The Pennsylvania State University. The Graduate School. Department of Dairy and Animal Science

Transcription

1 The Pennsylvania State University The Graduate School Department of Dairy and Animal Science DIETARY ALTERATIONS AND THEIR INFLUENCE ON RUMEN DEVELOPMENT IN NEONATAL DAIRY CALVES A Thesis in Animal Science by Keith Eric Lesmeister 2003 Keith Eric Lesmeister Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2003

2 The thesis of Keith Eric Lesmeister has been reviewed and approved* by the following: Arlyn J. Heinrichs Professor of Dairy and Animal Science Thesis Advisor Gabriella A. Varga Distinguished Professor of Animal Science Peter R. Tozer Assistant Professor of Animal Science Lester C. Griel Professor of Veterinary Science Harold W. Harpster Associate Professor of Animal Science Terry D. Etherton Distinguished Professor of Animal Nutrition Head of the Department of Dairy and Animal Science *Signatures are on file in the Graduate School

3 ABSTRACT A procedure for rumen tissue sampling was developed to determine treatment effects on rumen development and papillae growth in young calves and to improve repeatability in rumen tissue sampling techniques. Rumens were collected from 42 male Holstein calves from three separate experiments. Rumen sampling areas (n = 9) included the caudal dorsal blind sac, cranial dorsal sac, cranial ventral sac, and the caudal and ventral portions of the caudal ventral blind sac. Right and left sides of the rumen were sampled. Five 1-cm 2 sections were removed from each area and measured for papillae length (n = 20/area), papillae width (n = 20/area), rumen wall thickness (n = 5/area), and number of papillae per cm 2 (n = 5/area). Correlations between areas, samples, and measurements were obtained, and comparisons between experiments, areas, samples, and measurements were performed for all variables. In addition, power analyses were conducted for all variables to determine the efficacy of the procedure in detecting treatment differences. Results indicate that samples should be taken from the caudal and cranial sacs of the dorsal and ventral rumen to sufficiently represent papillae growth and development throughout the entire rumen. The procedure is capable of detecting treatment differences for papillae length and papillae width, has a decreased but acceptable capability of detecting treatment differences for rumen wall thickness, but appears limited in ability to detect treatment differences for papillae per cm 2. In conclusion, rumen tissue sampling to determine extent of rumen development in calves can be performed in a nonbiased and repeatable manner utilizing a limited number of calves. iii

4 Yeast culture (Saccharomyces cerevisiae culture) was added to a texturized calf starter at 0 (control), 1%, and 2% of DM to determine effects on intake, growth, blood parameters, and rumen development. Seventy-five Holstein calves (38 male and 37 female) were started on the experiment at 2 ± 1 d of age and studied for 42 d. Starter intake was measured and fecal scoring conducted daily. Growth and blood parameter measurements were recorded at weekly intervals. A subset of 6 male calves (2/treatment) was euthanized at 6 wk of age and rumen tissue sampled for rumen epithelial growth measurements. An additional 6 male calves were euthanized at 5 wk of age for rumen epithelial growth measurements. Inclusion of yeast culture at 2% of the starter ration significantly increased starter and total dry matter intake, average daily gain, and daily hip width change when compared to the control treatment. Daily change in hip height was also significantly greater for calves receiving 2% supplemental yeast culture than for calves receiving 1%. No significant treatment differences were observed for all other variables. These data suggest that the addition of yeast culture in a dairy calf starter at 2% enhances dry matter intake, growth, and slightly improves rumen development in dairy calves. A texturized calf starter containing 5 (control) or 12% molasses (dry matter basis) was fed to dairy calves to determine effects on intake, growth, blood parameters, and rumen development. Forty-six Holstein calves (26 male and 20 female) were started at 2 ± 1 d of age and studied for 42 days. Starter dry matter intake was measured and fecal scoring conducted daily. Growth and blood parameter measurements were conducted weekly. A subset of 6 male calves (3/treatment) was euthanized at 4 wk of age and rumen tissue sampled for rumen epithelial growth measurements. Starter sugar content was significantly increased in the starter containing extra molasses. Post- iv

5 weaning and overall starter dry matter intake, overall total dry matter intake, daily heart girth change, and final heart girth were significantly decreased while overall average daily gain tended to decrease when calves received starter containing 12% molasses. However, blood volatile fatty acid concentrations were significantly increased when calves received a starter containing 12% molasses. No significant differences were observed between calves receiving starters containing 5 or 12% molasses for all other variables. The data indicates that adding extra molasses to a texturized calf starter decreases intake and structural growth, possibly causing decreased weight gain, but increases blood volatile fatty acid concentrations and slightly increases rumen development. However, feed handling and physical prehension problems in addition to the negative influences on calf growth and intake do not support increasing starter molasses content to 12% of the ration. Neonatal Holstein calves were fed texturized calf starters containing 33% whole, dry-rolled, roasted-rolled, or steam-flaked corn to investigate how altering the corn processing method affects intake, growth, rumen and blood metabolites, and rumen development. In experiment 1Ninety-two Holstein calves (52 male and 40 female) were started at 2 ± 1 d of age and studied for 42 days. Starter dry matter intake was measured and fecal scoring conducted daily. Growth and blood parameter measurements were conducted weekly. A subset of 12 male calves (3/treatment) was euthanized at 4 wk of age and rumen tissue sampled for rumen epithelial development measurements. Experiment 2 consisted of 12 male Holstein calves ruminally cannulated at 7 ± 1 d of age. Rumen fluid and blood samples were collected at multiple time points over a 22 h period during weeks 2 to 6. In the first experiment, post-weaning and overall starter and total dry mater intake was significantly higher in calves fed starter with dry- v

6 rolled corn than in calves fed starter with roasted-rolled or steam flaked corn. Postweaning and overall starter and overall total dry matter intake was significantly higher in calves fed starter with whole corn than in calves fed starter with steam-flaked corn. Post-weaning average daily gain was significantly greater in calves fed starter with dryrolled corn than in calves fed starter with steam-flaked corn. Daily structural growth was greatest in calves receiving starter with roasted-rolled corn. Blood volatile fatty acid concentrations were significantly higher in calves fed starter with steam-flaked corn than in calves fed all other treatments. Papillae length and rumen wall thickness was significantly greater in calves fed starter with steam-flaked corn than in calves fed starter with dry-rolled and whole corn, respectively. In experiment 2, calves fed starter with whole corn had higher rumen ph and lower rumen volatile fatty acid concentrations than calves fed all other starters. Rumen propionate production was increased in calves receiving starter with steam-flaked corn, however, rumen butyrate production was higher in calves fed starter with roasted-rolled corn. Results from the current study indicate that the type of processed corn incorporated into calf starter can influence intake, growth, and rumen parameters in neonatal calves. Calves consuming starter containing roasted-rolled corn had similar body weight, feed efficiency, and rumen development but increased structural growth and ruminal butyrate production when compared to the other three corn processing treatments. vi

7 TABLE OF CONTENTS ABSTRACT...iii LIST OF FIGURES...xi LIST OF TABLES...xiv ACKNOWLEDGEMENTS...xviii CHAPTER INTRODUCTION...1 REFERENCES...3 CHAPTER REVIEW OF RUMEN DEVELOPMENT LITERATURE...4 Rudimentary Reticulo-Rumen...5 Changes in Rumen Epithelium...6 Liquids Feeds and Epithelial Development...8 Solid Feeds and Epithelial Development...8 Physical Structure and Epithelial Development...11 Changes in Rumen Muscularization and Volume...12 REVIEW OF YEAST CULTURE LITERATURE...14 Yeast Culture and the Rumen Environment...15 Yeast Culture and Lactic Acid...15 Yeast Culture and Ruminal ph...15 Yeast Culture and Rumen Microbes...17 Yeast Culture and Volatile Fatty Acids...18 Yeast Culture and Solid Component Digestion...20 Yeast Culture and Intake, Gain, and Efficiency...21 Yeast Culture and Health...22 REVIEW OF MOLASSES LITERATURE...23 Molasses and Intake, Gain, and Efficiency...24 Molasses and Diet Digestibility...26 Molasses and Dry Matter or Organic Matter Digestibility...26 Molasses and Structural Component Digestibility...27 Molasses and Crude Protein Digestibility...28 Molasses and the Rumen Environment...29 Molasses and Volatile Fatty Acids...29 Molasses and Rumen ph...31 REVIEW OF GRAIN PROCESSING LITERATURE...32 Grain Processing and the Rumen Environment...33 Grain Processing and Volatile Fatty Acids...33 Grain Processing and Rumen Ammonia...34 Grain Processing and Diet Digestibility...35 vii

8 Grain Processing and Starch Digestibility...35 Grain Processing and Structural Component Digestibility...36 Grain Processing and Intake, Gain, and Efficiency...38 REFERENCES...40 CHAPTER DEVELOPMENT AND ANALYSIS OF A RUMEN TISSUE SAMPLING PROCEDURE...50 ABSTRACT...50 INTRODUCTION...51 MATERIALS AND METHODS...52 Rumen Tissue Sampling...52 Statistical Analysis...54 Correlations...54 Comparisons...55 Regression Analysis...57 Power Analysis...57 RESULTS...61 Experiment Analysis...61 Area Analysis...61 Correlations...61 Comparisons...62 Sample and Measurement Analysis...63 Correlations...63 Comparisons...63 Rumen Variable Relationships...64 Power Analysis...64 Calves, Samples, and Measurements Required...64 LSN and Power when N = LSN...65 DISCUSSION...66 Correlations...66 Rumen Variable Relationships...67 Comparison and Power Analyses...67 Papillae Length...67 Papillae Width...68 Rumen Wall Thickness...69 Papillae per cm Calf Numbers, Samples, and Measurements...70 CONCLUSION...71 REFERENCES...73 CHAPTER EFFECTS OF SUPPLEMENTAL YEAST CULTURE (Saccharomyces cerevisiae culture) ON RUMEN DEVELOPMENT, GROWTH CHARACTERISTICS, AND BLOOD PARAMETERS IN NEONATAL DAIRY CALVES...79 viii

9 ABSTRACT...79 INTRODUCTION...80 MATERIALS AND METHODS...81 Animals, Housing, and Diet...81 Starter Nutrient Composition...82 Fecal Scoring and Experimental Measurements...83 Rumen Tissue Sampling...84 Statistical Analyses...84 RESULTS AND DISCUSSION...86 Intake and Weight Gain...86 Structural Growth...89 Blood Parameters...90 Days Scoured...90 Rumen Development...91 CONCLUSION...92 REFERENCES...93 CHAPTER EFFECTS OF ADDING EXTRA MOLASSES TO A TEXTURIZED CALF STARTER ON RUMEN DEVELOPMENT, GROWTH CHARACTERISTICS, AND BLOOD PARAMETERS IN NEONATAL DAIRY CALVES ABSTRACT INTRODUCTION MATERIALS AND METHODS Animals, Housing, and Diet Starter Nutrient Composition and Particle Size Fecal Scoring and Experimental Measures Rumen Tissue Sampling Statistical Analyses RESULTS AND DISCUSSION Diet Composition Intake and Weight Gain Structural Growth Blood Parameters and Days Scoured Rumen Development CONCLUSION REFERENCES CHAPTER EFFECTS OF CORN PROCESSSING ON GROWTH CHARACTERISTICS, RUMEN DEVELOPMENT, AND RUMEN PARAMETERS IN NEONATAL DAIRY CALVES ABSTRACT INTRODUCTION ix

10 MATERIALS AND METHODS Animals, Housing, and Diet Starter Nutrient Composition and Particle Size Fecal Scoring, Health Monitoring, and Experimental Measurements Rumen Tissue Sampling Rumen Cannulation Experiment Statistical Analyses RESULTS AND DISCUSSION Diet Composition Experiment Intake and Weight Gain Structural Growth Blood Parameters Days Scoured, Respiratory, and General Appearance Rumen Development Experiment Starter Intake Rumen ph Rumen NH 3 Concentration Total VFA Concentration Rumen Acetate Concentration Rumen Propionate Concentration Rumen Butyrate Concentration Rumen Butyrate + Propionate:Acetate Ratio Plasma βhba CONCLUSION REFERENCES APPENDIX A Supporting Tables for Chapter APPENDIX B Supporting Graphs for Chapter APPENDIX C Supporting Graphs for Chapter APPENDIX D Supporting Tables and Graphs for Chapter x

11 LIST OF FIGURES Figure 3-1: Example of a rumen dissected utilizing the described procedure depicting the physical areas of the rumen sampled and corresponding labels. (A) caudal portion of the caudal ventral blind sac; (RB) right side and (LB) left side caudal dorsal sac; (RC) right side and (LC) left side cranial dorsal sac; (RD) right side and (LD) left side cranial ventral sac; and (RE) right side and (LE) left side ventral portion of caudal ventral blind sac...75 Figure 6-1: Graphic representation of in vitro starch disappearance over time for whole and dry-rolled ( ), roasted-rolled ( ), and steam-flaked ( ) corn utilized in the current experiment Figure Appendix B-1: Daily starter DMI for Holstein calves receiving 0 ( ), 1% ( ), or 2% ( ) supplemental yeast culture in a texturized calf starter Figure Appendix B-2: Weekly body weight for Holstein calves receiving 0 ( ), 1% ( ), or 2% ( ) supplemental yeast culture in a texturized calf starter Figure Appendix B-3: Weekly hip height for Holstein calves receiving 0 ( ), 1% ( ), or 2% ( ) supplemental yeast culture in a texturized calf starter Figure Appendix B-4: Weekly wither height for Holstein calves receiving 0 ( ), 1% ( ), or 2% ( ) supplemental yeast culture in a texturized calf starter Figure Appendix B-5: Weekly hip width for Holstein calves receiving 0 ( ), 1% ( ), or 2% ( ) supplemental yeast culture in a texturized calf starter Figure Appendix B-6: Weekly heart girth for Holstein calves receiving 0 ( ), 1% ( ), or 2% ( ) supplemental yeast culture in a texturized calf starter Figure Appendix B-7: Weekly percent blood hematocrit for Holstein calves receiving 0 ( ), 1% ( ), or 2% ( ) supplemental yeast culture in a texturized calf starter Figure Appendix B-8: Weekly plasma total protein for Holstein calves receiving 0 ( ), 1% ( ), or 2% ( ) supplemental yeast culture in a texturized calf starter Figure Appendix B-9: Plasma βhba from weeks 4, 5, and 6 for Holstein calves receiving 0 ( ), 1% ( ), or 2% ( ) supplemental yeast culture in a texturized calf starter Figure Appendix C-1: Daily starter DMI for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter Figure Appendix C-2: Weekly body weight for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter xi

12 Figure Appendix C-3: Weekly hip height for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter Figure Appendix C-4: Weekly wither height for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter Figure Appendix C-5: Weekly hip width for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter Figure Appendix C-6: Weekly heart girth for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter Figure Appendix C-7: Weekly percent blood hematocrit for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter Figure Appendix C-8: Weekly plasma total protein for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter Figure Appendix C-9: Plasma βhba from weeks 3-6 for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter Figure Appendix C-10: Blood total VFA concentration from weeks 4 and 5 for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter Figure Appendix C-11: Blood acetate concentration from weeks 4 and 5 for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter Figure Appendix C-12: Blood propionate concentration from weeks 4 and 5 for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter Figure Appendix C-13: Blood butyrate concentration from weeks 4 and 5 for Holstein calves receiving 5 ( ) or 12% ( ) molasses in a texturized calf starter Figure Appendix D-1: Daily starter DMI of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter (Growth Experiment) Figure Appendix D-2: Weekly body weight of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter Figure Appendix D-3: Weekly hip height of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter xii

13 Figure Appendix D-4: Weekly wither height of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter Figure Appendix D-5: Weekly hip width of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter Figure Appendix D-6: Weekly heart girth of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter Figure Appendix D-7: Weekly percent blood hematocrit of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter Figure Appendix D-8: Weekly plasma total protein of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter Figure Appendix D-9: Plasma βhba for weeks 3-6 of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter Figure Appendix D-10: Total blood VFA concentration for weeks 4 and 5 of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter Figure Appendix D-11: Blood acetate concentration for weeks 4 and 5 of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter Figure Appendix D-12: Blood propionate concentration for weeks 4 and 5 of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter Figure Appendix D-13: Blood butyrate concentration for weeks 4 and 5 of Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter Figure Appendix D-14: Daily starter DMI of cannulated Holstein calves receiving whole ( ), dry-rolled ( ), roasted-rolled ( ), or steam-flaked ( ) corn in a texturized calf starter (Cannulation Experiment) xiii

14 LIST OF TABLES Table 3-1: Least squares means for papillae length (PL), papillae width (PW), rumen wall thickness (RWT), and papillae per cm 2 (PC) for different experiments...76 Table 3-2: Least squares means for papillae length (PL), papillae width (PW), rumen wall thickness (RWT), and papillae per cm 2 (PC) for different areas of the rumen across all calves and experiments Table 3-3: Results of power analysis for number of calves, samples, and measurements required for papillae length, papillae width, rumen wall thickness, and papillae per cm Table 4-1: Ingredient composition of texturized calf starter containing 0 (C), 1% (1YC), or 2% (2YC) supplemental yeast culture Table 4-2: Nutrient composition of texturized calf starter containing 0 (C), 1% (1YC), or 2% (2YC) supplemental yeast culture Table 4-3: Least squares means for intake and BW of Holstein calves receiving 0 (C), 1% (1YC), or 2% (2YC) supplemental yeast culture in a texturized calf starter Table 4-4: Least squares means for structural growth measurements of Holstein calves receiving 0 (C), 1% (1YC), or 2% (2YC) supplemental yeast culture in a texturized calf starter...99 Table 4-5: Least squares means for blood parameter measurements and days scoured of Holstein calves receiving 0 (C), 1% (1YC), or 2% (2YC) supplemental yeast culture in a texturized calf starter Table 4-6: Least squares means for rumen development measurements of Holstein calves receiving 0 (C), 1% (1YC), or 2% (2YC) supplemental yeast culture in a texturized calf starter Table 5-1: Ingredient composition and particle size distribution of texturized calf starter containing 5 (C) or 12% (EM) molasses Table 5-2: Nutrient composition of texturized calf starter containing 5 (C) or 12% (EM) molasses Table 5-3: Least squares means for intake and BW of Holstein calves receiving 5 (C) or 12% (EM) molasses in a texturized calf starter Table 5-4: Least squares means for structural growth measurements of Holstein calves receiving 5 (C) or 12% (EM) molasses in a texturized calf starter xiv

15 Table 5-5: Least squares means for blood parameter measurements and days scoured of Holstein calves receiving 5 (C) or 12% (EM) molasses in a texturized calf starter Table 5-6: Least squares means for rumen development measurements of Holstein calves 1 receiving 5 (C) or 12% (EM) molasses in a texturized calf starter Table 6-1: Ingredient composition of texturized calf starter containing whole, dryrolled, roasted-rolled, or steam-flaked corn Table 6-2: Nutrient composition and particle size distribution of texturized calf starter containing whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn Table 6-3: Least squares means for intake and BW of Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table 6-4: Least squares means for structural growth measurements of Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table 6-5: Least squares means for blood parameter measurements, days scoured, respiratory score, and general appearance score of Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table 6-6: Least squares means for rumen development measurements of Holstein calves 1 receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table 6-7: Least squares means for average daily starter DMI (g/d) treatment by week effect from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table 6-8: Least squares means for rumen ph and NH 3 treatment by week effect from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table 6-9: Least squares means for total rumen VFA, acetate, propionate, and butyrate concentrations from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table Appendix A-1: Correlations between papillae length measurements taken from different areas of the rumen across calves and trials xv

16 Table Appendix A-2: Correlations between papillae width measurements taken from different areas of the rumen across calves and trials Table Appendix A-3: Correlations between rumen wall thickness measurements taken from different areas of the rumen across calves and trials Table Appendix A-4: Correlations between papillae per cm 2 measurements taken from different areas of the rumen across calves and trials Table Appendix A-5: Correlations between measurements of papillae length, papillae width, rumen wall thickness, and papillae per cm 2 from different samples taken across areas, calves, and trials Table Appendix A-6: Least squares means and SEM for papillae length 1 (PL), papillae width 1 (PW), rumen wall thickness 1 (RWT), and papillae per cm 2 (PC) for different samples taken across all areas, calves, and trials Table Appendix A-7: Correlations between different measurements of papillae length and papillae width taken across samples, areas, calves, and trials Table Appendix A-8: Least squares means and SEM for papillae length 1 (PL) and papillae width 1 (PW) for different measurements taken across all samples, areas, calves, and trials Table Appendix D-1: In vitro starch disappearance of whole and dry-rolled (WC/DRC), roasted-rolled (RC), or steam-flaked (SFC) corn 1 utilized in the current study Table Appendix D-2: Least squares means for rumen ph treatment and treatment by hour effects from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table Appendix D-3: Least squares means for rumen NH 3 1 treatment and treatment by hour effects from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table Appendix D-4: Least squares means for rumen total VFA 1 treatment and treatment by hour effects from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table Appendix D-5: Least squares means for rumen acetate 1 treatment and treatment by hour effects from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter xvi

17 Table Appendix D-6: Least squares means for rumen propionate 1 treatment and treatment by hour effects from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table Appendix D-7: Least squares means for rumen butyrate 1 treatment and treatment by hour effects from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table Appendix D-8: Least squares means for rumen butyrate + propionate:acetate 1 ratio treatment, treatment by week, and treatment by hour effects from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roasted-rolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table Appendix D-9: Least squares means for plasma β-hydroxybutyrate 1 treatment, treatment by week, and treatment by hour effects from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roastedrolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table Appendix D-10: Week 5 least squares means for blood total VFA concentration 1 treatment and treatment by hour effects from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roastedrolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table Appendix D-11: Week 5 least squares means for blood acetate concentration 1 treatment and treatment by hour effects from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roastedrolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table Appendix D-12: Week 5 least squares means for blood propionate concentration 1 treatment and treatment by hour effects from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roastedrolled (RC), or steam-flaked (SFC) corn in a texturized calf starter Table Appendix D-13: Week 5 least squares means for blood butyrate concentration 1 treatment and treatment by hour effects from ruminally cannulated Holstein calves receiving whole (WC), dry-rolled (DRC), roastedrolled (RC), or steam-flaked (SFC) corn in a texturized calf starter xvii

18 ACKNOWLEDGEMENTS If nobody ever said anything unless he knew what he was talking about, a ghastly hush would descend upon the earth. Sir Alan Herbert First and foremost, I want to thank God. Without His help, support, provision, gifts, and love, none of this would be possible. Let alone worthwhile. A large amount of work, blood, sweat, and even tears have gone into this Doctoral program, and there are so many that have helped me along the way. Without them, I would not have been able to do this, either. I could not have asked for a better advisory committee. Each member of my committee has provided for me in so many ways, and they have enabled me to work through problems that should not have been difficult to solve, with minimal to know exasperation. They have been more than guides on this trip; they have been friends, confidantes, and comrades inside and outside the realm of animal science. I greatly appreciate all of the opportunities that Dr. A. Jud Heinrichs has provided for me. If he had not pulled some strings, I would not have even been here. He has constantly strived to stimulate deeper thought on the subjects that I was covering, and would not accept a cursory overview of topics. He has taught me more about calf nutrition than I thought I would ever learn, and in my opinion has more than adequately prepared me for the next career stage. In addition, his little piece of Central Pennsylvania provided a means of escape when things became more than I could handle. He likely feels that I xviii

19 was there more than I should be, and he is probably right. However, he never made it much of an issue, and I deeply thank him for that. I am deeply honored and pleased to have Dr. Gabriella Varga on my advisory committee. Who could desire a more knowledgeable and able advisor? She has a vast wealth of understanding and experience, and I actually fear that I did not utilize her knowledge to its full extent. I thank Dr. Peter Tozer for all of his statistical assistance. With the level of advice and support offered by Dr. Tozer, he probably deserves a cochair position on my committee. He was never unwilling to help, but never spoon-fed me the answers either, and under his instruction I have learned more about statistics than I ever thought I could or would. However, he may not totally agree with the previous statement. Dr. Lester Griel was an indispensable committee member, and without his surgical expertise, my final trial would not have run as smoothly as it did. In addition, he has provided an aspect of calf health to my program that I desperately needed but otherwise would have missed. Finally, I thank Dr. Harold Harpster for providing an applied background to my program. His addition to my advisory committee enabled me to keep my feet on the ground, and strive to find applicability for everything I did. Unfortunately, his knowledge was also another well that I did not dip into deep enough. I could not have performed this work if it was not for the help and support of the many friends in my life. I thank Dr. Matt Gabler and his lovely and wonderful wife MaraLee for all of their love and support during this process. They have been an inestimable asset to me and I will forever cherish them and their friendship. Matt has provided companionship, physical, emotional, and educational support, and someone with whom I could pursue my love for hunting and fishing. I am honored and blessed to have made his acquaintance. I also thank all the Animal Science graduate students for xix

20 their help and support, especially Liz Groff, Jana Edwards, Sally Flowers, Michelle Picket, Dana Pape, Emily Landis, Susan Kress, Adam Kauf, Paul Kononoff, Sylvia Wawrzyniak, and Jamie Delahoy. It has been wonderful to be a part of this graduate student group. I extend my appreciation and thanks to Clayton Hetrick and his family, Jeromy and Laureen Knepp, Brad and Brooke Biehl, Pete and Jill Reitz, Kyle and Jen Eastman, Kenny Carnes, Blaine Kemna and his family, and David (Cat) Dickey. There friendships and support have blessed me more than I could ever indicate. There have been numerous others that have supported me and made this work possible. In particular, I thank my Dad, Harold Lesmeister, and my Mom, Beth Baugh, for always being supportive of my goals and desires and for encouraging me to pursue them. Jim Dugan also deserves my heartfelt thanks for also encouraging me to pursue my dreams and for supporting me throughout my pursuits. Lastly, but definitely not least, I thank my excellent and wonderful wife, Esther. Earning my Doctorate degree was not one of my life goals when I began my college career. However, meeting my wife provided, for me, a reason to pursue my potential to the fullest. Pursuing my Doctorate degree has been a very long and difficult process, saturated with anxieties, tears, joys, and uncertainties. However, Esther has stuck by and supported me throughout it all, and that fact speaks more to the character of my wife than I ever could. Esther, I thank you and love you for this. That is the simplest and deepest way that I can explain how I feel about what you have done for me. To all those included above and to those that I have likely missed. Thank You xx

21 CHAPTER 1 INTRODUCTION Rumen development studies in neonatal calves began in 1956 with publications from Brownlee (1956), and Warner et al. (1956) indicating that access to different types of feed altered growth rates, rumen volume, and papillae growth in calves. These publications sparked a dramatically increased interest in this field of research and led to numerous papers throughout the late 1950 s and into the 1960 s (Flatt et al., 1958; Harrison et al., 1960; Stobo et al., 1966a; Stobo et al., 1966b; Sutton et al., 1963; Tamate et al., 1962). This research distinguished influencing factors of rumen development and papillae growth, delving into the biochemical and physiological attributes of rumen development. Concomitant with this research was the idea that acceleration in rumen development could enable decreased age at weaning, an attribute having economic (Gabler et al., 2000) and health advantages (Morrill, 1992) for the dairy heifer replacement industry and the dairy industry as a whole. Publication of research addressing neonatal rumen development has continued, but at a decreased frequency. Primary factors affecting rumen development were distinguished in the initial papers, whereas subsequent papers have focused on alterations in the chemical or physical structure of the diet or inclusion of novel dietary additives. Later publications have resulted in a divergence in research as to which attributes of neonatal ruminant diets actually optimize rate and extent of rumen development, with a split occurring between the physical and chemical attributes. It is likely that the optimal answer is not solely a physical or chemical dietary attribute, but rather a combination of both. In addition, 1

22 continued development and increased availability of new dietary byproducts, novel dietary additives, and feeding programs demands additional research in rumen development. The objectives of this literature review and the subsequent research are to revisit the groundwork that created interest in and current theories of rumen development, compile the available information from yeast culture, supplementary molasses, and grain processing research, and determine their possible influence on rumen development and overall nutrition of the neonatal dairy calf. Research was conducted on these three factors due to indications throughout the literature of their influence on intake and rumen metabolite production, variables reported to be important for rumen development in neonatal dairy calves. 2

23 REFERENCES Brownlee, A The development of rumen papillae in cattle fed on different diets. Brit. Vet. J. 112: Warner, R. G., W. P. Flatt, and J. K. Loosli Dietary factors influencing the development of the ruminant stomach. J. Agric. Food Chem. 4: Flatt, W. P., R. G. Warner, and J. K. Loosli Influence of purified materials on the development of the ruminant stomach. J. Dairy Sci. 41: Gabler, M. T., P. R. Tozer, and A. J. Heinrichs Development of a cost analysis spreadsheet for calculating the costs to raise a replacement dairy heifer. J. Dairy Sci. 83: Harrison, H. N., R. G. Warner, E. G. Sander, and J. K. Loosli Changes in the tissue and volume of the stomachs of calves following the removal of dry feed or consumption of inert bulk. J. Dairy Sci. 43: Morrill, J. L The Calf: Birth to 12 Weeks. Large Dairy Herd Management. H. H. Van Horn and C. J. Wilcox, editors. American Dairy Science Association, Champaign, IL. Chapter 41: Stobo, I. J. F., J. H. B. Roy, and H. J. Gaston. 1966a. Rumen development in the calf. 1. The effect of diets containing different proportions of concentrates to hay on rumen development. Br. J. Nutr. 20: Stobo, I. J. F., J. H. B. Roy, and H. J. Gaston. 1966b. Rumen development in the calf. 2. The effect of diets containing different proportions of concentrates to hay on digestive efficiency. Br. J. Nutr. 20: Sutton, J. D., A. D. McGilliard, and N. L. Jacobson Functional development of rumen mucosa. I. Absorptive ability. J. Dairy Sci. 46: Tamate, H., A. D. McGilliard, N. L. Jacobson, and R. Getty Effect of various dietaries on the anatomical development of the stomach in the calf. J. Dairy Sci. 45:

24 CHAPTER 2 REVIEW OF RUMEN DEVELOPMENT LITERATURE Neonatal ruminants are unique in the aspect that at birth they are physically and functionally two different types of animals, with respect to their gastro-intestinal system. To clarify, at birth the physical attributes distinguishing a ruminant from a monogastric animal, i.e. the reticulum, rumen, and omasum, are present. However, the rudimentary state of the reticulo-rumen and omasum, presence of the esophageal groove (Church, 1988), plus the developing abomasal and intestinal enzymatic state forces neonatal ruminants to function as monogastric animals (Longenbach and Heinrichs, 1998), subsisting on milk-based diets, which they do quite efficiently (Davis and Drackley, 1998; Van Soest, 1994). However, decreased milk-based diet utilization as the calf ages, due to digestive enzymatic changes (Longenbach and Heinrichs, 1998), coupled with the high daily costs of maintaining a preweaned calf (Gabler et al., 2000) results in a necessity to transition the calf from a monogastric animal to a ruminant animal (Church, 1988; Davis and Drackley, 1998). A smooth transition from a monogastric to ruminant animal, with minimal loss in growth, requires adequate size and development of the reticulo-rumen for efficient utilization of dry and forage-based diets. Therefore, understanding the factors responsible for initiating, driving, and establishing rumen development and function in the neonatal calf is of primary importance. 4

25 Rudimentary Reticulo-Rumen At birth, the reticulum, rumen, and omasum are undeveloped, nonfunctional, small in size when compared to the abomasum, and disproportionate to the adult digestive system (Tamate et al., 1962), papillary growth, rumen muscularization, and rumen vascularization is minimal to nonexistent, the rumen wall is thin and slightly transparent, and reticulo-rumen volume is minimal (Flatt et al., 1958; Harrison et al., 1960; Sander et al., 1959; Tamate et al., 1962; Warner et al., 1956). In addition, Lane et al. (2000) indicated that the rumen epithelial cells of sheep younger than 42 days of age were incapable of converting butyrate to β-hydroxybutyrate, suggesting a metabolically inactive epithelium at birth. However, rumen epithelial metabolic activity in calves has been shown to occur at a younger age than reported in lambs, and to increase with age (Nocek et al., 1980; Sutton, 1963). Ruminant animals require a physically and functionally developed rumen to meet the demands of an innate desire to consume forages and dry feeds (Van Soest, 1994). However, the neonatal rumen will remain undeveloped if the necessary diet requirements of rumen development are not provided (Brownlee, 1956; Harrison et al., 1960; Van Soest, 1994). Rumen development appears to be greatly affected by diet and drastic dietary changes (Brownlee, 1956; Harrison et al., 1960). Furthermore, the influence of dietary factors on rumen development may vary and development of rumen epithelium, rumen muscularization, and increases in rumen volume have been found to occur independently (Brownlee, 1956; Flatt et al., 1958; Harrison et al., 1960; Stobo et al., 1966a). These findings suggest that dietary factors influencing papillary growth and development may not affect rumen muscularization or rumen volume. 5

26 Changes in Rumen Epithelium Proliferation and growth of squamous epithelial cells increases papillae length, papillae width, and thickness of the interior rumen wall (Church, 1988). Prior to transitioning from a pre-ruminant to ruminant phase, growth and development of the ruminal absorptive surface area (papillae), is necessary to enable absorption and utilization of microbial digestion end products, specifically rumen volatile fatty acids (Church, 1988; Sutton et al., 1963; Warner et al., 1956; Van Soest, 1994). Presence and absorption of volatile fatty acids is indicated to stimulate rumen epithelial metabolism and may be key in initiating rumen epithelial development (Baldwin and McLeod, 2000; Sander et al., 1959; Sutton et al., 1963; Tamate et al., 1962;). However, it has been suggested that rumen epithelial ketogenesis, indicating metabolic activity, may occur independently of volatile fatty acid production (Sutton et al., 1963). Nevertheless, numerous researchers have indicated that ingestion of dry feeds and the resultant microbial end products sufficiently stimulates rumen epithelial development (Brownlee, 1956; Flatt et al., 1958; Greenwood et al., 1997; Nocek et al., 1984; Stobo et al., 1966a; Warner et al., 1956). However, the stimulatory effects of different volatile fatty acids are not equal, with butyrate being most stimulatory, followed by propionate (Flatt et al., 1958; Harrison et al., 1960; Sander et al., 1959; Stobo et al., 1966a; Sutton et al., 1963; Tamate et al., 1962). Low activity of the Acetyl CoA Synthetase enzyme appears to limit rumen epithelial metabolism of acetate; thereby limiting acetate s ability to stimulate epithelial development (Ash and Baird, 1973; Harmon et al., 1991). Conversely, Baldwin and McLeod (2000) indicated comparable acetate and butyrate metabolism in sheep, stating that animal energy status may influence individual volatile fatty acid metabolism rate. However, the millimolar concentration of acetate was higher 6

27 than butyrate in this study, indicating a possible conditioning of the rumen epithelium to acetate use, due to decreased butyrate availability (Baldwin and McLeod, 2000). Rumen epithelial conditioning to specific volatile fatty acid utilization has been previously reported (Rickard and Ternouth, 1965). Furthermore, epithelial butyrate metabolism appears to increase concomitantly with decreasing rumen ph and increasing butyrate concentrations (Baldwin and McLeod, 2000; Krehbiel et al., 1992; Stevens and Stettler, 1966; Sutton et al., 1963; Weigand et al., 1975). A continuous presence of volatile fatty acids maintains rumen papillae growth, size, and function (Harrison et al., 1960; Warner et al., 1956). Therefore, it is likely that diets composed of milk, concentrates, or forages, affect the rate and extent of rumen epithelial growth differently, and has been reported as such (Harrison et al., 1960; Stobo et al., 1966a; Tamate et al., 1962;Warner et al., 1956). Papillae length and width are the most obvious factors influencing absorptive surface area, but changes in papillae density should also be considered. Dietary and age differences have been found to alter papillae density of the developing rumen, however, significant differences due to dietary treatment are seldom reported for papillae density in calves (Klein et al., 1987; Lane et al., 2000; Nocek et al., 1984; Zitnan et al., 1998; Zitnan et al., 1999). Papillae density is commonly reported as the number of papillae in a fixed area (usually 1-cm 2 ), regardless of rumen volume, and rumen volume has been shown to increase with age (Stobo et al., 1966a). Therefore, minimal treatment influence on papillae density may be explained by a confounding effect of rumen volume. In addition, McGavin and Morrill (1976) reported intra-rumen variation for papillae measurements, suggesting that papillae growth may not be universal in all rumen areas. However, the physiological mechanics and genetics of papillae growth have not been thoroughly elucidated in the literature. 7

28 Liquids Feeds and Epithelial Development Milk or milk replacer is initially the primary diet of neonatal dairy calves, however, its chemical composition in addition to the shunting effect of the esophageal groove limits its ability to stimulate rumen development (Brownlee, 1956; Harrison et al., 1960; Lane et al., 2000; Stobo et al., 1966a; Tamate et al., 1962; Warner et al., 1956). Numerous researchers have reported minimal rumen development in calves receiving solely milk/milk replacer (Brownlee, 1956; Stobo et al., 1966a; Warner et al., 1956), even up to 12 wks of age (Tamate et al., 1962), and others have reported a regression, or stasis, of rumen development when calves were switched from a dry to milk/milk replacer diet (Harrison et al., 1960). Furthermore, calves receiving only milk/milk replacer exhibit minimal rumen epithelial metabolic activity and volatile fatty acid absorption, which once again does not increase with age (Sutton et al., 1963). However, ruminal size of the milk-fed calf, regardless of rumen development, has been shown to increase proportionately with body size (Flatt et al., 1958; Stobo et al., 1966a; Tamate et al., 1962; Warner et al., 1956). Therefore, while milk/milk replacer diets can result in rapid and efficient growth, it does little to prepare the pre-ruminant calf for weaning or utilization of grain and forage based diets. Solid Feeds and Epithelial Development Solid feeds, unlike liquid feeds, are preferentially directed to the reticulo-rumen for digestion (Church, 1988; Van Soest, 1994). Solid feed intake stimulates rumen microbial proliferation and production of microbial end products, volatile fatty acids, which have been shown to initiate rumen epithelial development (Beharka et al., 1998; 8

29 Harrison et al., 1960; Hibbs et al., 1956; Pounden and Hibbs, 1948a; Pounden and Hibbs, 1948b; Warner et al., 1956). However, solid feeds differ in their efficacy to stimulate rumen development. Chemical composition of the feeds, and the resultant microbial digestion end products, has the greatest influence on epithelial development (Harrison et al., 1960; Flatt et al., 1958; Stobo et al., 1966b; Warner et al., 1956). Multiple chemical characteristics of solid feeds appear to influence rumen epithelial growth. Concentrates (Brownlee, 1956; Harrison et al., 1960; Stobo et al., 1966a; Stobo et al., 1966b; Tamate et al., 1962; Warner et al., 1956) and purified diets containing casein, starch, cerelose, and minerals (Flatt et al., 1958) have increased the rate of rumen development when compared to forage sources. Sodium butyrate, when introduced into the rumen as purified sodium salts, had the greatest influence on rumen epithelial development, followed by sodium propionate, while sodium acetate and glucose had minimal affects (Flatt et al., 1958; Sander et al., 1959; Tamate et al., 1962). In addition, research has indicated butyrate and propionate as the volatile fatty acids most readily absorbed by rumen epithelium, especially when present at physiological concentrations (Baldwin and McLeod, 2000; Sander et al., 1959). Furthermore, the chemical composition of concentrates causes a shift in the microbial population, subsequently increasing butyrate and propionate production at the expense of acetate (Hibbs et al., 1956; Pounden and Hibbs, 1948a; Pounden and Hibbs, 1948b; Stobo et al., 1966b). Increased microbial production of stronger rumen acids, i.e. lactate, butyrate, and propionate, also decreases rumen ph (Hibbs et al., 1956; Stobo et al., 1966b). Stevens and Stettler (1966) indicated that only free state volatile fatty acids were absorbed into and across the rumen epithelium, and decreased rumen ph increases the free H + available, subsequently increasing absorbable free volatile fatty acids (Sutton et al., 1963). Therefore, a lower rumen ph and its affect on volatile fatty 9

30 acid absorption may be the catalyst driving rumen epithelial growth (Sutton et al., 1963). However, dietary type (Stobo et al., 1966b; Zitnan et al., 1998), microbial population present, and volatile fatty acids produced (Hibbs et al., 1956) greatly influence ruminal ph and cannot be removed from the equation. Conversely, forages have an increased ability to maintain a higher ruminal ph, due to a larger particle size and increased fiber content (Hibbs et al., 1956; Stobo et al., 1966b; Zitnan et al., 1998). Larger particle size increases ruminal salivary flow through greater initial mastication and increased subsequent rumination in mature and immature ruminants (Beauchemin and Rode, 1997; Hibbs et al., 1956). Maintenance of a higher ruminal ph supports microbial populations typically associated with forages, which in turn shift volatile fatty acid production from butyrate and propionate to acetate (Hibbs et al., 1956). Furthermore, there is evidence of higher NH 3 concentrations with forage diets (Sutton et al., 1963; Zitnan et al., 1998), resulting in H + sinks and effectively decreasing free volatile fatty acid availability (Stevens and Stettler, 1966). Some studies have indicated minimal differences between concentrates and forages for volatile fatty acid proportions and rumen development (Anderson et al., 1982; Baldwin and McLeod, 2000; Klein et al., 1987; Zitnan et al., 1998). However, confounding factors such as sampling of rumen volatile fatty acids via esophageal tube, age at slaughter, and dietary particle size may have influenced results from these studies. Furthermore, studies indicating increased rumen epithelial development when concentrates (Anderson et al., 1982; Brownlee, 1956; Stobo et al., 1966a; Warner et al., 1956; Zitnan et al., 1998; Zitnan et al., 1999), purified diets (Harrison et al., 1960), or fatty acid salts of butyrate and propionate (Sander et al., 1959; Tamate et al., 1962) were compared with forages are numerous and cannot be ignored. 10

31 Increased butyrate and propionate absorption and utilization, for rumen epithelial energy demands, over acetate supports evidence of the former volatile fatty acids stimulating epithelial development (Baldwin and McLeod, 2000; Sander et al., 1959). Whether the actual stimulant for epithelial development is increased butyrate and propionate production (Flatt et al., 1958; Harrison et al., 1960; Sander et al., 1959; Sutton et al., 1963; Tamate et al., 1962), a decreased ruminal ph concomitant with stronger ruminal acid production (Stevens and Stettler, 1966), or a combination; concentrates appear to result in greater rumen epithelial development than forages (Brownlee et al., 1956; Nocek et al., 1984; Stobo et al., 1966a; Stobo et al., 1966b; Warner et al., 1956; Zitnan et al., 1998). Physical Structure and Epithelial Development Rumen epithelial development cannot be thoroughly discussed without covering the influence of parakeratosis on papillae development and absorptive ability. Parakeratosis covers epithelial squamous cells with a hardened keratin layer due to a diet s inability to continuously remove degenerating epithelial cells (Bull et al., 1965; Hinders and Owen, 1965). Parakeratosis creates a physical barrier, restricting absorptive surface area and volatile fatty acid absorption, reducing epithelial blood flow, rumen motility, and causing papillae degeneration and sloughing in extreme cases (Anderson et al., 1982; Beharka et al., 1998; Brownlee, 1956; Bull et al., 1965; Hinders and Owen, 1965; McGavin and Morrill, 1976; Nocek et al., 1984). Initial evidence of parakeratosis is papillae clumping and branching, followed by papillae degeneration and sloughing (Anderson et al., 1982; Beharka et al., 1998; Brownlee, 1956; Bull et al., 1965; Hinders and Owen, 1965; McGavin and Morrill, 1976; Nocek et al., 1984; Zitnan et al., 11

32 1998). Concentrate diets having small particle size and low abrasive value (Beharka et al., 1998; Bull et al., 1965; Greenwood et al., 1997; McGavin and Morrill, 1976), increased volatile fatty acid production, decreased rumen buffering capacity, and subsequently decreased rumen ph (Anderson et al., 1982; Hinders and Owen, 1965) are factors commonly associated with occurrences of parakeratosis. Abrasive value is defined as a feed s efficacy in physically removing keratin and/or dead epithelial cells from the rumen epithelium (Greenwood et al., 1997). Therefore, increased feed particle size, especially with forages or coarsely-ground concentrates, maintains epithelial and papillae integrity and absorptive ability via physical removal of the keratin layer (Greenwood et al., 1997; McGavin and Morrill, 1976), increased rumination and rumen motility (Brownlee, 1956), subsequently increased salivary flow and buffering capacity (Bull et al., 1965; Hinders and Owen, 1965), and development of a mature type rumen function and environment (Hibbs et al., 1956). However, factors such as animal susceptibility, intake differences, passage rate, rumination rate, and salivary production may also contribute to occurrences of parakeratosis (Anderson et al., 1982; Zitnan et al., 1999). Changes in Rumen Muscularization and Volume Feed physical structure has the greatest influence on development of rumen muscularization and volume. Stimulation of rumen motility is governed by the same factors, particle size and effective fiber, in the neonatal ruminant as in the adult ruminant (Beauchemin and Rode, 1997; Mertens, 1997; Van Soest, 1994). In contrast to concentrate s advantages for epithelial development (Brownlee et al., 1956; Nocek et al., 1984; Tamate et al., 1962; Warner et al., 1956), forages appear to be the primary 12

33 stimulators of rumen muscularization development and increased rumen volume (Hibbs et al., 1956; Stobo et al., 1966a; Zitnan et al., 1998). Large particle size, high effective fiber content, and increased bulk of forages or high fiber sources physically increase rumen wall stimulation, subsequently increasing rumen motility, muscularization, and volume (Brownlee et al., 1956; Hibbs et al., 1956; Nocek et al., 1984; Stobo et al., 1966a; Tamate et al., 1962; Warner et al., 1956; Zitnan et al., 1998). As stated earlier, increases in rumen muscularization and volume have occurred independently of epithelial development (Brownlee, 1956; Flatt et al., 1958; Harrison et al., 1960; Stobo et al., 1966a). Supporting evidence for independent muscle and epithelial growth is found in studies determining the effects of inert material (sponges, toothbrush bristles, or bedding) on rumen epithelial, muscular, and capacity development (Brownlee, 1956; Flatt et al., 1958; Harrison et al., 1960). Inert materials were found ineffective for stimulating papillae growth, but capable of significantly increasing rumen capacity and muscularization (Brownlee, 1956; Flatt et al., 1958; Harrison et al., 1960). However, solid feeds other than forages or bulky feedstuffs can be effective in influencing rumen capacity and muscularization. Coarsely or moderately ground concentrate diets have been shown to increase rumen capacity and muscularization more than finely ground or pelleted concentrate diets, indicating that extent of processing and/or concentrate particle size affects the ability of concentrates to stimulate rumen capacity and muscularization increases (Beharka et al., 1998; Greenwood et al., 1997). Therefore, concentrate diets with increased particle size may be the most desirable feedstuff for overall rumen development, due to their ability to stimulate epithelial, rumen capacity, and rumen muscularization development. The basics of rumen development have been elucidated in the literature Brownlee, 1956; Flatt et al., 1958; Sutton et al., 1963). Current rumen development 13

34 research focuses on dietary manipulation, attempting to optimize the rate and extent of rumen development. Increased availability of feed by-products, development of new feed additives, and differences in calf starter particle size all provide areas for future rumen development research. However, standardization of procedures utilized in rumen development research could aid this field tremendously. In addition, understanding the cellular biology and physiological changes that occur during rumen development, clarifying neonatal calf digestion kinetics, and development of low-impact or non-invasive research procedures could be instrumental in advancing this area. REVIEW OF YEAST CULTURE LITERATURE Supplemental yeast culture incorporated into rations is derived from Saccharomyces cerevisiae grown in a medium containing ground corn, hominy feed, corn gluten feed, wheat and rye middlings, corn syrup, cane molasses, and other high energy substrates (Cole et al., 1992; Harrison et al., 1988; Mutsvangwa et al., 1992; Williams et al., 1991). The entire mixture (yeast plus medium) is subsequently dried at a low temperature to maintain yeast cell viability, however, total live yeast cell counts vary between products and growth batches (Williams et al., 1991). This variability in live yeast cell counts in addition to yeast culture inclusion levels ranging from % (Garcia et al., 2000) to 1.6% (Malcolm and Kiesling, 1990) may explain the variable results found throughout the yeast culture literature (Williams et al., 1991). However, research indicates that including yeast culture in mature and immature ruminant diets can alter the rumen environment (Erasmus et al., 1992; Yoon and Stern, 1996), and therefore may influence neonatal calf rumen development (Quigley et al., 1992; Williams et al., 1991). 14

35 Yeast Culture and the Rumen Environment Yeast Culture and Lactic Acid An altered ruminal lactic acid concentration is a notable effect of yeast culture on the rumen environment, and yeast culture has reduced rumen lactic acid concentration in both mature (Erasmus et al., 1992; Mir and Mir, 1994; Williams et al., 1991) and immature ruminant studies (El Hassan et al., 1996; Koul et al., 1998; Quigley et al., 1992). Yeast culture has been indicated to stimulate growth of lactic acid utilizing microbes, such as Selenomonas ruminantium and Megaspera elsdenii, and lactic acid uptake by these microbes (Koul et al., 1998; Mir and Mir, 1994; Nisbet and Martin, 1991; Quigley et al., 1992). Stimulation of lactic acid utilizing microbes may result from increased levels of L-malate, which serves as an electron sink for Selenomonas ruminantium, or by providing B-vitamins and amino acids for Selenomonas ruminantium growth (Nisbet and Martin, 1991). Furthermore, Williams et al. (1991) suggested that yeast culture might also limit lactic acid production through utilization of lactic acid precursors. However, others have suggested that yeast culture directly limits lactic acid production or stimulates significant increases in lactic acid utilizing bacteria (Putnam et al., 1997; Yoon and Stern, 1996). Reduced lactic acid production has subsequent effects on rumen ph, rumen microbial populations, volatile fatty acid production, rumen degradation and passage rates, and intake (Williams et al., 1991). Yeast Culture and Ruminal ph Rumen ph is governed by concentration and strength of the major ruminal acids, i.e. acetate, propionate, butyrate, and lactate, listed in increasing order of strength 15

36 (Church, 1988; Van Soest, 1994). In addition, dietary type or composition influences ruminal acid production, with concentrate rations resulting in greater production of butyrate and lactic acid than forages (Church, 1988; Van Soest, 1994). Young calf rations are predominantly concentrate based, resulting in greater lactic acid production and a subsequently lower rumen ph (Hibbs et al., 1956; Quigley et al., 1992; Stobo et al., 1966b; Sutton et al., 1963; Zitnan et al., 1998). A lower rumen ph may be necessary for rumen development, however, extreme acidity can have deleterious effects, resulting in parakeratosis (Beharka et al., 1998; Bull et al., 1965; Greenwood et al., 1997; Hinders and Owen, 1965; McGavin and Morrill, 1976), altering microbial populations (Hibbs et al., 1956), and limiting intake (Anderson et al., 1982; Quigley et al., 1992; Stobo et al., 1966b; Sutton et al., 1963; Zitnan et al., 1998). Therefore, inclusion of yeast culture in calf rations to limit ruminal lactic acid and extreme rumen acidity could aid rumen development through maintained intake and reduced parakeratosis (Anderson et al., 1982; Beharka et al., 1998; Bull et al., 1965; Greenwood et al., 1997; Williams et al., 1991). Research has indicated an increase in rumen ph (Adams et al., 1981; El Hassan et al., 1996; Kumar et al., 1997; Koul et al., 1998; Quigley et al., 1992) or decreased ph depression (Enjalbert et al., 1999; Williams et al., 1991) when yeast culture was included in ruminant diets. However, others have found no change (Erasmus et al., 1992; Garcia et al., 2000; Malcolm and Kiesling, 1990; Mutsvangwa et al., 1992; Olson et al., 1994; Putnam et al., 1997; Wagner et al., 1990; Wiedmeier et al., 1987; Yoon and Stern, 1996) or decreased rumen ph (Corona et al., 1999; Harrison et al., 1988) with supplemental yeast culture, indicating that yeast culture may not have the same effect in all diets or conditions. There is some indication that dietary composition may influence the extent of ph alteration by yeast culture, and that ingredients utilized to maintain ph could mask yeast culture s effects (El Hassan et al., 1996; Quigley et al., 1992; Koul et al., 1998; 16

37 Wagner et al., 1990). In addition, Williams et al. (1991) indicated that yeast culture has a greater influence when included with high concentrate rations than with forage-based rations, possibly explaining limited results when yeast culture was included in highforage diets fed to cows. Changes in rumen ph, with yeast culture supplementation, have been attributed to altered rumen microbial populations or stimulation of microbial growth (Kumar et al., 1997), decreased ruminal lactic acid (Koul et al., 1998), and shifts in volatile fatty acid production (Harrison et al., 1988). Yeast Culture and Rumen Microbes Yeast culture has been indicated to benefit lactic acid utilizing microbes (Koul et al., 1998; Mir and Mir, 1994; Nisbet and Martin, 1991; Quigley et al., 1992). In addition, yeast culture is suggested to also increase rumen microbes in the cellulolytic, amylolytic, proteolytic, and anaerobic classes, and to increase total and total viable microbes (El Hassan et al., 1996; Harrison et al., 1988; Koul et al., 1998; Kumar et al., 1997; Mutsvangwa et al., 1992; Weidmeier et al., 1987; Yoon and Stern, 1996). An increased cellulolytic bacterial population, or viability, could additionally indicate an altered rumen ph, and may mark yeast culture s influence on ph as the causative factor for increased rumen microbial populations (El Hassan et al., 1996; Koul et al., 1998; Kumar et al., 1997; Williams et al, 1991). In vivo and in vitro studies using viable, autoclaved, irradiated, and supernatant of yeast culture have indicated that viable yeast cells or heat labile components of yeast are important in microbial population changes (Dawson et al., 1990; Koul et al., 1998) and that an altered ph does not completely account for these changes (Kumar et al., 1997). Conversely, yeast culture has been found to decrease ruminal protozoa in sheep (Corona et al., 1999; Garcia et al., 2000) and to decrease, or 17

38 not alter, certain rumen microbe classes in cows (Erasmus et al., 1992; Putnam et al., 1997; Yoon and Stern, 1996). However, Yoon and Stern (1996) stated that statistically significant changes in microbial enumerations are not always biologically significant, unless large differences occur. Yeast Culture and Volatile Fatty Acids Impacts of supplemental yeast culture and an altered rumen environment on rumen volatile fatty acid production have been reported in numerous studies, but results are inconsistent. Yeast culture has increased total volatile fatty acid production in all age classes of bovine ruminants receiving both forage and concentrate based rations (El Hassan et al., 1996; Enjalbert et al., 1999; Koul et al., 1998; Kumar et al., 1997; Mutsvangwa et al., 1992; Quigley et al., 1992; Wiedmeier et al., 1987) and in continuous culture fermenters (Nisbet and Martin, 1991). Conversely, decreases or no changes in total volatile fatty acid production have been reported in both ovine (Corona et al., 1999; Garcia et al., 2000) and bovine ruminants receiving forage and concentrate diets supplemented with yeast culture (Adams et al., 1981; Dawson et al., 1990; Harrison et al., 1988; Kung et al., 1997; Olson et al., 1994; Wagner et al., 1990; Williams et al., 1991; Yoon and Stern, 1996). Results for individual volatile fatty acids are also varied, with increases in acetate, propionate, and butyrate reported for mature (Adams et al., 1981; Enjalbert et al., 1999; El Hassan et al., 1996; Erasmus et al., 1992; Harrison et al., 1988; Malcolm and Kiesling, 1990; Mutsvangwa et al., 1992; Wiedmeier et al., 1987; Mir and Mir, 1994) and immature (Kumar et al., 1997; Quigley et al., 1992) ruminants. However, decreases or no influence on individual volatile fatty acids have been reported in bovine (El Hassan et al., 1996; Enjalbert et al., 1999; Erasmus et al., 1992; Harrison 18

39 et al., 1988; Kung et al., 1997; Malcolm and Kiesling, 1990; Mir and Mir, 1994; Olson et al., 1994; Putnam et al., 1997; Quigley et al., 1992; Wagner et al, 1990; Wiedmeier et al., 1987; Williams et al., 1991; Yoon and Stern, 1996) and ovine (Adams et al., 1981; Corona et al., 1999; Garcia et al., 2000) ruminants and with continuous culture fermenters (Dawson et al., 1990; Kumar et al., 1997). Of particular interest to calf rumen development are reported increases in butyrate and propionate (Adams et al., 1981; El Hassan et al., 1996; Enjalbert et al., 1999; Erasmus et al., 1992; Harrison et al., 1988; Kumar et al., 1997; Mir and Mir, 1994; Mutsvangwa et al., 1992; Quigley et al., 1992; Wiedmeier et al., 1987). Total or individual volatile fatty acid increases are attributed to concentrate-based rations (Quigley et al., 1992), altered rumen microbial populations subsequently increasing activity and hydrogen utilization (Enjalbert et al., 1999; Kumar et al., 1997), rumen or fermentation stability, increased fermentation rate, decreased amylolytic bacteria depression, and delayed starch digestion (Harrison et al., 1988; Mutsvangwa et al., 1992). A lack of response to supplemental yeast culture has been attributed to low forage diets, low forage quality, or low intake (Quigley et al., 1992; Olson et al., 1994; Wagner et al., 1990). Furthermore, variations in yeast culture inclusion rates, different dietary types or ingredients, feeding procedures, environmental conditions, or variability in sampling procedures or sampling times have varied total or individual volatile fatty acid results (El Hassan et al., 1996; Enjalbert et al., 1999; Malcolm and Kiesling, 1990; Mutsvangwa et al., 1992; Putnam et al., 1997; Wagner et al., 1990; Williams et al., 1991; Yoon and Stern, 1996). 19

40 Yeast Culture and Solid Component Digestion Dietary yeast culture inclusion has been found to increase in situ dry or organic matter disappearance (Erasmus et al., 1997; Yoon and Stern, 1996), especially during the early hours of incubations (Williams et al., 1991), with increased disappearance attributed to decreased lag time (Kumar et al., 1997). However, yeast culture has also been shown to decrease dry matter disappearance and increase lag time, and studies reporting increased early dry matter disappearance have also found an opposite occurrence past hours of incubation (Enjalbert et al., 1999; Erasmus et al., 1997; Kumar et al., 1997; Mir and Mir, 1994; Williams et al., 1991). Koul et al. (1998) found that yeast culture had maximum stimulatory effects at 2 4 hours with decreasing activity up to 12 hours, indicating a necessity for frequent addition of yeast culture to optimize effectiveness. Increased disappearance/digestibility of ruminal neutral detergent fiber (Corona et al., 1999; Mir and Mir, 1994; Olson et al., 1994), total tract hemicellulose (Wiedmeier et al., 1987), and apparent crude fiber (LeGendre et al., 1957) have been reported with supplemental yeast culture, however, results appear diet related. Conversely, Enjalbert et al. (1999) indicated no effect of yeast culture on in situ neutral or acid detergent fiber disappearance. Lastly, yeast culture inclusion appears to have no influence on energy component digestibility, which is somewhat surprising given the aforementioned yeast culture effects on lactic acid, rumen ph, and microbial populations (Erasmus et al., 1997; Harrison et al., 1988; Putnam et al., 1997). However, Williams et al. (1991) indicated that a primary benefit of yeast culture is its ability to alter conditions detrimental to cellulolysis, suggesting that yeast culture has minimal influence on concentrate diet digestibility, but may alleviate negative associative effects between concentrates and forages. In addition, Enjalbert et al., 1999, suggested that increased 20

41 rumen protozoa, with yeast culture supplementation, may decrease availability of starch and delay starch digestion. Yeast Culture and Intake, Gain, and Efficiency Stabilization of the rumen environment is reported as a benefit of supplemental yeast culture, and some previously mentioned findings appear to agree (Harrison et al., 1988; Williams et al., 1991). Increased intake may occur concomitant with a more stable rumen, and numerous studies have reported positive influences of yeast culture on intake (Adams et al., 1981; Erasmus et al., 1997; Mutsvangwa et al., 1992; Phillips and VonTungeln, 1985; Williams et al., 1991). Of additional importance is increased intake during times of stress (Hughes, 1988), such as infective challenges (Cole et al., 1992), and increased starter intake in preweaned calves (Quigley et al., 1992). Williams et al. (1991) attributed increased intake in calves, with supplemental yeast culture, to rumen ph stabilization via reduced lactic acid concentration. Quigley et al. (1992) found that yeast culture increased starter intake prior to weaning, but decreased intake after weaning and over the entire 12-week period. There are more studies reporting a limited influence of yeast culture on intake, than studies reporting positive or negative affects (Cole et al., 1992; El Hassan et al., 1996; Garcia et al., 2000; Kung et al., 1997; Malcolm and Kiesling, 1990; Mir and Mir, 1994; Putnam et al., 1997; Seymour et al., 1995; Wagner et al., 1990; Yoon and Stern, 1996). However, decreased intakes have been reported (Adams et al., 1981; LeGendre et al., 1957). Once again, basal ration type, different dietary ingredients, or varied inclusion rates appear to account for variations in results (Adams et al., 1981; El Hassan et al., 1996; LeGendre et al., 1957; Mir and Mir, 1994; Putnam et al., 1997; Seymour et al., 1995; Wagner et al., 1990). Most studies 21

42 report no advantage in average daily gain with the addition of yeast culture; however, no studies indicate a negative affect (Kung et al., 1997; Mir and Mir, 1994; Mutsvangwa et al., 1992; Quigley et al., 1992; Seymour et al., 1995; Wagner et al., 1990; Williams et al., 1991). A few studies indicated increased average daily gain or a tendency to lose less weight following an infective challenge, and attributed these findings to increased intake (Adams et al., 1981; Cole et al., 1992; El Hassan et al., 1996; Phillips and VonTungeln, 1985). Positive (Adams et al., 1981; El Hassan et al., 1997; Quigley et al., 1992), negative (Cole et al., 1992), and no affects (Cole et al., 1992; Mir and Mir, 1994; Mutsvangwa et al., 1992; Wagner et al., 1990) on feed efficiency (gain:feed) with yeast culture have been reported, with results appearing to vary due to different inclusion rates or diet. Yeast Culture and Health Yoon and Stern (1996) indicated increased yeast culture response during stressful situations, possibly indicating an applicability of yeast culture to influence health parameters. Additionally, Cole et al. (1992) reported decreased morbidity and mortality rate and number of treated sick days, when yeast culture was included in feeder calf diets. However, in a second experiment, they found increased morbidity but significantly fewer treated sick days, indicating increased recovery time (Cole et al., 1992). Furthermore, the previously covered findings of increased intake and decreased rate of weight loss following infective challenge, also hints at some health attributes of yeast culture (Cole et al., 1992). Inclusion of yeast culture in dairy calf rations has decreased occurrences of scours, electrolyte and antibiotic treatments, and occurrences of elevated body temperature (Seymour et al., 1996). Provision of nutrients from yeast culture, 22

43 resulting in increased growth of beneficial gut microorganisms, establishment of normal gut fermentation, and subsequent reduction in stress and digestive upset, was suggested as the causative effect for these findings (Seymour et al., 1996). Due to the absence of research exploring yeast culture s influence on rumen development in the literature, the advantages or disadvantages have yet to be determined. The variability in reported findings from yeast culture studies is unfortunate. To arrive at a definitive conclusion on the advantages or disadvantages of yeast culture supplementation would require that a meta-analysis be conducted on the results from the available literature, due to differences in research materials and methods. However, yeast culture supplementation does appear to hold some advantages. In addition, differences in yeast culture strain and/or growth substrates utilized during yeast proliferation and manufacturing may explain some variability in literature results. Perhaps standardization of research materials and methods in future research will enable a better understanding of dietary yeast culture s influence. REVIEW OF MOLASSES LITERATURE Molasses refers to any liquid feed ingredient containing more than 43% sugar (Curtin, 1983). Dietary molasses is a byproduct of the sugar, starch, and paper industries and is derived from cane, sugar beets, citrus pulp, starch manufacturing, and hemicellulose extract from the paper industry (Curtin, 1983). The geographical location of these industries, plant source from which molasses is derived, and soil makeup where the plants are grown all determine the chemical composition of molasses, which is quite varied (Curtin, 1983). Molasses has become a generic feed ingredient that is included more for its physical characteristics, acting as an appetizer, binder, and dust settler, than 23

44 for its dietary characteristics (Morales et al., 1989). However, research has indicated alterations in digestive and production parameters with molasses inclusion. In addition, carbohydrates found in molasses are rapidly fermented by rumen microbes (Murphy, 1999) and dietary molasses influences have been suggested to mimic effects seen with dietary sucrose inclusion (Owen et al., 1967). Therefore, literature reporting dietary sucrose effects is included in this review. Molasses and Intake, Gain, and Efficiency Dietary molasses inclusion is indicated to influence dry/organic matter intake in numerous studies and feeding regimes. Increasing dietary levels of sucrose or molasses have increased dry/organic matter intake, with most increases occurring at a dietary molasses level of 10-20% (Bohman et al., 1954; Bond and Rumsey, 1973; Bouchard and Conrad, 1973; Brown, 1993; Brown and Johnson, 1991; Brown et al., 1987, Morales et al., 1989; Murphy, 1999). However, increased intake appears to depend on amount and/or quality of dietary forage, with greater intakes observed using low quality forages (Brown et al., 1987; Lofgreen and Otagaki, 1960; Morales et al., 1989). Depressed dry/organic matter intakes were found with greater than 20% molasses inclusion rates, high dietary forage content or quality, and when forages and concentrates were fed separately (Komkris et al., 1965; Lofgreen and Otagaki, 1960; Morales et al., 1989). Numerous researchers have also indicated no influence on dry/organic matter intake with dietary molasses, attributing these findings to low molasses inclusion rates (< 10%) or high quality dietary forage (Bond and Rumsey, 1973; Brown and Johnson, 1991; Brown et al., 1987; Heinemann and Hanks, 1977; 24

45 Komkris et al., 1965; Lofgreen and Otagaki, 1960; Morales et al., 1989; Wing and Powell, 1969; Wing et al., 1988). Molasses inclusion has increased body weight gains, with improved gains attributed to increased intake or improved N utilization via increased dietary energy (Bond and Rumsey, 1973; Brown, 1993; Brown and Johnson, 1991; Brown et al., 1987). However, high dietary molasses content (> 20%), lower energy yield, and depressed N utilization have decreased gains to the same extent as restricted intake (Bohman et al, 1954; Bond and Rumsey, 1973; Heinemann and Hanks, 1977; Lofgreen and Otagaki, 1960). In addition, Mather and Bender (1951) and Heinemann and Hanks (1977) suggested that replacing concentrates with molasses, as a dietary energy source, may result in an inadequate protein supply, subsequently reducing gain. However, proper ration balancing when incorporating molasses may overcome protein limitations. Other researchers have indicated no differences in body weight gain with molasses addition (Heinemann and Hanks, 1977; Mather and Bender, 1951; Morales et al., 1989; Wing and Powell, 1969; Wing et al., 1988). Intake and gain differences will inevitably alter feed utilization efficiency (feed:gain) and numerous studies have reported changes in feed efficiency with molasses inclusion. Increased feed efficiency is reported with molasses inclusion and attributed to increased dietary energy (Brown, 1993; Brown et al., 1987; Kellogg and Owen, 1969; Morales et al., 1989). Brown et al. (1987) indicated that efficiency increased when molasses was included in low energy diets, such as low quality forage, suggesting that molasses may correct an energy deficiency in such diets. Conversely, high molasses inclusion rates or inclusion in high energy diets decreased feed utilization efficiency, with results partially attributed to decreased energy digestibility and energy utilization efficiency (Heinemann and Hanks, 1977; Lofgreen and Otagaki, 1960; Martin and Wing, 1966; Wing et al., 1988). Non-significant differences in feed 25

46 utilization efficiency have also been found, and partially attributed to molasses inclusion with high quality forages (Brown et al., 1987; Owen et al., 1967; Wing and Powell, 1969). Molasses and Diet Digestibility Molasses and Dry Matter or Organic Matter Digestibility Reported differences in dry/organic matter digestibility may partially explain observed intake changes. Molasses inclusion has been reported to influence ruminal and total tract dry/organic matter digestibility in numerous trials. Komkris et al. (1965) reported increased organic matter digestibility when molasses was included in a complete diet, but no change when concentrates were fed separately. In addition, increased dry/organic matter digestibility has been observed with dietary molasses content at less than 10% and when molasses-soluble protein mixes were studied (Brown, 1993; Wing et al., 1988). However, others have reported no advantage in dry/organic matter digestibility with molasses-soluble protein mixes (Brown and Johnson, 1991; Brown et al., 1987). Conversely, decreased dry/organic matter digestibility, with molasses inclusion, has been reported; with low quality forages and/or inclusion rates greater than 10% indicated as some of the causative effects (Bohman et al., 1954; Bouchard and Conrad, 1973; Martin and Wing, 1966; Mould et al., 1983; Williams, 1925; Wing et al., 1988). 26

47 Molasses and Structural Component Digestibility Molasses supplementation appears to largely influence dietary fiber digestibility, subsequently influencing intake. Molasses has decreased neutral detergent fiber digestibility in numerous studies (Brown, 1993; Brown and Johnson, 1991; Brown et al., 1987; Wiedmeier et al., 1992). However, Wiedmeier et al. (1992) found no differences between molasses and other dietary energy supplements for neutral detergent fiber digestibility, suggesting that the observed decreases may have been related to energy supplementation and not molasses. Energy supplementation has been indicated to negatively affect fiber digestion, especially in low quality forages, and this depression has been attributed to low crude protein availability (Burroughs et al., 1949, Mould et al., 1983). In addition, microbial substrate substitution of fiber with highly soluble carbohydrates found in molasses has been shown to result in production of stronger volatile fatty acids, subsequently reduced ph, and other negative associative affects between concentrates and forages (Burroughs et al., 1949; Hoover, 1986; Mould et al., 1983). Further, Wiedmeier et al. (1992) found decreased neutral detergent fiber digestibility with a complete ration (concentrates + forages) but no change with an all forage diet, additionally supporting a substrate substitution theory. Increases in neutral detergent fiber digestibility have not been reported with dietary molasses inclusion. Multiple studies have found decreased acid detergent fiber digestibility with supplementary molasses, attributing these occurrences to factors that also decreased neutral detergent fiber digestibility (Brown, 1993; Brown and Johnson, 1991; Brown et al., 1987; Wiedmeier et al., 1992; Wing et al., 1988). Conversely, dietary molasses has not been reported to increase acid detergent fiber digestibility in the literature. However, 27

48 some studies utilized in this review predate neutral and acid detergent analyses and only reported crude fiber digestibility. Therefore, these are included in the next section. Williams (1925) reported increased crude fiber digestibility when molasses was included at 25% of the ration, but found decreased crude fiber digestibility with lower inclusion rates. Others have reported decreased cellulose, hemicellulose, and/or crude fiber digestibility with supplemental molasses (Bohman et al., 1954; Brown, 1993; Brown et al., 1987; Martin and Wing, 1966). Martin and Wing (1966) suggested that including molasses in high concentrate:forage diets increased occurrences of depressed cellulose digestibility. Furthermore, Komkris et al. (1965) found decreased fiber digestibility when a complete ration contained 20% molasses, but reported no molasses effect on fiber digestibility with separate concentrate and forage feeding. However, different feeding procedures (once per day for complete and twice per day for separate) may have confounded these results (Komkris et al., 1965). Molasses and Crude Protein Digestibility Molasses driven alterations in crude protein digestibility may be related to the differences in N utilization attributed to varying results for gain. Bouchard and Conrad (1973) indicated that molasses inclusion increased crude protein digestibility subsequently increasing N absorption, partitioning, and decreasing urinary N excretion. In addition, Wiedmeier et al. (1992) found increased total tract crude protein digestibility when all forage diets were supplemented with molasses. Conversely, others have reported decreased total tract crude protein digestibility when molasses was included in a complete diet and when concentrates and forages were fed separately (Komkris et al., 1965; Wiedmeier et al., 1992; Williams, 1925; Wing et al., 1988). In addition, Bohman et 28

49 al. (1954) reported increased urinary and fecal N excretion with supplemental molasses. However, Martin and Wing (1966), who found no influence of molasses on total tract crude protein digestibility, indicated that depressions in N digestion may be related to feeding regime and not molasses inclusion. Molasses and the Rumen Environment Molasses and Volatile Fatty Acids Changes in dietary constituent digestibility, in addition to a high soluble carbohydrate content of molasses, could subsequently alter rumen parameters. Only a few studies have reported molasses effects on total volatile fatty acid production, and results indicating increased (Wiedmeier et al., 1992), decreased (Bond and Rumsey, 1973), and unchanged total volatile fatty acid production were found (Owen et al., 1967). However, results of altered individual volatile fatty acid production by molasses inclusion are found in numerous studies. Alterations in rumen butyrate production by molasses inclusion are of particular interest to neonatal calf rumen development. Molasses increased butyrate production in numerous studies, with greater changes observed in rations containing forages and concentrates when compared to all forage rations (Bond and Rumsey, 1973; Kellogg and Owen, 1969; Owen et al., 1967; Wiedmeier et al., 1992, Wing et al., 1988). Murphy (1999) also found increased plasma β-hydroxybutyrate levels, possibly indicating increased rumen butyrate production. Additionally, Waldo and Schultz (1960) indicated that sucrose moderately increased butyrate production, preceded only by glycogenic solutions containing lactate. Glycogenic solutions containing lactate were most effective 29

50 for increasing propionate production, followed by butyrate, and least effective for increasing acetate production (Waldo and Schultz, 1960). Some have found no differences in butyrate production with molasses supplementation; however, nonsignificant results may be related to feeding regime and/or diet composition (Bond and Rumsey, 1973; Komkris et al., 1965; Martin and Wing, 1966). Molasses has been reported to reduce rumen propionate production (Bond and Rumsey, 1973; Kellogg and Owen, 1969; Wing et al., 1988). Waldo and Schultz (1960) found sucrose the least effective glycogenic solution for increasing propionate production, however, a minimal increase was reported. Others have indicated no differences in propionate production with molasses inclusion (Bond and Rumsey, 1973; Komkris et al., 1965; Martin and Wing, 1966; Owen et al., 1967; Wiedmeier et al., 1992). Separate concentrate and forage feeding in addition to high molasses inclusion rates have increased rumen acetate production (Komkris et al., 1965; Wing et al., 1988). Additionally, Waldo and Schultz (1960) indicated that sucrose increased ruminal acetate production more than other glycogenic solutions. Furthermore, increases in acetate production were larger in magnitude than increases in propionate or butyrate, when sucrose was utilized as a glycogenic solution (Waldo and Schultz, 1960). Conversely, decreased acetate production is reported for supplementary molasses, with greater decreases observed in forage plus concentrate rations than all forage rations (Bond and Rumsey, 1973; Wiedmeier et al., 1992). However, Komkris et al. (1965) found no differences in acetate production when molasses was included in a complete ration. Others have also reported limited molasses influence on acetate production (Bond and Rumsey, 1973; Kellogg and Owen, 1969; Martin and Wing, 1966; Owen et al., 1967). Possible increases in ruminal butyrate production, intake, and diet palatability may be beneficial to neonatal calf growth and rumen development. However, the effects 30

51 of additional molasses appear strongly diet dependent, with negative influences on high quality rations, rations containing forages and concentrates, or rations with high nutrient concentrations. Calf starter rations are typically high in quality and nutrient concentration; therefore, positive molasses effects on volatile fatty acid production may be limited in calf starters. Molasses and Rumen ph Subsequent rumen ph changes, due to altered volatile fatty acid profiles, have been reported with increased dietary molasses content. Wing et al. (1988) found increased rumen ph when molasses was included at approximately 20% of the ration, a finding that is contradictory to typically observed ph depressions with highly soluble carbohydrate feedstuffs (Van Soest, 1994). Conversely, Owen et al. (1967) found decreased rumen ph when 6% molasses was included in a high concentrate diet, but indicated the high concentrate diet as the likely causative factor and not molasses. Most studies have indicated minimal rumen ph fluctuations with molasses supplementation, however most rations studied contained high forage content, possibly masking any effects that molasses may have on rumen ph (Bond and Rumsey, 1973; Kellogg and Owen, 1969; Martin and Wing, 1966; Wiedmeier et al., 1992). Few studies have reported microbial populations changes with molasses inclusion. Kellogg and Owen (1969) suggested that increased availability of a readily fermentable carbohydrate source may lead to increased microbial populations. However, the previously covered findings of decreased nutrient digestibility, especially fiber, may refute this statement. 31

52 REVIEW OF GRAIN PROCESSING LITERATURE Whole grains are protected by an outer cover, called the pericarp, which is resistant to ruminal digestion and microbial attachment (Huntington, 1997; Owens et al., 1997; Theurer, 1986). In addition, strong crosslinks between starch granules and the presence of a protein matrix surrounding these granules limits digestibility, even upon microbial invasion (Hale, 1973; McNeill et al., 1975; Theurer, 1986). Processing of grains through applications of heat, moisture, and mechanical action are utilized to fracture the seed pericarp, increase surface area, disrupt the starch-protein matrix, and gelatinize starch granules (Hale, 1973; Harbers, 1975; Huntington, 1997; Joy et al., 1997; Owens et al., 1997; Rooney and Pflugfelder, 1986; Theurer, 1986). Starch gelatinization occurs through cleavage of H + bonds between starch molecules and formation of new starch-water bonds, resulting in disorderly structured starch conglomerates that are more readily solubolized (Hale, 1973; Harbers, 1975; Joy et al., 1997; McNeill et al., 1975). The different forms of processed grains found in the literature are distinguished by processing method(s) applied and the extent of application. Dry-ground and dryrolled processing involves a mechanical destruction of dry grains by large rollers or hammers, resulting in reduced particle size and increased surface area exposed, but little to no changes in chemical structure (Theurer et al., 1999). Steam-rolled grains are exposed to steam to increase moisture content and then passed through cold rollers, fracturing the pericarp, increasing surface area and internal starch exposure, disrupting the starch-protein matrix, and slightly gelatinizing starch (Frederick et al., 1973; Theurer et al., 1999). Steam flaking involves increasing internal moisture to 18-20%, via steaming, then flaking of the grains through preheated rollers, a distinctive difference 32

53 between steam rolling and steam flaking (Frederick et al., 1973; Theurer et al., 1999). Processing in this manner fractures the pericarp, increases surface area exposed, gelatinizes starch, and disrupts the starch-protein matrix to a greater extent than steam rolling (Frederick et al., 1973; Galyean et al., 1981; Harbers, 1975; Rooney and Pflugfelder, 1986, Theurer et al., 1999). The order, from largest to smallest, of mean particle sizes obtained with different corn processing methods has been reported as steam-rolled, steam-flaked, dry-rolled, and dry-ground (Yu et al., 1998). However, Pritchard and Stateler (1997) indicated that flaked grains are more fragile and extensive handling can easily reduce particle size. The bulk of grain processing research has been conducted with mature ruminants, with little to no literature studying grain processing effects in neonatal calves. Furthermore, a previous indication of differing digestive kinetics, rumen microbial populations, and rumen capacities between mature and immature ruminants limits postulation (Vazquez-Anon et al., 1993). Therefore, the following findings may not be wholly applicable to ruminally developing calves. Grain Processing and the Rumen Environment Grain Processing and Volatile Fatty Acids Changes in the rumen environment, resulting from altered physical and chemical characteristics of processed grains and concomitant changes in digestibility, have been reported. Steam flaking of grains increased propionate and valerate production, at the expense of acetate and butyrate, and decreased the acetate:propionate ratio when compared to dry-rolled grains (Crocker et al., 1998; Joy et al., 1997; Plascencia and Zinn, 1996). Knowlton et al. (1998) reported that dry rolling corn increased butyrate 33

54 production and decreased acetate and propionate production when compared with coarsely-ground corn, a finding that is partially contradictory to the previous indication. Additionally, dry rolling corn increased butyrate concentration, at the expense of acetate and propionate, when compared to whole corn (Murphy et al., 1994). Total volatile fatty acid production and rumen ph were not affected in comparisons between steam-flaked, steam-rolled, dry-rolled, and coarse-ground grains (Crocker et al., 1998; Joy et al., 1997; Knowlton et al., 1998; Plascencia and Zinn, 1996; Reis and Combs, 2000). However, Murphy et al., 1994, reported increased total volatile fatty acid concentrations and decreased ruminal ph with dry-rolled corn when compared to whole corn; and Trei et al. (1970) indicated greater in vitro gas production as the extent of grain processing increased, with in vitro gas production increasing in the order of whole, dry-rolled, and steam-flaked grains. In addition, the possibility of diet ingredients other than the processed grains dictating rumen volatile fatty acid production cannot be ignored. Grain Processing and Rumen Ammonia Rumen ammonia concentrations were decreased when corn was steam-flaked or coarsely-ground when compared to dry-rolled corn (Crocker et al., 1998; Knowlton et al., 1998). However, Joy et al. (1997) found no difference between steam-flaked and dryrolled corn for rumen ammonia. Rumen microbial protein production and microbial protein reaching the small intestine has been shown to increase as grain processing increased, with one study reporting an 18% increase in microbial protein production with steam-flaked over dry-rolled grain (Crocker et al., 1998; Plascencia and Zinn, 1996; Theurer et al., 1999). Increases in microbial protein production and rumen microbial escape have been attributed to increased urea and N cycling (Crocker et al., 1998; 34

55 Plascencia and Zinn, 1996; Theurer et al., 1999). However, others have found no difference between steam-flaked, dry-rolled, or coarsely-ground corn for microbial protein production or microbial nitrogen flow (Joy et al., 1997; Knowlton et al., 1998). Grain Processing and Diet Digestibility Grain Processing and Starch Digestibility Increased surface area exposure and chemical alteration of grains has been shown to change digestibility of all feed constituents in numerous research reports. An altered starch digestibility is the most notable, and possibly most important, aspect of grain processing (Huntington, 1997; Owens et al., 1997; Theurer, 1986; Theurer et al., 1999). Increases in starch digestibility have been reported to occur ruminally, intestinally, and in the total digestive tract (Chen et al., 1994; Crocker et al., 1998; Galyean et al., 1981; Huntington, 1997; Knowlton et al., 1998; Owens et al., 1997; Plascencia and Zinn, 1996; Theurer, 1986; Yu et al., 1998; Zinn, 1990). Extent of starch digestibility occurring with different processing methods, decreasing from greatest to least, appears to be steam-flaked, finely ground, dry-rolled, with whole grains having lowest digestibility (Chen et al., 1994; Crocker et al., 1998; Galyean et al., 1981; Huntington, 1997; Knowlton et al., 1998; Plascencia and Zinn, 1996; Theurer, 1986; Yu et al., 1998; Zinn, 1990). Processing shifts the site of starch digestion from the small intestine to the rumen, subsequently increasing starch digestion in both sites (Theurer, 1986; Theurer et al., 1999). Percent ruminally digested starch of differently processed corn, averaged across numerous trials, was reported by Huntington (1991) as 84.8 ± 4.1 for steam-flaked, 76.2 ± 7.9 for dry-rolled, 72.1 for steam-rolled, and 49.5 for coarsely- 35

56 ground. Increases in ruminal starch digestibility inevitably decrease the percent of ruminally escaping starch. A review by Theurer (1986) found an average of 10-25% of steam-flaked corn starch escaping rumen digestion, while 30-45% of dry-rolled corn starch escaped, with numerous others supporting these values (Crocker et al., 1998; Huntington, 1997; Plascencia and Zinn, 1996; Theurer et al., 1999). However, some have reported no differences in starch digestibility between steam-flaked or steam-rolled corn and dry-rolled corn (Joy et al., 1997; Reis and Combs, 2000). Concomitant with altered starch digestibility is a change in energy values for differently processed grains. The ordering for metabolizable energy of differently processed grains, reported by Owens et al. (1997) was steamed grains (flaked and rolled), whole grains, and dry-rolled grains, listed in decreasing value. The suggested reasons for greater metabolizable energy in whole grain than dry-rolled grain were a decreased negative associative effect between concentrates and roughages and/or a shift in digestion site to the small intestine, with whole grains (Owens et al., 1997). Alternately, net energy for lactation of steam-flaked corn has been reported as being 6% greater than steam-rolled and 20-33% greater than dry-rolled (Chen et al., 1994; Plascencia and Zinn, 1996; Theurer et al., 1999). Grain Processing and Structural Component Digestibility Grain processing alterations to dietary structural component digestibility appear negatively associated with changes in starch digestibility. Steam flaking has resulted in decreased rumen digestibility of neutral and acid detergent fiber and cellulose, when compared to steam or dry rolling (Crocker et al., 1998; Joy et al., 1997; Plascencia and Zinn, 1996; Yu et al., 1998). Further, ruminal and total tract neutral detergent fiber 36

57 digestibility was reported as lower for coarse-ground than dry-rolled corn (Knowlton et al., 1998). Decreases in structural component digestibility, with increased processing, have been attributed to increased availability of an alternate and more readily digested carbohydrate source (Hoover, 1986; Yu et al., 1998). Conversely, some have indicated increased structural component digestibility with increased processing (Chen et al., 1994; Reis and Combs, 2000). Differences in dry or organic matter digestibility with different processing methods have also been reported. Yu et al. (1998) indicated decreases in dietary organic matter digestibility with differently processed corn, in the order of steam-rolled, finely ground, dry-rolled, with steam-flaked having the lowest dietary organic matter digestibility. Murphy et al. (1994) indicated that dry rolling corn increased dry and organic matter digestibility over whole corn when intake was limited. However, an opposite occurrence was found with unrestricted intake and attributed to differences in ruminal passage rate and particle size (Murphy et al., 1994). Alternately to Yu et al. (1998), others have found increased dry matter digestibility with steam-flaked grains when compared to steam and dry-rolled grains (Chen et al., 1994; Galyean et al., 1981). Additionally, others have reported that processing method did not influence dry matter digestibility (Crocker et al., 1998; Joy et al., 1997). Furthermore, Knowlton et al. (1998) reported greater ileal and duodenal dry matter flow with coarse-ground than dry-rolled corn, indicating greater rumen dry matter digestibility of dry-rolled corn. However, total tract dry matter digestibility was greater for coarse-ground than dry-rolled corn (Knowlton et al., 1998). 37

58 Grain Processing and Intake, Gain, and Efficiency Different methods and extent of grain processing have been reported to influence dry matter intake with greatest intake for dry-rolled, followed by whole, steam-rolled, steam-flaked, with finely ground resulting in the lowest intake (Owens et al., 1997; Reinhardt et al., 1998; Reis and Combs, 2000; Yu et al., 1997; Yu et al., 1998). However, others have indicated increased dry matter intake for steam-flaked grains compared with dry or steam-rolled grains, and that processing has diminished intake effects with grazing cattle (Bargo et al., 2003; Chen et al., 1994; Delahoy et al., 2003); Plascencia and Zinn, 1996). Additionally, Crocker et al. (1997) and Joy et al. (1997) indicated no difference in dry matter intake between steam-flaked and dry-rolled corn. Whole, dry-rolled, and steam-rolled corn have been indicated to result in similar rates of gain (Owens et al., 1997; Theurer, 1986). However, increased average daily gains have been reported for steam-flaked or steam-rolled corn, despite decreased dry matter intake, possibly indicating an advantage in feed efficiency for extensively processed grains (Owens et al., 1997; Reinhardt et al., 1998). Increases in feed utilization efficiency (feed:gain or feed:milk) have been reported for different processing methods, with efficiency decreasing in the order of finely ground, steam-flaked, steam-rolled, whole, with dry-rolled being the least efficient (Owens et al., 1997; Reinhardt et al., 1998; Theurer, 1986; Yu et al., 1997; Yu et al., 1998). However, Chen et al. (1994) reported no difference in feed utilization efficiency between steam-flaked and steam-rolled corn. Increased digestibility typically associated with increasing processing level may be advantageous in the neonatal calf. The opportunity for greater butyrate and propionate production may increase rumen development rate. However, concomitant with increased starch digestibility is increased lactic acid production and subsequently 38

59 decreased ph, thereby increasing the possibility of parakeratosis. Therefore, determining an optimal grain processing level for use in calf starters must take into account both positive and negative affects of grain processing. 39

60 REFERENCES Adams, D. C., M. L. Galyean, H. E. Kiesling, J. D. Wallace, and M. D. Finkner Influence of viable yeast culture, sodium bicarbonate, and monensin on liquid dilution rate, rumen fermentation, and feedlot performance of growing steers and digestibility in lambs. J. Anim. Sci. 53: Anderson, M. J., M. Khoyloo, and J. L. Walters Effect of feeding whole cottonseed on intake, body weight, and reticulorumen development of young Holstein calves. J. Dairy Sci. 65: Ash, R., and G. D. Baird Activation of volatile fatty acids in bovine liver and rumen epithelium. Biochem. J. 136: Baldwin, R. L., VI., and K. R. McLeod Effects of diet forage:concentrate ratio and metabolizable energy intake on isolated rumen epithelial cell metabolism in vitro. J. Anim. Sci. 78: Bargo, F., L. D. Muller, E. S. Kolver, and J. E. Delahoy Production and digestion of supplemented dairy cows on pasture. J. Dairy Sci. 86:1-42. Beharka, A. A., T. G. Nagaraja, J. L. Morrill, G. A. Kennedy, and R. D. Klemm Effects of form of the diet on anatomical, microbial, and fermentative development of the rumen of neonatal calves. J. Dairy Sci. 81: Beauchemin, K. A., and L. M. Rode Minimum versus optimum concentrations of fiber in dairy cow diets based on barley silage and concentrates of barley or corn. J. Dairy Sci. 80: Bohman, V. R., G. W. Trimberger, J. K. Loosli, and K. L. Turk The utilization of molasses and urea in the rations of growing dairy cattle. J. Dairy Sci. 37: Bond, J., and T. S. Rumsey Liquid molasses-urea or biuret (NPN) feed supplements for beef cattle: Wintering performance, ruminal differences, and feeding patterns. J. Anim. Sci. 37: Bouchard, R., and H. R. Conrad Sulfur requirements of lactating dairy cows. II. Utilization of sulfates, molasses and lignin-sulfonate. J. Dairy Sci. 56: Brown, W. F Cane molasses and cottonseed meal supplementation of ammoniated tropical grass hay for yearling cattle. J. Anim. Sci. 71:

61 Brown, W. F., and D. D. Johnson Effects of energy and protein supplementation of ammoniated tropical grass hay on the growth and carcass characteristics of cull cows. J. Anim. Sci. 69: Brown, W. F., J. D. Phillips, and D. B. Jones Ammoniation or cane molasses supplementation of low quality forages. J. Anim Sci. 64: Brownlee, A The development of rumen papillae in cattle fed on different diets. Brit. Vet. J. 112: Bull, L. S., L. J. Bush, J. D. Friend, B. Harris, Jr., and E. W. Jones Incidence of ruminal parakeratosis in calves fed different rations and its relation to volatile fatty acid absorption. J. Dairy Sci. 48: Burroughs, W., P. Gerlaugh, B. H. Edgington, and R. M. Bethke The influence of corn starch upon roughage digestion in cattle. J. Anim. Sci. 8: Chen, K. H., J. T. Huber, C. B. Theurer, R. S. Swingle, J. Simas, S. C. Chan, Z. Wu, and J. L. Sullivan Effect of steam flaking of corn and sorghum grains on performance of lactating cows. J. Dairy Sci. 77: Church, D. C The Ruminant Animal: Digestive Physiology and Nutrition. Prentice-Hall, Inc. Englewood Cliffs, New Jersey. Cole, N. A., C. W. Purdy, and D. P. Hutcheson Influence of yeast culture on feeder calves and lambs. J. Anim. Sci. 70: Corona, L., G. D. Mendoza, F. A. Castrejon, M. M. Crosby, and M. A. Cobos Evaluation of two yeast cultures (Saccharomyces cerevisiae) on ruminal fermentation and digestion in sheep fed a corn stover diet. Small Rum. Res. 31: Crocker, L. M., E. J. DePeters, J. G. Fadel, H. Perez-Monti, S. J. Taylor, J. A. Wyckoff, and R. A. Zinn Influence of processed corn grain in diets of dairy cows on digestion of nutrients and milk composition. J. Dairy Sci. 81: Curtin, L. V Molasses in Animal Nutrition. National Feed Ingredients Association. West Des Moines, Iowa. Davis, C. L., and J. K. Drackley The Development, Nutrition, and Management of the Young Calf. Iowa State University Press. Ames, Iowa. Dawson, K. A., K. E. Newman, and J. A. Boling Effects of microbial supplements containing yeast and lactobacilli on roughage-fed ruminal microbial activities. J. Anim. Sci. 68: Delahoy, J. E., L. D. Muller, F. Bargo, T. W. Cassidy, and L. A. Holden Supplemental carbohydrates sources for lactating dairy cows on pasture. J. Dairy Sci. 86:

62 El Hassan, S. M., C. J. Newbold, I. E. Edwards, J. H. Topps, and R. J. Wallace Effect of yeast culture on rumen fermentation, microbial protein flow from the rumen and live-weight gain in bulls given high cereal diets. Anim. Sci. 62: Enjalbert, F., J. E. Garrett, R. Moncoulon, C. Bayourthe, and P. Chicoteau Effects of yeast culture (Saccharomyces cerevisiae) on ruminal digestion in nonlactating dairy cows. An. Feed Sci. and Tech. 76: Erasmus, L. J., P. M. Botha, and A. Kistner Effect of yeast culture supplement on production, rumen fermentation, and duodenal nitrogen flow in dairy cows. J. Dairy Sci. 75: Flatt, W. P., R. G. Warner, and J. K. Loosli Influence of purified materials on the development of the ruminant stomach. J. Dairy Sci. 41: Frederick, H. M., B. Theurer, and W. H. Hale Effect of moisture, pressure, and temperature on enzymatic starch degradation of barley and sorghum grain. J. Dairy Sci. 56: Gabler, M. T., P. R. Tozer, and A. J. Heinrichs Development of a cost analysis spreadsheet for calculating the costs to raise a replacement dairy heifer. J. Dairy Sci. 83: Galyean, M. L., D. G. Wagner, and F. N. Owens Dry matter and starch disappearance of corn and sorghum as influenced by particle size and processing. J. Dairy Sci. 64: Garcia, C. C. G., M. G. D. Mendoza, M. S. Gonzalez, P. M. Cobos, C. M. E. Ortega, and L. R. Ramirez Effect of a yeast culture (Saccharomyces cerevisiae) and monensin on ruminal fermentation and digestion in sheep. Anim. Feed Sci. and Tech. 83: Greenwood, R. H., J. L. Morrill, E. C. Titgemeyer, and G. A. Kennedy A new method of measuring diet abrasion and its effect on the development of the forestomach. J. Dairy Sci. 80: Hale, W. H Influence of processing on the utilization of grains (starch) by ruminants. J. Anim. Sci. 37: Harbers, L. H Starch granule structural changes and amylolytic patterns in processed sorghum grain. J. Anim. Sci. 41: Harmon, D. L., K. L. Gross, C. R. Krehbiel, K. K. Kreikemeir, M. L. Bauer, and R. A. Britton Influence of dietary forage and energy intake on metabolism and acyl-coa synthetase activity in bovine ruminal epithelial tissue. J. Anim. Sci. 69:

63 Harrison, G. A., R. W. Hemken, K. A. Dawson, R. J. Harmon, and K. B. Barker Influence of addition of yeast culture supplement to diets of lactating cows on ruminal fermentation and microbial populations. J. Dairy Sci. 71: Harrison, H. N., R. G. Warner, E. G. Sander, and J. K. Loosli Changes in the tissue and volume of the stomachs of calves following the removal of dry feed or consumption of inert bulk. J. Dairy Sci. 43: Heinemann, W. W., and E. M. Hanks Cane molasses in cattle rations. J. Anim. Sci. 45: Hibbs, J. W., H. R. Conrad, W. D. Pounden, and N. Frank A high roughage system for raising calves based on early development of rumen function. VI. Influence of hay to grain ratio on calf performance, rumen development, and certain blood changes. J. Dairy Sci. 39: Hinders, R. G. and F. G. Owen Relation of ruminal parakeratosis development to volatile fatty acid absorption. J. Dairy Sci. 48: Hoover, W. H Chemical factors involved in ruminal fiber digestion. J. Dairy Sci. 69: Hughes, J The effect of high-strength yeast culture in the diet of early-weaned calves. Anim. Prod. 46:526. Huntington, G. B Starch utilization by ruminants: From basics to the bunk. J. Anim. Sci. 75: Joy, M. T., E. J. DePeters, J. G. Fadel, and R. A. Zinn Effects of corn processing on the site and extent of digestion in lactating cows. J. Dairy Sci. 80: Kellogg, D. W., and F. G. Owen Relation of ration sucrose level and grain content to lactation performance and rumen fermentation. J. Dairy Sci. 62: Klein, R. D., R. L. Kincaid, A. S. Hodgson, J. H. Harrison, J. K. Hillers, and J. D. Cronrath Dietary fiber and early weaning on growth and rumen development of calves. J. Dairy Sci. 70: Knowlton, K. F., B. P. Glenn, and R. A. Erdman Performance, ruminal fermentation, and site of starch digestion in early lactation cows fed corn grain harvested and processed differently. J. Dairy Sci. 81: Komkris, T., R. W. Stanley, and K. Morita Effect of feeds containing molasses fed separately and together with roughage on digestibility of rations, volatile fatty acids produced in the rumen, milk production, and milk constituents. J. Dairy Sci. 48:

64 Koul, V., U. Kumar, V. K. Sareen, and S. Singh Mode of action of yeast culture (YEA-SACC 1026) for stimulation of rumen fermentation in buffalo calves. J. Sci. Food Agric. 77: Krehbiel, C. R., D. L. Harmon, and J. E. Schnieder Effect of increasing ruminal butyrate on portal and hepatic nutrient flux in steers. J. Anim. Sci. 70: Kumar, U., V. K. Sareen, and S. Singh Effect of yeast culture supplementation on ruminal microbial populations and metabolism in buffalo calves fed a high roughage diet. J. Sci. Food Agric. 73: Kung, L. Jr., E. M. Kreck, R. S. Tung, A. O. Hession, A. C. Sheperd, M. A. Cohen, H. E. Swain, and J. A. Z. Leedle Effects of a live yeast culture and enzymes on in vitro ruminal fermentation and milk production of dairy cows. J. Dairy Sci. 80: Lane, M. A., R. L. Baldwin, and B. W. Jesse Sheep rumen metabolic development in response to age and dietary treatments. J. Anim. Sci. 78: LeGendre, J. R., R. Totusek, and W. D. Gallup Effect of live-cell yeast on nitrogen retention and digestibility of rations by beef cattle. J. Anim. Sci. 16: Lofgreen, G. P., and K. K. Otagaki The net energy of blackstrap molasses for lactating dairy cows. J. Dairy Sci. 43: Longenbach, J. I., and A. J. Heinrichs A review of the importance and physiological role of curd formation in the abomasum of young calves. Anim. Feed Sci. and Tech. 73: Malcolm, K. J., and H. E. Kiesling Effects of whole cottonseed and live yeast culture on ruminal fermentation and fluid passage rate in steers. J. Anim. Sci. 68: Martin, R. J., and J. M. Wing Effect of molasses level on digestibility of a high concentrate ration and on molar proportions of volatile fatty acids produced in the rumen of dairy steers. J. Dairy Sci. 49: Mather, R. E., and C. B. Bender Molasses as a replacement for grain in the rations of growing heifers and milking cows. J. Anim. Sci. 10:1056. McGavin, M. D., and J. L. Morrill Scanning electron microscopy of ruminal papillae in fed various amounts and forms of roughage. Am. J. Vet. Res. 37: McNeill, J. W., G. D. Potter, J. K. Riggs, and L. W. Rooney Chemical and physical properties of processed sorghum grain carbohydrates. J. Anim. Sci. 40:

65 Mertens, D. R Creating a system for meeting the fiber requirements of dairy cows. J. Dairy Sci. 80: Mir, Z. and P. S. Mir Effect of the addition of live yeast (Saccharomyces cerevisiae) on growth and carcass quality of steers fed high-forage or high-grain diets and on feed digestibility and in situ degradability. J. Anim. Sci. 72: Morales, J. L., H. H. Van Horn, and J. E. Moore Dietary interaction of cane molasses with source of roughage: Intake and lactation effects. J. Dairy Sci. 72: Mould, F. L., E. R. Orskov, and S. O. Mann Associative effects of mixed feeds. I. Effects of type and level of supplementation and the influence of the rumen fluid ph on cellulolysis in vivo and dry matter digestion of various roughages. Anim. Feed Sci. Technol. 10: Murphy, J. J The effects of increasing the proportion of molasses in the diet of milking dairy cows on milk production and composition. Anim. Feed Sci. Technol. 78: Murphy, T. A., F. L. Fluharty, and S. C. Loerch The influence of intake level and corn processing on digestibility and ruminal metabolism in steers fed allconcentrate diets. J. Anim. Sci. 72: Mutsvangwa, T., I. E. Edwards, J. H. Topps, and G. F. M. Paterson The effect of dietary inclusion of yeast culture (Yea-Sacc) on patterns of rumen fermentation, food intake and growth of intensively fed bulls. Anim. Prod. 55: Nisbet, D. J., and S. A. Martin Effect of a Saccharomyces cerevisiae culture on lactate utilization by the ruminal bacterium Selenomonas ruminantium. J. Anim. Sci. 69: Nocek, J. E., J. H. Herbein, and C. E. Polan Influence of ration physical form, ruminal degradable nitrogen and age on rumen epithelial propionate acetate transport and some enzymatic activities. J. Nutr. 110: Nocek, J. E., C. W. Heald, and C. E. Polan Influence of ration physical form and nitrogen availability on ruminal morphology of growing bull calves. J. Dairy Sci. 67: Olson, K. C., J. S. Caton, D. R. Kirby, and P. L Norton Influence of yeast culture supplementation and advancing season on steers grazing mixed-grass prairie in the Northern Great Plains: II. Ruminal fermentation, site of digestion, and microbial efficiency. J. Anim. Sci. 72: Owen, F. G., D. W. Kellogg, and W. T. Howard Effect of molasses in normaland high-grain rations on utilization of nutrients for lactation. J. Dairy Sci. 50:

66 Owens, F. N., D. S. Secrist, W. J. Hill, and D. R. Gill The effect of grain source and grain processing on performance of feedlot cattle: A review. J. Anim. Sci. 75: Phillips, W. A. and D. L. VonTungeln The effect of yeast culture on the poststress performance of feeder calves. Nut. Rep. Int. 32: Plascencia, A., and R. A. Zinn Influence of flake density on the feeding value of steam-processed corn in diets for lactating cows. J Anim. Sci. 74: Pounden, W. D. and J. W. Hibbs. 1948a. The influence of the ration and rumen inoculation on the establishment of certain microorganisms in the rumens of young calves. J. Dairy Sci. 31: Pounden, W. D. and J. W. Hibbs. 1948b. The influence of the ratio of grain to hay in the ration of dairy calves on certain rumen microorganisms. J. Dairy Sci. 31: Pritchard, R. H., and D. A. Stateler Grain processing: Effects on mixing, prehension, and other characteristics of feeds. J. Anim. Sci. 75: Putnam, D. E., C. G. Schwab, M. T. Socha, N. L. Whitehouse, N. A. Kierstead, and B. D. Garthwaite Effect of yeast culture in the diets of early lactation dairy cows on ruminal fermentation and passage of nitrogen fractions and amino acids to the small intestine. J. Dairy Sci. 80: Quigley, J. D., L. B. Wallis, H. H. Dawlen, and R. N. Heitman Sodium Bicarbonate and yeast culture effects on ruminal fermentation, growth, and intake in dairy calves. J. Dairy Sci. 75: Reinhardt, C. D., R. T. Brandt, Jr., T. P. Eck, and E. C. Titgemeyer Performance, digestion, and mastication efficiency of Holstein steers fed whole or processed corn in limit- or full-fed growing-finishing systems. J. Anim. Sci. 76: Reis, R. B., and D. K. Combs Effects of corn processing and supplemental hay on rumen environment and lactation performance of dairy cows grazing grasslegume pasture. J. Dairy Sci. 83: Rickard, M. D., and J. H. Ternouth The effect of the increased dietary volatile fatty acids on the morphological and physiological development of lambs with particular reference to the rumen. J. Agric. Sci. 65: Rooney, L. W., and R. L. Pflugfelder Factors affecting starch digestibility with special emphasis on sorghum and corn. J. Anim. Sci. 63:

67 Sander, E. G., R. G. Warner, H. N. Harrison, and J. K. Loosli The stimulatory effect of sodium butyrate and sodium propionate on the development of rumen mucosa in the young calf. J. Dairy Sci. 42: Seymour, W. M., J. E. Nocek, and J. Sciliano-Jones Effect of a colostrum substitute and of dietary brewer s yeast on the health and performance of dairy calves. J. Dairy Sci. 78: Stevens, C. E., and B. K. Stettler Factors affecting the transport of volatile fatty acids across rumen epithelium. Am. J. Physiol. 210(2): Stobo, I. J. F., J. H. B. Roy, and H. J. Gaston. 1966a. Rumen development in the calf. 1. The effect of diets containing different proportions of concentrates to hay on rumen development. Br. J. Nutr. 20: Stobo, I. J. F., J. H. B. Roy, and H. J. Gaston. 1966b. Rumen development in the calf. 2. The effect of diets containing different proportions of concentrates to hay on digestive efficiency. Br. J. Nutr. 20: Sutton, J. D., A. D. McGilliard, and N. L. Jacobson Functional development of rumen mucosa. I. Absorptive ability. J. Dairy Sci. 46: Tamate, H., A. D. McGilliard, N. L. Jacobson, and R. Getty Effect of various dietaries on the anatomical development of the stomach in the calf. J. Dairy Sci. 45: Theurer, C. B Grain processing effects on starch utilization by ruminants. J. Anim. Sci. 63: Theurer, C. B., J. T. Huber, A. Delgado-Elorduy, and R. Wanderley Invited Review: Summary of steam-flaking corn or sorghum grain for lactating dairy cows. J. Dairy Sci. 82: Trei, J., W. H. Hale, and B. Theurer Effect of grain processing on in vitro gas production. J. Anim. Sci. 30: Van Soest, P. J Nutritional Ecology of the Ruminant. 2 nd Ed. Cornell University Press, Ithaca, NY. Vazquez-Anon, M., A. J. Heinrichs, J. M. Aldrich, and G. A. Varga Postweaning age effects on rumen fermentation end-products and digesta kinetics in calves weaned at 5 weeks of age. J. Dairy Sci. 76: Wagner, D. G., J. Quinonez, and L. J. Bush The effect of corn- or wheat-based diets and yeast culture on performance, ruminal ph, and volatile fatty acids in dairy calves. Agri-Practice 11:7-9. Waldo, D. R., and L. H. Schultz Blood and rumen changes following the intraruminal administration of glycogenic materials. J. Dairy Sci. 43:

68 Warner, R. G., W. P. Flatt, and J. K. Loosli Dietary factors influencing the development of the ruminant stomach. J. Agric. Food Chem. 4: Weigand, E., J. W. Young, and A. D. McGilliard Volatile fatty acid metabolism by rumen mucosa from cattle fed hay or grain. J. Dairy Sci. 58: Wiedmeier, R. D., B. H. Tanner, J. R. Bair, H. T. Shenton, M. J. Arambel, and J. L. Walters Effects of a new molasses byproduct, concentrated separator byproduct, on nutrient digestibility and ruminal fermentation in cattle. J. Anim. Sci. 70: Wiedmeier, R. D., M. J. Arambel, and J. L Walters Effect of yeast culture and Aspergillus oryzae fermentation extract on ruminal characteristics and nutrient digestibility. J. Dairy Sci. 70: Williams, P The effect of cane molasses on the digestibility of a complete ration fed to dairy cows. J. Dairy Sci. 8: Williams, P. E. V., C. A. G. Tait, G. M. Innes, and C. J. Newbold Effects of the inclusion of yeast culture (Saccharomyces cerevisiae plus growth medium) in the diet of dairy cows on milk yield and forage degradation and fermentation patterns in the rumen of steers. J. Anim. Sci. 69: Wing, J. M., and G. W. Powell Response of lactating cows to two levels of millrun blackstrap molasses from cane grown on organic soils. J. Dairy Sci. 52: Wing, J. M., H. H. Van Horn, S. D. Sklare, and B. Harris, Jr Effects of citrus molasses distillers solubles and molasses on rumen parameters and lactation. J. Dairy Sci. 71: Yoon, I. K., and M. D. Stern Effects of Saccharomyces cerevisiae and Aspergillus oryzae cultures on ruminal fermentation in dairy cows. J. Dairy Sci. 79: Yu, P., J. T. Huber, C. B. Theurer, K. H. Chen, L. G. Nussio, and Z. Wu Effect of steam-flaked or steam-rolled corn with or without Aspergillus oryzae in the diet on performance of dairy cows fed during hot weather. J. Dairy Sci. 80: Yu, P., J. T. Huber, F. A. P. Santos, J. M. Simas, and C. B. Theurer Effects of ground, steam-flaked, and steam-rolled corn grains on performance of lactating cows. J. Dairy Sci. 81: Zinn, R. A Influence of steaming time on site of digestion of flaked corn in steers. J. Anim. Sci. 68: Zitnan, R., J. Voigt, U. Schonhusen, J. Wegner, M. Kokardova, H. Hagemeister, M. Levkut, S. Kuhla, and A. Sommer Influence of dietary concentrate to 48

69 forage ratio on the development of rumen mucosa in calves. Arch. Anim. Nutr. 51: Zitnan, R., J. Voigt, J. Wegner, G. Breves, B. Schroder, C. Winckler, M. Levkut, M. Kokardova, U. Schonhusen, S. Kuhla, H. Hagemeister, and A. Sommer Morphological and functional development of the rumen in the calf: Influence of the time of weaning. Arch. Anim. Nutr. 52:

70 CHAPTER 3 DEVELOPMENT AND ANALYSIS OF A RUMEN TISSUE SAMPLING PROCEDURE ABSTRACT A procedure for rumen tissue sampling was developed to determine treatment effects on rumen development and papillae growth in young calves and to improve repeatability in rumen tissue sampling techniques. Rumens were collected from 42 male Holstein calves from three separate experiments. Rumen sampling areas (n = 9) included the caudal dorsal blind sac, cranial dorsal sac, cranial ventral sac, and the caudal and ventral portions of the caudal ventral blind sac. Right and left sides of the rumen were sampled. Five 1-cm 2 sections were removed from each area and measured for papillae length (n = 20/area), papillae width (n = 20/area), rumen wall thickness (n = 5/area), and number of papillae per cm 2 (n = 5/area). Correlations between areas, samples, and measurements were obtained, and comparisons between experiments, areas, samples, and measurements were performed for all variables. In addition, power analyses were conducted for all variables to determine the efficacy of the procedure in detecting treatment differences. Results indicate that samples should be taken from the caudal and cranial sacs of the dorsal and ventral rumen to sufficiently represent papillae growth and development throughout the entire rumen. The procedure is capable of detecting treatment differences for papillae length and papillae width, has a decreased but acceptable capability of detecting treatment differences for rumen wall thickness, but appears limited in ability to detect treatment differences for papillae per cm 2. In 50

71 conclusion, rumen tissue sampling to determine extent of rumen development in calves can be performed in a nonbiased and repeatable manner utilizing a limited number of calves. Abbreviations: A = caudal portion of caudal ventral blind sac, α = P value, δ = treatment difference, LB = left side caudal dorsal sac, LC = left side cranial dorsal sac, LD = left side cranial ventral sac, LE = left side ventral portion of caudal ventral blind sac, LSN = least significant number, PL = papillae length, PW = papillae width, PC = papillae per cm 2, RB = right side caudal dorsal sac, RC = right side cranial dorsal sac, RD = right side cranial ventral sac, RE = right side ventral portion of caudal ventral blind sac, σ = root mean square error, RWT = rumen wall thickness. INTRODUCTION The ruminal conditions necessary for papillae development have been previously elucidated (Brownlee, 1956; Flatt et al., 1958; Sutton et al., 1963). Alterations in dietary chemical and structural composition have influenced rumen papillae length (PL) and width (PW), rumen wall thickness (RWT), and papillae density (PC) (Brownlee, 1956; Harrison et al., 1960; Tamate et al., 1962). In addition, some have suggested that the rumen epithelium and ruminal muscle grow and develop independent of each other, indicating that dietary factors influencing one may not affect the other (Brownlee, 1956; Harrison et al., 1960). Continued research focusing on dietary manipulation to optimize the rate and extent of ruminal VFA production and development is still needed and could provide economic and health advantages to the dairy replacement industry. However, previous rumen sampling techniques have primarily obtained samples from the cranial 51

72 ventral and caudal dorsal sacs of the rumen, corresponding to areas indicated to have the largest and most numerous papillae (Nocek et al., 1984; Tamate et al., 1962; Van Soest, 1994). Accounting for the importance of rumen papillae growth and the apparent within rumen variation, development of a procedure that standardizes rumen tissue sampling, measurement, and analysis could greatly aid this field of research and fill a void in the available literature (Brownlee, 1956; McGavin and Morrill, 1976b). In addition, samples taken from multiple areas of the rumen may result in a more representative description of rumen growth and development. However, the possibility arises that some areas of the rumen and/or some measures of development may be better suited for determination of treatment differences than others, or that a single area of the rumen may be representative of multiple areas. The removal of highly variable areas from the analysis could also reduce procedural error, creating the possibility of decreasing the number of observations per area and/or calves required per treatment. The objectives of this study were threefold, 1) to develop a repeatable and unbiased procedure for rumen tissue sampling, 2) to determine the areas of the rumen best suited for identifying treatment differences, and 3) to determine the proposed procedure s efficacy in rumen development research. MATERIALS AND METHODS Rumen Tissue Sampling Reticulo-rumens were obtained from 42 male Holstein calves utilized in four separate experiments between 2000 and 2002, and representing 11 different dietary treatments. Experimental protocols and procedures were similar across experiments 52

73 with the exception of dietary treatments, weaning age, and age at slaughter. Briefly, all calves received 4 L of colostrum within 12 h of birth, and were fed a 20% all-milk protein and 20% fat milk replacer in two equal feedings totaling 10% of birthweight until abrupt weaning (4 wks for one experiment and 5 wks for three experiments). A calf starter of similar composition, with the exception of the dietary additives unique to each experiment, was offered ad libitum daily. Six calves from the first experiment conducted during the summer of 2000 were slaughtered at 6 wk of age and six calves from the second experiment conducted during the fall of 2000 and winter of 2001 were slaughtered at 5 wk of age. Fifteen calves from the third experiment conducted through the winter of 2001 to the winter of 2002 were slaughtered at 4 wk of age, and the final fifteen calves from the fourth experiment conducted during the summer of 2002 were slaughtered at 5 wk of age. Calves were euthanized via captive bolt stunning and exsanguination, reticulo-rumens were harvested, emptied, and rinsed with cold water, then transported immediately to the laboratory for the following rumen dissection and tissue sampling procedure. The reticulo-rumen was placed on its left side, esophageal groove facing away, and an incision made around the circumference of the emptied and rinsed reticulo-rumen in line with the esophageal groove. A 6-cm section of the caudal portion of the caudal ventral blind sac was maintained intact. The rumen pillars were incised in line with the initial incision, and the muscles forming the rumen pillars separated. The reticulo-rumen was then opened and laid flat, creating a roughly symmetrical right and left side separated by the portion of the rumen maintained intact. The rumen pillars separate the rumen into nine distinct sampling areas, shown in Figure 1 and labeled as (A) caudal portion of the caudal ventral blind sac; (RB) right side and (LB) left side caudal dorsal sac; (RC) right side and (LC) left side cranial dorsal sac; (RD) right side and (LD) left side cranial ventral sac; and (RE) right side and (LE) left 53

74 side ventral portion of caudal ventral blind sac. A 1-cm 2 tissue sample was removed from the center of each area and four 1-cm 2 sections were removed 1 cm (for small areas or rumens) or 2 cm (for large areas or rumens) diagonally and distally from the four corners of the center sample (n = 5), creating a checkerboard pattern. Tissue samples were fixed in a 30% formaldehyde solution for subsequent measurements. Four randomly selected papillae per sample (20 per area) were measured for PL and PW, and one measurement per sample (5 per area) was recorded for RWT and PC. Visual measurements were taken using a Bausch and Lomb Stereo Zoom 4 dissecting microscope fitted with a measuring eyepiece at 11.25x magnification. Data from all 42 calves were pooled for procedure analysis. Statistical Analysis Correlations Correlations for PL, PW, RWT, and PC between areas, samples, and measurements were obtained using the CORRELATION procedure of SAS (1999) and calculated as σ ij ρ ij = where σ σ i j ρ ij = the correlation between PL, PW, RWT, or PC of the i th and j th areas, samples, or measurements σ ij = the covariance of the i th and j th areas, samples, or measurements for PL, PW, RWT, or PC 54

75 σ i, (σ j ) = the standard deviation of the i th (j th ) area, sample, or measurement for PL, PW, RWT, or PC (σ i σ j ) i, j = A, RB, RC, RD, RE, LB, LC, LD, or LE for area, 1 to 5 for sample, or 1 to 4 for measurement (i j). Comparisons Comparisons between experiments, areas, samples, and measurements for PL, PW, RWT, and PC were conducted across all calves and experiments using the MIXED procedure of SAS (1999) with a repeated measures statement. A separate model was utilized for PL and PW analysis than for RWT and PC analysis. The model for PL and PW analysis was y tasm = µ + α t + β a + γ s + λ m + e tasm where y tasm = an observed value for PL or PW for the m th measurement, taken from the s th sample, collected from the a th area from a calf in the t th experiment µ = the overall mean of the population α t = the fixed effect of the t th experiment where t = 1 to 4 β a = the random effect of the a th area where a = A, RB, RC, RD, RE, LB, LC, LD, or LE γ s = the random effect of the s th sample where s = 1 to 5 λ m = the random effect of the m th measurement where m = 1 to 4 e tasm = the error associated with the m th measurement, taken from the s th sample, collected from the a th area from a calf in the t th experiment; e tasm id 2 N(0, σ ). e 55

76 Calf nested within experiment and sample nested within area were included in the RANDOM statement of the model and measure was utilized as the repeated variable. The model for RWT and PC analysis was y tas = µ + α t + β a + γ s + e tas where y tas = an observed value for RWT or PC for the s th sample, collected from the a th area from a calf in the t th experiment µ = the overall mean of the population α t = the fixed effect of the t th experiment where t = 1 to 4 β a = the random effect of the a th area where a = A, RB, RC, RD, RE, LB, LC, LD, or LE γ s = the random effect of the s th sample where s = 1 to 5 e tas = the error associated with the s th sample, collected from the a th area from a calf in the t th 2 experiment; e N(0, σ ). tas id Calf nested within experiment was included in the RANDOM statement and sample was utilized as the repeated variable. Treatment influence was accounted for by including treatment in all comparison models. Age variation is accounted for by the inclusion of an experiment effect in the model. Differences between experiments were considered significant at P < 0.10 and between areas, samples, or measurements at P < A strict P value was utilized for area, sample, and measurement comparisons to insure that significant differences were also physiologically measurable differences. e 56

77 Regression Analysis The REGRESSION procedure of SAS (1999) was utilized to determine relationships between PL, PW, RWT, and PC across all calves and experiments. Linear models were initially fit, subsequently followed by higher order models if applicable. Power Analysis To determine the ability of the procedure to distinguish differences between rumen developmental levels, the necessary number of calves per treatment, samples per calf, measurements per calf, and the resultant power of each test, a power analysis was conducted for each described variable using a power macro (version 1.2) obtained from SAS (Latour, 2003). The power macro enabled the description of the desired P value (α), root mean square error (σ), and treatment difference or effect size (δ) calculated as δ = [SS(Hyp)/n] 0.5 where SS = sum of squares for the comparison of interest Hyp = hypotheses stating that the effect size between two observed measurements for PL, PW, RWT, or PC equals 0 (null hypothesis) or that the effect size is not equal to 0 and is some non-zero number (alternate hypothesis) n = number of observations within the comparison of interest. Effect size is determined by the difference between the observed effect size and the null hypothesis effect size. Levels for α were set at 0.01, 0.05, and Levels for σ and δ were not defined by the researchers, but were calculated by the power macro through 57

78 incorporation of the GENERAL LINEAR MODEL procedure of SAS (1999) using the following model: y pr = µ + α p + β r + e pr where y pr = an observed value for PL, PW, RWT, or PC from the r th calf, sample, or measurement in the p th rumen development group µ = the overall mean of the population α p = the fixed effect of the p th rumen development group where p = low or high β r = the random effect of the r th calf, sample, or measurement where r = 1 to 42 for calf; 1 to 5 for sample; or 1 to 4 for measurement e pr = the error associated with the r th calf, sample, or measurement in the p th 2 rumen development group; e N(0, σ ). pr id The power of the test at n, the least significant number (LSN), and the power of the test when n = LSN were provided for each α level using the estimated σ and δ values. The LSN is defined as the sample size required to produce a significant test for a sample having a α, σ, and δ values equal to those of the dataset used. Average values for PL, PW, RWT, and PC were calculated for each calf. The average values of PL, PW, and RWT were sorted from lowest to highest, with low average measurements considered as indicative of a low level of rumen development and high average measurements representative of a high level of rumen development. Average values for PC were sorted from highest to lowest with high PC values indicative of low rumen development (Anderson et al., 1982; Klein et al., 1987; Zitnan et al., 1999). The calves were then divided into two treatment groups, a low or high rumen development group, based on average variable measurements. Consideration was given to simply selecting the 21 lowest and highest averages and their corresponding calves, as this method e 58

79 would possibly decrease the observed variance for each rumen development variable. However, with the desire to make the results of this paper as realistic and applicable as possible, a selection criterion was developed. Division into treatment groups was as follows: 1) The 21 calves with the lowest average measurement for PL were marked as the low group, and the 21 calves with the highest average measurement for PL were marked as the high group. 2) For a calf to remain marked as low or high group required at least two average measurements for PW, RWT, or PC within the 21 lowest or highest average measurements, respectively. 3) If a calf did not have two additional average measurements in the group corresponding with its PL, then an average measurement within the same group as PL was used to determine grouping. The order of importance for average measurements within the same group as PL was PW, RWT, and then PC. Papillae length was the primary grouping factor due to analyses indicating a high level of correlation between PL throughout the rumen and stronger relationships between PL and the other three variables than between PW, RWT, or PC and the other three variables. The importance order in step 3 was developed using the same reasoning and statistics, with higher correlation values and stronger relationships for PW, followed by RWT, with PC having the lowest correlation values and weakest relationships. The power analysis step was conducted last to avoid any influence on previous analyses due to the selection criteria used. The power analyses utilized all 42 calves and included observations from all areas. Calves from all four experiments were present in both the low and high group accounting for experiment and age differences. All σ and δ values 59

80 presented were calculated by the power analyses and are unique to the dataset utilized. Each calf, in the initial dataset, had 180 observations for PL and PW (labeled 1 to 180) and 45 observations for RWT and PC (labeled 1 to 45) taken from 45 tissue samples (labeled 1 to 45) within the rumen. Average PL, PW, RWT, and PC were calculated for each calf to create the CALF power analysis dataset. Average PL, PW, RWT, and PC were calculated for each sample (1 to 45) across calves within the given treatment group to create the SAMPLE power analysis dataset. For creation of the MEASUREMENT dataset, average PL and PW for each measurement (1 to 180) were calculated across calves within the treatment group. Datasets for SAMPLE and MEASUREMENT power analyses essentially represented two rumens, one with a high level of rumen development and one with a low level of rumen measurement, and contained 45 and 180 observations per treatment, respectively. Values averaged across calves within treatments were utilized due to a separate power analysis being conducted for each effect of interest (calf, sample, and measurement); therefore each effect analyzed was considered a main effect within their respective power analysis (Dawson and Lagakos, 1993). Calves within treatment and observations within calves were the same for all datasets. Variation attributable to each of the dependant variables of interest (i.e. calves, samples, measurements) was captured by the manner in which averages were calculated for each distinct dataset and the number of observations within each dataset. 60

81 RESULTS Experiment Analysis Values for least squares means, standard error, and comparisons for PL, PW, RWT, and PC from the four experiments are presented in Table 3-1. Values appear to be influenced by age across experiments with significant differences (P < 0.10) occurring between experiment three and experiment one for PL and PW, between experiment three and all other experiments for RWT, and between experiment two and experiment one for PC. Rumens from experiment three were collected from calves at 4 wks of age and represent the youngest group of animals in the dataset, therefore, lower values for PL, PW, and RWT were not unexpected. However, similar values between samples obtained at 5 and 6 wk of age were not expected and may indicate a rapidly developing rumen prior to 5 wk of age, with a slower rate of development afterwards, under the calf rearing conditions utilized. Area Analysis Correlations Correlation values for PL and PW between areas were relatively high in all cases, ranging from 0.42 to 0.67 for PL and 0.38 to 0.60 for PW. All correlation values for PL and PW were significantly different (P < 0.001) from 0. Correlations for RWT and PC between areas were not as strong and were highly variable with values ranging from 0.18 to 0.49 for RWT and 0.06 to 0.44 for PC. All correlation values for RWT were 61

82 significantly different (P < 0.05) from 0, but not as consistent as observed for PL and PW. Correlation values for PC were not all significantly different from 0. Comparisons Values for least squares means, standard error, and comparisons of PL, PW, RWT, and PC for areas across calves and experiments are presented in Table 3-2. Significant differences (P < 0.001) were observed between some areas for PL, PW, RWT, and PC. Occurrences of difference between areas may be primarily explained by intra-rumen variation. In addition, standard errors for PL and PW made up a larger portion of the mean than standard errors for RWT and PC, indicating greater variation within the former rumen development parameters. Similar values and minimal significant differences between areas corresponding to the caudal portion of the caudal ventral blind sac and the caudal dorsal sac (A and B) were observed, indicating that samples from either area could represent the caudal rumen. Numerous differences between right and left rumen areas were observed for PL, with only one difference observed for PW and RWT, and no right and left rumen area differences seen for PC. More differences between right and left rumen areas for PL may be explained by higher variation within PL when compared to PW, RWT, and PC. 62

83 Sample and Measurement Analysis Correlations All correlation values for variables PL, PW, RWT, and PC between different samples across areas, calves, and experiments were consistently high and significantly different (P < 0.001) from 0, showing a strong correlation between samples for these variables. Correlation values range from 0.70 to 0.77 for PL, 0.57 to 0.61 for PW, 0.44 to 0.58 for RWT, and from 0.55 to 0.65 for PC. Similar correlation results were observed between measurements taken across samples, areas, calves, and experiments. Measurement correlation values for variables PL and PW were all significantly different (P < 0.001) from 0, and ranged from 0.90 to 0.92 for PL and from 0.72 to 0.76 for PW, indicating a strong correlation between measurements for PL and PW. Comparisons Values for least squares means of PL (average 1.11 mm), PW (average 0.70 mm), RWT (average 1.38 mm), and PC (average 75.86) were similar for different samples across areas, calves, and experiments with no significant differences between samples. Standard errors as a percent of means were highest for variable PL (0.09) followed by PW (0.05), PC (3.30), then RWT (0.04), as observed in the area analysis. No significant differences were observed between least squares means of PL (average 1.11) and PW (average 0.70) from measurements taken across samples, areas, calves, and experiments. Standard errors as a percent of means were once 63

84 again highest for variable PL (0.09) followed by PW (0.04), as seen in the area and sample analyses. Rumen Variable Relationships All calves and experiments were included in the regression analyses. Relationships between most rumen variable pairs were minimal, except for PL and PW. There was a significant linear and quadratic relationship between PL and PW. The linear regression was: PL = (± 0.02) (± 0.03) PW with R 2 = The quadratic relationships between PL and PW were: PL = (± 0.04) (± 0.12) PW 0.17 (± 0.09) PW 2 with R 2 = 0.58 and PW = 1.16 (± 0.01) (± 0.02) PL 0.15 (± 0.01) PL 2 with R 2 = Power Analysis Calves, Samples, and Measurements Required Table 3-3 presents the results of power analyses conducted to determine the number of calves, samples, and measurements necessary to find significant differences between treatments with a α of 0.01, 0.05, or 0.10 for PL, PW, RWT, and PC. Results of the power analysis for PL, PW, and RWT indicate the ability of the procedure to detect treatment differences, at an acceptably high power, with a dataset containing 21 calves per treatment, 45 samples, and 180 measurements per calf. Results of the power 64

85 analysis for PC indicate an inability of the procedure to detect significant differences between treatments with the number of samples utilized. LSN and Power when N = LSN Values for LSN indicate the total number of calves, samples per calf, and measurements per calf necessary to detect treatment difference. As two treatments were utilized in the analyses, the value for LSN should be divided by 2 to determine the lowest number of calves per treatment, samples per calf, and measurements per calf necessary to detect treatment differences. However, the power of the test when n = LSN may not be sufficiently high to warrant acceptability. Therefore, values for LSN should be considered threshold numbers to detect difference. In addition, as the observed σ, δ, and LSN values are unique to this dataset, LSN values may not be universally applicable, but do provide a starting point for research design and planning. With the observed α, σ, and δ values, the results from Table 3-3 suggest 5, 3, or 3 calves per treatment, 5, 3, or 3 samples per calf, and 9, 6, or 4 measurements per calf to detect treatment differences for PL with an α of 0.01, 0.05, and 0.10, respectively. With the same format, the results suggest 6, 4, or 3 calves per treatment, 3, 3, or 2 samples per calf, and 5, 3, or 3 measurements per calf to detect treatment difference for PW. Results for RWT suggest 18, 11, or 8 calves per treatment and 13, 8, or 6 samples/measurements per calf to detect treatment differences with a α of 0.01, 0.05, or 0.10 respectively. Values of LSN for RWT are higher than for PL and PW, possibly due to low δ values (observed difference between treatments) for this variable. Due to low δ 65

86 values and low relationship between δ and σ, values of LSN for PC are high, suggesting that PC may not be a feasible variable in rumen development research. DISCUSSION Correlations Strong area, sample, and measurement correlations suggest a high probability that a change in PL, PW, RWT, or PC from any area, sample, or measurement will reflect a similar change for any other area, sample, or measurement, more with PL, PW, and RWT than PC. This indicates the possibility that samples taken from some areas of the rumen will represent rumen development as effectively as samples taken from all areas of the rumen. In addition, results also indicated high similarity between multiple samples and/or measurements taken from the same area, suggesting the possibility for reduced samples and/or measurements. Correlations between similar areas on the right and left sides of the rumen were also high, indicating that samples from either side could be representative of that entire area of the rumen. In addition, similarities between areas A, RB, and LB suggest that samples taken from any one of these areas will be representative of caudal rumen development. As stated, previous research has commonly obtained rumen tissue samples from the caudal dorsal and/or cranial ventral sacs of the rumen, which correspond to areas RB, LB, RD, and LD, respectively, indicating results from previous research to be sufficiently representative of rumen development (Anderson et al., 1982; Nocek et al., 1984; Tamate et al., 1962). Representation of area E (caudal ventral blind sac) is limited in previous research. Lack of representation of area E is not surprising, as it appears that this area is less 66

87 developed in the young ruminant calf. However, inclusion of area E, even if less developed, may aid in distinguishing treatment effects on development of the entire rumen. Rumen Variable Relationships The relationship among and explained variation of the rumen variables is greatest for PL, followed by PW, then RWT, with PC having little to no relationship with the other variables. In addition, the regression of PL, PW, and RWT on PC indicated a slight negative relationship. This suggests that as PC increases, values for PL, PW, and RWT decrease, a trait reported in previous research (Anderson et al., 1982; Klein et al., 1987; Zitnan et al., 1999). This relationship is not surprising as increased values for PL, PW, and RWT are indicative of increased rumen and papillary growth and development. An increase in the size of papillae in a fixed 1-cm 2 section of the rumen wall must result in a decrease in the number of papillae in that finite area. In addition, an increased rumen volume, possibly represented by an increased RWT, enlarges the area covered by 1-cm 2 at birth over a larger area at a later age, thereby decreasing the number of papillae in the finite section once again. Comparison and Power Analyses Papillae Length Values for PL indicate a greater occurrence of treatment and area differences and high variability within PL. The high variation and differences may explain the 67

88 increased power of the procedure to detect treatment differences in PL, as presented in Table 3-3. Therefore, PL may be the most important variable for rumen development research, and may represent the greatest influence of treatment on rumen development. However, high PL variability may also increase the number of areas that require sampling to sufficiently represent development of the entire rumen. Previous research has detected treatment differences for PL when weaning date or the dry portion of the ration was chemically or physically altered (Greenwood et al., 1997; Nocek et al., 1984; Zitnan et al., 1999). These previous findings are expected as rumen development is greatly influenced by age and the presence of butyrate from microbial and protozoal degradation of readily fermentable carbohydrate sources in the rumen (Brownlee, 1956; Klein et al., 1987; Warner et al., 1956). Other research has not detected treatment differences in PL, but has detected age differences (Anderson et al., 1982; Klein et al., 1987). Papillae Width Values for PW indicate a lower occurrence of treatment and area differences and decreased variability within PW than PL, but higher than RWT and PC. These values possibly explain why the power of PW falls between the power for PL and RWT (Table 3-3). However, the power of the procedure to detect treatment differences for PW is also very high. Therefore, these results suggest that PW is likely a secondarily important rumen variable for rumen development research. Detection of differences in PW has been limited in previous research, and weaning age appears to have a greater affect than chemical or physical alteration of the dry ration (Greenwood et al., 1997; Klein et al., 1987; Zitnan et al., 1999). 68

89 Rumen Wall Thickness It appears that RWT is tertiary in importance as a rumen development variable. Statistical treatment differences can be obtained with the procedure, but at a decreased ability than indicated for PL and PW, possibly due to insufficient observations per area or the inability of treatment differences to influence this variable to the same extent as PL and PW. Previous research has not detected significant differences between treatments for rumen wall and/or epithelial thickness when measurements were obtained in areas similar to those described for this procedure (Anderson et al., 1982; Greenwood et al., 1997). In addition, it has been suggested that an increase in rumen muscularization may occur independently of rumen epithelial growth (Brownlee, 1956; Harrison et al., 1960). However, as no attempts were made to separate the rumen muscle from the rumen epithelium in this procedure, the results in these analyses may be confounded. Papillae per cm 2 Results for PC indicate a low power, or possible inability, of the procedure to detect treatment differences in PC, suggesting inapplicability of PC as a rumen development variable. The inability of the procedure to detect treatment differences is likely a result of high σ values and low δ values. It is also possible that the described procedure is incapable of detecting treatment differences for PC due to sampling error. In addition, the possibility of high genetic control over PC, subsequently limiting environmental influence, should also be considered. Age of the calf, age at weaning, and subsequent length of time that concentrates make up an appreciable portion of the daily ration are likely the environmental factors having the greatest influence on 69

90 detectable differences in PC and therefore have the highest possibility of confounding results (Klein et al., 1987; Zitnan et al, 1998; Zitnan et al., 1999). Furthermore, age of the calf and related rumen volume has a tremendous effect on the finite area of the rumen sampled, as described in the rumen variable relationships section. However, some researchers have reported significant differences between treatments for this variable (Anderson et al., 1982; Nocek et al., 1984). Calf Numbers, Samples, and Measurements It is apparent from the results that the areas sampled, the number of tissue samples taken, and the number of measurements per area can be reduced from that originally suggested by this procedure. Papillae length is the only variable appearing to require sampling from the right and left side of the rumen. However, a high power of the test for PL may overcome the need for right and left side representation. In addition, the occurrence of only one right and left side difference for PW and RWT, and no differences for PC, coupled with relatively high correlations between right and left rumen areas, indicates a limited need for sampling both the right and left side of the rumen. Therefore, the rumen dissection procedure reported by McGavin and Morrill (1976a) may be effective for dissecting and sampling the reticulo-rumen. However, the visible guides utilized for dissection may not be readily apparent in the young calf, increasing the difficulty of the McGavin and Morrill (1976a) dissecting procedure. Due to the results indicated, it is recommended that tissue samples be obtained from the cranial and caudal sacs of the ventral and dorsal rumen (area n = 4), three random tissue samples collected from each area (sample n = 12), and two measurements per sample for PL, PW, and RWT recorded (measurement n = 24). For purposes of representing 70

91 physiological growth throughout the entire rumen, multiple sampling sites are suggested. However, for statistical purposes, samples collected from a specific rumen area may also be acceptable, provided that rumen sampling site is identical across calves and treatments. Twenty four measurements per calf reduces the originally suggested sample numbers by 87% for PL and PW and by 47% for RWT, and should result in statistically acceptable power for PL and PW, but may be limited in statistical power for RWT. Therefore, more RWT measurements per calf may be desirable, but will require additional tissue samples. However, the possible inapplicability of RWT may not warrant additional time spent on this variable, and PL or PW may sufficiently address rumen epithelial changes. It is not suggested to record PC measurements due to the high numbers required to find statistical differences for this variable. The required calves per treatment to find differences range from 3 to 18; however, these values result in detectable treatment difference with a possibly unacceptable power. In addition, it is not suggested to use less than three calves per treatment to avoid outlier influence and maintain degrees of freedom within treatment. It appears that three calves per treatment should result in an analysis of sufficient power for PL and PW, valid indicators of rumen development, and minimally limited power for RWT. CONCLUSION This rumen sampling procedure appears to have sufficient ability to detect treatment differences for PL and PW, with reduced, but possibly acceptable, ability for RWT. In contrast, there appears to be limited to no ability for detecting differences in PC, possibly due to inadequate observations, high variability, or a decreased treatment influence due to high genetic influence. In addition, the degree of variability for PL and 71

92 PW appears to be relatively high, even with a dataset of this size. Conversely, variability for RWT and PC is lower when compared to PL and PW, and may indicate decreased ability to influence the former parameters. It is hoped that the procedure and information presented in this paper will provide a means to increase the comparability of future rumen development research, and aid in the understanding of treatment effects on rumen development in young dairy calves. 72

93 REFERENCES Anderson, M. J., M. Khoyloo, and J. L. Walters Effect of feeding whole cottonseed on intake, body weight, and reticulorumen development of young Holstein calves. J. Dairy Sci. 65: Brownlee, A The development of rumen papillae in cattle fed on different diets. Brit. Vet. J. 112: Dawson, J. D., and S. W. Lagakos Size and power of two-sample tests of repeated measures data. Biometrics. 49: Flatt, W. P., R. G. Warner, and J. K. Loosli Influence of purified materials on the development of the ruminant stomach. J. Dairy Sci. 41: Greenwood, R. H., J. L. Morrill, E. C. Titgemeyer, and G. A. Kennedy A new method of measuring diet abrasion and its effect on the development of the forestomach. J. Dairy Sci. 80: Harrison, H. N., R. G. Warner, E. G. Sander, and J. K Loosli Changes in the tissue and volume of the stomachs of calves following removal of dry feed or consumption of inert bulk. J. Dairy Sci. 43: Klein, R. D., R. L. Kincaid, A. S. Hodgson, J. H. Harrison, J. K. Hillers, and J. D. Cronrath Dietary fiber and early weaning on growth and rumen development of calves. J. Dairy Sci. 70: Latour, K. R %Power: A simple macro for power and sample size calculations. Accessed May 20, McGavin, M. D., and J. L. Morrill. 1976a. Dissection technique for examination of the bovine ruminoreticulum. J. Anim. Sci. 42: McGavin, M. D., and J. L. Morrill. 1976b. Scanning electron microscopy of ruminal papillae in calves fed various amount and forms of roughage. Am. J. Vet. Res. 37: Nocek, J. E., C. W. Heald, and C. E. Polan Influence of ration physical form and nitrogen availability on ruminal morphology of growing bull calves. J. Dairy Sci. 67: SAS SAS/STAT User s Guide (Version 8.01 Edition). SAS Inst. Inc., Cary, NC. Sutton, J. D., A. D. McGilliard, and N. L. Jacobson Functional development of rumen mucosa. I. Absorptive ability. J. Dairy Sci. 46:

94 Tamate, H., A. D. McGilliard, N. L. Jacobson, and R. Getty Effect of various dietaries on the anatomical development of the stomach in the calf. J. Dairy Sci. 45: Van Soest, P. J Nutritional Ecology of the Ruminant. 2 nd Ed. Cornell University Press, Ithaca, NY. Warner, R. G., W. P. Flatt, and J. K. Loosli Dietary factors influencing the development of the ruminant stomach. J. Agric. Food Chem. 4: Zitnan, R., J. Voigt, U. Schonhusen, J. Wegner, M. Kokardova, H. Hagemeister, M. Levkut, S. Kuhla, and A. Sommer Influence of dietary concentrate to forage ratio on the development of rumen mucosa in calves. Arch. Anim. Nutr. 51: Zitnan, R., J. Voigt, J. Wegner, G. Breves, B. Schroder, C. Winckler, M. Levkut, M. Kokardova, U. Schonhusen, S. Kuhla, H. Hagemeister, and A. Sommer Morphological and functional development of the rumen in the calf: Influence of the time of weaning. Arch. Anim. Nutr. 52:

95 Figure 3-1 RC RB A LB LC RE LE LD RD Reticulum Figure 3-1: Example of a rumen dissected utilizing the described procedure depicting the physical areas of the rumen sampled and corresponding labels. (A) caudal portion of the caudal ventral blind sac; (RB) right side and (LB) left side caudal dorsal sac; (RC) right side and (LC) left side cranial dorsal sac; (RD) right side and (LD) left side cranial ventral sac; and (RE) right side and (LE) left side ventral portion of caudal ventral blind sac. 75