Su0St40 0 0.1161 0.0322 0.00000.0000 — — -0.1954 — — — — — — — — — — — 2 33 Su0St80 0 0.1160 0.0640 0.00000.0000 — — -0.1479 — — — — — — — — — — —

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209Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 34 Su0St80 0 0.1158 0.0641 0.00000.0000 — — 0.1479 — — — — — — — — — — — 2 35 Su0St120 0 0.1161 0.0957 0.00000.0000 — — -1.0243 — — — — — — — — — — — 2 36 Su0St120 0 0.1159 0.0959 0.00000.0000 — — 1.0247 — — — — — — — — — — — 2 37 Su40St80 0 0.1161 0.0641 0.03850.0000 — — -1.2297 — — — — — — — — — — — 2 38 Su40St80 0 0.1159 0.0639 0.03840.0000 — — 1.2270 — — — — — — — — — — — 2 39 Su80St40 0 0.1161 0.0319 0.07630.0000 — — -0.1448 — — — — — — — — — — — 2 40 Su80St40 0 0.1160 0.0319 0.07650.0000 — — 0.1450 — — — — — — — — — — — 2 41 BL 0 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.20340.5709 2 42 BL 0 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.26970.5288 2 43 NDF 0 0.1159 0.0000 0.00000.0000 — — — 0.5433-0.4985-0.0206 -0.35500.00000.20490.01810.00700.08100.21440.5087 2 44 NDF 0 0.1158 0.0000 0.00000.0000 — — — -0.45950.4008 0.0019 -0.28290.00000.1126-0.0988-0.02210.07160.22320.4168 2 45 Su40St0 0 0.1161 0.0000 0.03800.0000 — — — 0.784511.39913.6992 20.59740.09530.4864-0.38120.0957-0.13770.23860.3498 2 46 Su40St0 0 0.1160 0.0000 0.03830.0000 — — — 0.263215.17824.2668 17.96900.14210.25800.18270.0162-0.02950.23150.4377 2 47 Su80St0 0 0.1161 0.0000 0.07620.0000 — — — 1.651010.60086.7997 60.83500.1580-0.08630.2572-0.08960.05200.22440.5150 2 48 Su80St0 0 0.1162 0.0000 0.07630.0000 — — — 0.343011.75506.0421 53.23000.16640.4934— 0.04990.01610.23040.5528 2 49 Su120St0 0 0.1161 0.0000 0.11440.0000 — — — 1.180112.71347.0150 95.07850.15930.50480.1377-0.62790.01990.21360.3597 2 50 Su120St0 0 0.1160 0.0000 0.11420.0000 — — — 0.426810.64275.8003 97.33640.13800.96950.13310.01670.04480.21840.4038 2 51 Su0St40 0 0.1158 0.0323 0.00000.0000 — — — 28.0873-0.5664-0.0206 -0.31090.00000.68620.61420.19350.03740.18630.4446 2 52 Su0St40 0 0.1158 0.0321 0.00000.0000 — — — 26.3869-0.19200.0025 -0.26180.00000.50550.05060.02410.05670.19590.3905 2 53 Su0St80 0 0.1159 0.0640 0.00000.0000 — — — 54.3615-0.5336-0.0206 -0.26500.00000.6786-0.00970.03940.04420.23920.3773 2 54 Su0St80 0 0.1162 0.0641 0.00000.0000 — — — 49.8752-0.55870.0075 -0.27200.00000.3153-0.15270.0153-0.13770.19330.5146 2 55 Su0St120 0 0.1159 0.0957 0.00000.0000 — — — 83.0925-0.5318-0.0206 -0.27030.00000.79850.2875-0.67500.04070.25680.4077 2 56 Su0St120 0 0.1158 0.0956 0.00000.0000 — — — 77.5924-0.5314-0.0206 -0.26520.00000.50460.03680.0388-0.13770.22450.3712 2 57 Su40St80 0 0.1157 0.0636 0.03840.0000 — — — 52.23088.4941 5.5787 23.06010.15590.44670.0645-0.0167-0.13770.00000.0000 2 58 Su40St80 0 0.1158 0.0639 0.03840.0000 — — — 55.62949.1267 4.7703 15.47950.16320.59170.25350.00140.05540.20600.4711 2 59 Su80St40 0 0.1158 0.0320 0.07610.0000 — — — 28.961611.30006.1695 47.71900.15020.49010.1061-0.02250.02990.23050.5151 2 60 Su80St40 0 0.1161 0.0321 0.07590.0000 — — — 25.901910.71505.6323 47.65870.16730.3256-1.5342-0.08790.01400.16490.3232 2 61 BL 4 0.0000 0.0000 0.00000.0000 7.15— — — — — — — — — — — — —

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210Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 62 BL 4 0.0000 0.0000 0.00000.0000 7.14— — — — — — — — — — — — — 2 63 NDF 4 0.1161 0.0000 0.00000.0000 7.1494.8469 — — — — — — — — — — — — 2 64 NDF 4 0.1160 0.0000 0.00000.0000 7.1494.8396 — — — — — — — — — — — — 2 65 Su40St0 4 0.1157 0.0000 0.03810.0000 6.9894.4716 — — — — — — — — — — — — 2 66 Su40St0 4 0.1160 0.0000 0.03850.0000 6.9693.5463 — — — — — — — — — — — — 2 67 Su80St0 4 0.1160 0.0000 0.07620.0000 6.8093.0135 — — — — — — — — — — — — 2 68 Su80St0 4 0.1157 0.0000 0.07640.0000 6.8093.0023 — — — — — — — — — — — — 2 69 Su120St0 4 0.1161 0.0000 0.11390.0000 6.6691.9180 — — — — — — — — — — — — 2 70 Su120St0 4 0.1159 0.0000 0.11420.0000 6.6493.1064 — — — — — — — — — — — — 2 71 Su0St40 4 0.1158 0.0318 0.00000.0000 7.1693.9612 — — — — — — — — — — — — 2 72 Su0St40 4 0.1159 0.0319 0.00000.0000 7.1693.7104 — — — — — — — — — — — — 2 73 Su0St80 4 0.1158 0.0641 0.00000.0000 7.1694.5657 — — — — — — — — — — — — 2 74 Su0St80 4 0.1160 0.0636 0.00000.0000 7.1693.5463 — — — — — — — — — — — — 2 75 Su0St120 4 0.1159 0.0960 0.00000.0000 7.1694.4007 — — — — — — — — — — — — 2 76 Su0St120 4 0.1162 0.0959 0.00000.0000 7.1593.3910 — — — — — — — — — — — — 2 77 Su40St80 4 0.1161 0.0636 0.03820.0000 6.9993.0379 — — — — — — — — — — — — 2 78 Su40St80 4 0.1159 0.0642 0.03820.0000 7.0292.9338 — — — — — — — — — — — — 2 79 Su80St40 4 0.1161 0.0319 0.07630.0000 6.8693.7270 — — — — — — — — — — — — 2 80 Su80St40 4 0.1158 0.0319 0.07630.0000 6.8893.3566 — — — — — — — — — — — — 2 83 NDF 4 0.1159 0.0000 0.00000.0000 — — -0.2922 — — — — — — — — — — — 2 84 NDF 4 0.1161 0.0000 0.00000.0000 — — -1.1806 — — — — — — — — — — — 2 85 Su40St0 4 0.1159 0.0000 0.03810.0000 — — 2.1058 — — — — — — — — — — — 2 86 Su40St0 4 0.1161 0.0000 0.03840.0000 — — 2.2808 — — — — — — — — — — — 2 87 Su80St0 4 0.1160 0.0000 0.07630.0000 — — 5.8030 — — — — — — — — — — — 2 88 Su80St0 4 0.1161 0.0000 0.07630.0000 — — 7.2649 — — — — — — — — — — — 2 89 Su120St0 4 0.1160 0.0000 0.11400.0000 — — 10.0095 — — — — — — — — — — — 2 90 Su120St0 4 0.1162 0.0000 0.11390.0000 — — 10.7887 — — — — — — — — — — — 2 91 Su0St40 4 0.1162 0.0321 0.00000.0000 — — -2.7834 — — — — — — — — — — —

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211Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 92 Su0St40 4 0.1160 0.0322 0.00000.0000 — — -2.1940 — — — — — — — — — — — 2 93 Su0St80 4 0.1158 0.0638 0.00000.0000 — — 0.5946 — — — — — — — — — — — 2 94 Su0St80 4 0.1160 0.0639 0.00000.0000 — — 0.2939 — — — — — — — — — — — 2 95 Su0St120 4 0.1158 0.0960 0.00000.0000 — — -0.2934 — — — — — — — — — — — 2 96 Su0St120 4 0.1157 0.0958 0.00000.0000 — — -1.1660 — — — — — — — — — — — 2 97 Su40St80 4 0.1158 0.0638 0.03810.0000 — — 0.3153 — — — — — — — — — — — 2 98 Su40St80 4 0.1160 0.0642 0.03830.0000 — — 2.3414 — — — — — — — — — — — 2 99 Su80St40 4 0.1157 0.0319 0.07640.0000 — — 8.2074 — — — — — — — — — — — 2 100 Su80St40 4 0.1160 0.0319 0.07650.0000 — — 7.9096 — — — — — — — — — — — 2 101 BL 4 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.18250.5306 2 102 BL 4 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.17080.0000 2 103 NDF 4 0.1161 0.0000 0.00000.0000 — — — -0.0046-0.06910.0372 0.1444 0.00000.41660.24420.47410.80140.30660.5674 2 104 NDF 4 0.1161 0.0000 0.00000.0000 — — — 0.33280.0509 0.2962 -0.05180.00000.45210.24610.33550.25820.26350.3334 2 105 Su40St0 4 0.1160 0.0000 0.03820.0000 — — — 3.40560.8738 0.0237 -0.05156.59762.40321.85030.75690.29770.25930.5173 2 106 Su40St0 4 0.1160 0.0000 0.03790.0000 — — — 3.76311.0837 0.0231 -0.04306.56152.37281.65000.79950.17340.25780.7393 2 107 Su80St0 4 0.1158 0.0000 0.07590.0000 — — — 3.8032-0.0345-0.0007 0.0394 16.33982.97932.20680.74410.24820.12070.4838 2 108 Su80St0 4 0.1160 0.0000 0.07650.0000 — — — 4.64941.8076 -0.0007 -0.051816.51193.31572.3860-0.18560.00000.00000.0000 2 109 Su120St0 4 0.1162 0.0000 0.11440.0000 — — — 3.72222.4498 0.0114 -0.051826.47283.51732.9295— 0.20940.25360.7143 2 110 Su120St0 4 0.1158 0.0000 0.11440.0000 — — — 4.5695-0.00770.1283 -0.047126.81713.99613.02783.85110.00000.29080.4206 2 111 Su0St40 4 0.1158 0.0319 0.00000.0000 — — — 23.74530.0866 -0.0007 0.0291 0.03470.62130.41490.58320.26900.31730.8346 2 112 Su0St40 4 0.1160 0.0323 0.00000.0000 — — — 24.13460.0742 0.0213 0.0115 0.00000.71850.45980.59340.73910.30380.5787 2 113 Su0St80 4 0.1159 0.0637 0.00000.0000 — — — 48.4369-0.10300.0056 0.4091 0.04341.10630.55720.61840.26410.22490.6967 2 114 Su0St80 4 0.1160 0.0637 0.00000.0000 — — — 47.05600.6993 0.0869 -0.05180.00000.73250.11290.36480.25760.20560.5406 2 115 Su0St120 4 0.1158 0.0956 0.00000.0000 — — — 60.07440.0659 -0.0007 -0.05180.03280.57900.53290.52150.25780.30970.6387 2 116 Su0St120 4 0.1161 0.0960 0.00000.0000 — — — 67.18400.0691 -0.0007 -0.05180.07021.39760.76420.58020.29540.24580.5629 2 117 Su40St80 4 0.1161 0.0636 0.03850.0000 — — — 38.7042-0.0986-0.0007 -0.01596.76462.82581.96010.90000.28190.26270.7037 2 118 Su40St80 4 0.1157 0.0641 0.03790.0000 — — — 44.18090.4536 -0.0007 -0.05186.47782.84851.88070.95730.28160.30380.5667 2 119 Su80St40 4 0.1160 0.0319 0.07650.0000 — — — 27.3890-0.06310.0288 0.0431 16.59663.30642.43390.85170.19290.26320.4044

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212Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 120 Su80St40 4 0.1159 0.0322 0.07610.0000 — — — 23.8911-0.10300.0071 -0.051816.21573.25112.74130.91870.30610.25430.4295 2 121 BL 8 0.0000 0.0000 0.00000.0000 7.17— — — — — — — — — — — — — 2 122 BL 8 0.0000 0.0000 0.00000.0000 7.23— — — — — — — — — — — — — 2 123 NDF 8 0.1161 0.0000 0.00000.0000 7.2390.5396 — — — — — — — — — — — — 2 124 NDF 8 0.1160 0.0000 0.00000.0000 7.2189.5803 — — — — — — — — — — — — 2 125 Su40St0 8 0.1161 0.0000 0.03820.0000 7.0688.8955 — — — — — — — — — — — — 2 126 Su40St0 8 0.1160 0.0000 0.03840.0000 7.0487.5111 — — — — — — — — — — — — 2 127 Su80St0 8 0.1162 0.0000 0.07620.0000 6.8689.1734 — — — — — — — — — — — — 2 128 Su80St0 8 0.1158 0.0000 0.07650.0000 6.9087.8295 — — — — — — — — — — — — 2 129 Su120St0 8 0.1159 0.0000 0.11390.0000 6.7988.1879 — — — — — — — — — — — — 2 130 Su120St0 8 0.1161 0.0000 0.11430.0000 6.7888.3860 — — — — — — — — — — — — 2 131 Su0St40 8 0.1161 0.0318 0.00000.0000 7.0388.9029 — — — — — — — — — — — — 2 132 Su0St40 8 0.1161 0.0322 0.00000.0000 7.1090.5396 — — — — — — — — — — — — 2 133 Su0St80 8 0.1160 0.0637 0.00000.0000 7.0791.5633 — — — — — — — — — — — — 2 134 Su0St80 8 0.1160 0.0639 0.00000.0000 7.0790.7873 — — — — — — — — — — — — 2 135 Su0St120 8 0.1161 0.0957 0.00000.0000 7.1190.8842 — — — — — — — — — — — — 2 136 Su0St120 8 0.1160 0.0956 0.00000.0000 7.0890.3562 — — — — — — — — — — — — 2 137 Su40St80 8 0.1158 0.0641 0.03820.0000 6.9389.5567 — — — — — — — — — — — — 2 138 Su40St80 8 0.1159 0.0638 0.03840.0000 6.9189.3097 — — — — — — — — — — — — 2 139 Su80St40 8 0.1161 0.0319 0.07640.0000 6.8789.3336 — — — — — — — — — — — — 2 140 Su80St40 8 0.1159 0.0319 0.07650.0000 6.8388.5330 — — — — — — — — — — — — 2 143 NDF 8 0.1158 0.0000 0.00000.0000 — — 1.3237 — — — — — — — — — — — 2 144 NDF 8 0.1158 0.0000 0.00000.0000 — — 1.0308 — — — — — — — — — — — 2 145 Su40St0 8 0.1157 0.0000 0.03830.0000 — — 6.4510 — — — — — — — — — — — 2 146 Su40St0 8 0.1160 0.0000 0.03800.0000 — — 6.2561 — — — — — — — — — — — 2 147 Su80St0 8 0.1160 0.0000 0.07620.0000 — — 9.9560 — — — — — — — — — — — 2 148 Su80St0 8 0.1156 0.0000 0.07590.0000 — — 10.9524 — — — — — — — — — — — 2 149 Su120St0 8 0.1159 0.0000 0.11450.0000 — — 12.4909 — — — — — — — — — — —

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213Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 150 Su120St0 8 0.1161 0.0000 0.11390.0000 — — 13.2809 — — — — — — — — — — — 2 151 Su0St40 8 0.1157 0.0322 0.00000.0000 — — 2.0637 — — — — — — — — — — — 2 152 Su0St40 8 0.1157 0.0321 0.00000.0000 — — 2.2620 — — — — — — — — — — — 2 153 Su0St80 8 0.1160 0.0637 0.00000.0000 — — 5.3277 — — — — — — — — — — — 2 154 Su0St80 8 0.1160 0.0642 0.00000.0000 — — 3.7532 — — — — — — — — — — — 2 155 Su0St120 8 0.1160 0.0957 0.00000.0000 — — 5.4208 — — — — — — — — — — — 2 156 Su0St120 8 0.1160 0.0960 0.00000.0000 — — 5.6100 — — — — — — — — — — — 2 157 Su40St80 8 0.1162 0.0639 0.03850.0000 — — 10.2957 — — — — — — — — — — — 2 158 Su40St80 8 0.1159 0.0642 0.03830.0000 — — 10.3030 — — — — — — — — — — — 2 159 Su80St40 8 0.1157 0.0319 0.07650.0000 — — 15.2869 — — — — — — — — — — — 2 160 Su80St40 8 0.1157 0.0319 0.07620.0000 — — 15.2918 — — — — — — — — — — — 2 161 BL 8 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.41240.5806 2 162 BL 8 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.29350.7520 2 163 NDF 8 0.1161 0.0000 0.00000.0000 — — — 0.2736-0.07470.0146 0.1148 0.00000.74210.16250.10170.19790.31020.6851 2 164 NDF 8 0.1158 0.0000 0.00000.0000 — — — 0.2861-0.05670.0000 0.0000 0.00000.4711-0.0574-0.01860.15080.23980.4714 2 165 Su40St0 8 0.1160 0.0000 0.03830.0000 — — — 2.73430.0486 0.0908 0.0000 4.62771.99022.48910.48170.16880.28230.7720 2 166 Su40St0 8 0.1158 0.0000 0.03820.0000 — — — 2.4503-0.05000.0536 0.0000 4.77634.57163.28370.78860.24630.18700.8229 2 167 Su80St0 8 0.1159 0.0000 0.07600.0000 — — — 3.2129-0.09160.0066 0.0998 13.53055.04114.46080.81430.22220.32570.8494 2 168 Su80St0 8 0.1158 0.0000 0.07620.0000 — — — 3.09120.8002 0.0000 0.0000 14.70605.13564.17920.7104-0.08020.29240.7355 2 169 Su120St0 8 0.1158 0.0000 0.11390.0000 — — — 3.68770.8653 0.0344 0.0000 23.97386.73895.76501.0547-0.01650.38320.9227 2 170 Su120St0 8 0.1158 0.0000 0.11410.0000 — — — 3.16910.9269 0.0931 0.1773 24.00706.32454.48480.85900.21400.23110.7305 2 171 Su0St40 8 0.1160 0.0320 0.00000.0000 — — — 14.9257-0.08350.0001 0.1279 0.00002.44001.30070.32670.13260.31370.5200 2 172 Su0St40 8 0.1159 0.0322 0.00000.0000 — — — 19.14610.0321 0.0000 0.0000 0.00002.00771.18570.1658-0.12550.14190.2960 2 173 Su0St80 8 0.1159 0.0642 0.00000.0000 — — — 33.3682-0.06770.0458 0.0311 0.00003.24871.51550.29040.20940.28330.8507 2 174 Su0St80 8 0.1162 0.0638 0.00000.0000 — — — 34.9633-0.03740.1412 0.0000 0.03493.95841.91670.42870.16620.32270.7978 2 175 Su0St120 8 0.1157 0.0957 0.00000.0000 — — — 58.1448-0.05710.0003 0.0000 0.14703.13251.67980.45000.05510.32190.7591 2 176 Su0St120 8 0.1157 0.0959 0.00000.0000 — — — 46.15230.0211 0.0328 0.0000 0.08063.74831.83960.42400.20290.32890.8189 2 177 Su40St80 8 0.1162 0.0636 0.03800.0000 — — — 23.0778-0.08460.0410 0.7407 5.11177.14805.34541.09970.24500.31790.8688

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214Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 178 Su40St80 8 0.1157 0.0637 0.03830.0000 — — — 14.0327-0.09160.3084 0.0000 5.1395— 4.57930.96530.35680.30670.7355 2 179 Su80St40 8 0.1161 0.0320 0.07650.0000 — — — 15.87720.7607 0.0652 0.0000 14.46017.93056.20951.18520.23520.30320.7325 2 180 Su80St40 8 0.1162 0.0322 0.07640.0000 — — — 5.08590.0265 0.0000 0.1384 14.04888.07726.58591.14110.26190.30420.7698 2 181 BL 12 0.0000 0.0000 0.00000.0000 7.23— — — — — — — — — — — — — 2 182 BL 12 0.0000 0.0000 0.00000.0000 7.23— — — — — — — — — — — — — 2 183 NDF 12 0.1161 0.0000 0.00000.0000 7.2681.2789 — — — — — — — — — — — — 2 184 NDF 12 0.1160 0.0000 0.00000.0000 7.2680.3119 — — — — — — — — — — — — 2 185 Su40St0 12 0.1161 0.0000 0.03850.0000 7.1579.5560 — — — — — — — — — — — — 2 186 Su40St0 12 0.1159 0.0000 0.03850.0000 7.1678.6529 — — — — — — — — — — — — 2 187 Su80St0 12 0.1161 0.0000 0.07610.0000 7.0478.8668 — — — — — — — — — — — — 2 188 Su80St0 12 0.1161 0.0000 0.07640.0000 7.0578.3499 — — — — — — — — — — — — 2 189 Su120St0 12 0.1161 0.0000 0.11420.0000 6.9079.2976 — — — — — — — — — — — — 2 190 Su120St0 12 0.1161 0.0000 0.11400.0000 6.8879.2114 — — — — — — — — — — — — 2 191 Su0St40 12 0.1160 0.0322 0.00000.0000 7.1680.3981 — — — — — — — — — — — — 2 192 Su0St40 12 0.1161 0.0322 0.00000.0000 7.1680.4175 — — — — — — — — — — — — 2 193 Su0St80 12 0.1159 0.0637 0.00000.0000 7.0380.5513 — — — — — — — — — — — — 2 194 Su0St80 12 0.1158 0.0642 0.00000.0000 6.9880.3593 — — — — — — — — — — — — 2 195 Su0St120 12 0.1159 0.0960 0.00000.0000 6.9981.7593 — — — — — — — — — — — — 2 196 Su0St120 12 0.1161 0.0960 0.00000.0000 6.8882.6573 — — — — — — — — — — — — 2 197 Su40St80 12 0.1157 0.0640 0.03840.0000 6.9578.6111 — — — — — — — — — — — — 2 198 Su40St80 12 0.1159 0.0639 0.03850.0000 6.9578.6529 — — — — — — — — — — — — 2 199 Su80St40 12 0.1160 0.0321 0.07650.0000 6.9377.9840 — — — — — — — — — — — — 2 200 Su80St40 12 0.1160 0.0319 0.07640.0000 6.9176.7769 — — — — — — — — — — — — 2 203 NDF 12 0.1158 0.0000 0.00000.0000 — — 4.5462 — — — — — — — — — — — 2 204 NDF 12 0.1159 0.0000 0.00000.0000 — — 4.5414 — — — — — — — — — — — 2 205 Su40St0 12 0.1161 0.0000 0.03820.0000 — — 8.3922 — — — — — — — — — — — 2 206 Su40St0 12 0.1160 0.0000 0.03850.0000 — — 8.5791 — — — — — — — — — — — 2 207 Su80St0 12 0.1158 0.0000 0.07590.0000 — — 14.0716 — — — — — — — — — — —

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215Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 208 Su80St0 12 0.1162 0.0000 0.07650.0000 — — 14.5313 — — — — — — — — — — — 2 209 Su120St0 12 0.1157 0.0000 0.11420.0000 — — 17.2864 — — — — — — — — — — — 2 210 Su120St0 12 0.1160 0.0000 0.11400.0000 — — 17.2845 — — — — — — — — — — — 2 211 Su0St40 12 0.1158 0.0319 0.00000.0000 — — 10.6627 — — — — — — — — — — — 2 212 Su0St40 12 0.1157 0.0319 0.00000.0000 — — 10.9620 — — — — — — — — — — — 2 213 Su0St80 12 0.1160 0.0639 0.00000.0000 — — 14.1114 — — — — — — — — — — — 2 214 Su0St80 12 0.1161 0.0639 0.00000.0000 — — 16.6476 — — — — — — — — — — — 2 215 Su0St120 12 0.1160 0.0956 0.00000.0000 — — 21.4394 — — — — — — — — — — — 2 216 Su0St120 12 0.1160 0.0958 0.00000.0000 — — 19.2850 — — — — — — — — — — — 2 217 Su40St80 12 0.1159 0.0639 0.03830.0000 — — 18.9050 — — — — — — — — — — — 2 218 Su40St80 12 0.1160 0.0636 0.03830.0000 — — 20.2776 — — — — — — — — — — — 2 219 Su80St40 12 0.1161 0.0320 0.07590.0000 — — 22.6112 — — — — — — — — — — — 2 220 Su80St40 12 0.1158 0.0322 0.07620.0000 — — 20.0693 — — — — — — — — — — — 2 221 BL 12 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.23651.1549 2 222 BL 12 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.47461.3227 2 223 NDF 12 0.1161 0.0000 0.00000.0000 — — — 0.5928-0.1623-0.0043 0.0201 0.00003.56731.47330.41790.16980.41891.2735 2 224 NDF 12 0.1162 0.0000 0.00000.0000 — — — 0.5255-0.1835-0.0036 -0.03580.00004.40021.61230.56650.16800.45790.7884 2 225 Su40St0 12 0.1159 0.0000 0.03800.0000 — — — 2.25840.2612 0.0015 -0.03971.75578.68405.12412.29780.51800.43121.4498 2 226 Su40St0 12 0.1161 0.0000 0.03790.0000 — — — 2.20650.1857 -0.0045 -0.03161.60698.36584.97922.31440.68450.46941.6386 2 227 Su80St0 12 0.1159 0.0000 0.07650.0000 — — — 2.65400.2676 -0.0222 -0.03837.13139.96387.47513.19850.72600.52390.9273 2 228 Su80St0 12 0.1158 0.0000 0.07620.0000 — — — 2.37020.1807 -0.0248 -0.01567.302610.90377.51043.18040.90340.42011.3262 2 229 Su120St0 12 0.1160 0.0000 0.11410.0000 — — — — -0.0218-0.0292 0.0203 — — — — — — — 2 230 Su120St0 12 0.1161 0.0000 0.11390.0000 — — — 2.44810.0035 -0.0338 -0.02494.612517.472416.04773.89941.22750.35601.2864 2 231 Su0St40 12 0.1160 0.0318 0.00000.0000 — — — 4.0744-0.1835-0.0354 0.0372 0.00008.77034.82271.13560.25420.59311.4600 2 232 Su0St40 12 0.1157 0.0319 0.00000.0000 — — — 3.3246-0.1835-0.0374 -0.02800.00008.86804.89311.10170.25080.63521.4401 2 233 Su0St80 12 0.1161 0.0636 0.00000.0000 — — — 4.1574-0.1835-0.0396 -0.00970.000013.53849.01341.33640.29600.52431.4643 2 234 Su0St80 12 0.1158 0.0641 0.00000.0000 — — — 5.0875-0.1835-0.0414 -0.03040.000013.96679.06261.44000.36720.52231.5346 2 235 Su0St120 12 0.1159 0.0957 0.00000.0000 — — — 13.0236-0.1835-0.0426 -0.04180.032416.518210.06981.86950.38550.52491.5286

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216Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 236 Su0St120 12 0.1159 0.0957 0.00000.0000 — — — 14.7571-0.1835-0.0426 -0.04180.000016.174310.64421.97570.41920.54061.5396 2 237 Su40St80 12 0.1161 0.0638 0.03800.0000 — — — 8.3550-0.1835-0.0426 -0.02220.337916.517412.39313.78580.87490.54451.6524 2 238 Su40St80 12 0.1157 0.0642 0.03810.0000 — — — 8.7555-0.1835-0.0426 -0.04180.044016.705612.60873.81220.87780.54251.7565 2 239 Su80St40 12 0.1160 0.0318 0.07630.0000 — — — 5.1919-0.1835-0.0426 -0.04186.068714.867111.71725.39041.59700.51450.9400 2 240 Su80St40 12 0.1157 0.0318 0.07650.0000 — — — 6.7589-0.1835-0.0331 -0.02167.278514.003310.72794.68771.48470.49621.2789 2 241 BL 16 0.0000 0.0000 0.00000.0000 7.34— — — — — — — — — — — — — 2 242 BL 16 0.0000 0.0000 0.00000.0000 7.38— — — — — — — — — — — — — 2 243 NDF 16 0.1160 0.0000 0.00000.0000 7.3472.3798 — — — — — — — — — — — — 2 244 NDF 16 0.1159 0.0000 0.00000.0000 7.3271.4909 — — — — — — — — — — — — 2 245 Su40St0 16 0.1161 0.0000 0.03840.0000 7.2370.8552 — — — — — — — — — — — — 2 246 Su40St0 16 0.1161 0.0000 0.03840.0000 7.2571.4583 — — — — — — — — — — — — 2 247 Su80St0 16 0.1160 0.0000 0.07600.0000 7.1572.2936 — — — — — — — — — — — — 2 248 Su80St0 16 0.1160 0.0000 0.07620.0000 7.0272.2074 — — — — — — — — — — — — 2 249 Su120St0 16 0.1159 0.0000 0.11400.0000 7.0471.7497 — — — — — — — — — — — — 2 250 Su120St0 16 0.1159 0.0000 0.11400.0000 7.0271.0594 — — — — — — — — — — — — 2 251 Su0St40 16 0.1157 0.0321 0.00000.0000 7.2272.3015 — — — — — — — — — — — — 2 252 Su0St40 16 0.1161 0.0322 0.00000.0000 7.2873.1812 — — — — — — — — — — — — 2 253 Su0St80 16 0.1157 0.0637 0.00000.0000 7.1673.2522 — — — — — — — — — — — — 2 254 Su0St80 16 0.1157 0.0639 0.00000.0000 7.1472.6472 — — — — — — — — — — — — 2 255 Su0St120 16 0.1157 0.0959 0.00000.0000 7.0774.4623 — — — — — — — — — — — — 2 256 Su0St120 16 0.1161 0.0957 0.00000.0000 7.0774.2149 — — — — — — — — — — — — 2 257 Su40St80 16 0.1161 0.0640 0.03850.0000 7.0871.1998 — — — — — — — — — — — — 2 258 Su40St80 16 0.1159 0.0642 0.03800.0000 7.0772.7852 — — — — — — — — — — — — 2 259 Su80St40 16 0.1161 0.0320 0.07610.0000 7.0567.3232 — — — — — — — — — — — — 2 260 Su80St40 16 0.1160 0.0322 0.07650.0000 7.0871.2590 — — — — — — — — — — — — 2 263 NDF 16 0.1161 0.0000 0.00000.0000 — — 3.9460 — — — — — — — — — — — 2 264 NDF 16 0.1157 0.0000 0.00000.0000 — — 3.4768 — — — — — — — — — — — 2 265 Su40St0 16 0.1157 0.0000 0.03800.0000 — — 8.5140 — — — — — — — — — — —

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217Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 266 Su40St0 16 0.1161 0.0000 0.03830.0000 — — 8.0951 — — — — — — — — — — — 2 267 Su80St0 16 0.1161 0.0000 0.07610.0000 — — 13.1786 — — — — — — — — — — — 2 268 Su80St0 16 0.1157 0.0000 0.07600.0000 — — 13.5832 — — — — — — — — — — — 2 269 Su120St0 16 0.1157 0.0000 0.11420.0000 — — 16.2122 — — — — — — — — — — — 2 270 Su120St0 16 0.1156 0.0000 0.11390.0000 — — 18.1738 — — — — — — — — — — — 2 271 Su0St40 16 0.1160 0.0319 0.00000.0000 — — 10.0702 — — — — — — — — — — — 2 272 Su0St40 16 0.1160 0.0319 0.00000.0000 — — 10.2685 — — — — — — — — — — — 2 273 Su0St80 16 0.1160 0.0641 0.00000.0000 — — 18.3055 — — — — — — — — — — — 2 274 Su0St80 16 0.1158 0.0637 0.00000.0000 — — 17.4416 — — — — — — — — — — — 2 275 Su0St120 16 0.1159 0.0957 0.00000.0000 — — 20.3654 — — — — — — — — — — — 2 276 Su0St120 16 0.1160 0.0957 0.00000.0000 — — 21.2401 — — — — — — — — — — — 2 277 Su40St80 16 0.1157 0.0639 0.03810.0000 — — 20.2850 — — — — — — — — — — — 2 278 Su40St80 16 0.1160 0.0641 0.03850.0000 — — 20.2565 — — — — — — — — — — — 2 279 Su80St40 16 0.1158 0.0321 0.07650.0000 — — 23.7765 — — — — — — — — — — — 2 280 Su80St40 16 0.1161 0.0318 0.07640.0000 — — 23.8769 — — — — — — — — — — — 2 281 BL 16 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.58741.9229 2 282 BL 16 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 1.10331.8552 2 283 NDF 16 0.1158 0.0000 0.00000.0000 — — — 0.3594-0.05500.0000 -0.00870.00006.36072.59540.61020.18480.57112.0101 2 284 NDF 16 0.1157 0.0000 0.00000.0000 — — — 0.2071-0.06620.0000 -0.00870.00005.93482.41173.18360.11700.57301.5940 2 285 Su40St0 16 0.1160 0.0000 0.03840.0000 — — — 1.7837-0.00090.0000 0.0169 0.000011.41525.69793.13130.73420.70601.9145 2 286 Su40St0 16 0.1157 0.0000 0.03830.0000 — — — 1.41090.0129 0.0042 0.0053 0.028511.20176.00032.84990.76380.65942.1862 2 287 Su80St0 16 0.1159 0.0000 0.07600.0000 — — — 2.0249-0.00930.0000 0.0045 0.222411.47868.13345.76852.67390.56481.8261 2 288 Su80St0 16 0.1159 0.0000 0.07640.0000 — — — 1.8103-0.02300.0000 -0.00081.642311.75817.81625.58982.42070.66251.9916 2 289 Su120St0 16 0.1161 0.0000 0.11390.0000 — — — 2.02490.0158 0.0000 -0.00870.000018.720016.79835.11682.79740.86702.1451 2 290 Su120St0 16 0.1159 0.0000 0.11400.0000 — — — 2.0499-0.00830.0000 -0.00870.000019.595317.91465.01362.58020.82911.9934 2 291 Su0St40 16 0.1157 0.0319 0.00000.0000 — — — 3.2871-0.03080.0000 -0.00650.000011.21985.95001.42990.40340.74502.2876 2 292 Su0St40 16 0.1160 0.0318 0.00000.0000 — — — 2.7686-0.04080.0000 -0.00660.000011.00115.87341.30890.34410.65192.2097 2 293 Su0St80 16 0.1160 0.0642 0.00000.0000 — — — 5.1881-0.01450.0000 -0.00870.000016.02659.88031.87000.53680.67562.4155

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218Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 294 Su0St80 16 0.1158 0.0637 0.00000.0000 — — — 5.8074-0.00840.0000 0.0038 0.000015.75749.49431.95760.38760.72212.3309 2 295 Su0St120 16 0.1158 0.0957 0.00000.0000 — — — 14.69310.0136 0.0000 0.0039 0.071618.619112.45692.69890.63440.76442.1866 2 296 Su0St120 16 0.1158 0.0955 0.00000.0000 — — — 7.80410.0116 0.0000 0.0611 0.054917.870712.25942.39460.63130.80982.3953 2 297 Su40St80 16 0.1162 0.0641 0.03850.0000 — — — 6.2206-0.03640.0000 0.0291 0.000018.592213.33584.46231.43190.86332.1862 2 298 Su40St80 16 0.1157 0.0638 0.03810.0000 — — — 6.5262-0.00540.0000 -0.00400.000019.461113.20814.41231.44540.89912.1920 2 299 Su80St40 16 0.1161 0.0319 0.07600.0000 — — — 2.8220-0.05530.0000 0.0280 0.000017.224012.42176.86173.08390.85022.0150 2 300 Su80St40 16 0.1159 0.0321 0.07650.0000 — — — 4.5354-0.01780.0000 -0.00530.000016.236812.13356.54983.30180.88182.3210 2 301 BL 20 0.0000 0.0000 0.00000.0000 7.33— — — — — — — — — — — — — 2 302 BL 20 0.0000 0.0000 0.00000.0000 7.41— — — — — — — — — — — — — 2 303 NDF 20 0.1159 0.0000 0.00000.0000 7.1563.5091 — — — — — — — — — — — — 2 304 NDF 20 0.1161 0.0000 0.00000.0000 7.3166.3326 — — — — — — — — — — — — 2 305 Su40St0 20 0.1158 0.0000 0.03830.0000 7.2364.6847 — — — — — — — — — — — — 2 306 Su40St0 20 0.1158 0.0000 0.03810.0000 7.2664.8574 — — — — — — — — — — — — 2 307 Su80St0 20 0.1158 0.0000 0.07650.0000 7.1663.3892 — — — — — — — — — — — — 2 308 Su80St0 20 0.1160 0.0000 0.07590.0000 7.1763.1114 — — — — — — — — — — — — 2 309 Su120St0 20 0.1162 0.0000 0.11450.0000 7.0763.1788 — — — — — — — — — — — — 2 310 Su120St0 20 0.1162 0.0000 0.11390.0000 7.0963.6953 — — — — — — — — — — — — 2 311 Su0St40 20 0.1157 0.0320 0.00000.0000 7.2765.5165 — — — — — — — — — — — — 2 312 Su0St40 20 0.1160 0.0323 0.00000.0000 7.1263.6287 — — — — — — — — — — — — 2 313 Su0St80 20 0.1161 0.0639 0.00000.0000 7.0964.0928 — — — — — — — — — — — — 2 314 Su0St80 20 0.1161 0.0642 0.00000.0000 7.1764.7819 — — — — — — — — — — — — 2 315 Su0St120 20 0.1158 0.0959 0.00000.0000 7.0564.7710 — — — — — — — — — — — — 2 316 Su0St120 20 0.1160 0.0960 0.00000.0000 7.0865.1806 — — — — — — — — — — — — 2 317 Su40St80 20 0.1158 0.0641 0.03840.0000 7.1063.4756 — — — — — — — — — — — — 2 318 Su40St80 20 0.1161 0.0636 0.03810.0000 7.0863.0538 — — — — — — — — — — — — 2 319 Su80St40 20 0.1157 0.0318 0.07640.0000 7.1365.4300 — — — — — — — — — — — — 2 320 Su80St40 20 0.1160 0.0322 0.07610.0000 7.1364.9220 — — — — — — — — — — — — 2 323 NDF 20 0.1158 0.0000 0.00000.0000 — — 2.5932 — — — — — — — — — — —

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219Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 324 NDF 20 0.1157 0.0000 0.00000.0000 — — 2.5003 — — — — — — — — — — — 2 325 Su40St0 20 0.1160 0.0000 0.03850.0000 — — 7.9932 — — — — — — — — — — — 2 326 Su40St0 20 0.1157 0.0000 0.03840.0000 — — 6.1539 — — — — — — — — — — — 2 327 Su80St0 20 0.1159 0.0000 0.07610.0000 — — 12.6959 — — — — — — — — — — — 2 328 Su80St0 20 0.1160 0.0000 0.07610.0000 — — 12.6931 — — — — — — — — — — — 2 329 Su120St0 20 0.1159 0.0000 0.11430.0000 — — 18.7448 — — — — — — — — — — — 2 330 Su120St0 20 0.1157 0.0000 0.11390.0000 — — 18.1717 — — — — — — — — — — — 2 331 Su0St40 20 0.1159 0.0323 0.00000.0000 — — 7.3272 — — — — — — — — — — — 2 332 Su0St40 20 0.1158 0.0319 0.00000.0000 — — 9.2986 — — — — — — — — — — — 2 333 Su0St80 20 0.1162 0.0638 0.00000.0000 — — 14.9874 — — — — — — — — — — — 2 334 Su0St80 20 0.1156 0.0638 0.00000.0000 — — 17.2492 — — — — — — — — — — — 2 335 Su0St120 20 0.1160 0.0959 0.00000.0000 — — 21.2360 — — — — — — — — — — — 2 336 Su0St120 20 0.1161 0.0959 0.00000.0000 — — 21.2338 — — — — — — — — — — — 2 337 Su40St80 20 0.1157 0.0642 0.03810.0000 — — 18.7138 — — — — — — — — — — — 2 338 Su40St80 20 0.1161 0.0639 0.03850.0000 — — 19.2827 — — — — — — — — — — — 2 339 Su80St40 20 0.1159 0.0323 0.07630.0000 — — 21.4317 — — — — — — — — — — — 2 340 Su80St40 20 0.1158 0.0322 0.07590.0000 — — 21.9295 — — — — — — — — — — — 2 341 BL 20 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.89412.5390 2 342 BL 20 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.87022.5797 2 343 NDF 20 0.1160 0.0000 0.00000.0000 — — — 0.2415-0.07990.0000 0.0039 0.000011.51514.56981.34530.52120.90892.7344 2 344 NDF 20 0.1161 0.0000 0.00000.0000 — — — 0.3214-0.09790.0000 0.0016 0.000010.94204.19731.41260.53510.94772.7512 2 345 Su40St0 20 0.1160 0.0000 0.03850.0000 — — — 0.8844-0.08220.0289 0.1329 0.000016.28807.98514.11401.35951.06532.7591 2 346 Su40St0 20 0.1158 0.0000 0.03790.0000 — — — 0.9681-0.10420.0010 0.0005 0.000015.62137.83613.93381.26900.99602.7848 2 347 Su80St0 20 0.1158 0.0000 0.07600.0000 — — — 1.0441-0.18170.0022 -0.00130.000017.87139.79517.20122.98470.92072.4168 2 348 Su80St0 20 0.1158 0.0000 0.07650.0000 — — — 1.1279-0.18170.0252 -0.00130.000018.25009.86197.76173.53030.93392.5573 2 349 Su120St0 20 0.1157 0.0000 0.11450.0000 — — — 0.9681-0.18170.0004 0.0005 0.000024.711417.74087.25023.64210.83062.1888 2 350 Su120St0 20 0.1161 0.0000 0.11450.0000 — — — 1.3713-0.18170.0000 -0.00130.000023.673118.51496.85123.55030.78742.2382 2 351 Su0St40 20 0.1161 0.0319 0.00000.0000 — — — 1.0460-0.14940.0028 -0.00030.000016.90957.85792.26720.61321.02362.7728

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220Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 352 Su0St40 20 0.1161 0.0320 0.00000.0000 — — — 1.4512-0.14230.0057 0.0149 0.000015.89257.71462.08430.53130.92822.6714 2 353 Su0St80 20 0.1158 0.0636 0.00000.0000 — — — 2.0025-0.17250.0000 0.0042 0.000022.824712.31942.86470.73311.05152.7358 2 354 Su0St80 20 0.1161 0.0637 0.00000.0000 — — — 1.9304-0.15050.0006 -0.00130.000022.917812.87302.90020.71441.12422.6368 2 355 Su0St120 20 0.1157 0.0956 0.00000.0000 — — — 2.8011-0.15410.0010 -0.00110.000026.803516.50373.48050.84291.01652.5402 2 356 Su0St120 20 0.1159 0.0957 0.00000.0000 — — — 2.2460-0.18170.0025 0.0074 0.000027.195116.49613.65130.81891.30772.5180 2 357 Su40St80 20 0.1160 0.0641 0.03800.0000 — — — 3.1225-0.14480.0000 -0.00130.000025.725115.91575.27031.49500.91062.4685 2 358 Su40St80 20 0.1161 0.0641 0.03790.0000 — — — 2.8109-0.14440.0000 0.0023 0.000026.470915.96135.45081.55560.96312.4020 2 359 Su80St40 20 0.1157 0.0320 0.07630.0000 — — — 2.2538-0.15440.0066 0.0146 0.075123.078713.80567.68273.18170.90112.2769 2 360 Su80St40 20 0.1157 0.0319 0.07640.0000 — — — 2.2538-0.17320.0000 -0.00130.000022.130113.08217.94503.20960.85842.2203 2 361 BL 24 0.0000 0.0000 0.00000.0000 7.58— — — — — — — — — — — — — 2 362 BL 24 0.0000 0.0000 0.00000.0000 7.58— — — — — — — — — — — — — 2 363 NDF 24 0.1160 0.0000 0.00000.0000 7.4662.8528 — — — — — — — — — — — — 2 364 NDF 24 0.1158 0.0000 0.00000.0000 7.4261.9211 — — — — — — — — — — — — 2 365 Su40St0 24 0.1161 0.0000 0.03850.0000 7.3461.6807 — — — — — — — — — — — — 2 366 Su40St0 24 0.1158 0.0000 0.03800.0000 7.3361.2302 — — — — — — — — — — — — 2 367 Su80St0 24 0.1162 0.0000 0.07630.0000 7.1559.5637 — — — — — — — — — — — — 2 368 Su80St0 24 0.1158 0.0000 0.07610.0000 7.2561.4029 — — — — — — — — — — — — 2 369 Su120St0 24 0.1161 0.0000 0.11400.0000 7.1259.8716 — — — — — — — — — — — — 2 370 Su120St0 24 0.1161 0.0000 0.11420.0000 7.1359.9577 — — — — — — — — — — — — 2 371 Su0St40 24 0.1157 0.0318 0.00000.0000 7.3261.7134 — — — — — — — — — — — — 2 372 Su0St40 24 0.1159 0.0321 0.00000.0000 7.3160.6615 — — — — — — — — — — — — 2 373 Su0St80 24 0.1160 0.0640 0.00000.0000 7.0559.3178 — — — — — — — — — — — — 2 374 Su0St80 24 0.1158 0.0640 0.00000.0000 7.2561.7484 — — — — — — — — — — — — 2 375 Su0St120 24 0.1159 0.0956 0.00000.0000 7.1361.5244 — — — — — — — — — — — — 2 376 Su0St120 24 0.1158 0.0955 0.00000.0000 7.0362.7847 — — — — — — — — — — — — 2 377 Su40St80 24 0.1157 0.0637 0.03850.0000 7.1760.5033 — — — — — — — — — — — — 2 378 Su40St80 24 0.1157 0.0641 0.03790.0000 7.1560.5898 — — — — — — — — — — — — 2 379 Su80St40 24 0.1161 0.0321 0.07640.0000 7.1862.0252 — — — — — — — — — — — —

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221Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 380 Su80St40 24 0.1161 0.0322 0.07600.0000 7.2159.9577 — — — — — — — — — — — — 2 383 NDF 24 0.1161 0.0000 0.00000.0000 — — -1.2782 — — — — — — — — — — — 2 384 NDF 24 0.1159 0.0000 0.00000.0000 — — 0.4890 — — — — — — — — — — — 2 385 Su40St0 24 0.1157 0.0000 0.03840.0000 — — 4.9333 — — — — — — — — — — — 2 386 Su40St0 24 0.1157 0.0000 0.03800.0000 — — 1.0437 — — — — — — — — — — — 2 387 Su80St0 24 0.1159 0.0000 0.07620.0000 — — 7.9570 — — — — — — — — — — — 2 388 Su80St0 24 0.1159 0.0000 0.07610.0000 — — 8.1552 — — — — — — — — — — — 2 389 Su120St0 24 0.1160 0.0000 0.11400.0000 — — 10.0095 — — — — — — — — — — — 2 390 Su120St0 24 0.1160 0.0000 0.11450.0000 — — 15.2718 — — — — — — — — — — — 2 391 Su0St40 24 0.1160 0.0319 0.00000.0000 — — 1.0376 — — — — — — — — — — — 2 392 Su0St40 24 0.1162 0.0318 0.00000.0000 — — 4.0642 — — — — — — — — — — — 2 393 Su0St80 24 0.1160 0.0636 0.00000.0000 — — 13.6792 — — — — — — — — — — — 2 394 Su0St80 24 0.1160 0.0637 0.00000.0000 — — 12.7003 — — — — — — — — — — — 2 395 Su0St120 24 0.1161 0.0958 0.00000.0000 — — 11.4220 — — — — — — — — — — — 2 396 Su0St120 24 0.1158 0.0956 0.00000.0000 — — 13.2900 — — — — — — — — — — — 2 397 Su40St80 24 0.1161 0.0637 0.03790.0000 — — 9.1038 — — — — — — — — — — — 2 398 Su40St80 24 0.1161 0.0638 0.03790.0000 — — 8.8079 — — — — — — — — — — — 2 399 Su80St40 24 0.1158 0.0319 0.07630.0000 — — 16.8984 — — — — — — — — — — — 2 400 Su80St40 24 0.1159 0.0320 0.07640.0000 — — 19.1397 — — — — — — — — — — — 2 401 BL 24 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 1.04062.8190 2 402 BL 24 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.99872.6317 2 403 NDF 24 0.1157 0.0000 0.00000.0000 — — — 0.0799-0.0105-0.0041 0.0209 0.00009.38044.21391.82330.45530.98852.8245 2 404 NDF 24 0.1159 0.0000 0.00000.0000 — — — 0.16560.0102 -0.0041 -0.00120.00008.81294.14371.44560.39280.94802.8552 2 405 Su40St0 24 0.1161 0.0000 0.03840.0000 — — — 0.71880.0334 -0.0041 -0.00120.000017.20478.09425.03701.36141.01522.7371 2 406 Su40St0 24 0.1161 0.0000 0.03810.0000 — — — 0.8026-0.0169-0.0018 0.0001 0.000015.99617.99404.94141.23070.92442.5526 2 407 Su80St0 24 0.1160 0.0000 0.07620.0000 — — — 1.0383-0.0087-0.0041 -0.00120.000018.11559.44037.73462.85200.77482.3511 2 408 Su80St0 24 0.1161 0.0000 0.07590.0000 — — — 1.04210.0196 -0.0027 -0.00120.000019.425010.10808.12352.98390.90062.3944 2 409 Su120St0 24 0.1159 0.0000 0.11450.0000 — — — 0.80450.0255 -0.0035 0.0058 0.000025.556617.42738.24613.75590.79262.2225

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222Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 2 410 Su120St0 24 0.1161 0.0000 0.11400.0000 — — — 0.7285-0.00530.0049 0.0116 0.000025.451216.99858.29213.70730.86672.2393 2 411 Su0St40 24 0.1158 0.0322 0.00000.0000 — — — 0.49090.0027 -0.0032 -0.00120.000017.94348.76863.55410.98941.17432.9219 2 412 Su0St40 24 0.1161 0.0320 0.00000.0000 — — — 0.96620.0094 -0.0041 -0.00120.000017.45408.16443.42340.92561.11412.9183 2 413 Su0St80 24 0.1159 0.0639 0.00000.0000 — — — 0.7344-0.00420.0154 0.0058 0.000026.226713.37464.32130.88801.19252.7058 2 414 Su0St80 24 0.1162 0.0642 0.00000.0000 — — — 1.2058-0.01690.0191 -0.00120.000026.336413.54214.28000.88851.17432.7130 2 415 Su0St120 24 0.1160 0.0957 0.00000.0000 — — — 1.29730.0204 -0.0032 -0.00120.000027.561217.08868.11421.07901.69251.6827 2 416 Su0St120 24 0.1158 0.0959 0.00000.0000 — — — 1.52910.0030 -0.0041 -0.00120.000031.077218.11734.86740.92381.16562.5473 2 417 Su40St80 24 0.1157 0.0642 0.03840.0000 — — — 0.8259-0.01490.0165 -0.00120.000030.245217.88556.45671.51251.11832.4269 2 418 Su40St80 24 0.1158 0.0639 0.03800.0000 — — — 1.7707-0.0169-0.0005 -0.00120.000026.475315.79376.18401.39120.97681.7231 2 419 Su80St40 24 0.1161 0.0319 0.07640.0000 — — — 0.65840.0186 -0.0041 -0.00120.000021.081514.664910.20094.23770.88212.2939 2 420 Su80St40 24 0.1159 0.0320 0.07630.0000 — — — 1.53300.0122 -0.0041 -0.00120.073626.521514.71208.94373.05420.96592.3015 3 1 BL 0 0.0000 0.0000 0.00000.0000 7.15— — — — — — — — — — — — — 3 2 BL 0 0.0000 0.0000 0.00000.0000 7.13— — — — — — — — — — — — — 3 3 NDF 0 0.1157 0.0000 0.00000.0000 7.1693.6505 — — — — — — — — — — — — 3 4 NDF 0 0.1157 0.0000 0.00000.0000 7.1593.3912 — — — — — — — — — — — — 3 5 St40Pe0 0 0.1159 0.0318 0.00000.0000 7.1393.4947 — — — — — — — — — — — — 3 6 St40Pe0 0 0.1159 0.0319 0.00000.0000 7.1892.8907 — — — — — — — — — — — — 3 7 St80Pe0 0 0.1160 0.0641 0.00000.0000 7.1791.5202 — — — — — — — — — — — — 3 8 St80Pe0 0 0.1161 0.0641 0.00000.0000 7.1392.4779 — — — — — — — — — — — — 3 9 St120Pe0 0 0.1157 0.0957 0.00000.0000 7.1593.4776 — — — — — — — — — — — — 3 10 St120Pe0 0 0.1157 0.0956 0.00000.0000 7.1592.8726 — — — — — — — — — — — — 3 11 St0Pe40 0 0.1159 0.0000 0.00000.0435 7.0593.4947 — — — — — — — — — — — — 3 12 St0Pe40 0 0.1158 0.0000 0.00000.0435 7.0792.3635 — — — — — — — — — — — — 3 13 St0Pe91 0 0.1161 0.0000 0.00000.0869 7.0693.2532 — — — — — — — — — — — — 3 14 St0Pe91 0 0.1157 0.0000 0.00000.0869 7.0893.9962 — — — — — — — — — — — — 3 15 St0Pe120 0 0.1160 0.0000 0.00000.1304 7.1393.4170 — — — — — — — — — — — — 3 16 St0Pe120 0 0.1160 0.0000 0.00000.1304 7.1493.5032 — — — — — — — — — — — — 3 17 St40Pe91 0 0.1157 0.0321 0.00000.0869 7.0795.2927 — — — — — — — — — — — —

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223Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 3 18 St40Pe91 0 0.1161 0.0322 0.00000.0869 7.0795.0623 — — — — — — — — — — — — 3 19 St80Pe40 0 0.1157 0.0641 0.00000.0435 7.0893.6505 — — — — — — — — — — — — 3 20 St80Pe40 0 0.1162 0.0642 0.00000.0435 7.0694.2948 — — — — — — — — — — — — 3 23 NDF 0 0.1160 0.0000 0.00000.0000 — — 0.4865 — — — — — — — — — — — 3 24 NDF 0 0.1159 0.0000 0.00000.0000 — — -0.4861 — — — — — — — — — — — 3 25 St40Pe0 0 0.1158 0.0322 0.00000.0000 — — 0.5322 — — — — — — — — — — — 3 26 St40Pe0 0 0.1159 0.0318 0.00000.0000 — — -0.5310 — — — — — — — — — — — 3 27 St80Pe0 0 0.1157 0.0637 0.00000.0000 — — 0.0133 — — — — — — — — — — — 3 28 St80Pe0 0 0.1161 0.0642 0.00000.0000 — — -0.0134 — — — — — — — — — — — 3 29 St120Pe0 0 0.1158 0.0960 0.00000.0000 — — -0.9761 — — — — — — — — — — — 3 30 St120Pe0 0 0.1161 0.0957 0.00000.0000 — — 0.9762 — — — — — — — — — — — 3 31 St0Pe40 0 0.1159 0.0000 0.00000.0435 — — -0.2410 — — — — — — — — — — — 3 32 St0Pe40 0 0.1161 0.0000 0.00000.0435 — — 0.2413 — — — — — — — — — — — 3 33 St0Pe91 0 0.1161 0.0000 0.00000.0869 — — -0.1027 — — — — — — — — — — — 3 34 St0Pe91 0 0.1157 0.0000 0.00000.0869 — — 0.1025 — — — — — — — — — — — 3 35 St0Pe120 0 0.1157 0.0000 0.00000.1304 — — -0.3846 — — — — — — — — — — — 3 36 St0Pe120 0 0.1162 0.0000 0.00000.1304 — — 0.3854 — — — — — — — — — — — 3 37 St40Pe91 0 0.1160 0.0320 0.00000.0869 — — 0.0990 — — — — — — — — — — — 3 38 St40Pe91 0 0.1162 0.0319 0.00000.0869 — — -0.0990 — — — — — — — — — — — 3 39 St80Pe40 0 0.1159 0.0639 0.00000.0435 — — -0.7291 — — — — — — — — — — — 3 40 St80Pe40 0 0.1162 0.0639 0.00000.0435 — — 0.7300 — — — — — — — — — — — 3 41 BL 0 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.19180.2521 3 42 BL 0 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.26870.2378 3 43 NDF 0 0.1159 0.0000 0.00000.0000 — — — 0.03950.0033 0.0821 0.2825 0.00000.1916-0.3264-0.33280.07900.17450.0446 3 44 NDF 0 0.1162 0.0000 0.00000.0000 — — — -0.1913-0.0002-0.0432 -0.23020.00000.2649-0.0714-0.33090.05070.17160.3349 3 45 St40Pe0 0 0.1161 0.0318 0.00000.0000 — — — 20.16000.0169 0.1556 0.7461 0.0000-0.0268-0.2375-0.3477-0.17130.17880.2187 3 46 St40Pe0 0 0.1161 0.0318 0.00000.0000 — — — 21.0790-0.0002-0.0475 -0.22730.00000.0605-0.1615-0.1797-0.06760.26700.2958 3 47 St80Pe0 0 0.1156 0.0638 0.00000.0000 — — — 37.36650.0141 0.1174 0.3096 0.00000.0347-0.1266-0.2321-0.35020.00000.0000

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224Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 3 48 St80Pe0 0 0.1161 0.0637 0.00000.0000 — — — 50.3659-0.0002-0.0502 -0.27690.00000.34020.0845-0.2983-0.35020.15560.1542 3 49 St120Pe0 0 0.1158 0.0960 0.00000.0000 — — — 55.83170.0176 0.1245 0.0766 0.00001.4305-1.1731-0.2614-0.25290.10560.0000 3 50 St120Pe0 0 0.1159 0.0960 0.00000.0000 — — — 71.7783-0.0002-0.0518 -0.25310.00000.10810.3416-0.2988-0.07590.21150.2444 3 51 St0Pe40 0 0.1157 0.0000 0.00000.0435 — — — 0.04520.0207 0.1111 0.1878 0.08500.47380.1724-0.00860.05080.12140.2280 3 52 St0Pe40 0 0.1159 0.0000 0.00000.0435 — — — -0.0972-0.0002-0.0525 -0.24780.11260.6104-1.4049-0.02200.33890.11080.2823 3 53 St0Pe91 0 0.1162 0.0000 0.00000.0869 — — — 0.43800.1426 0.0561 0.4964 0.34751.79530.4377-0.21370.03410.07080.2515 3 54 St0Pe91 0 0.1161 0.0000 0.00000.0869 — — — 0.2963-0.00020.0299 -0.25680.22100.72280.4476-0.21570.03240.08890.2556 3 55 St0Pe120 0 0.1156 0.0000 0.00000.1304 — — — 0.59700.3698 0.1718 0.5122 0.21450.4619-0.2017-0.26360.01680.15050.2405 3 56 St0Pe120 0 0.1158 0.0000 0.00000.1304 — — — 11.47670.8776 0.2152 -0.27680.23170.8296-0.2887-0.3117-0.00650.03960.3052 3 57 St40Pe91 0 0.1161 0.0322 0.00000.0869 — — — 15.52930.1689 0.1683 0.2834 0.13460.45550.2329-0.2152-0.10690.09050.2557 3 58 St40Pe91 0 0.1161 0.0323 0.00000.0869 — — — 11.0121-0.00020.0115 -0.28420.15010.32500.2393-0.2640-0.06200.07020.2834 3 59 St80Pe40 0 0.1157 0.0642 0.00000.0435 — — — 21.5777-0.0002-0.0887 0.6106 0.07430.0822-0.0514-0.2578-0.05920.11230.4097 3 60 St80Pe40 0 0.1162 0.0640 0.00000.0435 — — — 37.6230-0.0002-0.0638 -0.26910.08020.27250.1349-0.22600.06990.12620.2184 3 61 BL 4 0.0000 0.0000 0.00000.0000 7.17— — — — — — — — — — — — — 3 62 BL 4 0.0000 0.0000 0.00000.0000 7.16— — — — — — — — — — — — — 3 63 NDF 4 0.1156 0.0000 0.00000.0000 7.1292.4310 — — — — — — — — — — — — 3 64 NDF 4 0.1159 0.0000 0.00000.0000 7.1292.2003 — — — — — — — — — — — — 3 65 St40Pe0 4 0.1157 0.0323 0.00000.0000 7.1490.6253 — — — — — — — — — — — — 3 66 St40Pe0 4 0.1162 0.0319 0.00000.0000 7.1792.3151 — — — — — — — — — — — — 3 67 St80Pe0 4 0.1158 0.0638 0.00000.0000 7.1991.7590 — — — — — — — — — — — — 3 68 St80Pe0 4 0.1160 0.0641 0.00000.0000 7.1792.1237 — — — — — — — — — — — — 3 69 St120Pe0 4 0.1161 0.0960 0.00000.0000 7.1690.6689 — — — — — — — — — — — — 3 70 St120Pe0 4 0.1161 0.0960 0.00000.0000 7.1792.3056 — — — — — — — — — — — — 3 71 St0Pe40 4 0.1157 0.0000 0.00000.0435 7.0593.3048 — — — — — — — — — — — — 3 72 St0Pe40 4 0.1162 0.0000 0.00000.0435 7.0393.0037 — — — — — — — — — — — — 3 73 St0Pe91 4 0.1160 0.0000 0.00000.0869 7.0192.0375 — — — — — — — — — — — — 3 74 St0Pe91 4 0.1157 0.0000 0.00000.0869 7.0492.5269 — — — — — — — — — — — — 3 75 St0Pe120 4 0.1159 0.0000 0.00000.1304 7.0294.7027 — — — — — — — — — — — —

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225Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 3 76 St0Pe120 4 0.1161 0.0000 0.00000.1304 7.1292.3056 — — — — — — — — — — — — 3 77 St40Pe91 4 0.1162 0.0321 0.00000.0869 7.0493.3480 — — — — — — — — — — — — 3 78 St40Pe91 4 0.1161 0.0323 0.00000.0869 7.0293.7701 — — — — — — — — — — — — 3 79 St80Pe40 4 0.1159 0.0640 0.00000.0435 7.0193.3221 — — — — — — — — — — — — 3 80 St80Pe40 4 0.1161 0.0642 0.00000.0435 7.0392.5641 — — — — — — — — — — — — 3 83 NDF 4 0.1161 0.0000 0.00000.0000 — — -0.2497 — — — — — — — — — — — 3 84 NDF 4 0.1160 0.0000 0.00000.0000 — — -0.2459 — — — — — — — — — — — 3 85 St40Pe0 4 0.1159 0.0322 0.00000.0000 — — 0.1874 — — — — — — — — — — — 3 86 St40Pe0 4 0.1162 0.0323 0.00000.0000 — — 0.1754 — — — — — — — — — — — 3 87 St80Pe0 4 0.1158 0.0637 0.00000.0000 — — -1.4057 — — — — — — — — — — — 3 88 St80Pe0 4 0.1158 0.0638 0.00000.0000 — — -1.3109 — — — — — — — — — — — 3 89 St120Pe0 4 0.1162 0.0959 0.00000.0000 — — -0.2516 — — — — — — — — — — — 3 90 St120Pe0 4 0.1161 0.0960 0.00000.0000 — — -0.2514 — — — — — — — — — — — 3 91 St0Pe40 4 0.1161 0.0000 0.00000.0435 — — 1.4619 — — — — — — — — — — — 3 92 St0Pe40 4 0.1160 0.0000 0.00000.0435 — — 1.2696 — — — — — — — — — — — 3 93 St0Pe91 4 0.1162 0.0000 0.00000.0869 — — 1.6037 — — — — — — — — — — — 3 94 St0Pe91 4 0.1161 0.0000 0.00000.0869 — — 1.6062 — — — — — — — — — — — 3 95 St0Pe120 4 0.1160 0.0000 0.00000.1304 — — 2.5870 — — — — — — — — — — — 3 96 St0Pe120 4 0.1157 0.0000 0.00000.1304 — — 2.5937 — — — — — — — — — — — 3 97 St40Pe91 4 0.1157 0.0322 0.00000.0869 — — 2.7872 — — — — — — — — — — — 3 98 St40Pe91 4 0.1157 0.0323 0.00000.0869 — — 2.6873 — — — — — — — — — — — 3 99 St80Pe40 4 0.1161 0.0640 0.00000.0435 — — 3.4155 — — — — — — — — — — — 3 100 St80Pe40 4 0.1161 0.0640 0.00000.0435 — — 3.4155 — — — — — — — — — — — 3 101 BL 4 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.22050.3756 3 102 BL 4 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.20070.3901 3 103 NDF 4 0.1159 0.0000 0.00000.0000 — — — -0.00660.0000 -0.0105 0.0669 0.00000.1343-1.17810.11530.00860.21100.4775 3 104 NDF 4 0.1159 0.0000 0.00000.0000 — — — 0.79710.0000 0.0097 -0.05920.00000.59440.33450.1821-0.03270.27840.3962 3 105 St40Pe0 4 0.1161 0.0319 0.00000.0000 — — — 22.86320.0000 -0.0105 -0.11770.00000.60520.32780.1653-0.01710.23110.4320

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226Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 3 106 St40Pe0 4 0.1158 0.0323 0.00000.0000 — — — 16.07210.0000 0.0094 -0.09040.08290.85000.27370.6595-0.08941.54120.4485 3 107 St80Pe0 4 0.1159 0.0642 0.00000.0000 — — — 40.61950.0181 0.1575 -0.11770.09150.9419-0.00240.18400.04300.21160.4829 3 108 St80Pe0 4 0.1160 0.0642 0.00000.0000 — — — 45.51840.0000 0.0075 -0.06120.05170.75180.29420.1830-0.11220.21670.3605 3 109 St120Pe0 4 0.1157 0.0959 0.00000.0000 — — — 57.98310.0207 0.1779 -0.11770.05250.51210.25590.1590-0.15850.21340.3747 3 110 St120Pe0 4 0.1157 0.0956 0.00000.0000 — — — 70.32090.0000 0.0023 -0.10400.0000-0.2368-1.1182-0.1776-0.47650.00000.0000 3 111 St0Pe40 4 0.1156 0.0000 0.00000.0435 — — — 0.62750.0180 0.1939 -0.11770.00003.04991.00680.08300.01050.13940.3915 3 112 St0Pe40 4 0.1159 0.0000 0.00000.0435 — — — 0.25380.0000 -0.0003 -0.09370.03913.14051.61650.4316-0.02110.24420.0800 3 113 St0Pe91 4 0.1159 0.0000 0.00000.0869 — — — 0.86510.0000 -0.0105 -0.11770.00003.30920.11170.1539-0.47650.00000.3482 3 114 St0Pe91 4 0.1161 0.0000 0.00000.0869 — — — 0.41120.0000 -0.0021 -0.10130.07354.40411.61230.16590.21120.05670.3944 3 115 St0Pe120 4 0.1162 0.0000 0.00000.1304 — — — 0.86130.0362 0.1769 -0.11770.17544.43651.52000.1991-0.11890.00000.3896 3 116 St0Pe120 4 0.1160 0.0000 0.00000.1304 — — — 0.02530.0000 0.0103 -0.10180.36694.05121.82800.1239-0.02230.03440.4335 3 117 St40Pe91 4 0.1157 0.0319 0.00000.0869 — — — 15.16710.0229 0.2326 0.0551 0.23423.90451.49760.13620.01050.07660.3792 3 118 St40Pe91 4 0.1160 0.0322 0.00000.0869 — — — 13.80690.0000 0.0121 -0.09940.00001.40590.09170.21100.02400.24670.6915 3 119 St80Pe40 4 0.1159 0.0642 0.00000.0435 — — — 19.48550.0226 0.2140 0.0418 0.13973.86711.50610.1715-0.08560.14680.4164 3 120 St80Pe40 4 0.1159 0.0639 0.00000.0435 — — — 42.85380.0000 0.0062 -0.11510.12774.23321.26750.2229-0.14710.09210.4219 3 121 BL 8 0.0000 0.0000 0.00000.0000 7.16— — — — — — — — — — — — — 3 122 BL 8 0.0000 0.0000 0.00000.0000 7.15— — — — — — — — — — — — — 3 123 NDF 8 0.1159 0.0000 0.00000.0000 7.2088.7056 — — — — — — — — — — — — 3 124 NDF 8 0.1159 0.0000 0.00000.0000 7.1888.7919 — — — — — — — — — — — — 3 125 St40Pe0 8 0.1159 0.0323 0.00000.0000 7.1088.7056 — — — — — — — — — — — — 3 126 St40Pe0 8 0.1162 0.0319 0.00000.0000 7.1388.7430 — — — — — — — — — — — — 3 127 St80Pe0 8 0.1158 0.0637 0.00000.0000 7.0988.8658 — — — — — — — — — — — — 3 128 St80Pe0 8 0.1157 0.0638 0.00000.0000 7.1089.3721 — — — — — — — — — — — — 3 129 St120Pe0 8 0.1157 0.0956 0.00000.0000 7.0788.5077 — — — — — — — — — — — — 3 130 St120Pe0 8 0.1161 0.0960 0.00000.0000 7.0489.8505 — — — — — — — — — — — — 3 131 St0Pe40 8 0.1157 0.0000 0.00000.0435 7.0289.1992 — — — — — — — — — — — — 3 132 St0Pe40 8 0.1157 0.0000 0.00000.0435 7.0389.2856 — — — — — — — — — — — — 3 133 St0Pe91 8 0.1158 0.0000 0.00000.0869 6.8889.5567 — — — — — — — — — — — —

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227Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 3 134 St0Pe91 8 0.1157 0.0000 0.00000.0869 6.8989.1128 — — — — — — — — — — — — 3 135 St0Pe120 8 0.1159 0.0000 0.00000.1304 6.6790.3451 — — — — — — — — — — — — 3 136 St0Pe120 8 0.1162 0.0000 0.00000.1304 6.6790.5506 — — — — — — — — — — — — 3 137 St40Pe91 8 0.1160 0.0322 0.00000.0869 6.8790.3562 — — — — — — — — — — — — 3 138 St40Pe91 8 0.1157 0.0323 0.00000.0869 6.8490.8414 — — — — — — — — — — — — 3 139 St80Pe40 8 0.1157 0.0642 0.00000.0435 6.9690.4093 — — — — — — — — — — — — 3 140 St80Pe40 8 0.1158 0.0637 0.00000.0435 6.9790.3340 — — — — — — — — — — — — 3 143 NDF 8 0.1161 0.0000 0.00000.0000 — — 0.8244 — — — — — — — — — — — 3 144 NDF 8 0.1159 0.0000 0.00000.0000 — — 0.8322 — — — — — — — — — — — 3 145 St40Pe0 8 0.1159 0.0323 0.00000.0000 — — 3.7000 — — — — — — — — — — — 3 146 St40Pe0 8 0.1159 0.0322 0.00000.0000 — — 3.7028 — — — — — — — — — — — 3 147 St80Pe0 8 0.1161 0.0640 0.00000.0000 — — 4.0448 — — — — — — — — — — — 3 148 St80Pe0 8 0.1157 0.0641 0.00000.0000 — — 4.0544 — — — — — — — — — — — 3 149 St120Pe0 8 0.1162 0.0955 0.00000.0000 — — 9.1345 — — — — — — — — — — — 3 150 St120Pe0 8 0.1158 0.0956 0.00000.0000 — — 6.7011 — — — — — — — — — — — 3 151 St0Pe40 8 0.1162 0.0000 0.00000.0435 — — 8.0992 — — — — — — — — — — — 3 152 St0Pe40 8 0.1157 0.0000 0.00000.0435 — — 8.6999 — — — — — — — — — — — 3 153 St0Pe91 8 0.1160 0.0000 0.00000.0869 — — 15.7679 — — — — — — — — — — — 3 154 St0Pe91 8 0.1157 0.0000 0.00000.0869 — — 15.8730 — — — — — — — — — — — 3 155 St0Pe120 8 0.1159 0.0000 0.00000.1304 — — 19.7756 — — — — — — — — — — — 3 156 St0Pe120 8 0.1158 0.0000 0.00000.1304 — — 20.2661 — — — — — — — — — — — 3 157 St40Pe91 8 0.1159 0.0321 0.00000.0869 — — 17.5297 — — — — — — — — — — — 3 158 St40Pe91 8 0.1159 0.0322 0.00000.0869 — — 17.0392 — — — — — — — — — — — 3 159 St80Pe40 8 0.1161 0.0642 0.00000.0435 — — 12.7865 — — — — — — — — — — — 3 160 St80Pe40 8 0.1161 0.0642 0.00000.0435 — — 12.7865 — — — — — — — — — — — 3 161 BL 8 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — — — 3 162 BL 8 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.37680.5172 3 163 NDF 8 0.1159 0.0000 0.00000.0000 — — — -0.62460.0012 0.0450 0.0563 0.0000-0.65480.43290.0658-0.02900.28190.6624

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228Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 3 164 NDF 8 0.1160 0.0000 0.00000.0000 — — — 0.6318-0.0037-0.0228 0.0013 0.1019-0.95350.27490.0964-0.00060.16740.3081 3 165 St40Pe0 8 0.1159 0.0319 0.00000.0000 — — — 13.05470.0120 0.1195 0.0856 0.00001.66781.34740.29410.08880.26830.5586 3 166 St40Pe0 8 0.1161 0.0323 0.00000.0000 — — — 17.8803-0.0037-0.0251 -0.00800.00001.45871.17990.3369-0.20220.23680.6535 3 167 St80Pe0 8 0.1157 0.0642 0.00000.0000 — — — 34.8957-0.00120.0522 0.0831 0.07320.91801.25470.2273-0.15830.25380.5331 3 168 St80Pe0 8 0.1158 0.0637 0.00000.0000 — — — 42.2874-0.0037-0.0251 -0.01160.09721.52241.54260.3337-0.04690.24510.6426 3 169 St120Pe0 8 0.1159 0.0957 0.00000.0000 — — — 51.15960.0102 0.0710 0.0874 0.09492.46191.58570.3267-0.05320.22770.5283 3 170 St120Pe0 8 0.1161 0.0957 0.00000.0000 — — — 59.8430-0.0037-0.0251 -0.01580.05522.17191.63090.3143-0.03130.23930.6782 3 171 St0Pe40 8 0.1158 0.0000 0.00000.0435 — — — 0.08620.0194 0.0899 0.1486 0.00009.89722.49310.05110.00120.07250.4036 3 172 St0Pe40 8 0.1157 0.0000 0.00000.0435 — — — 1.65880.1139 -0.0251 -0.01580.000010.54962.66320.10880.00280.17010.4389 3 173 St0Pe91 8 0.1158 0.0000 0.00000.0869 — — — 0.40040.0066 0.1065 0.1143 0.032216.06143.62740.1673-0.35040.11540.6037 3 174 St0Pe91 8 0.1157 0.0000 0.00000.0869 — — — 2.36720.3042 0.0133 -0.01580.000017.73734.05170.20160.11950.11200.4880 3 175 St0Pe120 8 0.1161 0.0000 0.00000.1304 — — — 0.40420.0059 0.0879 0.3557 0.088217.51254.38340.2866-0.08210.10920.3544 3 176 St0Pe120 8 0.1157 0.0000 0.00000.1304 — — — 1.89690.3113 0.0153 -0.01580.063818.38674.24140.3134-0.08960.08560.2071 3 177 St40Pe91 8 0.1159 0.0319 0.00000.0869 — — — 9.52000.0196 0.0007 0.2962 0.072319.38934.81090.3813-0.10900.00000.3570 3 178 St40Pe91 8 0.1157 0.0319 0.00000.0869 — — — 13.47270.9398 0.0127 -0.01580.090119.20734.88230.4032-0.03100.11560.2197 3 179 St80Pe40 8 0.1161 0.0637 0.00000.0435 — — — 24.36990.0295 0.0061 0.1904 0.353212.54244.34990.7084-0.42140.20340.4675 3 180 St80Pe40 8 0.1162 0.0637 0.00000.0435 — — — 30.24510.8643 -0.0251 -0.01580.474711.97194.70940.8228-0.03360.11100.4783 3 181 BL 12 0.0000 0.0000 0.00000.0000 7.26— — — — — — — — — — — — — 3 182 BL 12 0.0000 0.0000 0.00000.0000 7.26— — — — — — — — — — — — — 3 183 NDF 12 0.1159 0.0000 0.00000.0000 7.2782.0182 — — — — — — — — — — — — 3 184 NDF 12 0.1158 0.0000 0.00000.0000 7.2682.5183 — — — — — — — — — — — — 3 185 St40Pe0 12 0.1160 0.0322 0.00000.0000 7.1182.4673 — — — — — — — — — — — — 3 186 St40Pe0 12 0.1159 0.0319 0.00000.0000 7.0681.0690 — — — — — — — — — — — — 3 187 St80Pe0 12 0.1161 0.0642 0.00000.0000 7.0482.4850 — — — — — — — — — — — — 3 188 St80Pe0 12 0.1161 0.0637 0.00000.0000 7.0182.7434 — — — — — — — — — — — — 3 189 St120Pe0 12 0.1161 0.0958 0.00000.0000 6.9083.1741 — — — — — — — — — — — — 3 190 St120Pe0 12 0.1157 0.0956 0.00000.0000 6.9582.5006 — — — — — — — — — — — — 3 191 St0Pe40 12 0.1162 0.0000 0.00000.0435 7.1284.9988 — — — — — — — — — — — —

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229Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 3 192 St0Pe40 12 0.1157 0.0000 0.00000.0435 7.1184.6614 — — — — — — — — — — — — 3 193 St0Pe91 12 0.1162 0.0000 0.00000.0869 7.0386.8063 — — — — — — — — — — — — 3 194 St0Pe91 12 0.1157 0.0000 0.00000.0869 7.0286.6494 — — — — — — — — — — — — 3 195 St0Pe120 12 0.1158 0.0000 0.00000.1304 6.8985.3682 — — — — — — — — — — — — 3 196 St0Pe120 12 0.1161 0.0000 0.00000.1304 6.9088.3429 — — — — — — — — — — — — 3 197 St40Pe91 12 0.1160 0.0320 0.00000.0869 6.9587.9853 — — — — — — — — — — — — 3 198 St40Pe91 12 0.1159 0.0319 0.00000.0869 6.9587.8859 — — — — — — — — — — — — 3 199 St80Pe40 12 0.1161 0.0642 0.00000.0435 6.8984.8971 — — — — — — — — — — — — 3 200 St80Pe40 12 0.1162 0.0642 0.00000.0435 6.9384.9988 — — — — — — — — — — — — 3 203 NDF 12 0.1160 0.0000 0.00000.0000 — — 2.1954 — — — — — — — — — — — 3 204 NDF 12 0.1160 0.0000 0.00000.0000 — — 2.5860 — — — — — — — — — — — 3 205 St40Pe0 12 0.1158 0.0322 0.00000.0000 — — 8.9789 — — — — — — — — — — — 3 206 St40Pe0 12 0.1158 0.0322 0.00000.0000 — — 8.4907 — — — — — — — — — — — 3 207 St80Pe0 12 0.1162 0.0638 0.00000.0000 — — 9.3205 — — — — — — — — — — — 3 208 St80Pe0 12 0.1160 0.0641 0.00000.0000 — — 9.8064 — — — — — — — — — — — 3 209 St120Pe0 12 0.1160 0.0956 0.00000.0000 — — 12.9456 — — — — — — — — — — — 3 210 St120Pe0 12 0.1158 0.0959 0.00000.0000 — — 12.0625 — — — — — — — — — — — 3 211 St0Pe40 12 0.1158 0.0000 0.00000.0435 — — 8.7945 — — — — — — — — — — — 3 212 St0Pe40 12 0.1160 0.0000 0.00000.0435 — — 8.7886 — — — — — — — — — — — 3 213 St0Pe91 12 0.1161 0.0000 0.00000.0869 — — 13.5195 — — — — — — — — — — — 3 214 St0Pe91 12 0.1160 0.0000 0.00000.0869 — — 12.8384 — — — — — — — — — — — 3 215 St0Pe120 12 0.1161 0.0000 0.00000.1304 — — 19.2829 — — — — — — — — — — — 3 216 St0Pe120 12 0.1159 0.0000 0.00000.1304 — — 19.0921 — — — — — — — — — — — 3 217 St40Pe91 12 0.1159 0.0319 0.00000.0869 — — 18.6106 — — — — — — — — — — — 3 218 St40Pe91 12 0.1157 0.0322 0.00000.0869 — — 18.8995 — — — — — — — — — — — 3 219 St80Pe40 12 0.1157 0.0637 0.00000.0435 — — 19.3453 — — — — — — — — — — — 3 220 St80Pe40 12 0.1160 0.0641 0.00000.0435 — — 19.4303 — — — — — — — — — — — 3 221 BL 12 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.00000.0000

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230Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 3 222 BL 12 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.36660.9720 3 223 NDF 12 0.1159 0.0000 0.00000.0000 — — — -0.03660.0002 -0.0063 -0.01850.00003.05071.46950.34920.46900.35900.8310 3 224 NDF 12 0.1157 0.0000 0.00000.0000 — — — 0.6730-0.00530.0177 -0.01300.00002.90281.79810.1733-0.00880.45790.9756 3 225 St40Pe0 12 0.1161 0.0319 0.00000.0000 — — — 2.87360.0171 -0.0076 0.2517 0.00008.55395.04961.11590.33260.37301.1604 3 226 St40Pe0 12 0.1162 0.0319 0.00000.0000 — — — 3.2742-0.0053-0.0031 -0.01440.00008.35064.94661.04950.35450.38921.1976 3 227 St80Pe0 12 0.1160 0.0637 0.00000.0000 — — — 9.71710.0045 0.0028 -0.01980.065111.39817.40322.34480.28090.43140.5897 3 228 St80Pe0 12 0.1158 0.0638 0.00000.0000 — — — 9.4172-0.0053-0.0212 -0.01990.000010.70107.76761.74340.23530.37861.1326 3 229 St120Pe0 12 0.1158 0.0956 0.00000.0000 — — — 20.40390.0119 -0.0196 -0.01820.225613.14019.44452.37660.38020.42011.0598 3 230 St120Pe0 12 0.1158 0.0959 0.00000.0000 — — — 17.6124-0.0053-0.0136 -0.01990.169914.46749.29332.29260.27910.26911.1243 3 231 St0Pe40 12 0.1159 0.0000 0.00000.0435 — — — 0.43660.0343 0.0005 0.1201 0.000013.65723.62370.74280.37630.21711.4254 3 232 St0Pe40 12 0.1162 0.0000 0.00000.0435 — — — 0.9936-0.0053-0.0212 -0.01990.000013.02904.07560.64190.37390.36130.6720 3 233 St0Pe91 12 0.1161 0.0000 0.00000.0869 — — — 0.67030.0599 -0.0021 0.1871 0.000028.84678.68900.89830.31550.47711.4258 3 234 St0Pe91 12 0.1159 0.0000 0.00000.0869 — — — 1.2278-0.0053-0.0212 -0.01990.038721.37936.05360.6877-0.00260.26981.2420 3 235 St0Pe120 12 0.1157 0.0000 0.00000.1304 — — — 0.59560.0708 0.0143 0.2024 0.051533.53139.79831.12760.20030.29671.3247 3 236 St0Pe120 12 0.1158 0.0000 0.00000.1304 — — — 1.86130.0077 -0.0212 -0.01990.000033.82839.29021.01520.10210.25170.6287 3 237 St40Pe91 12 0.1162 0.0323 0.00000.0869 — — — 6.01950.0546 -0.0024 0.0729 0.034730.46129.11761.66350.44890.45150.9855 3 238 St40Pe91 12 0.1157 0.0320 0.00000.0869 — — — 5.1689-0.0053-0.0212 -0.01990.030928.39929.31721.69470.36960.31441.3633 3 239 St80Pe40 12 0.1162 0.0642 0.00000.0435 — — — 14.66390.0528 0.0002 0.2241 0.098620.29129.97302.26240.26620.34441.2460 3 240 St80Pe40 12 0.1158 0.0642 0.00000.0435 — — — 11.4713-0.00530.0063 0.0038 0.000020.43399.10642.00350.34510.00000.9381 3 241 BL 16 0.0000 0.0000 0.00000.0000 7.38— — — — — — — — — — — — — 3 242 BL 16 0.0000 0.0000 0.00000.0000 7.32— — — — — — — — — — — — — 3 243 NDF 16 0.1161 0.0000 0.00000.0000 7.2672.9658 — — — — — — — — — — — — 3 244 NDF 16 0.1161 0.0000 0.00000.0000 7.0770.3814 — — — — — — — — — — — — 3 245 St40Pe0 16 0.1157 0.0322 0.00000.0000 7.1173.8141 — — — — — — — — — — — — 3 246 St40Pe0 16 0.1159 0.0323 0.00000.0000 7.1072.6558 — — — — — — — — — — — — 3 247 St80Pe0 16 0.1160 0.0642 0.00000.0000 6.9271.3883 — — — — — — — — — — — — 3 248 St80Pe0 16 0.1161 0.0637 0.00000.0000 6.9574.3442 — — — — — — — — — — — — 3 249 St120Pe0 16 0.1161 0.0956 0.00000.0000 6.8674.9472 — — — — — — — — — — — —

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231Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 3 250 St120Pe0 16 0.1157 0.0960 0.00000.0000 6.8674.7648 — — — — — — — — — — — — 3 251 St0Pe40 16 0.1161 0.0000 0.00000.0435 7.1977.4454 — — — — — — — — — — — — 3 252 St0Pe40 16 0.1162 0.0000 0.00000.0435 7.1977.8115 — — — — — — — — — — — — 3 253 St0Pe91 16 0.1159 0.0000 0.00000.0869 7.1279.4726 — — — — — — — — — — — — 3 254 St0Pe91 16 0.1160 0.0000 0.00000.0869 7.1178.6306 — — — — — — — — — — — — 3 255 St0Pe120 16 0.1161 0.0000 0.00000.1304 7.0080.2882 — — — — — — — — — — — — 3 256 St0Pe120 16 0.1161 0.0000 0.00000.1304 6.9782.0973 — — — — — — — — — — — — 3 257 St40Pe91 16 0.1160 0.0320 0.00000.0869 7.0080.3550 — — — — — — — — — — — — 3 258 St40Pe91 16 0.1157 0.0319 0.00000.0869 6.9979.5187 — — — — — — — — — — — — 3 259 St80Pe40 16 0.1157 0.0638 0.00000.0435 6.9676.3206 — — — — — — — — — — — — 3 260 St80Pe40 16 0.1156 0.0639 0.00000.0435 6.9476.4708 — — — — — — — — — — — — 3 263 NDF 16 0.1157 0.0000 0.00000.0000 — — 2.4511 — — — — — — — — — — — 3 264 NDF 16 0.1160 0.0000 0.00000.0000 — — 3.0254 — — — — — — — — — — — 3 265 St40Pe0 16 0.1162 0.0321 0.00000.0000 — — 8.7254 — — — — — — — — — — — 3 266 St40Pe0 16 0.1160 0.0320 0.00000.0000 — — 8.7343 — — — — — — — — — — — 3 267 St80Pe0 16 0.1157 0.0637 0.00000.0000 — — 13.4890 — — — — — — — — — — — 3 268 St80Pe0 16 0.1157 0.0641 0.00000.0000 — — 12.9894 — — — — — — — — — — — 3 269 St120Pe0 16 0.1157 0.0956 0.00000.0000 — — 18.0799 — — — — — — — — — — — 3 270 St120Pe0 16 0.1158 0.0956 0.00000.0000 — — 17.5891 — — — — — — — — — — — 3 271 St0Pe40 16 0.1159 0.0000 0.00000.0435 — — 9.0357 — — — — — — — — — — — 3 272 St0Pe40 16 0.1160 0.0000 0.00000.0435 — — 9.2281 — — — — — — — — — — — 3 273 St0Pe91 16 0.1160 0.0000 0.00000.0869 — — 15.0355 — — — — — — — — — — — 3 274 St0Pe91 16 0.1161 0.0000 0.00000.0869 — — 14.2518 — — — — — — — — — — — 3 275 St0Pe120 16 0.1160 0.0000 0.00000.1304 — — 19.7246 — — — — — — — — — — — 3 276 St0Pe120 16 0.1160 0.0000 0.00000.1304 — — 20.4081 — — — — — — — — — — — 3 277 St40Pe91 16 0.1158 0.0319 0.00000.0869 — — 19.9314 — — — — — — — — — — — 3 278 St40Pe91 16 0.1161 0.0319 0.00000.0869 — — 20.6075 — — — — — — — — — — — 3 279 St80Pe40 16 0.1162 0.0638 0.00000.0435 — — 19.2852 — — — — — — — — — — —

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232Ferm1 Tube ID Trt Hr iNDF (g DM) Starch (g DM) Sucrose (g DM) Pectin (g DM) pH rNDF (g OM) MCP (mg) GLY (mg) rGlc (mg) rFruc (mg) rSuc (mg) Lac (m M ) C2 (m M ) C3 (m M ) C4 (m M ) Val (m M ) Isobut (m M ) Isoval + 2MB (m M ) 3 280 St80Pe40 16 0.1159 0.0638 0.00000.0435 — — 19.6815 — — — — — — — — — — — 3 281 BL 16 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.45981.4304 3 282 BL 16 0.0000 0.0000 0.00000.0000 — — — — — — — — — — — — 0.51321.2932 3 283 NDF 16 0.1156 0.0000 0.00000.0000 — — — 0.4317-0.0086-0.0004 0.0759 0.00003.86922.48120.00970.12110.44661.3526 3 284 NDF 16 0.1160 0.0000 0.00000.0000 — — — 0.90890.0021 -0.0135 -0.07570.00005.07742.6912-0.48610.24890.51201.5709 3 285 St40Pe0 16 0.1158 0.0323 0.00000.0000 — — — 3.02770.0151 0.0166 0.1513 0.000011.25706.11851.70790.45390.59931.7921 3 286 St40Pe0 16 0.1161 0.0323 0.00000.0000 — — — 3.5907-0.01870.0072 -0.07570.000011.68286.05951.59600.20820.50811.8524 3 287 St80Pe0 16 0.1161 0.0640 0.00000.0000 — — — 4.44550.0142 0.0236 0.1552 0.000014.51239.50372.40860.48920.49361.8521 3 288 St80Pe0 16 0.1157 0.0640 0.00000.0000 — — — 3.3565-0.0187-0.0148 -0.07570.000016.00579.64072.47980.34070.55171.8674 3 289 St120Pe0 16 0.1160 0.0956 0.00000.0000 — — — 7.19860.0166 0.0242 0.1305 0.000019.097513.28643.79520.60890.59431.4856 3 290 St120Pe0 16 0.1158 0.0958 0.00000.0000 — — — 6.0364-0.0187-0.0148 -0.07570.000018.979012.70193.51840.46580.45821.8350 3 291 St0Pe40 16 0.1157 0.0000 0.00000.0435 — — — 0.66740.0167 0.0117 0.0933 0.000013.55593.92620.8570-0.25350.47621.7465 3 292 St0Pe40 16 0.115
Permanent Link: http://ufdc.ufl.edu/UFE0008120/00001

Material Information

Title: Effect of Nonfiber Carbohydrates on Product Yield and Fiber Digestion in Fermentations with Mixed Ruminal Microbes
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008120:00001

Permanent Link: http://ufdc.ufl.edu/UFE0008120/00001

Material Information

Title: Effect of Nonfiber Carbohydrates on Product Yield and Fiber Digestion in Fermentations with Mixed Ruminal Microbes
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008120:00001


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EFFECT OF NONFIBER CARBOHYDRATES ON PRODUCT YIELD AND FIBER
DIGESTION IN FERMENTATIONS WITH MIXED RUMINAL MICROBES














By

LUCIA HOLTSHAUSEN


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


2004

































Copyright 2004

by

Lucia Holtshausen
































This dissertation is dedicated to Heidi Bissell for her friendship during the last three and a
half years. It was her support, endless patience and encouragement that carried me
through my PhD program.















ACKNOWLEDGMENTS

I would like to express my appreciation to all the people who made valuable

contributions throughout my PhD program. First, I would like to thank Dr. Mary Beth

Hall, chair of my supervisory committee, for helping me further develop as a scientist and

for her encouragement. I would also like to thank the other members of my committee:

Dr. Ramon Littell for his assistance with the statistical analysis, Dr. Adegbola Adesogan

for acting as assistant chair on short notice and for always being available to answer even

the most trivial of questions, Dr. Christian Cruywagen for continued academic and moral

support and for always taking a keen interest in my academic future, and the late Dr. Bill

Kunkle for providing valuable practical insight at the onset of my program. Next I would

like to thank all the people who helped at various stages with the in vitro studies and

laboratory analyses: Alexandra Amorocho, Heidi Bissell, Jocelyn Croci, Faith Cullens,

Bruno Amaral, Ashley Hughes, Celeste Kearney, Colleen Larson, Sergei Sennikov and

Tina Sheedy. Without their help I would not have been able to do the studies on such a

large scale. I would like to thank Dr. Paul Weimer and Christine Odt for the analysis of

organic acids and their patience in answering my questions, Dr. Glen Broderick for

amino acid and ammonia nitrogen analyses, and Hangxin Hou from US Sugar in

Clewiston (Florida) for sugar analysis. I would also like to thank Sabrina Robinson for

various administrative favors throughout my PhD program. Last but not least I would

like to thank the Liquid Feed Committee of the American Feed Industry Association and

the Florida Milk Check-Off Program for funding this research.
















TABLE OF CONTENTS


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

LIST OF TABLES .......................................... viii

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

LIST OF A BBREV IA TION S ..................................... ......................... .............. xxiv

A B S T R A C T ..................................................................................................................... x x v

CHAPTER

1 REVIEW OF LITERATURE: MICROBIAL FERMENTATION OF NON-
NEUTRAL DETERGENT FIBER CARBOHYDRATES, AND HOW IT MAY
RELATE TO ANIMAL PERFORMANCE .............. ...................................

Introdu action .................................................... ...... ..... .... ............................ 1
Chemical Structure and degradation of NFC Sources............................................2...
Sucrose .............. ....................................................... ...............2 2..... ........... 2
S ta rc h ......................................................... ............................................... . 3
Pectic Substances................. .......... ............... ........ 5
NFCs and M icrobial Ferm entation Products........................................... ...............5...
O rg an ic A cid s ................................................................................................. .. 6
T total volatile fatty acids ...................................................... ...............6......
A c etic a cid ............................................................................................... .
P ropionic acid ................................................................... . ............ .9
B utyric acid .................................................................. ........................ 11
Branched chain volatile fatty acids ......................................... ................ 12
L a ctic a c id .................................................................................................... 13
M icrob ial M ass .................................................................................................... 14
M icrobial com position ....................................................... 14
M icrobial protein yield ............................................................ .............. 15
M icrobial a-glucan ................................................ .... ......... .. ........ .... 17
Other Factors Which Influence Microbial Product Yield ................ ..................... 18
N itrogen Source ............................................................................................. 18
Ferm entation pH ...................................................................... ..... ............................. 21
NFCs and Ruminal pH, Fiber Digestion and Animal Performance ........................22
Rum inal pH and Fiber D igestion.................................................... ................ 22
A nim al P perform ance ........................................... ......................... ................ 24


v









D ry M atter Intake .......... ....................... ................................................. 25
M ilk Production and Com position ................................................. ................ 25


2 EFFECT OF PH ON MICROBIAL YIELD AND NEUTRAL DETERGENT FIBER
DIGESTION FROM IN VITRO FERMENTATIONS OF SUCROSE AND
ISOLATED NEUTRAL DETERGENT RESIDUE ........................ ..................... 34

Introduction ................................................................................... ...................... 34
M materials and M ethods .. ..................................................................... ................ 35
S u b stra te s .............................................................................................................3 5
M edium and reducing solution ....................................................... ................ 35
Ferm entation................................................................................... ................. 36
Sample Handling and Subsequent Analyses .................................. ................ 37
Statistical A analysis .............. .................. .............................................. 39
R results and D discussion ................ .............. ............................................ 41
R esidual Sucrose .............................. ............................................ 4 1
M icrobial G ly cogen ........................................................................ ...............43
Fermentation pH ...................................................................... 44
O organic A cids ......................................................................................................45
Protein D egradation Products......................................................... ................ 47
N eutral D etergent Fiber D igestion ................................................. ................ 48
Microbial Crude Protein Yield and Efficiency ..............................................49
C o n c lu sio n s............................................................................................................... .. 5 0


3 EFFECT OF NITROGEN SOURCE ON MICROBIAL YIELD AND NEUTRAL
DETERGENT FIBER DIGESTION FROM IN VITRO FERMENTATIONS OF
SUCROSE AND ISOLATED NEUTRAL DETERGENT RESIDUE...................... 58

In tro d u ctio n ............................................................................................................... .. 5 8
M materials and M ethods .. ..................................................................... ................ 59
S u b state s ............................................................................................................. 5 9
M e d iu m ............................................................................................................ . 6 0
Ferm entation ........................................................................... .......... ............... 60
Sample Handling and Subsequent Analyses .................................. ................ 61
Statistical A analysis .............. .................. .............................................. 64
R results and D discussion ................ .............. ............................................ 66
R esidual Substrate ................ .............. ............................................ 66
M icrobial G ly cogen ........................................................................ ...............67
Fermentation pH ...................................................................... 67
O organic A cids ...................................................................................................... 68
Protein D egradation Products......................................................... ................ 69
N eutral D etergent Fiber D igestion ................................................. ................ 70
Microbial Crude Protein Yield and Efficiency ..............................................71
C o n c lu sio n s............................................................................................................... .. 7 2









4 MICROBIAL PRODUCT YIELD AND NEUTRAL DETERGENT FIBER
DIGESTION FROM IN VITRO FERMENTATIONS WITH SUCROSE, STARCH
AND PECTIN IN COMBINATION WITH ISOLATED BERMUDAGRASS
NEU TRAL DETERGEN T RESIDUE ....................................................................... 83

In tro d u ctio n ............................................................................................................... .. 8 3
M materials and M ethods .. ..................................................................... ................ 84
Sub states and T reatm ents .............................................................. ................ 84
Ferm entation ........................................................................... ......... ............... 86
Sample Handling and Subsequent Analyses .................................. ................ 88
Statistical A analysis ..................................................... ...... .... ........................ 91
Treatment mean comparisons and temporal pattern descriptions .............91
Comparisons of maxima, minima and 24 h data.....................................92
Com prisons of N FC m ixtures................................................ ................ 94
R results and D discussion ................................................................... ... ... .. .......... 95
Residual Sugars (Sucrose, Glucose, Fructose)............................... ................ 95
M icrobial G ly cogen Y ield .............................................................. ................ 96
Fermentation pH ......................................................... ............. 97
O organic A cids ............................................................................................. 99
Neutral Detergent Fiber Digestion ....... ... ......................... 104
M icrobial Crude Protein Yield ...... ......... .. ........ ..................... 106
C o n c lu sio n s.............................................................................................................. 10 7


5 CONCLUSION S ................... .. ............................... ...... .... ............... 121

APPENDIX

A ADDITIONAL FIGURES FOR CHAPTER 2...... .......................................124

B ADDITIONAL FIGURES FOR CHAPTER 3......... ....................................126

C FIG U R E S FO R CH A P TER 4 ...................................................................................128

D ADDITIONAL TABLE FOR CHAPTER 4...... .... ......................................166

E CH A PTER 2 R A W D A TA ........................................... ....................... ............... 168

F C H A PTER 3 R A W D A TA ........................................... ....................... ............... 179

G CH A PTER 4 R AW D A TA ..................................... ....................... ................ 193

LIST O F REFEREN CE S ... ................................................................... ................ 281

BIOGRAPH ICAL SKETCH .................. .............................................................. 293















LIST OF TABLES


Table page

1-1 The effects of NFC source on ruminal or fermentation pH and organic acid profile.28

1-2 Effects of NFC source on dry matter intake, milk production and milk composition.31

2-1 Type and number of fermentation tubes per medium for each sampling hour,
indicating the substrate and analysis for which tubes were reserved in an 24 h in
vitro ferm entation of sucrose and iN D F.............................................. ................ 51

2-2 Residual glucose, fructose, unhydrolyzed sucrose, monosaccharide sucrose
equivalent (glucose+fructose) and sucrose equivalent at 0, 4 and 8 h, and averaged
for 24 h in vitro fermentations of sucrose and isolated bermudagrass neutral
detergent residue with initial medium pH of 6.8 or 5.6. ..................... ................ 51

3-1 Type and number of fermentation tubes per medium for one sampling hour,
indicating the substrate and analysis for which tubes were reserved in a 16 h in
vitro fermentation of sucrose and isolated neutral detergent fiber........................73

3-2 Residual glucose, fructose, sucrose, monosaccharide sucrose equivalent (glucose +
fructose) and sucrose equivalent at 0, 4 and 8 h, and averaged for 16 h in vitro
fermentations of sucrose and isolated bermudagrass neutral detergent residue with
different sources of nitrogen in m edia................................................. ................ 74

3-3 Organic acid concentrations (least squares means) at 16 h (corrected for blank
fermentations) for in vitro fermentations of sucrose and isolated bermudagrass
neutral detergent residue with different sources of nitrogen in media.................. 75

3-4 Maximum microbial crude protein (MCP) yield (hour of maximum) and efficiency
of MCP yield at the point of maximum MCP yield for in vitro fermentations of
iNDF and of SuNDF with different source of nitrogen in media. Values are least
sq u are s m ean s........................................................................................................... 7 5

4-1 Layout of treatments and substrate amounts for a series of three 24 h in vitro
fermentations (performed in duplicate) of a mixed batch culture ........................108

4-2 Residual glucose, fructose, unhydrolyzed sucrose and sucrose equivalent (mg) at 0
and 4 h for 24 h in vitro fermentations of iNDF, NFC sources (sucrose, starch and
pectin), and combinations of NFCs. Values are least squares means .................109









4-3 Maximum microbial glycogen (GLY) yield (mg), hour of maximum yield and
temporal patterns for 24 h in vitro fermentations of iNDF, NFC sources (sucrose
and pectin), and combinations of NFCs. Values are least squares means. ..........110

4-4 Main effects and regression coefficients for maximum microbial glycogen (GLY)
yield (mg) for increasing hexose equivalent amounts of NFCs (sucrose, starch and
pectin) fermented, and regression coefficients for fermentations of NFC
com binations. ........................................................................................ 111

4-5 Fermentation pH (mean, minimum, hour of minimum and temporal pattern) for 24
h in vitro fermentations of iNDF, NFC sources (sucrose, starch and pectin), and
combinations of NFCs. Values are least squares means....................................112

4-6 Main effects and regression coefficients for minimum fermentation pH for
increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin)
fermented, and regression coefficients for fermentations of NFC combinations... 113

4-7 Volatile fatty acid concentrations at 24 h and maximum lactate concentrations
(hour of maximum indicated) for 24 h in vitro fermentations of iNDF, NFC sources
(sucrose, starch and pectin), and combinations of NFCs. Values are least squares
m e a n s ........................................................................................................... .... 1 1 4

4-8 Main effects and regression coefficients for maximum organic acid concentrations
for increasing hexose equivalent amounts of NFCs (sucrose, starch and pectin)
fermented, and regression coefficients for fermentations of NFC combinations... 115

4-9 Residual NDFOM at 24 h and temporal patterns for 24 h in vitro fermentations of
iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs.
V alues are least squares m eans. ...... ......... ......... .....................1... 17

4-10 Main effects and regression coefficients for residual NDFOM at 24 h for increasing
hexose equivalent amounts of NFCs (sucrose, starch and pectin) fermented, and
regression coefficients for fermentations of NFC combinations. .........................118

4-11 Microbial crude protein (MCP) yield (mean, maximum, hour of maximum and
temporal pattern) for 24 h in vitro fermentations of iNDF, NFC sources (sucrose,
starch and pectin), and combinations of NFCs. Values are least squares means. .119

4-12 Main effects and regression coefficients for maximum microbial crude protein
(MCP) yield for increasing hexose equivalent amounts of NFCs (sucrose, starch
and pectin) fermented, and regression coefficients for fermentations of NFC
co m b in atio n s. ......................................................................................................... 12 0

D-1 Temporal patterns of organic acid concentrations for 24 h in vitro fermentations of
iNDF, NFC sources (sucrose, starch and pectin), and combinations of NFCs ......167









E-1 Data used for statistical analysis in evaluating the effect of pH on microbial yield
and neutral detergen fiber digestion from in vitro fermentations of sucrose and
isolated bermudagrass neutral detergent residue......................... .................. 168

F-i Data used for statistical analysis in evaluating the effect of nitrogen source on
microbial yield and neutral detergen fiber digestion from in vitro fermentations of
sucrose and isolated bermudagrass neutral detergent residue............................. 179

G-1 Data used for statistical analysis in evaluating the effect on microbial yield and
neutral detergen fiber digestion from in vitro fermentations of different sources
(sucrose, starch and pectin), amounts (0, 40, 80 and 120 mg nominal hexose
equivalents) and combinations (sucrose+starch, starch+pectin and sucrose+pectin)
o f N F C s .............................................................................................................. 1 9 3















LIST OF FIGURES


Figure page

2-1 Microbial glycogen yield (LSmeans standard error) for 24 h in vitro
fermentations of SuNDF with initial medium pH of 6.8 (m) or 5.6 (A). SuNDF =
sucrose + isolated bermudagrass neutral detergent residue. ....................................52

2-2 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (A, o) and SuNDF (A, m) with an initial medium pH of 6.8 (m or o) or 5.6
(A or A). iNDF = isolated bermudagrass neutral detergent residue; SuNDF =
su cro se+ iN D F ........................................................................................................... 52

2-3 Acetate concentrations (LSmeans standard error) for 24 h in vitro fermentations
containing no substrate (o, x) or SuNDF (A, m) with an initial medium pH of 6.8
(m or o) or 5.6 (A or x). SuNDF = sucrose + isolated bermudagrass neutral
d eterg ent residue e. ..................................................................................................... 53

2-4 Propionate concentrations (LSmeans standard error) for 24 h in vitro
fermentations containing no substrate (o, x) or SuNDF (A, m) with an initial
medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF = sucrose + isolated
berm udagrass neutral detergent residue .............................................. ................ 53

2-5 Butyrate concentrations (LSmeans standard error) for 24 h in vitro fermentations
containing no substrate (o, x) or SuNDF (A, m) with an initial medium pH of 6.8
(m or o) or 5.6 (A or x). SuNDF = sucrose + isolated bermudagrass neutral
d eterg ent residue e. ..................................................................................................... 54

2-6 Total volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro
fermentations containing no substrate (o, x) or SuNDF (A, m) with an initial
medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF = sucrose + isolated
berm udagrass neutral detergent residue .............................................. ................ 54

2-7 Lactate concentrations (LSmeans standard error) for 24 h in vitro fermentations
containing no substrate (o, x) or SuNDF (A, m) with an initial medium pH of 6.8
(m or o) or 5.6 (A or x). SuNDF = sucrose + isolated bermudagrass neutral
d eterg ent residue e. ..................................................................................................... 5 5









2-8 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24
h in vitro fermentations containing no substrate (o, x) or SuNDF (A, m) with an
initial medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF = sucrose + isolated
berm udagrass neutral detergent residue .............................................. ................ 55

2-9 Ammonia nitrogen concentration (LSmeans standard error) for 24 h in vitro
fermentations containing no substrate (o, x) or SuNDF (A, m) with an initial
medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF = sucrose + isolated
berm udagrass neutral detergent residue .............................................. ................ 56

2-10 Total free amino acid concentration (LSmeans standard error) for 24 h in vitro
fermentations containing no substrate (o, x) or SuNDF (A, m) with an initial
medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF = sucrose + isolated
berm udagrass neutral detergent residue .............................................. ................ 56

2-11 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of
(iNDF; A, o) and SuNDF (A, m) with an initial medium pH of 6.8 (m or o) or 5.6
(A or A). iNDF = isolated bermudagrass neutral detergent residue; SuNDF =
su cro se + iN D F ......................................................................................................... 5 7

2-12 Microbial crude protein yield (LSmeans standard error) for 24 h in vitro
fermentations of SuNDF with an initial medium pH of 6.8 (m) or 5.6 (A). SuNDF
= sucrose + isolated bermudagrass neutral detergent residue. .................................57

3-1 Microbial glycogen yield (least squares means standard error) for 16 h in vitro
fermentations of SuNDF with media containing nitrogen in the form of non-protein
nitrogen + true protein (m), true protein only (A) or non-protein nitrogen only (*).
SuNDF = sucrose + isolated bermudagrass neutral detergent residue..................76

3-2 Fermentation pH (least squares means standard error) for 16 h in vitro
fermentations of iNDF (o, A, o) and SuNDF (m, A, e) with media containing
nitrogen in the form of non-protein nitrogen + true protein (m or o), true protein
only (A or A) or non-protein nitrogen only (e or o). iNDF = isolated
bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF ......................76

3-3 Total volatile fatty acid concentrations (LSmeans standard error) for 16 h in vitro
fermentations of SuNDF with media containing nitrogen in the form of non-protein
nitrogen + true protein (m), true protein only (A) or non-protein nitrogen only (*).
SuNDF = sucrose + isolated bermudagrass neutral detergent residue..................77

3-4 Acetate concentrations (LSmeans standard error) for 16 h in vitro fermentations
of SuNDF with media containing nitrogen in the form of non-protein nitrogen +
true protein (m), true protein only (A) or non-protein nitrogen only (e). SuNDF =
sucrose + isolated bermudagrass neutral detergent residue. ...............................77









3-5 Propionate concentrations (LSmeans standard error) for 16 h in vitro
fermentations of SuNDF with media containing nitrogen in the form of non-protein
nitrogen + true protein (m), true protein only (A) or non-protein nitrogen only (*).
SuNDF = sucrose + isolated bermudagrass neutral detergent residue..................78

3-6 Butyrate concentrations (LSmeans standard error) for 16 h in vitro fermentations
of SuNDF with media containing nitrogen in the form of non-protein nitrogen +
true protein (m), true protein only (A) or non-protein nitrogen only (e). SuNDF =
sucrose + isolated bermudagrass neutral detergent residue. ...............................78

3-7 Lactate concentrations (LSmeans standard error) for 16 h in vitro fermentations
of SuNDF with media containing nitrogen in the form of non-protein nitrogen +
true protein (m), true protein only (A) or non-protein nitrogen only (e). SuNDF =
sucrose + isolated bermudagrass neutral detergent residue. ...............................79

3-8 Ammonia nitrogen concentration (LSmeans standard error) for 16 h in vitro
fermentations with no substrate (o, A, o) or SuNDF as the substrate (m, A,*) and
media containing nitrogen in the form of non-protein nitrogen + true protein (o, m),
true protein only (A, A) or non-protein nitrogen only (o, e). SuNDF =
sucrose+isolated bermudagrass neutral detergent residue. .................................79

3-9 Total free amino acid concentration (LSmeans standard error) for 16 h in vitro
fermentations with no substrate (o, A, o) or SuNDF as the substrate (m, A,*) and
media containing nitrogen in the form of non-protein nitrogen + true protein (o, m),
true protein only (A, A) or non-protein nitrogen only (o, e). SuNDF =
sucrose+isolated bermudagrass neutral detergent residue. .................................... 80

3-10 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 16
h in vitro fermentation with no substrate (o, A, o) or SuNDF as the substrate
(m, A,*) and media containing nitrogen in the form of non-protein nitrogen + true
protein (o, m), true protein only (A, A) or non-protein nitrogen only (o, *).
SuNDF = sucrose+isolated bermudagrass neutral detergent residue....................80

3-11 Residual NDFOM for 16 h in vitro fermentations of iNDF (A; o, A, o) and SuNDF
(B; m, A, e) with media containing nitrogen in the form of non-protein nitrogen +
true protein (o, m), true protein only (A, A) or non-protein nitrogen only (o, *).
iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose+iNDF.81

3-12 Microbial crude protein yield for 16 h in vitro fermentations of iNDF (o, A, o) and
SuNDF (m, A, e) with media containing nitrogen in the form of non-protein
nitrogen + true protein (m or o), true protein only (A or A) or non-protein nitrogen
only (e or o). iNDF = isolated bermudagrass neutral detergent residue; SuNDF =
su cro se+ iN D F ........................................................................................................... 82

A-i Residual glucose content (LSmeans standard error) for 24 h in vitro fermentations
of SuNDF with initial medium pH, before addition of reducing solution or
inoculum, of 6.8 (m) or 5.6 (A). SuNDF = sucrose + isolated bermudagrass neutral
detergent residue ... .. .............................. ......... ........ ............... 124









A-2 Residual fructose content (LSmeans standard error) for 24 h in vitro
fermentations of SuNDF with initial medium pH, before addition of reducing
solution or inoculum, of 6.8 (m) or 5.6 (A). SuNDF = sucrose + isolated
bermudagrass neutral detergent residue. ...... ... ......................................... 125

A-3 Residual sucrose content (LSmeans standard error) for 24 h in vitro fermentations
of SuNDF with initial medium pH, before addition of reducing solution or
inoculum, of 6.8 (m) or 5.6 (A). SuNDF = sucrose + isolated bermudagrass neutral
detergent residue .......... ............... ......... ........ ............... 125

B-i Residual glucose content (LSmeans standard error) for 16 h in vitro fermentations
of SuNDF with media containing nitrogen in the form of non-protein nitrogen +
true protein (m), true protein only (A) or non-protein nitrogen only (e). SuNDF =
sucrose + isolated bermudagrass neutral detergent residue. ..............................126

B-2 Residual fructose content (LSmeans standard error) for 24 h in vitro
fermentations of SuNDF with media containing nitrogen in the form of non-protein
nitrogen + true protein (m), true protein only (A) or non-protein nitrogen only (*).
SuNDF = sucrose + isolated bermudagrass neutral detergent residue .................127

B-3 Residual sucrose content (LSmeans standard error) for 24 h in vitro fermentations
of SuNDF with media containing nitrogen in the form of non-protein nitrogen +
true protein (m), true protein only (A) or non-protein nitrogen only (e). SuNDF =
sucrose + isolated bermudagrass neutral detergent residue. ..............................127

C-i Residual glucose content (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 128

C-2 Residual glucose content (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + starch at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 129

C-3 Residual glucose content (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 129

C-4 Residual glucose content (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + sucrose (o) or starch (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate .............................................. 130









C-5 Residual glucose content (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + pectin (o) or starch (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate ............................................... 130

C-6 Residual glucose content (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate. ...... ......... ...................................... 131

C-7 Residual fructose content (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 3 1

C-8 Residual fructose content (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + starch at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 3 2

C-9 Residual fructose content (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 3 2

C-10 Residual fructose content (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + sucrose (o) or starch (A) at 120 mg
hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40
(m) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue;
NFC = non-neutral detergent fiber carbohydrate. ..................... ....................133

C-11 Residual fructose content (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + pectin (o) or starch (A) at 120 mg hexose
equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate ............................................... 133

C-12 Residual fructose content (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120 mg
hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40
(m) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue;
NFC = non-neutral detergent fiber carbohydrate. ..................... ....................134









C-13 Residual sucrose content (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 134

C-14 Residual sucrose content (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + starch at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 135

C-15 Residual sucrose content (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 135

C-16 Residual sucrose content (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + sucrose (o) or starch (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate ............................................... 136

C-17 Residual sucrose content (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + pectin (o) or starch (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate ............................................... 136

C-18 Residual sucrose content (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate .............................................. 137

C-19 Microbial glycogen yield (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 3 7

C-20 Microbial glycogen yield (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 3 8

C-21 Microbial glycogen yield (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120 mg
hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40
(m) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue;
NFC = non-neutral detergent fiber carbohydrate. ..................... .................... 138









C-22 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......139

C-23 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (120 mg air dry; x), and iNDF + starch at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......139

C-24 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 140

C-25 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (120 mg air dry) + sucrose (o) or starch (A) at 120 mg hexose equivalent, and
iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for sucrose:starch.
iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral
detergent fiber carbohydrate...................................................... ............... 140

C-26 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (120 mg air dry) + pectin (o) or starch (A) at 120 mg hexose equivalent, and
iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for pectin:starch.
iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral
detergent fiber carbohydrate...................................................... ................141

C-27 Fermentation pH (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate .............................................. 141

C-28 Acetate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 142

C-29 Acetate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + starch at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 142

C-30 Acetate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 143

C-31 Acetate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + sucrose (o) or starch (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate .............................................. 143









C-32 Acetate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + pectin (o) or starch (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate ............................................... 144

C-33 Acetate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate ............................................... 144

C-34 Propionate concentrations (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 4 5

C-35 Propionate concentrations (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + starch at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 4 5

C-36 Propionate concentrations (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 4 6

C-37 Propionate concentrations (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + sucrose (o) or starch (A) at 120 mg
hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40
(m) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue;
NFC = non-neutral detergent fiber carbohydrate. ..................... ....................146

C-38 Propionate concentrations (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + pectin (o) or starch (A) at 120 mg hexose
equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate ............................................... 147

C-39 Propionate concentrations (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120 mg
hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40
(m) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue;
NFC = non-neutral detergent fiber carbohydrate. ..................... ....................147


xviii









C-40 Butyrate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 148

C-41 Butyrate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + starch at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 148

C-42 Butyrate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 149

C-43 Butyrate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + sucrose (o) or starch (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate ............................................... 149

C-44 Butyrate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + pectin (o) or starch (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate ............................................... 150

C-45 Butyrate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate ............................................... 150

C-46 Total volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 5 1

C-47 Total volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + starch at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 5 1

C-48 Total volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 5 2









C-49 Total volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + sucrose (o) or starch (A) at 120 mg
hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40
(m) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue;
NFC = non-neutral detergent fiber carbohydrate. ..................... .................... 152

C-50 Total volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + pectin (o) or starch (A) at 120 mg hexose
equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate .............................................. 153

C-51 Total volatile fatty acid concentrations (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120 mg
hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40
(m) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue;
NFC = non-neutral detergent fiber carbohydrate. ..................... .................... 153

C-52 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24
h in vitro fermentations of iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o),
80 (A) or 120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral
detergent residue .... ............... .. ......... .............. ...............154

C-53 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24
h in vitro fermentations of iNDF (120 mg air dry; x), and iNDF + starch at 40 (o),
80 (A) or 120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral
detergent residue .... ............... .. ......... .............. ...............154

C-54 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24
h in vitro fermentations of iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o),
80 (A) or 120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral
detergent residue e. ..................................................................................... ........... 155

C-55 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24
h in vitro fermentations of iNDF (120 mg air dry) + sucrose (o) or starch (A) at 120
mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and
80:40 (m) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent
residue; NFC = non-neutral detergent fiber carbohydrate. ................................. 155

C-56 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24
h in vitro fermentations of iNDF (120 mg air dry) + pectin (o) or starch (A) at 120
mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and
80:40 (m) for pectin:starch. iNDF = isolated bermudagrass neutral detergent
residue; NFC = non-neutral detergent fiber carbohydrate. ...............................156









C-57 Branched chain volatile fatty acid concentrations (LSmeans standard error) for 24
h in vitro fermentations of iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120
mg hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and
80:40 (m) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent
residue; NFC = non-neutral detergent fiber carbohydrate. ...............................156

C-58 Lactate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 157

C-59 Lactate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + starch at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 157

C-60 Lactate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue.......158

C-61 Lactate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + sucrose (o) or starch (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate. ...... ... ....................................... 158

C-62 Lactate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + pectin (o) or starch (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate .............................................. 159

C-63 Lactate concentrations (LSmeans standard error) for 24 h in vitro fermentations
of iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate .............................................. 159

C-64 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 160

C-65 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (120 mg air dry; x), and iNDF + starch at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 160

C-66 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o), 80 (A) or 120 mg (0)
hexose equivalent. iNDF = isolated bermudagrass neutral detergent residue....... 161









C-67 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (120 mg air dry) + sucrose (o) or starch (A) at 120 mg hexose equivalent, and
iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for sucrose:starch.
iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral
detergent fiber carbohydrate...................................................... ............... 161

C-68 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (120 mg air dry) + pectin (o) or starch (A) at 120 mg hexose equivalent, and
iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for pectin:starch.
iNDF = isolated bermudagrass neutral detergent residue; NFC = non-neutral
detergent fiber carbohydrate...................................................... ............... 162

C-69 Residual NDF OM (LSmeans standard error) for 24 h in vitro fermentations of
iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120 mg hexose equivalent,
and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate ............................................... 162

C-70 Microbial crude protein yield (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + sucrose at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 6 3

C-71 Microbial crude protein yield (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + starch at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 6 3

C-72 Microbial crude protein yield (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry; x), and iNDF + pectin at 40 (o), 80 (A) or
120 mg (o) hexose equivalent. iNDF = isolated bermudagrass neutral detergent
re sid u e ........................................................................................................... .. 1 6 4

C-73 Microbial crude protein yield (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + sucrose (o) or starch (A) at 120 mg
hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40
(m) for sucrose:starch. iNDF = isolated bermudagrass neutral detergent residue;
NFC = non-neutral detergent fiber carbohydrate. ..................... ....................164

C-74 Microbial crude protein yield (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + pectin (o) or starch (A) at 120 mg hexose
equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40 (m) for
pectin:starch. iNDF = isolated bermudagrass neutral detergent residue; NFC =
non-neutral detergent fiber carbohydrate ............................................... 165









C-75 Microbial crude protein yield (LSmeans standard error) for 24 h in vitro
fermentations of iNDF (120 mg air dry) + sucrose (o) or pectin (A) at 120 mg
hexose equivalent, and iNDF + NFC combinations in ratios of 40:80 (A) and 80:40
(m) for sucrose:pectin. iNDF = isolated bermudagrass neutral detergent residue;
NFC = non-neutral detergent fiber carbohydrate. ..................... .................... 165


xxiii
















LIST OF ABBREVIATIONS


ADF acid detergent fiber
ApH acidic pH medium
B non-protein nitrogen+ true protein medium
BCVFA branched chain volatile fatty acid
BW body weight
C true protein only medium
CP crude protein
DM dry matter
DMI dry matter intake
GLY microbial glycogen
iNDF isolated neutral detergent residue
MCP microbial crude protein
MCPeff microbial crude protein efficiency
N Nitrogen
NDF neutral detergent fiber
NDFCP neutral detergent fiber crude protein
NDFOM neutral detergent organic matter
NFC non-neutral detergent fiber carbohydrate
NH3-N ammonia nitrogen
NpH neutral pH medium
NPN non-protein nitrogen
NRC National Research Council
NSC non-structural carbohydrate
OM organic matter
OMD organic matter digested
OMI organic matter intake
Pe Pectin
RDP rumen degradable protein
St Starch
Su Sucrose
SuNDF sucrose + isolated neutral detergent residue
TCA trichloroacetic acid
U non-protein nitrogen only medium
VFA volatile fatty acid


xxiv















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

EFFECT OF NONFIBER CARBOHYDRATES ON PRODUCT YIELD AND FIBER
DIGESTION IN FERMENTATIONS WITH MIXED RUMINAL MICROBES

By

Lucia Holtshausen

December 2004

Chair: Mary Beth Hall
Major Department: Animal Sciences

Effects of nonfiber carbohydrate (NFC) supplementation on fiber digestion and

fermentation product yields were examined in three in vitro fermentation studies. Studies

1 and 2 respectively examined the effects of medium pH (5.6 vs. 6.7) and nitrogen source

(non-protein nitrogen (U) vs. true protein (C) vs. mixture (B)) on fermentation of isolated

neutral detergent residue (iNDF) with or without sucrose (Su). Study 3 examined the

effect of supplementing iNDF with starch, sucrose, pectin or their combinations.

Anaerobic fermentations of 24 h (Studies 1 and 3) and 16 h (Study 2) were performed in

batch culture with Goering and Van Soest medium and rumen inoculum. Fermentation

samples were analyzed for residual substrate, pH, iNDF digestion, and microbial

fermentation products.

In Study 1, maximum microbial crude protein (MCP) and glycogen (GLY) yields

for Su+iNDF were greater at pH 6.7 (MCP: 19.4 mg; GLY:6.0 mg) than those at pH 5.7

(MCP: 11.1 mg; GLY:3.5mg). At pH 6.7, 24 h iNDF digestion was greater for Su+iNDF









(42.4%) than for iNDF (26.4%) and the reverse was true at pH 5.6 (Su+iNDF: 2.2%;

iNDF: 7.8%).

In Study 2, maximum MCP yields from fermentations of Su+NDF in media

containing C (15.7 mg), and B (14.3 mg) were greater than those containing U (8.05 mg).

At 16 h, iNDF digestion for Su+iNDF was lower for U vs. B and C, with no difference

among treatments for iNDF. Maximum GLY was similar among nitrogen treatments.

In Study 3, sucrose decreased pH more than NFC combinations (sucrose+starch,

starch+pectin, sucrose+pectin) followed by pectin, starch and iNDF (6.83, 6.87, 6.89,

7.03 and 7.13, respectively). Residual iNDF was increased by sucrose (60.6%), not

affected by starch (61.5%), and decreased by pectin (65.7%) and NFC combinations

(63.6 %) compared to iNDF fermented alone (61.8%). Maximum MCP yield was

greatest for NFC combinations followed by pectin, starch, sucrose and iNDF fermented

alone (22.4, 18.6, 16.6, 14.8 and 5.12 mg, respectively).

The various types of NFCs as well as pH and nitrogen source altered the yield of

microbial products and extent of fiber digestion. Treatment of NFCs as a uniform entity

in ruminant nutrition is not warranted.


xxvi














CHAPTER 1
REVIEW OF LITERATURE: MICROBIAL FERMENTATION OF NON-NEUTRAL
DETERGENT FIBER CARBOHYDRATES, AND HOW IT MAY RELATE TO
ANIMAL PERFORMANCE

Introduction

In order to meet the nutritional needs of high producing beef and dairy cattle, it is

necessary to supplement diets with energy and protein rich feeds. Grain and byproduct

feeds, such as molasses, have been used to increase the energy density of ruminant diets

(Huntington, 1997; Lykos et al., 1997). Other byproduct feeds, such as soybean hulls

(Royes et al., 2001), almond hulls (Grasser et al., 1995), sugar beet pulp (Mansfield et al.,

1994) and citrus pulp (Ammerman et al., 1963; Van Horn et al., 1975; Fegeros et al.,

1995; Leiva et al., 2000; Arthington et al., 2002), have been used as substitutes for grains.

These byproduct feeds have lower starch contents and higher neutral detergent soluble

fiber (NDSF) contents (compared to grain feeds), and may have high sugar contents

(Hall, 1998). Starch, sugars (mono- and oligosaccharides) and NDSF (non-starch, non-

neutral detergent fiber polysaccharides) are three of the major components of the

carbohydrate fraction of feeds referred to as non-neutral detergent fiber carbohydrates

(NFCs).

The NFC fraction of feedstuffs is estimated from the following calculation: NFC =

100 crude protein ether extract ash neutral detergent fiber + neutral detergent

insoluble crude protein (National Research Council [NRC], 2001). The terms non-

structural carbohydrates (NSCs) and NFCs have at times been used interchangeably for

the fraction derived by this calculation. However, NSCs refer to cell contents, and









include organic acids (which are not carbohydrates), mono- and oligosaccharides, starch

and fructans, whilst NFCs also include soluble fiber: pectic substances, P-glucans and

galactans (Van Soest et al., 1991). Therefore, NFCs include structural and non-structural

carbohydrates, as well as fibrous and non-fibrous carbohydrates (Hall, 1998).

From a nutritional standpoint, some of the carbohydrates in plants are also

designated as dietary fiber. This term refers to the non-starch polysaccharides that are

not digestible by mammalian enzymes. Dietary fiber encompasses both NDSF and

neutral detergent fiber (NDF), thus, including both cell wall carbohydrates and some cell

contents. Cellulose, hemicelluloses, lignin, pectic substances, P-glucans, fructans and

galactans are all dietary fiber. Pectic substances, P-glucans, fructans and galactans are

soluble in neutral detergent solution, and thus are included in the NFC fraction.

The NRC (2001) applies a total digestible nutrient content of 98% to the NFC

fraction and it can therefore play a major role in the nutrient supply to the animal. The

rest of this discussion will focus on sucrose, starch and pectic substances (more

specifically, pectin), as three of the major components of NFCs.

Chemical Structure and degradation of NFC Sources

When trying to understand or predict animal responses to supplementation of NFC

sources it is necessary to keep in mind that this carbohydrate fraction is by no means a

homogenous entity, chemically or nutritionally. Some of the fundamental differences of

the individual components are found in their chemical structure.

Sucrose

Sucrose is the primary vehicle for energy transport in plants and the majority of

plants convert sucrose into polymeric forms for storage (Van Soest, 1994). Sucrose, and

its constituent monosaccharides glucose and fructose, are the predominant saccharides of









the mono- and oligosaccharide component of NFC and are found in byproduct feeds such

as molasses (Kunkle et al., 2000), sugar beet pulp (Hall, 2002) and citrus pulp (Ben-

Ghedalia et al., 1989). Sucrose is a disaccharide consisting of single glucose and fructose

monomers linked through an a-1--2 linkage. It is considered to be 100% degradable in

the rumen (Sniffen et al., 1992) and is reported to be fermented at a rate as high as

300%/h (Sniffen et al., 1983). Sucrose is hydrolyzed to glucose and fructose by the

enzyme sucrase (Van Soest, 1994). Ruminal bacteria that ferment sucrose include

Streptococcus bovis, Lachnospira multiparus, Lactobacillus ruminis, Lactobacillus

vitulinis, Clostridium longisporum, Eubacterium cellulosolvens, and some strains of

Eubacterium ruminantium, Butyrivibriofibrisolvens, Ruminococcus albus, Ruminococcus

flavefaciens, Megaspaera elsdenii, Prevotella spp., Selenomonas ruminantium and

Succinivibrio dextrinosolvens (Stewart et al., 1997).

Starch

Starch is a polymer of glucose molecules and is the major storage carbohydrate in

most cereal grains. It consists of amylose, a predominantly linear a-(1--4) linked

polymer, and amylopectin, an a-(1--4) linked polymer with a-(1--6) linked branches,

which can be present in various ratios. Amylopectin comprises 70 to 80% of most cereal

starches and amylose 20 to 30% (Rooney and Pflugfelder, 1986). The proportions of

these two polysaccharides appear to affect the digestion characteristics of starch. There

are contradictions in the literature as to the ease of hydrolysis of amylose as compared to

amylopectin. Rooney and Pflugfelder (1986) described starch granules as having

crystalline and amorphous areas, with amylopectin comprising the majority of the

crystalline region and amylose that of the amorphous region. According to Rooney and

Pflugfelder (1986) amylase attack starts in the amorphous region and hydrolysis of the









crystalline areas occurs more slowly. However, the author also stated that the

digestibility of starch is inversely proportional to the amylose content. According to Piva

and Masoero (1996) amylose is slowly degraded in the rumen, whereas amylopectin is

more rapidly degraded. The reason for the conflicting statements may be because

amylose and amylopectin both contain crystalline areas and the crystalline area of

amylose has greater crystal strength (Van Soest, 1994). Therefore, hydrolysis may start

in the amorphous region of amylose, exposing the amylopectin for hydrolysis, while the

crystalline region of amylose hydrolyzes more slowly. Nonetheless, starch fermentation

in the rumen is considered to be extensive, but can vary from 40 to 90% (NRC, 2001)

depending on factors such as structure (amylose/amylopectin ratio; (Piva and Masoero,

1996), plant source (Rooney and Pflugfelder, 1986) and processing or physical form

(Baldwin and Allison, 1983).

Starch can be degraded by ruminal microbial enzymes as well as enzymes in the

small intestine of the animal. A series of enzymes are required to degrade amylose and

amylopectin to glucose in both the rumen and small intestine. These include randomly

acting endo-a-amylases releasing maltodextrins from amylose and P-amylases removing

maltose units from the non-reducing end of the chain. Approximately 50% of

amylopectin can be degraded by P-amylase to maltose. The residue is hydrolyzed by

glucoamylase (cleaving a-(1--4) linkages), and a-dextrin-6-glucanohydrolase and

isoamylase (cleaving at the a-(1--6) linked branch points). Maltose and maltodextrins

are degraded to glucose by a-glucosidase (Chesson and Forsberg, 1997). There are

numerous amylolytic ruminal bacteria, which include Ruminobacter amylophilus,









Prevotella ruminicola, Succinimonas amylolytica, S. bovis, S. ruminantium, B.

fibrisolvens, E. ruminantium and Clostridium spp. (Cotta, 1988).

Pectic Substances

Pectic substances are found in the middle lamella and other cell wall layers (Van

Soest, 1994), but are not covalently linked to the lignified portions. They are almost

completely digested (90-100%) in the rumen (Nocek and Tamminga, 1991; NRC, 2001).

Pectic substances are a family of complex molecules that contain a great variety of

monomers and potential branch-points. The pectin backbone consists of galacturonic

acid monomers linked via a-(1--4) linkages, and rhamnose inserts. With the addition of

neutral sugar side chains, made up largely of arabinose and galactose, bound to the

rhamnose inserts, these complex molecules are referred to as pectic substances (Jarvis,

1984). Various degrees of methoxylation (Jarvis, 1984) and acetylation (Marounek and

Dugkova, 1999) of the galacturonic acid backbone are possible. The acid groups in the

backbone can also be associated with calcium ions (Van Soest, 1994).

Animals do not have the enzymes to digest pectin, but microorganisms in the

rumen do (McDonald et al., 1995). At least two enzymes, a methylesterase and

polygalacturonidase, are required for pectin hydrolysis (Baldwin and Allison, 1983).

Pectin-utilizing bacteria include some of the prominent ruminal populations such as

Fibrobacter succinogenes, P. ruminicola, B. fibrisolvens, S. bovis and Lachnospira

multiparus (Czerkawski and Breckenridge, 1969; Gradel and Dehority, 1972; Baldwin

and Allison, 1983).

NFCs and Microbial Fermentation Products

Microbial fermentation products play an important part in the nutrient supply to the

ruminant animal and thus also in animal performance. Major products of microbial









fermentation of carbohydrates include organic acids, which are absorbed through the

ruminal wall and serve as a source of energy for the animal, and microbial mass, which

provides potentially metabolizable nutrients in the form of protein, glycogen, and lipids

to the animal when they pass to the small intestine. The NFC fraction is considered a

good source of readily available energy for microbial growth (Ariza et al., 2001). It has

been suggested that microbial growth is directly proportional to the rate of carbohydrate

degradation (Russell et al., 1992). As documented in the Cornell Net Carbohydrate and

Protein System model, simple sugars are considered to have a fast degradation rate, and

starch and pectin an intermediate rate (Sniffen et al., 1992). This would imply that

supplementation with sugars would yield more microbial mass and other microbial

fermentation products compared to starch and pectin. There is evidence, both in vitro and

in vivo, of differences in fermentation characteristics among individual NFC components

(Table 1-1). Sometimes these results have not been consistent with the notion that

individual NFC components with faster degradation rates result in greater microbial yield

(Hall and Herejk, 2001). It is important to understand how individual NFC components

differ in their effect on microbial product yield and efficiency of yield to help predict and

explain some of the variation seen in animal performance when supplementing with

NFCs. Differences among NFC components regarding microbial fermentation may also

imply that the complement of NFCs in a particular feedstuff is important when predicting

animal response.

Organic Acids

Total volatile fatty acids

Total volatile fatty acid (VFA) production is generally similar among different

NFC sources (Table 1-1) both in vitro (Mansfield et al., 1994; Ariza et al., 2001) and in









vivo (Ben-Ghedalia et al., 1989; Khalili and Huhtanen, 1991a; Chamberlain et al., 1993;

Moloney et al., 1994; O'Mara et al., 1997a; Leiva et al., 2000; Sannes et al., 2002;

Voelker and Allen, 2003c). However, Bach et al. (1999) reported an increase (P > 0.05)

in total VFA concentration for cracked corn compared with beet pulp and molasses in a

continuous culture study. In contrast, total VFA concentration increased (P = 0.01) or

tended to increase (P = 0.07) in lactating dairy cows fed a total mixed ration (TMR)

containing dried citrus pulp and high moisture ear corn in a 50:50 ratio compared to cows

receiving a TMR containing high moisture ear corn or cracked shelled corn, respectively

(Broderick et al., 2002b). The varying results in these studies may be due to the fact that

the combinations of NFCs that were compared differed, which may imply that the effect

on VFA production from supplementation with individual NFC sources may not be

additive.

Acetate, propionate and butyrate are the major VFAs included in the total VFA

concentration. Despite giving relatively similar total VFA yields there may be

differences in the relative proportions of individual VFAs from different NFC sources.

Acetic acid

Acetate (acetic acid) is a lipogenic nutrient, a precursor of fatty acid synthesis and

ultimately of milk fat synthesis in the mammary gland (Van Soest, 1994). Starch and

sucrose (Sutton, 1979; Khalili and Huhtanen, 1991a; Chamberlain et al., 1993; Moloney

et al., 1994; Heldt et al., 1999) have been associated with relative decreases in ruminal

acetate concentration, whereas pectin had either no effect (Van Vuuren et al., 1993; Leiva

et al., 2000) or increased (Broderick et al., 2002b; Voelker and Allen, 2003c) acetate in

the rumen (Table 1-1). In vitro fermentations of different carbohydrates showed a greater









acetate production from pectin compared to starch and sucrose (P < 0.05), which did not

differ from each other (P > 0.05; [Strobel and Russell, 1986]).

The effect of sugars on the ruminal molar proportion of acetate in vivo may depend

on the amount of sucrose or glucose included in the diet (Table 1-1). Sucrose

supplementation at 10% of silage DM intake did not affect ruminal acetate molar

proportion for sheep compared to those on the silage control diet (P > 0.05; [Charmely et

al., 1991]). When sucrose was substituted for corn at 3.2% of diet DM in a diet for

lactating dairy cows, acetate production was also not affected (P = 0.15; [Sannes et al.,

2002]). Dextrose (glucose) at 5.6% of diet DM did not affect ruminal acetate proportions

in heifers compared to a medium concentrate diet containing 39.7% ground barley, or the

control diet containing 10% ground barley (P > 0.05; [Piwonka et al., 1994]). However,

when starch or sucrose was supplemented at 200g/d (approximately 5% of daily diet DM)

to a grass silage diet, ruminal acetate proportions for sheep on the starch-supplemented

diet did not differ compared to those on the control diet, whereas sheep on the sucrose-

supplemented diet had decreased ruminal acetate proportions (P < 0.05; [Chamberlain et

al., 1993]). When cane molasses, a source of sugars, was fed to steers at 61% of DM

intake, ruminal acetate proportions was decreased compared to steers fed a diet with the

same amount of barley, a starch source (P < 0.01; [Moloney et al., 1994]).

Pectin is reported to ferment primarily to acetate (Czerkawski and Breckenridge,

1969; Marounek et al., 1985). When citrus pectin was fermented in cultures of B.

fibrisolvens 787 and P. ruminicola AR29, 73.7 and 57.3% of metabolite carbon was

captured in acetate, respectively (Marounek and Dugkova, 1999). Citrus pulp can contain

25.2 to 43.7% neutral detergent-soluble fiber (Hall, 2002), of which pectin is a major









component. Several continuous culture studies reported an increase in acetate proportion

for fermentations of beet pulp and citrus pulp compared to corn (Table 1-1), whether it

was included at the same concentration as corn (P < 0.05; [Bach et al., 1999]) and P =

0.03; [Ariza et al., 2001]) or replaced a portion of the corn (P < 0.05; [Mansfield et al.,

1994]). This effect was also seen in vivo when increasing concentrations of citrus pulp

substituted for high moisture corn in lactating dairy cow diets resulted in a linear increase

(P < 0.01) in the ruminal acetate proportion (Voelker and Allen, 2003c). Ben-Ghedalia et

al. (1989) also reported increased ruminal acetate proportions for cannulated rams fed

dried citrus pulp compared to those fed barley (P < 0.05). It would appear that

fermentation of pectin in general increases the molar proportion of acetate compared to

fermentation of sugars and starch, while sugars often decrease the acetate proportion

when compared to starch.

Propionic acid

Propionate propionicc acid) is a precursor for glucose synthesis in the liver and thus

important for the glucogenic energy supply to the ruminant. In vitro fermentation with

mixed ruminal bacteria yielded similar propionate concentrations from starch and sucrose

(P > 0.05; [Strobel and Russell, 1986]), Table 1-1). The effect of sugars compared to

starch on ruminal propionate proportion varies among in vivo studies. In some in vivo

studies ruminal molar proportions of propionate did not differ between sugars and starch,

whether small amounts (5.6% dextrose; Piwonka et al., 1994) or larger amounts (61%

molasses; Moloney et al., 1994) of sugar were added to the diet. In contrast, ruminal

propionate molar proportions in sheep on a starch-supplemented diet was similar to that

of sheep fed the control ryegrass silage diet (P > 0.05), whereas ruminal propionate

proportions increased (P < 0.05) for sheep fed a sucrose supplementation (Chamberlain et









al., 1993). In another contrasting study, ruminal molar proportions of propionate tended

to increase with starch supplementation (P = 0.11) compared to supplementation of

sugars (sucrose, glucose and fructose) when a low amount of ruminally degradable

protein (RDP; 0.031% BW/d) was supplemented to steers, and increased (P < 0.01)

ruminal propionate proportions when a higher amount (0.122% BW/d) of RDP was

supplemented (Heldt et al., 1999). It may be that other components of the diet such as

protein alter the yield of propionate from NFCs.

Pectin yielded less (P < 0.05) propionate compared to starch and sucrose when

fermented in vitro with mixed ruminal bacteria (Strobel and Russell, 1986, Table 1-1).

Citrus pectin fermented by a P. ruminicola AR29 in vitro culture yielded a small amount

of propionate (3.2 mmol/L), while no propionate production was detected in the culture

with B. fibrisolvens 787 (Marounek and Dugkova, 1999). Corn additions increased

propionate molar proportions in continuous culture fermentations when compared to

similar amounts of citrus pulp (P = 0.02; [Ariza et al., 2001] and beet pulp (P < 0.05;

[Bach et al., 1999]). Broderick et al. (2002b) also reported higher ruminal propionate

proportions in lactating dairy cows fed high moisture ear corn (P < 0.01) and cracked

shelled corn (P = 0.04) compared to cows fed a diet in which citrus pulp substituted for

50% of high moisture ear corn. However, when beet pulp was substituted for 50% of the

corn in a continuous culture study no difference was found for the molar proportion of

propionate (P > 0.05, [Mansfield et al., 1994]). Other researchers also reported no effect

on ruminal propionate proportions in lactating dairy cows when replacing beet pulp for

ground corn (O'Mara et al., 1997a) and replacing dried citrus pulp for corn hominy

(Leiva et al., 2000). The varied propionate response when feeding citrus and beet pulp









could be a result of the variation in composition of these feedstuffs, especially in the ratio

of sugars to neutral detergent soluble fiber. It would appear that pectin yields less

propionate than sugars and starch, with no clear difference between the latter two NFCs.

Butyric acid

Butyrate (butyric acid) supplies energy to the animal, mainly to the heart and

skeletal muscle, in the form of P-hydroxybutyrate (a ketone body; McDonald et al.,

1995). It is lipogenic and can be used for the production of fat. Ruminally-produced

butyrate is converted to P-hydroxybutyrate in the ruminal epithelial cells, and is

considered more effective than propionate or acetate in enhancing development of

ruminal papillae (Van Soest, 1994). Overall, it would appear that sucrose yields more

butyrate than other NFCs (Table 1-1). In vitro fermentations with mixed ruminal

microorganisms yielded more butyrate from sucrose compared to starch (P < 0.05),

which in turn yielded more butyrate than pectin (P < 0.05; [Strobel and Russell, 1986]).

Several in vivo studies also reported increased butyrate production from sucrose

compared to starch. Ruminal butyrate proportions in cannulated steers increased with

sugar (sucrose, glucose and fructose) supplementation compared to supplementation with

starch (Heldt et al., 1999). Khalili and Huhtanen (1991a) reported greater ruminal molar

proportions of butyrate for bulls consuming a sucrose-supplemented diet compared to a

grass silage and barley-based diet. Steers fed a molasses-based diet also had increased

ruminal butyrate proportions compared to those fed a barley-based diet (Moloney et al.,

1994).

Studies that have evaluated the fermentation of pectin or feeds that are reported to

contain substantial amounts of pectin have shown differences among microorganisms in

the yield of butyrate. Pectin fermentation in a B. fibrisolvens 787 culture yielded a small









amount of butyrate (2.6 mmol/L), while no butyrate production was detected in a culture

with P. ruminicola AR29 (Marounek and Dugkova, 1999). In vivo comparisons of the

fermentation of feeds high in starch and those that typically contain a high proportion of

pectin (citrus and beet pulps) have shown no difference (Ben-Ghedalia et al., 1989; Leiva

et al., 2000) or an increase in ruminal butyrate concentration (Broderick et al., 2002b;

Voelker and Allen, 2003c) for animals consuming diets containing pulps. Citrus pulp can

contain between 12.5 and 40.2% sugars, and sugar beet pulp between 12.8 and 24.7%

(Hall, 2002). The increase in the proportion of butyrate in these studies may be a result

of the fermentation of sugar rather than of the soluble fiber content. This emphasizes the

need to know the NFC complement of a feedstuff when evaluating the effect of

supplementation on fermentation and animal performance.

Branched chain volatile fatty acids

The branched chain volatile fatty acids (BCVFAs), isobutyric, iso-valeric and 2-

methylbutyric acid result from the deamination of valine, leucine and iso-leucine,

respectively (Van Soest, 1994). Branched chain VFAs serve as carbon skeletons to

ruminal microorganisms for the synthesis of microbial crude protein (MCP) from

ammonia. In fact, the value of amino acids to cellulolytic organisms that have an

obligate need for BCVFAs appears to be mainly as a source of BCVFAs (Stern, 1986).

Sheep fed diets supplemented with sucrose or starch showed decreased proportions

of ruminal BCVFAs compared to those fed a silage control diet (Chamberlain et al.,

1993, Table 1-1), and the proportion of BCVFAs for sheep fed the starch-supplemented

diet (2.8 mol/100 mol) was numerically higher than for those fed the sucrose supplement

(1.8 mol/100 mol; difference was not statistically tested). The ruminal concentrations of

BCVFAs for lactating dairy cows were greater for animals fed a corn control diet









compared to those receiving a diet with sucrose (P = 0.02) substituted for corn at 3.2 %

of diet DM (Sannes et al., 2002). Fermentation of corn by mixed ruminal

microorganisms in continuous culture studies gave higher proportions of BCVFAs as

compared to citrus pulp (P = 0.03; [Ariza et al., 2001]) and sugar beet pulp (P > 0.05;

[Mansfield et al., 1994] and P < 0.05; [Bach et al., 1999]). The apparently consistent

thread here is that BCVFA concentrations are less for diets with more sucrose relative to

starch.

Lactic acid

Compared to acetate, butyrate and propionate (average pKa = 4.8), lactate (lactic

acid, pKa = 3.1) is a 10-fold stronger acid (Dawson et al., 1997). An increase in lactate

concentration therefore has a greater potential to decrease ruminal pH. The fermentation

of sugars and starch can yield lactate (Strobel and Russell, 1986), whereas pectin

fermentation is generally not associated with lactate production (Strobel and Russell,

1986; Hatfield and Weimer, 1995). In vitro fermentations of sucrose with mixed ruminal

microorganisms gave a higher lactate concentration compared to fermentations with

starch (P < 0.05; [Strobel and Russell, 1986], Table 1-1). Heldt et al. (1999) also

reported higher ruminal proportions of lactate for steers fed sugar supplements (sucrose,

glucose, fructose) compared to those fed starch. However, in a study with cannulated

steers, animals fed a barley-based diet tended to have higher ruminal concentrations of L-

lactate compared to those receiving a molasses-based diet (P = 0.09; [Moloney et al.,

1994]). This difference in lactate production response may have been a result of a

difference in the source of starch (corn starch vs. barley) and sugar (sucrose, glucose and

fructose vs. molasses) supplemented in the two studies.









Although pectin is generally not associated with the production of lactate, pectin

fermentation has been shown to yield small amounts of lactate (Czerkawski and

Breckenridge, 1969). Cultures of B. fibrisolvens 787 and P. ruminicola AR29 both

produced small amounts of lactate (1.5mmol/L and 0.4mmol/L, respectively) from the

fermentation of citrus pectin (Marounek and Dugkova, 1999). In vivo studies showed no

effect on lactate production in animals fed diets containing citrus pulp (P = 0.34; [Leiva

et al., 2000]) or sugar beet pulp (P = 0.72; [Voelker and Allen, 2003c]) compared to those

fed corn hominy and high moisture corn supplements, respectively. In general, pectin is

not expected to yield lactate to the extent that sugars and starch may.

Microbial Mass

Microbial composition

The microbial mass that flows from the rumen to the small intestine forms a major

part of the metabolizable nutrient supply to the ruminant animal. The composition of the

microorganisms determines the potential specific nutrient contribution to the small

intestine. Bacteria typically contain 50% protein, 20% RNA, 3% DNA, 9% lipid and

18% carbohydrate, but this composition can change dramatically (Nocek and Russell,

1988). The two main components of microbial mass that contribute to the metabolizable

nutrient supply in the small intestine are MCP and microbial storage carbohydrate (a-

glucan). Bacterial amino nitrogen as a percentage of total nitrogen has been considered

as relatively constant, but it can range from 54.9 to 86.7%, with an average of 66.5%

(Clark et al., 1992). Large changes may also be seen in microbial glycogen content,

especially when cultures are starved for nutrients other than energy (McAllan and Smith,

1974; Nocek and Russell, 1988).









There are a variety of factors that can affect microbial growth, of which supply of

carbohydrate and nitrogen (Stern, 1986; Hoover and Stokes, 1991; Clark et al., 1992)

appear to be the most important. Another factor that may affect the efficiency of

microbial growth is pH (Russell et al., 1992). The rest of this discussion will focus on the

synthesis of MCP and glycogen from fermentation of starch, sucrose and pectin. The

effect of nitrogen source (ammonia nitrogen, amino acids) and pH on microbial

fermentation product yield from NFCs will also be considered.

Microbial protein yield

Microbial crude protein has been reported to supply from 34 (Clark et al., 1992);

summary of 31 articles) to 80 (Owens and Bergen, 1983; Stern, 1986) or even 89%

(Clark et al., 1992) of the total amino acid nitrogen entering the small intestine of

ruminants (Owens and Bergen, 1983; Stern, 1986). Microbial crude protein is considered

to have a good amino acid balance relative to the animal's requirements (Clark et al.,

1992) with a mean true digestibility of 84.7% (Storm et al., 1983). Accordingly, MCP is

an important source of true protein to the animal.

Carbohydrate fermentation in the rumen provides both energy in the form of ATP,

and carbon skeletons for MCP synthesis. The amount of carbohydrate and its rate of

fermentation regulate microbial metabolism and are in turn regulated by the physical and

chemical form of carbohydrates (Stern et al., 1994). Hall and Herejk (2001) compared

the MCP yield from sucrose, starch and pectin in an in vitro fermentation with mixed

ruminal microorganisms. Microbial growth was initiated most rapidly on sucrose,

followed by pectin and starch. Maximal yield was greatest for starch compared to

sucrose and pectin (P < 0.01), which did not differ from each other (P = 0.30). An

explanation offered for the proportional difference between maximal yields of MCP for









pectin and starch was the difference between the NFCs in the amount of hexose, and

relative amounts of carbon available to the microorganisms. However, this approach still

overestimated the theoretical maximal yield of MCP from sucrose.

In an in vivo study, Cameron et al. (1991) found no effect on microbial nitrogen

flow to the small intestine and efficiency of microbial crude protein synthesis (MCPeff)

in response to starch and dextrose supplementation to mid-lactation Holstein cows.

Dijkstra et al. (1992) suggested that variation in the results on MCP yield and efficiency

might be due to the lack of correction for microbial a-glucan reaching the small intestine.

For lack of a convenient method to distinguish between dietary starch that escapes

degradation in the rumen and a-glucan (glycogen) stored in ruminal microorganisms,

starch measured as glucose hydrolyzed from a-glucan in the duodenal digesta will

include both fractions. Therefore undigested feed starch content of digesta in the

duodenum will be overestimated, and ruminal digestion of starch will be underestimated,

and that of dextrose, overestimated. Expressing MCP yield as a proportion of the

carbohydrate digested in the rumen, with an underestimation of starch digestion in the

rumen, will lead to overestimation of the MCPeff for starch. The efficiency of microbial

yield from dextrose would be underestimated, because the unfermented dextrose in the

form of glycogen would not be accounted for.

In a continuous culture system, Mansfield et al. (1994) found an increase (P < 0.05)

in non-ammonia nitrogen flow when beet pulp was substituted for corn, but there was no

effect on MCPeff (P > 0.05) and bacterial crude protein production (P > 0.05). If there

was less degradation of crude protein from the feed, this may have resulted in a lower

ammonia nitrogen concentration and less available nitrogen for MCP synthesis, which









could explain why no difference in bacterial crude protein production was reported

between beet pulp and corn. In the study by Mansfield et al. (1994) there were indeed

decreased ammonia nitrogen concentrations in fermentations of beet pulp compared to

fermentations of corn (P < 0.05).

Microbial a-glucan

Many species of ruminal bacteria produce polysaccharide, and some can store large

amounts as an intracellular reserve (Thomas, 1960; John, 1984; Lou et al., 1997).

However, studies with P. ruminicola B14 (Russell, 1992) and F. succinogenes S85

(Maglione and Russell, 1997) indicated that ruminal bacteria might have a limited

capacity to store polysaccharides as shown by a decrease of the viable cell number when

the polysaccharide:protein ratio of the cultures exceeded 1.0.

Little information exists on microbial a-glucan production from different

carbohydrate sources. As mentioned earlier, it is difficult to distinguish microbial a-

glucan from dietary starch that passed through the rumen undegraded. McAllan and

Smith (1974) reported a higher microbial a-glucan content for animals on a diet with

more than 70% concentrates (barley and flaked corn). In the same study, time after

feeding also had an effect, with an increased microbial a-glucan content from feeding

through four to six hours after feeding. McAllan and Smith (1974) established a ratio of

individual carbohydrates to nucleic acids in samples of ruminal bacteria to use as an

estimate of the contribution of microbial carbohydrate in the duodenum. This may not be

a very accurate way to quantify microbial a-glucan content. Craig et al. (1987)

confirmed the change in microbial a-glucan content with time after feeding, and added

that particle-associated microbial populations had higher a-glucan content compared to

the liquid-associated populations in the rumen. Therefore, the ratio of individual









carbohydrates to nucleic acids will depend on the method of sampling and isolating

ruminal bacteria, as well as time of sampling.

Ruminal microorganisms may incorporate and store carbohydrate as a-glucan

under conditions of excess available carbohydrate (shortly after feeding) and potentially

limiting nitrogen supply. McAllan and Smith (1974) reported diurnal variations in the a-

glucan content of ruminal bacteria. It is possible that when the supply of available

dietary carbohydrate runs out, microorganisms utilize the storage carbohydrate as a

source of energy. This stored carbohydrate can also become available to other

microorganisms upon cell lysis or it can pass to the small intestine and become part of the

glucose supply to the animal.

Other Factors Which Influence Microbial Product Yield

Nitrogen Source

Most ruminal microorganisms can synthesize MCP with a non-protein nitrogen

(NPN) source such as urea, as the sole source of nitrogen (Oltjen, 1969). In a review of

several studies Wallace et al. (1997) concluded that microbial nitrogen derived from

ruminal ammonia nitrogen can range from 40 to 100%. Maximal in vitro microbial

growth has been reported at ammonia nitrogen concentrations of 5 to 8 mg/100 ml (Satter

and Slyter, 1974) in continuous culture studies with purified substrates (starch, cerelose,

wood pulp), a concentrate based diet (cracked corn) or a forage-concentrate combination

(cracked corn, cerelose, lucerne hay, timothy hay). Hume (1970) reported maximum in

vivo MCP synthesis at a ruminal ammonia nitrogen concentration of approximately 9

mg/100ml in sheep fed diets containing urea only or a 50:50 ratio of urea and protein

(casein, gelatin or zein). Ruminal ammonia is a source of nitrogen for MCP synthesis for

both structural and non-structural carbohydrate fermenting microorganisms. In the









presence of increased levels of NFCs potential competition between NFC-utilizing

bacteria and cellulolytic bacteria for ammonia could lead to an increase in the theoretical

optimum ammonia concentration required for maximum growth of cellulolytic bacteria.

However, over the longer time of the feeding cycle this apparent negative impact of

increasing NFCs might be alleviated by cross-feeding among different ruminal microbial

populations (McAllister et al., 1994). An example of cross-feeding was shown by Miura

et al. (1980) when Megasphaera elsdenii (lactate utilizer) deaminated protein resulting

from the lysis of Ruminobacter amylophilus (starch utilizer) which provided BCVFA for

the growth of Ruminococcus albus (cellulose and hemicellulose utilizer).

Several studies reported no difference in ruminal ammonia concentration when

comparing different NFC sources (Chamberlain et al., 1993; O'Mara et al., 1997a; Sannes

et al., 2002). When interpreting the response to ruminal ammonia concentration it is

important to keep in mind that this concentration is the net result of protein degradation

in the rumen, ammonia absorption across the ruminal wall, ammonia passage from the

rumen and ammonia incorporation into MCP. Therefore decreased ruminal ammonia

concentration may indicate increased utilization by ruminal microorganisms and potential

increased MCP synthesis. In this case a decrease in ammonia concentration might be

viewed as a positive response. On the other hand a decrease in ammonia concentration

may also indicate a decrease in protein degradation, in which case nitrogen might become

limited and MCP synthesis decreased. If a decrease in ammonia concentration is

accompanied by increased MCP synthesis, it may provide needed nutrients to support

higher milk protein production.









Although MCP synthesis can occur with urea as the sole source of nitrogen, the

efficiency of yield may be lower compared to when peptides or amino acids are supplied.

Maeng et al. (1976) reported an optimum ratio of urea nitrogen to amino acid nitrogen of

75:25 for ruminal microbial growth in a series of in vitro fermentations with mixed

ruminal microorganisms in which glucose, cellobiose and starch were the carbohydrate

sources. However, nitrogen used for microbial growth may be derived from sources

other than. Amino acids and peptides, which are breakdown products of dietary protein,

may be directly incorporated into microbial crude protein. Alternatively, amino acids

from dietary origin can be degraded in the rumen to BCVFAs and ammonia, which can

then be used for MCP synthesis.

Both peptides and amino acids have been shown to stimulate MCP synthesis when

substituted for ammonia in vivo (Rooke and Amstrong, 1989) and in vitro (Russell and

Strobel, 1993). The importance of amino acids and peptides from dietary protein

degradation for increasing both MCP production and energetic efficiency has been shown

in several studies with batch culture fermentations (Maeng et al., 1976; Maeng and

Baldwin, 1976a, 1976b). Russell and Sniffen (1984) reported an increase of 18.7% in

ruminal bacteria yield with the addition of amino acids to mixed cultures with

theoretically adequate ammonia concentrations. A yield increase of 28% in vivo (sheep)

has also been reported when true protein was added to urea-containing diets (Hume and

Purser, 1974).

The advantages of peptides and amino acids over NPN for microbial protein

growth may depend on the species of bacteria and energy source (Cruz Soto et al., 1994).

There appears to be a higher requirement for amino acids and peptides by amylolytic









organisms (Maeng and Baldwin, 1976a, 1976b) and sugar-utilizing organisms (Hungate,

1966). However, proteins have also been shown to be superior to urea for maintenance

of fiber digestion despite the fact that cellulolytic organisms primarily use ammonia as a

nitrogen source. This may indicate that cellulolytic bacteria may have some requirement

for amino acids or peptides (Hoover, 1986), which may be related to the supply of

BCVFAs. Varga et al. (1988) showed that decreased BCVFAs are responsible for

depressed fiber digestion in continuous culture fermentations of formaldehyde-treated

soybean meal. Gorosito et al. (1985), however, suggested that amino acids or peptides

might increase cell wall digestion more than BCVFAs alone. Pectin-fermenting ruminal

bacteria include species that ferment both structural and non-structural carbohydrates

(e.g. F. succinogenes and P. ruminicola, respectively) and would therefore utilize

ammonia, amino acids or peptides. It appears to be beneficial to supply nitrogen in the

form of amino acids and peptides, whether to be incorporated directly or as source of

BCVFAs, in addition to ammonia to optimize microbial growth.

Fermentation pH

As mentioned previously, NFC supplementation has the potential to decrease

ruminal pH. Low pH in turn may decrease MCPeff (Russell et al., 1992), which may be

related to energy spilling strategies of ruminal microorganisms to cope with excess

available carbohydrate and low pH (Russell and Strobel, 1993). One example of an

energy spilling strategy involves the ability of S. bovis to ferment glucose to lactate

which only yields 2 ATP molecules per glucose molecule as opposed to acetate, format

and ethanol which yield approximately 3 ATP molecules per glucose molecule (Russell

and Baldwin, 1979). At a low pH, S. bovis decreases its intracellular pH, which favors

lactate production (Russell and Hino, 1985). Low pH has also been shown to increase









the maintenance energy cost of ruminal microorganisms and thus decrease microbial cell

yield (Shi and Weimer, 1992).

It appears that cellulolytic bacteria are especially sensitive to low ruminal pH.

However, a moderate decrease in pH from 6.8 to 6.0 does not always affect cellulolytic

numbers (Slyter et al., 1970; Mackie et al., 1978; Leedle et al., 1982; Van der Linden et

al., 1984) and isolated fibrolytic enzyme activity remains high in this range (Stanley and

Kesler, 1959; Smith et al., 1973). On the other hand, a decrease in pH below 6.0 has

been reported to result in loss of fibrolytic activity and decreased numbers of cellulolytic

bacteria in vitro and in vivo (Slyter et al., 1970; Stewart, 1977; Crawford et al., 1980;

Hoover et al., 1984; Mould and Orskov, 1984; Mould et al., 1984). At a pH between 4.5

and 5.0 there is virtually complete inhibition of fiber digestion (Stewart, 1977; Hoover et

al., 1984; Mould et al., 1984). Russell and Dombrowski (1980) observed washout of

cellulolytic bacteria in continuous culture fermentations at a pH below 6.0. Huhtanen

and Khalili (1992) reported a decrease in cellulolytic and hemicellulolytic enzymes at

decreased ruminal pH, when sucrose was supplemented to cattle on grass-silage based

diets. It is thus not surprising that one of the major results of decreased ruminal pH has

been a decrease in fiber digestion, reported both in vitro and in vivo (Terry et al., 1969;

Mould and Orskov, 1984; Mould et al., 1984).

NFCs and Ruminal pH, Fiber Digestion and Animal Performance

Ruminal pH and Fiber Digestion

Decreased fiber digestion in vivo is often associated with supplementation of

forage diets with readily fermentable carbohydrate sources. Cameron et al. (1991)

reported decreases in ruminal neutral detergent fiber (NDF) and acid detergent fiber

(ADF) digestion for lactating dairy cows receiving supplements of starch and dextrose









(glucose). Heldt et al. (1999) reported a decrease in total tract NDF digestion relative to

control diet-fed animals in steers supplemented at 0.3% BW of DM/d with starch,

sucrose, glucose or fructose with low-quality, tallgrass-prairie hay. The diets in this

study were supplemented with degradable intake protein at 0.031% BW of DM/d, which

may have been below the amount needed to meet ruminal microbe requirements for a

degradable nitrogen source. In a second study, with the same NFC sources, but with

supplemental degradable intake protein of 0.122% BW of DMId, total tract NDF

digestion increased with NFC supplementation (Heldt et al., 1999). Also, ruminal pH

decreased more in animals consuming the starch diet, and these animals had a lower total

tract NDF digestion compared to animals fed the sugar (sucrose, glucose and fructose)

diets. Some of the decreases noted for fiber digestion may be the result of competition

between NFC and fiber utilizing microorganisms for the nitrogen supply.

A decrease in ruminal fiber digestion is often attributed to a decrease in ruminal pH

(Hoover, 1986), caused by rapid fermentation of NFCs and production of VFAs by

ruminal microorganisms. Several studies have contradicted this concept and reported no

effect on pH and varying effects on fiber digestion as a result of supplementation with

starch or sucrose (Cameron et al., 1991; Aldrich et al., 1993; Casper et al., 1999). In a

two-part study by Khalili and Huhtanen (1991 la, b), a decrease in both pH and NDF

digestion was reported in animals fed a grass silage and barley-based diet with

supplementation of sucrose at 1 kg DM/day. However, the negative effect on pH and

NDF digestion was alleviated when sodium bicarbonate was supplemented in

combination with sucrose.









The effect of NFC supplementation (especially sucrose and starch) on fiber

digestion might involve more than just the decrease of ruminal pH. It was Mould and

Orskov (1984) who first coined the term "carbohydrate effect" to describe the initial

impaired fiber digestion at a pH of approximately 6.2. The authors suggested that a

series of events takes place: 1) ruminal microorganisms exhibit a preference for

carbohydrate sources that are more readily available, 2) fermentation of readily available

carbohydrates produce organic acids and ruminal pH decreases and 3) ruminal pH

decreases below 5.5 resulting in a decrease of cellulolytic microorganisms and potentially

completely inhibits fiber digestion. Piwonka and Firkins (1996) also suggested that there

might be a carbohydrate effect related to microbially produced inhibitors, which is

independent from pH.

Substitution of dried, pelleted beet pulp for high moisture corn did not affect

ruminal pH and increased both extent and rate of NDF digestion (Voelker and Allen,

2003b) when fed to Holstein cows on an alfalfa and corn silage diet in early lactation. In

several other studies increased NDF digestion as a result of supplementing sugar beet

pulp (Van Vuuren et al., 1993) or citrus pulp (Zinn and Owens, 1993; Miron et al., 2002)

for barley or corn has been reported.

Animal Performance

There are a multitude of animal studies that compared feedstuffs that differed in

types of NFCs that predominated. The challenge to interpreting these studies is that the

diets were rarely characterized for their carbohydrate fractions and proportional

substitution of carbohydrates was often not equal. Accordingly, one should view these

studies in a general sense to try and understand the impact of various carbohydrates on

ruminal and animal performance, but the results are anything but clear-cut.









Dry Matter Intake

Supplementation with NFCs is generally thought to result in decreased dry matter

intake. However, total dry matter intake was not affected by form of NFC

supplementation in several studies (Table 1-2; Charmely et al., 1991; Nombekela and

Murphy, 1995; O'Mara et al., 1997a; O'Mara et al., 1997b; Leiva et al., 2000; McCormick

et al., 2001; Broderick et al., 2002b; Ordway et al., 2002; Sannes et al., 2002; Cherney et

al., 2003; Delahoy et al., 2003). The effect of NFC supplementation on forage intake

alone also gave varied responses (Charmely et al., 1991; O'Mara et al., 1997b; Heldt et

al., 1999; Delahoy et al., 2003). The only report of a decrease (P < 0.05) in forage intake

with lactating dairy cows was in a study by O'Mara et al. (1997b) where perennial

ryegrass was supplemented with molassed beet pulp. Several studies reported no effect

of NFC type on forage intake for a variety of forages and animals, including hay for

steers (Heldt et al., 1999), pasture for lactating dairy cows (Delahoy et al., 2003) and

silage for sheep (Charmely et al., 1991). The varying responses in forage and dry matter

intake among studies investigating the effect of NFC supplementation may be due to

differences in the amount, source and combination of NFC supplemented. However, it

would appear that in general the different NFC sources do not differ from each other in

their effect on dry matter intake, and potentially also forage intake.

Milk Production and Composition

The effect of different NFC sources on milk yield is inconsistent (Table 1-2). In

some studies, substituting sucrose for some portion of corn had no effect on milk yield

(Nombekela and Murphy, 1995; Ordway et al., 2002; Cherney et al., 2003), while Sannes

et al. (2002) reported a decrease (P = 0.02) in milk production. In three studies,

substituting beet pulp for ground corn (Delahoy et al., 2003) and citrus pulp for corn









(Solomon et al., 2000) or corn hominy (Leiva et al., 2000) had no effect on milk

production. In other studies, substituting citrus pulp for high moisture ear corn or

cracked shelled corn (P = 0.01 and P = 0.02, respectively; [Broderick et al., 2002b]) or

corn meal (P < 0.01; [Leiva et al., 2000]) decreased milk production.

Milk composition changes in response to different NFC sources also varies (Table

1-2). Sucrose substituted for corn (starch) had no effect on milk fat concentration

(Nombekela and Murphy, 1995; Ordway et al., 2002; Sannes et al., 2002; Cherney et al.,

2003), while it decreased (P = 0.04; [Sannes et al., 2002]), tended to increase (P = 0.07;

[Nombekela and Murphy, 1995] and P = 0.06; [Ordway et al., 2002]), or did not affect

(Cherney et al., 2003) milk fat yield. Cows fed beet pulp substituted for corn had similar

milk fat yields, but increased (P < 0.05) milk fat concentrations (Mansfield et al., 1994).

In contrast cows fed citrus pulp substituted for corn (Solomon et al., 2000), and corn

hominy or corn meal (Leiva et al., 2000) had similar milk fat yields and concentrations.

Varied responses to NFC supplementation in these studies may be due to differences in

carbohydrate composition of feedstuffs such as beet pulp and citrus pulp, and also

variation in composition within a particular feedstuff.

Sucrose supplementation has decreased both milk protein yield (Sannes et al.,

2002) and milk protein percentage (Nombekela and Murphy, 1995) compared to corn.

Beet pulp (Mansfield et al., 1994) and citrus pulp (Solomon et al., 2000; Broderick et al.,

2002b) substituted for corn also decreased milk protein yield and milk protein

concentration. In at least some of the studies (Mansfield et al., 1994; Broderick et al.,

2002b) decreased milk protein yield might have been due to a lower total milk yield.









Starch and sucrose appear to be similar in their effects on milk yield, and starch

may increase milk yield compared to pectin. There does not seem to be a consistent

effect on milk composition from different NFC sources.

With the effects of NFC on animal and microbial responses reported in the

literature, and the variation in these responses, it is clear that further knowledge of the

microbial fermentation product yields from different NFC could be a vital resource in

understanding and predicting animal response. It is also necessary to consider factors

that may, in combination with NFC supplementation, affect substrate utilization,

microbial product yield and nutrient supply to the ruminant animal.

In this light, three in vitro fermentation studies with mixed ruminal cultures were

conducted to determine the effect of fermentation pH, nitrogen source and NFC

supplementation on NDF digestion and fermentation product yields. The first two studies

examined the effects of pH and nitrogen source on the fermentation of sucrose and

isolated neutral detergent residue (iNDF). The final study examined the fermentation of

iNDF together with starch, sucrose, pectin and their combinations.












Table 1-1. The effects of NFC source on ruminal or fermentation pH and organic acid profile.
Reference and treatment pH Total VFA1 C2 C3 C4 BCVFA Val Lac
(% of diet DM or fermentation substrate) mM ---------------------- mol/100 mol -------------
(Ariza et al., 2001), continuous culture
Citrus pulp (23.6%) --- 104.2 68.9 16.7 11.4 3.0 --- ---
Hominy feed (25.3%) --- 101.2 62.6 22.7 11.0 3.7 --- ---
(Bach et al., 1999), continuous culture
Control (lush pasture) 6.10 126.4 71.4 17.8 9.6 0.0 --- ---
Beet pulp with molasses (44.7%) 6.04 124.3 60.9 20.2 15.6 0.9 --- ---
Cracked corn (44.7%) 6.02 141.9 54.3 22.1 18.8 1.2 --- ---
(Ben-Ghedalia et al., 1989), 4 cannulated rams
Barley (76.5%) 6.18 82.4 65.0 17.6 14.3 1.9 1.4 ---
Dried citrus pulp (84.4%) 6.42 74.4 69.1 14.4 14.2 1.5 1.0
(Broderick et al., 2002b), 6 cannulated lactating cows
High moisture ear corn (38.4%) 6.14 102.0 63.3 20.6 11.2 1.6 1.9 ---
Cracked shelled corn (38.7%) 6.21 104.1 63.7 19.6 11.7 1.7 1.8 ---
High moisture ear corn (19.1%) + Citrus pulp ---
(19.1%) 6.14 107.5 64.0 18.7 12.7 1.4 1.8
(Chamberlain et al., 1993), 6 sheep
Grass silage control (4 kg/d) 6.43 56.8 62.7 24.5 7.8 3.5 1.5 ---
Sucrose (200 g/d) 6.34 57.2 57.3 27.8 11.6 1.8 1.5 ---
Starch (200g/d) 6.25 65.4 62.8 24.1 8.9 2.8 1.4 ---


(Charmely et al., 1991), 8 sheep
Alfalfa silage
Alfalfa silage + Sucrose (10% of silage DM intake)


6.73
6.63


84.9
90.6


70.1 19.2
65.8 21.6












Table 1-1 Continued.
Reference and treatment
(% of diet DM or fermentation substrate)
(Heldt et al., 1999), 20 cannulated steers
0.031% BW/d RDP supplement
Control
Starch (0.3% BW of DMId)
Glucose (0.3% BW of DMId)
Fructose (0.3% BW of DMId)
Sucrose (0.3% BW of DMId)
0.122% BW/d RDP suoolement


pH Total VFA
mM


6.40
6.36
6.28
6.36
6.23


C2 C3 C4 BCVFA Val Lac
---------------------- mol/100 mol ----- -------


76.1
70.3
59.3
58.8
56.3


13.3
17.1
15.5
13.5
15.5


9.0
10.0
18.6
19.7
20.9


Control 6.56 --- 73.5 14.0 10.6 1.2 0.5 0.3
Starch (0.3% BW of DMId) 6.13 --- 69.5 16.4 10.3 2.0 1.4 0.5
Glucose (0.3% BW of DMId) 6.16 --- 61.5 14.1 17.5 1.7 1.7 3.5
Fructose (0.3% BW of DMId) 6.29 --- 59.8 14.2 18.9 1.6 1.7 3.8
Sucrose (0.3% BW of DM/d) 6.22 --- 59.7 14.4 19.5 1.5 1.8 3.0
(Khalili and Huhtanen, 1991a), 4 cannulated bulls
Control (starch) 6.28 105.0 63.6 17.8 14.9 --- 14.8 4.4
Sucrose (1 kg/d) 6.03 104.0 58.9 16.5 19.7 --- 23.5 12.5
(Leiva et al., 2000), 11 lactating cows (3 cannulated)
Corn hominy diet (25.3%) 6.24 106.1 67.4 21.4 11.2 --- --- 1.5
Citrus pulp diet (23.6%) 6.19 116.4 67.7 20.8 11.5 --- --- 0.6
(Mansfield et al., 1994), continuous culture
Corn (30.2%) --- 112.7 58.7 21.7 15.3 2.3 2.0 0.9
Corn (15.5%) + Beet pulp (15.3%) --- 109.5 61.6 20.5 14.0 1.9 2.0 0.9
(Marounek et al., 1985), in vitro; inoculum from goats
Starch (9 g/150 ml, 8 h incubation) --- 76.3 64.5 29.1 6.4 --- --- ---
Pectin (2 g/150 ml, 6 h incubation) --- 61.5 81.8 14.8 3.4 --- --- ---













Table 1-1. Continued.
Reference and treatment
(% of diet DM or fermentation substrate)
(Moloney et al., 1994), 6 cannulated steers
Barley (61% of DM intake)
AM 61% 1 DM0i- rlk


pH Total VFA


6.94
I< QI


mM

71.2
'71 '7


C2 C3 C4 BCVFA Val Lac
------------------- mol/100 mol ------------ mg/dl


66.5 15.8 14.0
5Q A 1 6 ')Q I 2


--- 3.3 42.3
Sn )QO


oVaUlslses IU1 /( U ULi JlVI ntILaUe). U. U / ./ 1.. 1U.U J. --- JU.. SJ,..
(Piwonka et al., 1994), 6 cannulated heifers ---------- mol/100 mol ----- -------
Control --- 82.4 70.0 16.7 9.6 --- --- ---
Dextrose (5.6%) --- 91.2 68.9 18.1 9.9 --- --- ---
Barley (39.7%) --- 90.5 68.7 16.7 11.1 --- --- ---
(Sannes et al., 2002), 16 lactating cows (4 cannulated) ----------------------------- mM------- ---------
Corn (20%) --- 131.0 85.34 26.3 16.2 1.9 --- ---
Corn (13.5%) + Sucrose (3.2%) --- 123.0 77.81 27.1 15.5 1.3 --- ---
(Strobel and Russell, 1986)
in vitro pH 6.7
Sucrose 6.70 --- 4.7 2.1 1.1 --- --- 3.7
Starch 6.70 --- 5.1 2.9 0.8 --- --- 0.9
Pectin 6.70 --- 10.1 1.3 0.2 --- --- ND2
in vitro pH 6.0
Sucrose 5.50 --- 1.7 1.1 0.7 --- --- 8.3
Starch 5.80 --- 2.7 1.1 0.7 --- --- 4.1
Pectin 5.80 --- 5.0 0.7 0.3 --- --- ND
(Voelker and Allen, 2003c), 8 cannulated lact. cows ------------------------ mol/100 mol ----- --------
Hi moisture corn (35.6%) 5.93 138.0 56.9 27.0 11.3 2.3 --- 0.3
Hi moisture corn (29.5%) + Citrus pulp (6%) 5.97 141.0 59.1 24.9 11.5 2.3 --- 0.3
Hi moisture corn (23.5%) + Citrus pulp (12%) 6.02 142.0 60.2 23.0 12.2 2.3 --- 0.3
Hi moisture corn (11.4%) + Citrus pulp (24%) 5.94 142.0 61.6 22.4 12.3 1.9 --- 0.1
1 VFA = volatile fatty acids; C2 = acetate; C3 = propionate; C4 = butyrate; BCVFA = branched chain VFA; Val = valerate; Lac = lactate
2 ND = not detected












Table 1-2. Effects of NFC source on dry matter intake, milk production and milk composition.
Reference and treatment DMI1 Milk Fat
(% of diet DM) kg/d


Protein


Fat Protein


(Broderick et al., 2002b), 48 lactating cows
High moisture ear corn (38.4%)
Cracked shelled corn (38.7%)
High moisture ear corn (19.1%) + Citrus pulp (19.1%)
(Charmely et al., 1991), 8 sheep
Alfalfa silage
Alfalfa silage + Sucrose (10% of silage DMI)
Silage intake only
Alfalfa silage
Alfalfa silage + Sucrose (10% of silage DMI)
(Cherney et al., 2003), 20 lactating cows
High moisture corn (35.7%)
High moisture corn (32.1%) + Sucrose (3.6%)
High moisture corn (19.2%)
High moisture corn (17.3%) + Sucrose (1.9%)
(Delahoy et al., 2003), 28 lactating cows
Ground corn (70.2%)
Ground corn (34.8%), Beet pulp (18.0%), Wheat middlings
(17.4%)


(Fegeros et al., 1995), 26 sheep
Maize (28.0%) + Barley (30.0%)
Maize (20.0%) + (Barley 15.0%) + Dried citrus pulp (30.0%)
(Friggens et al., 1995), 18 lacating cows
Molassed sugar beet pulp (74.5%)
Molassed sugar beet pulp (37.3%) + Grain (38.2%)
Grain (Barley and Corn 76.4%)


20.9
21.4
19.7


35.2
35.1
32.1


1.24
1.19
1.07


1.04
1.06
0.88


2.94
3.02
2.85


3.53
3.38
3.44


1.25
1.22

1.25
1.07


21.7
21.4
20.1
20.6


39.8
39.5
37.8
38.9


1.28
1.29
1.30
1.33


1.02
1.02
0.95
0.97


2.58
2.59
2.52
2.49


3.24
3.27
3.44
3.47


20.3 27.6 1.05 0.96 3.23 3.53

20.2 27.4 1.08 0.95 3.19 3.63


--- 0.82
--- 0.78

--- 14.3
--- 14.9
--- 14.5


0.60
0.57
0.58


0.48
0.51
0.50


5.36
5.32

3.51
3.41
3.56


7.04
7.27

4.19
4.05
3.98












Table 1-2. Continued.
Reference and treatment
(% of diet DM)


DMI Milk Fat Protein
kg/d


Fat Protein
%


(Leiva et al., 2000)
11 lactating cows (3 cannulated)
Corn hominy diet (25.3%) 21.4 32.8 1.12 0.93 2.83 3.43
Citrus pulp diet (23.6%) 20.9 31.3 1.11 0.85 2.71 3.54
184 lactating cows
Corn meal diet (19.5%) 19.5 31.8 1.02 0.96 3.08 3.27
Citrus pulp diet (20.5%) 18.9 27.9 0.97 0.88 3.13 3.45
(Mansfield et al., 1994), 46 lactating cows
Corn (30.2%) 21.5 32.2 1.18 0.97 3.64 3.01
Corn (15.5%) + Beet pulp (15.3%) 20.3 31.9 1.21 0.92 3.82 2.90
(McCormick et al., 2001), 32 lactating cows
Ground corn (75.0%) 22.8 39.6 1.28 1.14 2.99 3.32
Ground corn (68.9%) + Brown sugar (5.0%) 22.9 38.7 1.30 1.13 2.97 3.39
(Nombekela and Murphy, 1995), 24 lactating cows
Ground corn (39.9%) 19.0 28.4 0.96 0.96 3.51 3.40
Ground corn (38.4%) + Sucrose (1.5%) 19.1 29.3 0.97 0.95 3.28 3.30
(O'Mara et al., 1997a), 36 lactating cows
Beet pulp (30.0%) 15.2 19.8 0.71 0.62 3.16 3.66
Beet pulp (10.6%) + Ground corn (20.0%) 13.7 21.2 0.78 0.64 3.04 3.64
(O'Mara et al., 1997b), 8 cannulated lactating cows
Perennial ryegrass 13.6 --- --- --- --- ---
Perennial ryegrass + Molassed beet pulp (3 kg/day) 14.2 --- --- --- --- ---
Grass intake only
Perennial ryegrass 13.6 --- --- --- --- ---
Perennial ryegrass + Molassed beet pulp (3 kg/day) 11.5 --- --- --- --- ---












Table 1-2. Continued.
Reference and treatment
(% of diet DM)
(Ordway et al., 2002). prepartum diets (carry over effect
measured; postpartum diet 15.1% ground corn)
Ground corn (11.5%)
Ground corn (8.8%) + Sucrose (2.7%)
(Sannes et al., 2002), 16 lactating cows (4 cannulated)
Corn (20.0%)
Corn (13.5%) + Sucrose (3.2%)
(Solomon et al., 2000), 20 lactating cows
Corn (23.7%)
Dried citrus pulp (23.9%)
(Valk et al., 1990)
Study 1 (18 lactating cows)
Beet pulp (82.5%)
Maize meal (47.5%) + Maize bran (50.0%)
Study 2 (27 lactating cows)
Beet pulp (78.4%)
Maize meal (87.5%)
Beet pulp (44.0%) + Maize meal (44.0%)
(Voelker and Allen, 2003a), 8 cannulated lactating cows
High moisture corn (35.6%)
High moisture corn (29.5%) + Dried citrus pulp (6%)
High moisture corn (23.5%) + Dried citrus pulp (12%)
High moisture corn (11.4%) + Dried citrus pulp (24%)
1 DMI = Dry Matter Intake


DMI Milk Fat Protein
kg/d


22.1
21.7

25.7
25.5

21.5
20.6


19.5
20.1

21.2
20.7
20.7

24.8
25.0
25.1
22.9


45.8 1.66
45.6 1.72

34.3 1.33
33.2 1.27

36.9 1.22
36.4 1.21


25.8 1.07
28.4 1.11


30.9
31.6
30.8

36.4
36.6
35.9
35.4


1.29
1.17
1.24

1.34
1.40
1.39
1.33


1.26
1.25

1.07
1.03

1.07
1.04


0.89
0.98

1.02
1.03
1.02

1.13
1.15
1.15
1.09


Fat Protein
%


2.68
2.72

3.14
3.12

2.94
2.88


3.43
3.48

3.32
3.27
3.31

3.21
3.21
3.22
3.10


3.54
3.76

3.88
3.83

3.33
3.34


4.15
3.92

4.19
3.70
4.05

3.72
3.84
3.90
3.81













CHAPTER 2
EFFECT OF PH ON MICROBIAL YIELD AND NEUTRAL DETERGENT FIBER
DIGESTION FROM IN VITRO FERMENTATIONS OF SUCROSE AND ISOLATED
NEUTRAL DETERGENT RESIDUE

Introduction

Dairy cattle diets are often supplemented with grains and byproduct feeds that have

a large concentration of non-neutral detergent fiber carbohydrates (NFCs) in order to

increase the energy intake of high producing ruminants. Sucrose, and its constituent

monosaccharides glucose and fructose, are the predominant saccharides of the mono- and

oligosaccharide component of NFCs and are found in byproduct feeds such as molasses

(Kunkle et al., 2000), sugar beet pulp (Hall, 2002) and citrus pulp (Ben-Ghedalia et al.,

1989). Sucrose is readily fermentable in the rumen (Sniffen et al., 1983) and has been

associated with a decrease in ruminal pH (Sutton, 1979; Khalili and Huhtanen, 1991 la).

Ruminal pH is considered one of the major modifiers of rumen fermentation (Hoover and

Stokes, 1991). A decrease in ruminal pH below 6.0 has been associated with decreases in

fiber digestion (Khalili and Huhtanen, 1991 la, b; Grant and Weidner, 1992), microbial cell

yield (Shi and Weimer, 1992) and efficiency of microbial protein synthesis (MCPeff;

[Russell et al., 1992]), as well as altered volatile fatty acid (VFA) profiles (Sutton, 1979;

Chamberlain et al., 1993; Araba et al., 2002). However, there is evidence that there are

effects of readily fermented carbohydrates that are not pH related (Mould and Orskov,

1984; Piwonka and Firkins, 1993).

The objectives of this study were to compare the effects of media with neutral or

acidic starting pHs on 1) the yield of microbial fermentation products and on neutral









detergent fiber (NDF) digestion, from the in vitro fermentation of sucrose and isolated

NDF, and 2) fermentation pH and NDF digestion of isolated NDF fermented with or

without sucrose supplementation.

Materials and Methods

Substrates

Substrates used were isolated bermudagrass (Cynodon dactylon L.) neutral

detergent residue (iNDF; 92.8% DM, 99.4% OM, 98.6% NDFOM, 5.5% NDFCP) and a

50:50 mixture of iNDF and sucrose (SuNDF). The iNDF was prepared as described by

Hall and Herejk (2001). Sucrose (S5-500, Fisher Scientific, Atlanta, GA; 99.98% DM,

100% OM) and iNDF were analyzed prior to the study for DM and OM (AOAC, 1980),

and iNDF for NDF using heat-stable c.-amylase (Termamyl 120L, Novo Nordisk

Biochem, Franklinton, NC; (Van Soest et al., 1991) and CP (AOAC, 1980). A total of

240 mg + 0.5 mg of substrate, with iNDF and sucrose weighed individually, were

transferred into duplicate 50 ml Nalgene high speed, low density, polyethylene centrifuge

tubes (05-562-13, Fisher Scientific, Atlanta, GA) or Nalgene high speed polypropylene

centrifuge tubes (05-562-10K, Fisher Scientific, Atlanta, GA) depending on the type of

analysis to be performed on the fermentation residues (Table 2-1).

Medium and reducing solution

The pH treatments consisted of one of two iso-nitrogenous media with neutral or

acidic pH, respectively. The Goering and Van Soest (1970) medium served as the neutral

medium (NpH) with initial pH of 6.8, and was modified by adding 4.4 ml of a 1 M citric

acid solution per 100 ml of Goering and Van Soest medium to obtain the acidic medium

(ApH), with initial pH of 5.6 (P. J. Weimer and D. R. Mertens, personal communication).

The media provided 6.85 mg non-protein nitrogen and 3.52 mg amino nitrogen per









fermentation tube. Casein acid hydrolysate (A-2427, Sigma Chemical Co., St. Louis,

MO) provided the amino nitrogen source in the medium. The reducing solution was

mixed according to a modification of the Goering and Van Soest (1970) procedure (P. J.

Van Soest, personal communication). For a volume of 100 ml, 0.625 g of L-cysteine

hydrochloride (C-7880, Sigma Chemical Co., St. Louis, MO) and approximately 10

pellets of KOH (P250-3, Fisher Scientific, Atlanta, GA) were dissolved with stirring in

50 ml of distilled water. In a separate 250 ml glass beaker 0.625 g sodium sulfide (S-

4766, Sigma Chemical Co., St. Louis, MO) was dissolved with stirring in 50 ml of

distilled water. The solutions were combined when the contents of both beakers were in

solution, and just before addition of the reducing solution to the fermentation tubes.

Fermentation

Duplicate 24 h in vitro fermentation runs using destructive sampling of mixed

batch cultures were performed according to the method of Goering and Van Soest (1970).

Ruminal inoculum was obtained approximately 3 h post-feeding from a ruminally

cannulated, non-pregnant, non-lactating Holstein cow under protocols approved by the

University of Florida Institutional Animal Care and Use Committee. The donor cow

received a diet of bermudagrass hay (10 kg DMIday), 48% crude protein soybean meal

(900 g/d) and free choice mineral supplement (Ca 17- 20%, P > 9%, NaCl < 25%, Mg >

0.25%, Cu > 0.15, Co > 0.01%, I > 0.01%, Mn > 0.2%, Se < 0.004%, Zn > 0.4%, Fl <

0.09%). The inoculum was filtered through four layers of cheesecloth and one layer of

glass wool, and maintained under anaerobic conditions at 390C. Twenty milliliters of the

appropriate medium, 1 ml of reducing solution and 5 ml of filtered rumen fluid were

added to each fermentation tube. After each addition, tube headspace was purged with

CO2. Fermentation tubes were capped with rubber stoppers fitted with gas release valves,









incubated (Equatherm Incubator Model C1487, Curtin Matheson Scientific, Inc.,

Houston, TX) under anaerobic conditions at 390C and destructively sampled at 0, 4, 8,

12, 16, 20 and 24 hours. Tubes were swirled individually every 4 hours.

Sample Handling and Subsequent Analyses

At each sampling hour the fermentation tubes for the specific hour were removed

from the incubator and placed in an ice bath to terminate the fermentation process.

Approximately 5 min after tubes were removed from the incubator pH was recorded on

tubes reserved for NDF analysis. The tubes used for NDF analysis were stored at 10C

and were analyzed for residual NDFOM (NDF on an ash-free basis) within two days of

completion of the fermentation. For NDF analysis, samples were allowed to equilibrate

to room temperature, the pH was adjusted up or down with minimal amounts of a 0.2 N

NaOH or 1 M citric acid solution, respectively, to obtain a pH of between 6.9 and 7.1.

Samples were quantitatively transferred to 600 ml Berzellius beakers and refluxed with

50 ml of neutral detergent solution and heat-stable a -amylase (Termamyl 120L, Novo

Nordisk Biochem, Franklinton, NC) for 1 h (Van Soest et al., 1991). To ensure

hydrolysis of a-glucan, three doses of 0.2 ml heat-stable a-amylase were used: one with

addition of detergent, one 10 min before removing the sample from the burner and one

added to the Gooch crucible during rinsing with boiling water.

Fermentation tubes reserved for microbial glycogen (GLY), residual sucrose

equivalents (sucrose, and its hydrolysis products: glucose and fructose), organic acids,

ammonia-nitrogen (NH3-N) and total free amino acid analyses were centrifuged at 15,000

x g for 30 min at 50C. The supernatant was transferred to scintillation vials and stored at

-200C until analysis for residual sucrose and organic acids by HPLC, and NH3-N and









total free amino acid analyses as described by Broderick et al. (2004). The HPLC for

analyzing residual sucrose was equipped with an anion exchange analytical column

(CarboPacTM PAl, Dionex, Sunnyvale, CA), the mobile phase used was 100 mMNaOH,

the flow rate 1.0 ml/min and the injection volume 10 [tL. The HPLC for analyzing

organic acids was equipped with an organic acid column (PHX-87H, Bio-Rad

Laboratories, Richmond, CA). The solvent used was 0.015 NH2SO4 / 0.0034MEDTA,

the flow rate 0.7 ml/min, the column temperature 450C and the injection volume 50 gL.

The pellets from the high-speed centrifugation were quantitatively transferred to 50

ml glass beakers using no more than 20 ml of a 0.2 NNaOH solution to rinse out the

fermentation tubes and stored at -200C until analysis for GLY. Beakers were removed

from the freezer and samples were allowed to equilibrate to room temperature.

Microorganisms were lysed with a 0.2 NNaOH solution (brought to a volume of 20 ml in

the 50 ml glass beakers) in a boiling water bath for 15 min. Samples were cooled to

room temperature and then neutralized to pH 7.0 + 0.1 with 6 NHC1. Samples were

quantitatively transferred from the glass beakers to funnels fitted with glass wool plugs

for filtration into 100 ml volumetric flasks. Beakers, glass wool and funnels were rinsed

with distilled de-ionized water (ddH20), and then samples were brought to volume with

ddH20. Four milliliters of a 0.1 M sodium acetate buffer (pH ~ 4.5) and 50 [tl of

amyloglucosidase (EC 3.2.1.3, A-3514, Sigma Chemical Co., St. Louis, MO) were added

to 4 ml of sample, incubated at 60C for 45 min, and analyzed for ca-glucan content as

released glucose corrected for free glucose (Karkalas, 1985).

Microbial crude protein (MCP) was estimated as trichloroacetic acid (TCA)-

precipitated crude protein. Fermentation tubes were individually removed from the ice









bath and 5.2 ml of a 120% (w/v) TCA solution were added in two equal increments to

achieve a final concentration of 20.0% TCA. Fermentation tubes were returned to the ice

bath for 45 min after which tubes were centrifuged at 7700 x g for 20 min at 50C. The

contents of each fermentation tube were then quantitatively transferred into Whatman

541 filter paper (09-851D, Fisher Scientific, Atlanta, GA) in veined funnels set in 125 ml

Erlenmeyer flasks, using approximately 50 ml of chilled 10% TCA to rinse the tubes,

filter and residue. Samples were allowed to filter under gravity. The filtrate for each

tube was filtered through a Whatman GF/A glass fiber filter (09-874-16D, Fisher

Scientific, Atlanta, GA), using 10% TCA to rinse the flask, filter and residue. Both

Whatman 541 and GF/A filters containing the TCA-precipitated material from one

fermentation tube were placed together in a beaker and dried for 24 h at 550C, before

analysis for crude protein content as Kjeldahl nitrogen content x 6.25 (AOAC, 1980).

Kjeldahl analysis blanks consisted of a Whatman 541 filter and a GF/A filter digested and

distilled together in one Kjeldahl flask. The MCP and GLY contents of each tube were

corrected for fermentation blanks at each hour, and MCP for its content by substrate at

hour 0.

Statistical Analysis

The experimental design was a split-split plot in time with a 2 x 2 factorial

arrangements of treatments (media and substrates). The data were analyzed using the

PROC MIXED procedure of SAS (1999) with fermentation run (R) as a random variable,

and medium (M) and substrate (S) as fixed variables. Fermentation hour (H) was used as

a class variable. Linear or quadratic temporal patterns within media and substrate

treatments were determined using orthogonal contrast statements.









The Kenward-Roger method was used to calculate the denominator degrees of

freedom for testing fixed effects. The contrast, NpH vs. ApH, was used for medium

comparisons within substrate (iNDF, SuNDF). All values presented are least squares

means. The model statement used was:

Yijk = [ + Mi + Sj + MSij + Hk + MHik + SHjk + MSHijk + ijkl

Where:

Yijk = the dependent variable

|t = overall mean

Mi = medium (i = NpH, ApH)

Sj = substrate (j = iNDF, SuNDF)

Hk = hour (k = 0, 4, 8, 12, 16, 20, 24)

MSij = interaction term for medium and substrate

MHik = interaction term for medium and hour

SHjk = interaction term for substrate and hour

MSHijk = interaction term for medium, substrate and hour

s;ijkl = residual error

A treatment term (T) consisting of the interaction between substrate and medium

was used in the random statement to obtain appropriate standard errors for the least

squares means. The random statement included the following terms:

R1 + RTim + RHik + RTHimk

Where:

RI = fermentation run (m = 1, 2)

Tm = treatment (n = 0, 1, 2, 3); number assigned to each M by S combination









RTim = interaction term for fermentation run and treatment

RHik = interaction term for fermentation run and hour

RTHImk = interaction term for fermentation run, treatment and hour

The sampling hour of maximum MCP or GLY yield within medium and

fermentation run was defined as the hour with the maximum least squares means for

these measures. Efficiency of MCP yield was expressed as maximum MCP (mg)/organic

matter digested (OMD, mg). Organic matter digested (mg) was calculated as total

sucrose (mg) minus residual sucrose equivalent (mg) plus NDFOM digested (mg), minus

GLY (mg). The sucrose equivalent at any specific sampling hour was calculated as

residual sucrose (mg) + 0.95 x (residual fructose (mg) + residual glucose (mg)). Since

NDF digestion, residual sucrose, MCP and GLY were not measured on the same

fermentation tube, the least squares means for these measurements, within fermentation

run at the hour of maximum MCP, were used to calculate MCP efficiency for individual

treatments. The "hour" term and its interaction terms were omitted from the above

mentioned model to compare minimum values, maximum values, and results at a single

sampling hour.

Results and Discussion

Residual Sucrose

Sucrose disappeared rapidly from the fermentation medium, with only 69 and 40%

of the original 120 mg sucrose recovered at 0 h in NpH and ApH, respectively (Table 2-

2). Even when residual glucose, fructose and the unhydrolyzed sucrose were expressed

as sucrose equivalent, only 83% of the original substrate was accounted for NpH and

ApH at 0 h. Residual sucrose amount, averaged over the 24 h fermentation, did not differ









for ApH and NpH, whereas glucose and sucrose equivalent contents were greater and

fructose content tended to be greater for ApH compared to NpH.

Amount of residual sucrose, and sucrose equivalent did not differ between media at

0 h. By 4 h, and through 24 h, no residual sucrose could be detected in either medium.

The ApH fermentation tended to contain more glucose and fructose at 0 h compared to

NpH. At 4 h fructose amount and residual monosaccharide sucrose equivalent (glucose

and fructose) were greater for ApH compared to NpH, whereas glucose was only

numerically higher for ApH. Glucose and fructose were not detected in NpH at 4 h, or in

ApH at 8 h, and in subsequent hours for both media. It appears that ruminal

microorganisms, depending on the pH of the fermentation, do not utilize glucose and

fructose similarly. For NpH, glucose and fructose disappeared from the fermentation by

the same sampling time (4 h). However, for ApH, fructose remained in the fermentation

for a longer period of time compared to glucose (8 and 4 h, respectively).

Early disappearance of sucrose from the fermentations is consistent with reports of

sugar degradation rates of up to 300%/h (Sniffen et al., 1983). In a study by Henning et

al. (1991) glucose was detected through 8 and 12 hours of fermentation when added at

12.5 g /L and 25 g/L of culture medium, respectively. In the current study the initial

concentration of sucrose (4.62 g/L) was only one-sixth to one-third of the amounts used

by Henning et al. (1991), which could explain why no residual sugars were detected as

early as 4 h in NpH. The present study and that of Henning et al. (1991) would indicate

that sucrose or glucose disappearance is rapid, but not instantaneous in fermentations

with ruminal microorganisms. Residual sugar concentrations in the current study









confirmed that sucrose is readily utilized by ruminal microorganisms at near neutral pH

(6.8) but may be more slowly utilized at a more acidic pH (5.6).

Microbial Glycogen

Although it is considered part of the microbial cell mass, glycogen represents

available substrate that has been stored, but not yet metabolized by the cells. Maximum

GLY yields for both media were recorded at 4 h, with 6.0 mg and 3.5 mg for NpH and

ApH, respectively (Figure 2-1). In the ApH fermentation, sucrose-utilizing

microorganisms converted less sucrose to GLY (P = 0.04 at 4 h). Though the temporal

pattern for GLY amount tended to differ between media (P = 0.06 for medium by hour

interaction), GLY appeared to decrease through 8 h for both ApH and NpH, and then

remained relatively constant through 24 h.

Ruminal microorganisms can store microbial glycogen under conditions of excess

carbohydrate supply (Thomas, 1960; John, 1984; Lou et al., 1997). Decreased pH has

been shown to increase the maintenance energy required by ruminal microorganisms (Shi

and Weimer, 1992). If more energy were diverted towards maintenance requirements in

the ApH fermentation, there might not have been a relative excess of available

carbohydrates, hence less microbial glycogen storage occurred at the lower pH.

However, the ratio of GLY to MCP (at the hour of maximum GLY yield) was 0.67 and

1.68 for NpH and ApH, respectively. Studies with Prevotella ruminicola B14 (Russell,

1992) and Fibrobacter succinogenes S85 (Maglione and Russell, 1997) indicated a

decreased viable cell number when the polysaccharide:protein ratio of the cultures

exceeded 1.0, which might offer partial explanation for the lower microbial protein yield

(Figure 2-12) and decreased NDF digestion (Figure 2-11) for ApH compared to NpH.









Fermentation pH

Temporal patterns for fermentation pH were somewhat similar in both media for

the two substrates (Figure 2-2). The pH of iNDF fermentations increased over the 24 h

fermentation (linear; P = 0.03 and P < 0.01 for NpH and ApH, respectively), whereas that

for SuNDF decreased in the early hours and then increased through 24 h (quadratic

pattern; P = 0.02 and P < 0.01 for NpH and ApH, respectively). However, substrates did

not follow parallel paths for the two media, as indicated by a medium by hour interaction

within substrate (P < 0.01 for both iNDF and SuNDF).

Minimum fermentation pH for ApH, with SuNDF as the substrate, was recorded at

8 h and was lower (P < 0.01) than that for NpH (5.25 and 6.71, respectively), which was

recorded at 4 h. Also, minimum fermentation pH for both media with SuNDF as the

substrate was achieved at the same sampling hour that lactate concentration peaked

(Figure 2-7). Thereafter the pH for these fermentations increased through 24 h.

Minimum fermentation pH for iNDF in both media was recorded at 0 h, and this was

lower (P < 0.01) for ApH compared to NpH (6.07 and 6.99, respectively). The

magnitude of pH change appeared to differ for the two media as well as for the substrates

within each medium. At their minima, the difference in pH between iNDF and SuNDF

was numerically greater for ApH, a difference of 1.16 pH units at 8 h, compared to the

difference of 0.31 pH units at 4 h for NpH. Strobel and Russell (1986) also reported a

decrease in pH after 10 h in vitro fermentation of sucrose when the initial fermentation

pH was 6.0, but not when the initial pH was 6.7.

One explanation for the larger numerical decrease in fermentation pH for SuNDF in

the current study could be the increased lactic (Figure 2-7) and acetic acid (Figure 2-3)

production at 8 h for ApH compared to NpH. Another explanation could be a decreased









buffering capacity due to the addition of citric acid to obtain a pH of 5.6 for the ApH

medium. However, Grant and Mertens (1992) reported that a 1 M citric acid solution

used to adjust a phosphate-bicarbonate buffer to a pH of 5.8 was effective in maintaining

the fermentation pH through 24 h for in vitro fermentations of alfalfa silage and corn

grain. It is possible that the alfalfa silage and 1:1 alfalfa silage:corn grain substrates used

in that study did not provide as much rapidly fermenting carbohydrate as did substrates in

the present study, and therefore did not yield as much VFA to decrease pH.

Organic Acids

Temporal patterns for all organic acid concentrations differed for fermentations

containing no substrate (blank fermentations; only inoculum) compared to those

containing SuNDF for ApH and NpH (P < 0.01 for medium by substrate by hour

interactions; Figures 2-3 to 2-7). Organic acid concentrations were not corrected at each

sampling hour for concentration in the blank fermentations (tubes with only medium and

inoculum, but no substrate) since higher acetate (Figure 2-3) and BCVFA (Figure 2-8)

concentrations in blank fermentations for ApH would have resulted in negative net

concentrations of these analytes from 8 h through the end of the fermentations. These

results raise questions about the appropriate use of fermentation blanks to adjust for

treatment values, especially when the fermentation pH is more acidic. It is not clear why

the ApH fermentation for iNDF and fermentation blanks yielded higher acetate

concentrations compared to the ApH fermentation with SuNDF. A partial explanation

could be that the fermentation of plant organic acids such as citric acid primarily yields

acetate (Russell and Van Soest, 1984).

At the end of the 24 h fermentation, with SuNDF as the substrate, there was no

difference between ApH and NpH (P = 0.36) for butyrate concentrations (Figure 2-5),









whereas acetate (Figure 2-3), propionate (Figure 2-4) and total VFA concentrations

(Figure 2-6) were greater for ApH (P < 0.01, P = 0.04 and P < 0.01, respectively). In

contrast, Strobel and Russell (1986) recorded decreased acetate and butyrate

concentrations at a more acidic pH and no difference in propionate concentration in

sucrose fermentations. In a continuous culture fermentation of alfalfa hay and corn grain,

Calsamiglia et al. (2002) reported no effect on butyrate and an increase in propionate

proportion at a more acidic pH (5.7). However, in contrast to the current study, the

authors reported decreased total VFA concentrations, and a decrease in the acetate

proportion at the more acidic pH. The differences among studies in VFA production may

be in part due to different fermentation methods (continuous vs. batch culture), and

different sources of inocula.

Lactate was not detected in fermentations with only inoculum. Maximum lactate

concentration did not differ (P = 0.64) between ApH and NpH with SuNDF as the

substrate (28.9 and 27.9 mM, respectively), and this occurred at 4 h for NpH and at 8 h

for ApH (Figure 2-7). It would appear that the lower pH resulted in delayed lactate

production. In contrast, Strobel and Russell (1986) recorded increased lactate

concentrations for sucrose fermented by mixed ruminal microorganisms at pH 6.0

compared to 6.7. In the study by Strobel and Russell (1986) the increased lactate

concentration was recorded after a 10 h fermentation, which would correspond with a

point in the current study where the lactate concentration appears to be greater for the

ApH than for the NpH fermentation.

For NpH, with SuNDF as the substrate, lactate concentration decreased from 4 h

through 12 h and remained at zero till the end of the fermentation. However, for the ApH









fermentation lactate was detectable in ApH until 16 h, and it was only at 20 and 24 h that

lactate could no longer be detected. It would appear that lactate utilization might be

delayed at a more acidic pH. Both D(+) and L(-) isomers of lactic acid are produced in

the rumen. However, D-lactate becomes the predominant isomer under more acidic pH

(pH < 6) conditions and is considered to be less degradable compared to L-lactate. Also,

Megasphaera elsdenii, one of the major lactate utilizers in the rumen, is inhibited when

the pH decreases below 5.5 (Dawson et al., 1997).

Protein Degradation Products

Temporal patterns for BCVFA concentrations differed for fermentations containing

no substrate (blank fermentations) compared to those containing SuNDF for ApH and

NpH (P < 0.01 for medium by substrate by hour interaction; Figure 2-8). Higher BCVFA

concentrations in the blank fermentations for ApH from 12 through 24 h could possibly

be due to increased cell lysis and de-amination of microbial protein, leading to higher (at

least numerically) NH3-N (Figure 2-9) and BCVFA concentrations. At the end of the 24

h fermentation of SuNDF, BCVFA concentration tended (P = 0.11) to be lower for ApH

compared to NpH. In contrast to the current study, Calsamiglia et al. (2002) reported

decreased BCVFA concentrations at a more acidic pH (5.7) in a continuous culture

fermentation of alfalfa hay and corn grain.

Total free amino acid and NH3-N concentrations are the net result of protein

breakdown and nitrogen utilization by ruminal microorganisms in vitro. Ammonia

nitrogen concentration, averaged across the 24 h fermentation, was higher (P < 0.01) for

NpH compared to ApH with SuNDF as the substrate (Figure 2-9). In the continuous

culture study by Calsamiglia et al. (2002) NH3-N concentration also decreased at a more

acidic pH (5.7). In another continuous culture fermentation, this time with soybean meal,









barley and corn silage as substrates, Erfle et al. (1982) reported decreased ammonia

concentrations at a pH below 6 and attributed this to decreased microbial deaminase

activity. In the same study, protease activity decreased at a more acidic pH, and as a

result free amino acid concentrations decreased. In the current study, total free amino

acid concentration was greater (P < 0.01) for ApH compared to NpH (Figure 2-10). The

lower total free amino acid concentration for NpH in this study may indicate greater

utilization of amino acids for MCP synthesis, which coincided with a higher MCP yield

(P < 0.01) compared to ApH (Figure 2-12).

Neutral Detergent Fiber Digestion

Temporal patterns for NDF digestion differed for substrates within ApH and NpH

(P < 0.01 for both), as well as for ApH and NpH within each substrate (P < 0.01 for

iNDF and SuNDF; Figure 2-11). At 24 h, NDF digestibility for NpH was greater for

SuNDF at 42.4% than for iNDF at 26.4% (P < 0.01). The reverse was true for ApH,

where 24 h NDF digestibility for iNDF (7.8%) tended to be greater (P = 0.15) compared

to SuNDF (2.2%). This would suggest that sucrose addition is more detrimental to fiber

digestion at a more acidic pH, but may actually have a positive effect under more neutral

conditions.

Fermentation pH for ApH decreased to 5.25 at 8 h. Decreased fiber digestion has

been associated with a more acidic pH (Khalili and Huhtanen, 1991b; Kennelly et al.,

1999). Several studies (Stewart, 1977; Hoover et al., 1984; Mould et al., 1984) have

reported almost complete inhibition of fiber digestion at pH 5.0. A decrease in pH or

supplementation of readily fermentable carbohydrates may decrease extent and increase

lag of fiber digestion (Grant, 1994). The current study did not include enough data points

in the early hours of the fermentation to adequately describe the lag phase. Nonetheless,









evaluation of the graphed data suggests that sucrose addition in the NpH fermentation

appeared to increase lag over the first 4 h of fermentation compared to fermentation of

iNDF as sole substrate (Figure 2-11).

Decreased fiber digestion can be attributed to a decrease in pH as a result of

increased VFA production, or a "carbohydrate effect" (Mould and Orskov, 1984). The

carbohydrate effect refers to a preference by ruminal microorganisms for more readily

available carbohydrates. An increased competition for nutrients such as nitrogen among

microbial populations fermenting NFC or NDF might also contribute to reduced fiber

digestion by fibrolytic microorganisms. Mould and Orskov (1984) suggested that the

carbohydrate effect might be a factor in reducing fiber digestion at a pH as high as 6.2.

However, the addition of sucrose to the NpH fermentation in the current study resulted in

increased fiber digestion. Heldt et al. (1999) also reported an increase in NDF digestion

when supplementing starch, sucrose, glucose or fructose to steers on a tallgrass-prairie

hay diet. In the study by Heldt et al. (1999) pH was only slightly decreased to 6.2, and in

the current study the pH for the neutral fermentation never decreased below 6.7.

Microbial Crude Protein Yield and Efficiency

Microbial crude protein yield was greater (P < 0.01) for NpH compared to ApH

over the 24 h fermentation (Figure 2-12). Maximum MCP yield was recorded at 12 h for

NpH (19.4 mg) and this was almost double the maximum amount achieved by ApH (11.1

mg at 20 h). Synthesis of MCP tended (P = 0.10) to be more efficient for NpH compared

to ApH (0.14 mg and 0.09 mg MCP/mg OMD, respectively).

Other studies have also shown a decrease in bacterial growth at a lower pH (Strobel

and Russell, 1986; Shi and Weimer, 1992; De Veth and Kolver, 2001). Decreased pH

has been associated with a decrease in the efficiency of microbial growth (Russell et al.,









1992; Russell and Wilson, 1996), which may be related to a potential increase in ruminal

microorganisms' energy requirements for maintenance at a lower pH (Shi and Weimer,

1992). If more energy is expended on maintenance it will lead to lower efficiency of

microbial protein synthesis.

Conclusions

The fermentation pH alters the yields of products and fermentation of NDF when

sucrose is included in the fermentation. Though sucrose appears to be readily fermented

regardless of pH, the utilization of the monosaccharide constituents (glucose and

fructose) changes depending on pH. Fructose utilization may be delayed at an acidic pH,

whereas glucose utilization does not appear to be affected. At a more neutral pH (6.7)

sucrose supplementation may increase fiber digestion, whereas at an acidic pH (5.6)

sucrose supplementation may decrease fiber digestion. To avoid the potential negative

effect of sucrose supplementation on ruminal pH and fiber digestion adequate effective

fiber should be provided in rations. Microbial protein yield and composition of microbial

mass (microbial protein:glycogen) changed with pH. The potential effects of sucrose

supplementation on ruminal fermentation as they change with pH could be important to

take into consideration when supplementing sucrose to ruminant diets that predispose the

animals to more acidic or more neutral ruminal pH.

Results from this study suggest that consideration be given to the interaction of

sucrose supplementation and ruminal pH for predicting fiber digestion and yield of

potentially metabolizable nutrients to the animal. Animal studies further evaluating the

effects of ruminal pH on substrate utilization and nutrient supply are warranted.









Table 2-1. Type and number of fermentation tubes per medium for each sampling hour,
indicating the substrate and analysis for which tubes were reserved in an 24 h
in vitro fermentation of sucrose and iNDF.
Analysis
pH and Microbial Microbial glycogen, residual
Substrat1 pH and Microbial 2
residual NDF crude protein sucrose, organic acids, NH3-N
and amino acid nitrogen
No substrate 2 x HSPP3 2 x LDPE4
iNDF 2 x HSPP
SuNDF 2 x HSPP 2 x HSPP 2 x LDPE
1 iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose + iNDF
2 NH3-N = ammonia nitrogen
3 HSPP = Nalgene high speed, polypropylene
4 LDPE = Nalgene high speed, low density, polyethylene

Table 2-2. Residual glucose, fructose, unhydrolyzed sucrose, monosaccharide sucrose
equivalent (glucose+fructose) and sucrose equivalent at 0, 4 and 8 h, and
averaged for 24 h in vitro fermentations of sucrose and isolated bermudagrass
neutral detergent residue with initial medium pH of 6.8 or 5.6.
Medium Unhydrolyzed Glucose+ Sucrose
Time Glucose Fructose sucrose Fructose1 eq. 2
pH Sucrose Fructose'- eq.
mg
Hour 0 6.8 8.45 9.31 83.4 16.9 100
5.6 27.0 27.4 48.5 51.6 100
SE3 3.09 3.68 25.0 6.43 18.7
P-value (medium) 0.11 0.15 0.36 0.13 1.00
Hour 4 6.8 -0.37 -0.02 -0.29 -0.37 -0.66
5.6 6.11 41.1 -0.24 44.8 44.6
SE 5.47 4.40 0.09 1.08 1.06
P-value (medium) 0.54 0.02 0.75 < 0.01 < 0.01
Hour 8 6.8 0.08 -0.05 -0.16 0.03 -0.13
5.6 -0.15 0.13 -0.17 -0.02 -0.19
SE 0.16 0.09 0.05 0.23 0.26
P-value (medium) 0.41 0.27 0.82 0.89 0.89
Avg. for 24 h 6.8 1.20 1.31 11.8 2.39 14.2
5.6 5.07 9.81 6.90 14.1 21.0
SE 0.90 1.14 3.60 0.94 2.95
P-value (medium) 0.01 0.11 0.37 < 0.01 < 0.01
P-value (medium x hour) 0.03 < 0.01 0.15 < 0.01 0.22
1 Calculated as residual (glucose + fructose) x 0.95 to give residual monosaccharide sucrose equivalent
2 Sucrose equivalent = residual (glucose + fructose) x 0.95 + unhydrolyzed sucrose
3 SE = standard error of least squares means
















,a OU
E
- 5.0

0 4.0
0
0)
S3.0

2 2.0
0


0 4 8 12 16 20 24
Fermentation hour


Figure 2-1. Microbial glycogen yield (LSmeans + standard error) for 24 h in vitro
fermentations of SuNDF with initial medium pH of 6.8 (m) or 5.6 (A).
SuNDF = sucrose + isolated bermudagrass neutral detergent residue.




8.0


7.5


c 7.0
0.0
4 6.5


6.0


5.5


5.0 -
0 4 8 12 16 20 24
Fermentation hour


Figure 2-2.Fermentation pH (LSmeans + standard error) for 24 h in vitro fermentations of
iNDF (A, o) and SuNDF (A, m) with an initial medium pH of 6.8 (m or o) or
5.6 (A or A). iNDF = isolated bermudagrass neutral detergent residue;
SuNDF = sucrose+iNDF.


























12 16 20
Fermentation hour


Figure 2-3. Acetate concentrations (LSmeans standard error) for 24 h in vitro
fermentations containing no substrate (o, x) or SuNDF (A, m) with an initial
medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF = sucrose + isolated
bermudagrass neutral detergent residue.


0 4 8 12 16 20 24
Fermentation hour


Figure 2-4.Propionate concentrations (LSmeans + standard error) for 24 h in vitro
fermentations containing no substrate (o, x) or SuNDF (A, m) with an initial
medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF = sucrose + isolated
bermudagrass neutral detergent residue.




























0 4 8 12 16 20 24
Fermentation hour


Figure 2-5.Butyrate concentrations (LSmeans standard error) for 24 h in vitro
fermentations containing no substrate (o, x) or SuNDF (A, m) with an initial
medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF = sucrose + isolated
bermudagrass neutral detergent residue.




140

120





100

4-
40


4 8 12 16 20 24
Fermentation hour


Figure 2-6. Total volatile fatty acid concentrations (LSmeans standard error) for 24 h in
vitro fermentations containing no substrate (o, x) or SuNDF (A, m) with an
initial medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF = sucrose +
isolated bermudagrass neutral detergent residue.










35

30 4

25



C 15

10

5
-5 /y






Fermentation hour


Figure 2-7.Lactate concentrations (LSmeans standard error) for 24 h in vitro
fermentations containing no substrate (o, x) or SuNDF (A, m) with an initial
medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF = sucrose + isolated
bermudagrass neutral detergent residue.




4.0

3.5

3.0



2.0
L-
1.5

1.0


0.0.

0 4 8 12 16 20 24
Fermentation hour


Figure 2-8.Branched chain volatile fatty acid concentrations (LSmeans + standard error)
for 24 h in vitro fermentations containing no substrate (o, x) or SuNDF (A,
m) with an initial medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF =
sucrose + isolated bermudagrass neutral detergent residue.










25



20



E 15



10


0 4 8 12 16 20 24
Fermentation hour


Figure 2-9. Ammonia nitrogen concentration (LSmeans standard error) for 24 h in vitro
fermentations containing no substrate (o, x) or SuNDF (A, m) with an initial
medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF = sucrose + isolated
bermudagrass neutral detergent residue.


4 8 12 16 20 24
Fermentation hour


Figure 2-10. Total free amino acid concentration (LSmeans standard error) for 24 h in
vitro fermentations containing no substrate (o, x) or SuNDF (A, m) with an
initial medium pH of 6.8 (m or o) or 5.6 (A or x). SuNDF = sucrose +
isolated bermudagrass neutral detergent residue.


























0 4 8 12 16 20 24
Fermentation hour


Figure 2-11. Residual NDF OM (LSmeans standard error) for 24 h in vitro
fermentations of (iNDF; A, o) and SuNDF (A, m) with an initial medium pH
of 6.8 (m or o) or 5.6 (A or A). iNDF = isolated bermudagrass neutral
detergent residue; SuNDF = sucrose + iNDF.


0 4 8 12 16 20 24
Fermentation hour


Figure 2-12. Microbial crude protein yield (LSmeans + standard error) for 24 h in vitro
fermentations of SuNDF with an initial medium pH of 6.8 (m) or 5.6 (A).
SuNDF = sucrose + isolated bermudagrass neutral detergent residue.














CHAPTER 3
EFFECT OF NITROGEN SOURCE ON MICROBIAL YIELD AND NEUTRAL
DETERGENT FIBER DIGESTION FROM IN VITRO FERMENTATIONS OF
SUCROSE AND ISOLATED NEUTRAL DETERGENT RESIDUE

Introduction

Many ruminal microorganisms can synthesize microbial protein with a non-protein

nitrogen (NPN) source such as urea, as the sole source of nitrogen (Oltjen, 1969). The

efficiency of microbial protein synthesis (MCPeff) with urea as the sole source of

nitrogen may be lower compared to when peptides or amino acids are supplied. The

importance of amino acids and peptides from dietary protein degradation for increasing

microbial protein (MCP) production and energetic efficiency has been shown in several

studies with batch culture fermentations (Maeng et al., 1976; Maeng and Baldwin, 1976a,

1976b). Russell and Sniffen (1984) reported an increase of 18.7% in ruminal bacteria

yield with the addition of amino acids to mixed cultures with theoretically adequate

ammonia concentrations. The advantages of peptides and amino acids may depend on

the species of bacteria and energy source (Cruz Soto et al., 1994). There appears to be a

higher requirement for amino acids or peptides by amylolytic organisms (Maeng and

Baldwin, 1976a, 1976b) and sugar-utilizing organisms (Hungate, 1966), relative to fiber

utilizers. Although cellulolytic organisms primarily use ammonia as nitrogen source,

amino acids and peptides have been shown to increase in situ fiber digestion compared to

ammonia nitrogen (NH3-N) (Yang, 2002). When a rapidly fermented carbohydrate

source, such as sucrose, is available, there could be competition between sugar-utilizing

bacteria and other microbial populations for available nitrogen. This has potential to









affect the microbial growth of the different populations and alter neutral detergent fiber

(NDF) digestion. The objective of the present study was to evaluate the effects of

different nitrogen sources on microbial fermentation products and NDF digestion from

the in vitro fermentation of isolated NDF and sucrose. A secondary objective was to

determine the effect of sucrose supplementation in combination with different nitrogen

sources on fermentation pH and NDF digestion compared to fermentation of isolated

NDF only.

Materials and Methods

Substrates

Substrates used were isolated bermudagrass (Cynodon dactylon L.) neutral

detergent residue (iNDF; 92.8% dry matter: DM, 99.4% organic matter: OM, 98.6%

neutral detergent fiber OM: NDFOM, 5.4% neutral detergent fiber crude protein:

NDFCP) and a 50:50 mixture of iNDF and sucrose (SuNDF). The iNDF was prepared as

described by Hall and Herejk (2001). Sucrose (S5-500, Fisher Scientific, Atlanta, GA;

99.98% DM, 100% OM) and iNDF were analyzed prior to the onset of the study for DM

and OM (AOAC, 1980), and iNDF for NDF using heat-stable a-amylase (Termamyl

120L, Novo Nordisk Biochem, Franklinton, NC (Van Soest et al., 1991) and crude

protein (AOAC, 1980). A total of 240 mg + 0.5 mg of substrate, with iNDF and sucrose

weighed individually, were transferred into duplicate 50 ml Nalgene high speed low

density polyethylene (LDPE) centrifuge tubes (05-562-13, Fisher Scientific, Atlanta, GA)

or Nalgene high speed polypropylene (HSPP) centrifuge tubes (05-562-10K, Fisher

Scientific, Atlanta, GA) depending on the type of analysis to be performed on

fermentation residues (Table 3-1).









Medium

Treatments consisted of one of three iso-nitrogenous media, containing different

nitrogen sources. The Goering and Van Soest (1970) medium contained NPN (3.52 mg

N) and true protein (6.85 mg N)(B), and was modified to contain only NPN (U) by

substituting urea (0.73 g urea/L of medium) for casein acid hydrolysate, or to contain

only true protein (C) by substituting casein acid hydrolysate (3.79 g casein acid

hydrolysate/L of medium) + sodium bicarbonate (4.25 g sodium bicarbonate/L of

medium) for ammonium bicarbonate.

Fermentation

Duplicate 16 h in vitro fermentation runs using destructive sampling of batch

cultures were performed according to the method of Goering and Van Soest (1970).

Casein acid hydrolysate (A-2427, Sigma Chemical Co., St. Louis, MO) was used as the

amino nitrogen source in B and C media. The reducing solution was mixed according to

a modification of the Goering and Van Soest (1970) procedure (P. J. Van Soest, personal

communication).The reducing solution was modified as described by Van Soest and

Robertson (1985). For a volume of 100 ml, 0.625 g ofL-cysteine hydrochloride (C-7880,

Sigma Chemical Co., St. Louis, MO) and approximately 10 pellets of KOH (P250-2,

Fisher Scientific, Atlanta, GA) were dissolved with stirring in 50 ml of distilled water. In

a separate glass beaker 0.625 g sodium sulfide (S-4766, Sigma Chemical Co., St. Louis,

MO) was dissolved with stirring in 50 ml of distilled water. The solutions were

combined when the contents of both beakers were in solution, and just before addition of

the reducing solution to the fermentation tubes.

Rumen inoculum was obtained approximately 3 h post feeding from a ruminally

cannulated, non-pregnant, non-lactating Holstein cow under approved protocols of the









University of Florida Institutional Animal Care and Use Committee. The donor cow

received a diet of bermudagrass hay (10 kg DM/day), 48% crude protein soybean meal

(900 g/d) and free choice mineral supplement (Ca 17- 20%, P > 9%, NaCl < 25%, Mg >

0.25%, Cu > 0.15, Co > 0.01 %, I > 0.01%, Mn > 0.2%, Se < 0.004%, Zn > 0.4%, Fl <

0.09%). The inoculum was filtered through four layers of cheesecloth and one layer of

glass wool and maintained under anaerobic conditions at 390C. Twenty milliliters of the

appropriate medium, 1 ml of reducing solution and 5 ml of inoculum were added to each

fermentation tube. After each addition, tube headspace was purged with CO2.

Fermentation tubes were capped with rubber stoppers fitted with gas release valves,

incubated (Equatherm Incubator Model C1487, Curtin Matheson Scientific, Inc.,

Houston, TX) under anaerobic conditions at 390C and destructively sampled at 0, 4, 8, 12

and 16 hours. Tubes were swirled individually to mix every 4 hours.

Sample Handling and Subsequent Analyses

At each sampling hour the fermentation tubes for the specific hour were removed

from the incubator and placed in an ice bath to terminate the fermentation process.

Approximately 5 min after tubes were removed from the incubator pH was recorded on

tubes reserved for NDF analysis. The tubes used for NDF analysis were stored at 10C

and were analyzed for residual NDF within two days of completion of the fermentation.

For NDF analysis, samples were allowed to equilibrate to room temperature, were

quantitatively transferred to 600 ml Berzellius beakers and refluxed with 50 ml of neutral

detergent solution and heat-stable a-amylase (Termamyl 120L, Novo Nordisk Biochem,

Franklinton, NC) for 1 h (Van Soest et al., 1991). To ensure removal of a-glucan, three

doses of 0.2 ml heat-stable a-amylase were used: one with addition of detergent, one 10









min before removing the sample from the burner and one added to the Gooch crucible

during rinsing with boiling water.

Fermentation tubes reserved for microbial glycogen (GLY), residual sucrose

equivalents (sucrose, and its hydrolysis products: glucose and fructose), organic acids,

NH3-N and amino acid analyses were centrifuged at 15,000 x g for 30 min at 50C. The

supernatant was transferred to scintillation vials and stored at -200C until analysis for

residual sucrose and organic acids by HPLC, and NH3-N and amino acids by flow-

injection analysis (Broderick et al., 2004). The HPLC for analyzing residual sucrose was

equipped with an anion exchange analytical column (CarboPacTM PAl, Dionex,

Sunnyvale, CA), the mobile phase used was 100 mMNaOH, the flow rate 1.0 ml/min and

the injection volume 10 pL. The HPLC for analyzing organic acids was equipped with

an organic acid column (PHX-87H, Bio-Rad Laboratories, Richmond, CA). The solvent

used was 0.015 NH2SO4 / 0.0034 MEDTA, the flow rate 0.7 ml/min, the column

temperature 450C and the injection volume 50 ptL.

The pellets from the high-speed centrifugation were quantitatively transferred to 50

ml glass beakers using no more than 20 ml of a 0.2 NNaOH solution to rinse out the

fermentation tubes. Glass beakers were stored at -200C until further analysis for GLY.

Beakers were removed from the freezer and samples were allowed to equilibrate to room

temperature. Microorganisms were lysed with a 0.2 NNaOH solution (brought to a

volume of 20 ml in the 50 ml glass beakers) in a boiling water bath for 15 min. Samples

were cooled to room temperature and then neutralized to pH 7.0 + 0.1 with 6 NHC1.

Samples were quantitatively transferred from the glass beakers to funnels fitted with glass

wool plugs for filtration into 100 ml volumetric flasks. Beakers, glass wool and funnels









were rinsed with distilled de-ionized water (ddH20), and then samples were brought to

volume with ddH20. Four milliliters of a 0.1 M sodium acetate buffer (pH ~ 4.5) and 50

[tl of amyloglucosidase (EC 3.2.1.3, A-3514, Sigma Chemical Co., St. Louis, MO) were

added to 4 ml of sample, incubated at 60 C for 45 min, and analyzed for a-glucan

content as released glucose corrected for free glucose (Karkalas, 1985).

Microbial crude protein was estimated as trichloroacetic acid (TCA)-precipitated

crude protein. Samples were individually removed from the ice bath and a total of 5.2 ml

of a 120% (w/v) TCA solution were added in two equal increments to achieve a final

concentration of 20.0% TCA. Samples were returned to the ice bath for 45 min after

which tubes were centrifuged at 7700 x g for 20 min at 50C. Each fermentation tube's

contents were then quantitatively transferred into Whatman 541 filter paper (09-85 ID,

Fisher Scientific, Atlanta, GA) in veined funnels set in 125 ml Erlenmeyer flasks, using

approximately 50 ml of chilled 10% TCA to rinse the tubes, filter and residue. Samples

were allowed to filter under gravity. The filtrate was filtered through a Whatman GF/A

glass fiber filter (09-874-16D, Fisher Scientific, Atlanta, GA), using 10% TCA to rinse

the flask, filter and residue. Both Whatman 541 and GF/A filters containing the TCA-

precipitated material from one fermentation tube were placed together in a beaker and

dried for 24 h at 550C, before analysis for crude protein content as Kjeldahl nitrogen

content x 6.25 (AOAC, 1980). Kjeldahl analysis blanks consisted of a Whatman 541

filter and a GF/A filter digested and distilled together in one Kjeldahl flask. The MCP

and GLY contents of each tube were corrected for fermentation blanks at each hour, and

MCP for its content by substrate at hour 0.









Statistical Analysis

The experimental design was a split-split plot in time with a 2 x 2 factorial

arrangements of treatments (media and substrates). The data were analyzed using the

PROC MIXED procedure of SAS (1999) with fermentation run (R) as a random variable,

and medium (M) and substrate (S) as fixed variables. Fermentation hour (H) was used as

a class variable. The Kenward-Roger method was used to calculate the denominator

degrees of freedom for testing fixed effects. Orthogonal contrasts, B and C vs. U, and B

vs. C, were used for medium comparisons across substrates (iNDF, SuNDF) as well as

within substrate. All values presented are least squares means. The model statement

used was:

Yij = + Mi + Sj + MSij + Hk + MHik + SHjk + MSHjk + Sijkl

Where:

Yijk = the dependent variable

|t = overall mean

Mi = medium (i = B, C, U)

Sj = substrate (j = iNDF, SuNDF)

Hk = hour (k = 0, 4, 8, 12, 16)

MSij = interaction term for medium and substrate

MHik = interaction term for medium and hour

SHjk = interaction term for substrate and hour

MSHijk = interaction term for medium, substrate and hour

sijkl = residual error









A treatment term (T) consisting of the interaction between substrate (S) and

medium (M) were used in the random statement to obtain appropriate standard errors for

the least squares means. The random statement included the following terms:

R1 + RTim + RHik + RTHimk

Where:

RI = fermentation run (m = 1, 2)

Tm = treatment (n = 0, 1, 2, 3, 4, 5); number assigned to M by S combinations

RTim = interaction term for fermentation run and treatment

RHik = interaction term for fermentation run and hour

RTHkIm = interaction term for fermentation run, treatment and hour

The sampling hour of maximum MCP or GLY yield within substrate, medium and

fermentation run were defined as the hour with the maximum least squares means for

these measures. The "hour" term and its interaction terms were omitted from the above

model to compare maximum MCP yield and MCP efficiency, GLY at 4 h, and residual

sucrose, fructose, glucose and sucrose equivalent at 0, 4 and 8 h. The MCP efficiency

was expressed as MCP (mg)/organic matter digested (OMD, mg). Organic matter

digested (mg) was calculated as the total sucrose (mg) minus residual sucrose equivalent

(mg) plus iNDFOM digested (mg), minus GLY (mg). Since NDF digestion, residual

sucrose, MCP and GLY were not measured on the same fermentation tube, the least

squares means for these measurements, within fermentation run at the hour of maximum

MCP, were used to calculate MCPeff for individual treatments. Sucrose equivalent was

calculated as residual sucrose (mg) + 0.95 x (residual fructose (mg) + residual glucose

(mg)). Total volatile fatty acids (VFA) is defined as the sum of acetate, propionate,









butyrate and valerate. Organic acid concentrations, but not protein degradation products

(branched chain VFA (BCVFA), total free amino acids and NH3-N), were corrected for

blank fermentations (no substrate, only inoculum). Orthogonal contrasts were used to

make comparisons among media across substrates (iNDF + SuNDF) as well as within

substrate.

Results and Discussion

Residual Substrate

Residual substrate, defined as the amount (mg) of glucose, fructose and sucrose

that could be detected in the supernatant, did not differ among media over the 16 h

fermentation (P = 0.60, 0.37 and 0.24, respectively). However, there were some

differences among media in the initial sampling hours (Table 3-2). At 0 h, U had more

residual fructose and tended to have more residual glucose than the other treatments.

There was no difference among the media for sucrose content at 0 h, though U was

numerically much smaller than B and C. The great variation and accordingly large

standard errors may have prevented detection of differences. Sucrose equivalents did not

differ among media at 0 h. The percentage of sucrose equivalent remaining at 0 h, as a

proportion of the initial 120 mg sucrose, was 74.6%, 77.9% and 50.9% for B, C and U,

respectively.

Glucose was not detected in either B or C at 4 h, and in U at 8 h, and at subsequent

hours for all media. No fructose was detected for B at 4 h and for C and U at 8 h, and at

subsequent hours for all media. Sucrose was readily degraded regardless of the source of

nitrogen, whereas ruminal microorganisms more readily utilized the monosaccharide

constituents (glucose and fructose) when provided with true protein compared to NH3-N

only. Fructose appeared to be utilized more slowly than glucose.









Microbial Glycogen

Maximum GLY accumulation for all treatments was achieved at 4 h, with a similar

steady decline thereafter (P = 0.46 for medium by hour interaction from 4 to 24 h; Figure

3-1). There was no detectable difference (P = 0.54) in maximal glycogen accumulation

among nitrogen sources (7.31, 7.17 and 6.84 mg for B, C and U, respectively). However,

over the 16 h fermentation, U tended to have a lower (P = 0.11) yield of GLY compared

to B and C (3.09, 3.48 and 3.54 mg, respectively), whereas B and C did not differ (P =

0.75).

Microbial glycogen accumulation may have been reduced in U as compared to B

and C as microorganisms had less preformed amino acids to incorporate into MCP and

thus had to expend more energy for MCP synthesis. Alternatively, a lack of sufficient

BCVFAs rather than nitrogen may have reduced the efficiency of substrate use by the

microorganisms provided only with NPN, and the limited amount of amino nitrogen

supplied by the inoculum (Russell and Sniffen, 1984)

Fermentation pH

Fermentation pH was lower (P < 0.01) with SuNDF as compared to iNDF (Figure

3-2). Among the media, mean fermentation pH for U was higher compared to B and C

for both SuNDF (P < 0.01) and iNDF (P < 0.01), while B and C did not differ (SuNDF, P

= 0.67; iNDF, P = 0.24). Aldrich et al. (1993) reported a decrease in ruminal pH for

cows fed a diet containing 65.7% compared to those fed a diet containing 52.4% rumen

available protein in combination with a rapidly degradable carbohydrates source (starch).

However, the comparison of different rumen degradable nitrogen sources (NPN and

amino acids or peptides) on fermentation pH needs further investigation.









The higher fermentation pH for U may be a result of the hydrolysis of the added

urea in the NPN fermentation to release ammonia (Figure 3-8), which is alkaline, and the

lower yield of organic acids for this medium. The lower fermentation pH with SuNDF

compared to iNDF as the substrate is likely due to increased organic acid production in

SuNDF fermentations which contained more readily fermented carbohydrate, however,

organic acid concentrations were not measured on iNDF fermentations.

Organic Acids

In general, organic acid production increased in the presence of amino acid

nitrogen compared to NH3-N only (Table 3-3). For fermentations containing SuNDF as

the substrate, concentrations of organic acids at 16 h, with the exception of lactate, were

greater or tended to be greater (acetate) for B and C as compared to U. The organic acid

concentrations of B and C media did not differ at 16 h. Organic acid concentrations did

not follow similar temporal patterns (Figures 3-3 to 3-7) over the 16 h fermentation as

indicated by medium by hour interactions (P < 0.01 for total VFA, propionate, butyrate,

valerate and lactate; P = 0.02 for acetate).

Maximum lactate concentration did not differ (P = 0.58) among media (27.6, 27.1

and 30.2 mM for B, C and U, respectively). However, maximum lactate concentration

was detected at 4 h for B and at 8 h for C and U (Figure 3-7). At the end of the

fermentation, lactate concentration was greater for U compared to B and C, and no lactate

was detected in B and C at 16 h (Table 3-3). Growth rates for Megasphaera elsdenii, a

major ruminal lactate-utilizer, may be stimulated in the presence of peptides and amino

acids (Cruz Soto et al., 1994). The relative decrease in lactate disappearance in

fermentations containing only NPN can be explained by impaired growth of lactate-









utilizers due to the lack of amino acids or BCVFAs (needed together with ammonia for

microbial protein synthesis).

Protein Degradation Products

Total free amino acid and NH3-N concentrations in fermentations are the net result

of amount supplied in the medium and inoculum, as well as protein breakdown and

nitrogen utilization by ruminal microorganisms in vitro. At various sampling hours

during the 16 h fermentation NH3-N (Figure 3-8) and total amino acid (Figure 3-9)

concentrations appeared to be higher for blank fermentations (no substrate, only

inoculum) compared to fermentations containing SuNDF. This may have resulted from

increased protein breakdown and decreased utilization in blank fermentations lacking in

fermentable carbohydrate substrates, or increased utilization of protein breakdown

products in fermentations with SuNDF as the substrate.

For the entire fermentation, and at the 16 h endpoint, the NH3-N concentration for

fermentations with SuNDF as the substrate was greater for U (P < 0.01) compared to B

and C, and B was greater than C (P < 0.01). Total free amino acid concentration for the

16 h fermentation, with SuNDF as the substrate, was greater (P < 0.01) for B and C

compared to U, and C was greater than B (P < 0.01). At 16 h, total free amino acid

concentration for B and C was greater (P = 0.02) than for U, and C tended to be greater

than B (P = 0.06). For the most part, relative differences in total amino acid and NH3-N

concentrations among the three fermentations reflected the differences in type of nitrogen

source supplied at the onset: the greater the amount of NPN in the medium, the more

NH3-N, and the more true protein, the more free amino acids.

Branched chain VFAs are also products of protein degradation. The concentration

of BCVFAs remained relatively low and similar among media through the initial 12 h of









fermentation (Figure 3-10). It was only at 16 h, with SuNDF as the substrate, that

BCVFA concentration was greater (P < 0.01) for B and C (2.09 and 3.07 mM,

respectively) compared to U (0.28 mM), and C was greater than B (P < 0.01). Increased

microbial lysis and degradation of microbial protein may occur when substrates

supporting growth and maintenance becomes limiting, which could explain the relative

increase in protein degradation products (BCVFA and ammonia) towards the end of the

fermentation.

Most research have been focused on comparing the effect of rumen degradable and

rumen undegradable nitrogen sources on animal performance and on protein breakdown

product concentrations in the rumen. Information on the effect of different rumen

degradable nitrogen sources (NPN vs. amino acids and peptides) on measurements such

as total free amino acid and ammonia nitrogen concentrations both in vitro and in the

rumen needs further investigation.

Neutral Detergent Fiber Digestion

Fermentation pH did not decline below 6.56 for any treatment, and so it is not

likely that pH had a negative effect on NDF digestion. At 16 h of fermentation, NDF

digestion (100 residual NDF) did not differ (P = 0.68) among media with iNDF as

substrate (18.5, 16.0 and 16.6% for B, C and U, respectively; Figure 3-11). At 16 h,

digestion of NDF for SuNDF was greater (P = 0.01) for B and C (21.0 and 19.5%,

respectively) compared to U (14.4%), and B and C did not differ (P = 0.45). Digestion of

NDF at 16 h, averaged across media, did not differ (P = 0.28) between iNDF and SuNDF

fermentations.

Proteins may be superior to urea for maintenance of fiber digestion despite the fact

that cellulolytic organisms primarily use ammonia as nitrogen source. This may indicate









that cellulolytic bacteria have some requirement for supplementation with amino acids or

peptides (Hoover, 1986), which may be related to the supply of BCVFAs. Gorosito et al.

(1985), however, suggested that amino acids or peptides might increase cell wall

digestion over BCVFAs alone.

There are not enough data points in the early hours to clearly define it, however, the

patterns of the lines suggest that the addition of sucrose increased the lag time of NDF

digestion in the early hours; iNDF declined below 95% residual NDF by 4 h, whereas,

SuNDF did not reach that point until after 8 h. The apparent lag noted for NDF digestion

may be the result of competition between NFC and fiber-utilizing microorganisms for the

nitrogen supply.

The addition of sucrose and true protein increased NDF digestion in the later hours

of the fermentation. Proteolytic activity by bacteria that ferment readily available

carbohydrates (e.g. sucrose; [Wallace et al., 1999]) could increase available ammonia and

BCVFAs, which are growth requirements for cellulolytic bacteria (Hoover, 1986).

Provision of limiting nutrients required by the microorganisms could enhance fiber

digestion.

Microbial Crude Protein Yield and Efficiency

The yield of MCP over the 16 h fermentation was lower for U compared to B and C

with SuNDF (P = 0.01) as the substrate, and tended to be lower when iNDF (P = 0.11)

was fermented alone (Figure 3-12). There was a greater MCP yield with SuNDF

compared to iNDF (P < 0.01) across all media. Maximum MCP yield was greater for B

and C compared to U, and did not differ between B and C, for SuNDF fermentations

(Table 3-4). For iNDF fermentations, however, there was no difference among media for









maximum MCP yield. Therefore the benefit of amino acids or peptides for maximum

MCP yield might only be apparent when sucrose is present in the fermentation.

Microbes receiving NPN alone (U) were only 64% as efficient in their yield of

MCP, with SuNDF as the substrate, as those receiving true protein (B and C), whereas B

and C did not differ from each other (Table 3-4). The greater MCP and MCPeff with the

addition of true protein is likely due to direct incorporation of amino acid or peptides into

microbial protein (Cotta and Russell, 1982), or increased availability of carbon skeletons

in the form of BCVFAs to support amino acid synthesis (Russell and Sniffen, 1984). The

importance of amino acids and peptides from dietary protein degradation for increasing

both microbial protein production and energetic efficiency has been shown in several

other studies with batch culture fermentations (Maeng et al., 1976; Maeng and Baldwin,

1976a, 1976b). Russell and Sniffen (1984) reported an increase of 18.7% in ruminal

bacteria yield in mixed culture fermentations with a mixture of carbohydrates (equal parts

of glucose, maltose, sucrose, cellobiose and soluble starch) and the addition of amino

acids with theoretically adequate ammonia concentrations.


Conclusions

Addition of sucrose and source of nitrogen affected in vitro yield of fermentation

products and NDF fermentation. Addition of true protein increased MCP yield and

efficiency of yield from ruminal microorganisms when sucrose was present and had a

positive effect on MCP yield from NDF alone. True protein addition increased NDF

digestion when sucrose was present, and increased total yield of organic acids.

Maximum accumulation of GLY was not affected by nitrogen source when sucrose and

NDF were fermented together. These results imply that the sources of ruminally









degradable nitrogen and the inclusion of sucrose may be important to consider in the

prediction of fiber digestion and metabolizable nutrient supply in ruminant diets. Several

animal studies have considered the effect of supplying ruminally degradable nitrogen

compared to ruminally undegradable nitrogen on animal performance. Animal studies

investigating the interaction of ruminally degradable nitrogen source and NFC source on

ruminal measures and animal performance are warranted.





Table 3-1. Type and number of fermentation tubes per medium for one sampling hour,
indicating the substrate and analysis for which tubes were reserved in a 16 h in
vitro fermentation of sucrose and isolated neutral detergent fiber.
Analysis
S. Microbial glycogen, residual
Substrat1 pH and Microbial 2
Substrate pH an Mia. sucrose, organic acids, NH3-N
residual NDF crude protein and amino acid nitrogen
and amino acid nitrogen
No substrate 4
No substrate2 x HSPP3 2 x LDPE4
(fermentation blank)
iNDF 2 x HSPP 2 x HSPP
SuNDF 2 x HSPP 2 x HSPP 2 x LDPE
1 iNDF = isolated bermudagrass neutral detergent residue; SuNDF = sucrose + iNDF
2 NH3-N = ammonia nitrogen
3 HSPP = Nalgene high speed, polypropylene
4 LDPE = Nalgene high speed, low density, polyethylene










Table 3-2. Residual glucose, fructose, sucrose, monosaccharide sucrose equivalent
(glucose + fructose) and sucrose equivalent at 0, 4 and 8 h, and averaged for
16 h in vitro fermentations of sucrose and isolated bermudagrass neutral
detergent residue with different sources of nitrogen in media.
Treatment' Glucose Fructose Sucrose Glucose+Fructose2 Sucrose eq.3


Hour 0
B
C
U
SE4

Treatment
Contrasts
B and C vs. U
B vs. C
Treatment'
Hour 4
B
C
U
SE

Treatment
Contrasts
B and C vs. U
B vs. C
Hour 8
B
C
U
SE

Treatment
Contrasts
B and C vs. U
B vs. C
Average for 16 h
B
C
U
SE


0.31
0.71
2.69
2.10


1.21
1.75
2.34
2.51


0.20 < 0.01


0.11
0.70
Glucose

-0.47
-0.72
3.14
1.88


0.32

0.18
0.92

0.13
0.61
0.05
0.11

0.06

0.09
0.05

0.35
0.77
1.18
0.73


<0.01
0.06
Fructose

0.02
5.92
13.9
8.62


0.51

0.33
0.61

0.06
-0.03
0.03
0.05

0.55

0.89
0.33

0.32
1.57
3.26
2.14


Least squares means (mg)
88.0 1.44
91.1 2.34
56.3 4.78
26.3 4.34
P-value


0.36

0.20
0.89
Sucrose


0.08

0.05
0.35
Glucose+Fructose2


Least squares means (mg)
-0.34 -0.43
-0.48 4.94
0.08 16.2
0.22 9.94
P-value


0.07

0.03
0.52


0.48

0.29
0.69


Least squares means (mg)
-0.07 0.18
-0.14 0.55
0.00 0.07
0.04 0.06
P-value


0.21

0.13
0.32


0.02

0.03
0.02


Least squares means (mg)
17.5 0.63
18.1 2.23
11.3 4.21
5.28 2.68
P-value


89.5
93.4
61.1
30.2

0.41

0.24
0.87
Sucrose eq.3

-0.77
4.46
16.3
10.0


0.46

0.28
0.69

0.11
0.41
0.07
0.05

0.02

0.04
0.02

18.1
20.3
15.5
7.95


Treatment 0.60 0.37 0.24 0.48 0.79
Contrasts
B and C vs. U 0.42 0.21 0.10 0.32 0.58
B vs. C 0.61 0.54 0.88 0.58 0.76
1 B = True protein + Non protein nitrogen; C = True protein only; U = Non-protein nitrogen only
2 Calculated as residual (glucose + fructose) x 0.95 to give residual monosaccharide sucrose equivalent
3 Sucrose equivalent = residual (glucose + fructose) x 0.95 + unhydrolyzed sucrose
4 SE = standard error of least squares means