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University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 8913617 Contribution of ciliate protozoa to the rumen fermentation and nutrition of the ruminant Ankrah, Peter, Ph.D. The Ohio State University, 1989 UMI 300 N. Zceb Rd. Ann Arbor, MI 48106 CONTRIBUTION OF CILIATE PROTOZOA TO THE RUMEN FERMENTATION AND NUTRITION OF THE RUMINANT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Peter Ankrah, B.Sc., M.Sc. ««**«*« The Ohio State University 1989 Dissertation Committee: Approved by S. C. Loerch B. A. Dehority W. L. Shockey J. L. Firkins Advisor Department of Animal Science i Dedicated to my father, the late J. 0. Ankrah and my two aons, Anthony and Peter 11 ACKNOWLEDGMENTS I would like to express my sincere appreciation to my advisors, Drs. Steven C. Loerch and B. A. Dehority, for their encouragement, guidance, patience, support and great interest throughout the course of my studies and during preparation of thi3 thesis. I would also like to thank Dr. Jack H. Cline for the part he played in getting me over to the U.S.A. for the Ph.D. program and for his relentless ef fort in getting my family to join me. I thank Drs. W. L. Shockey and J. L. Firkins for serving on my committee and for their useful suggestions and contributions to this thesis. Thanks is extended to Bev Fisher for her expertise in the typing of thi3 thesis. My appreciation is extended to Fay for her assistance in caring for the experimental animals. The laboratory assistance by Peggy, Pat, Diane and Tony is greatly appreciated. Thanks also go to my fellow graduate students in Animal and Dairy Science departments for formal and informal discussions and for their encouragement. My deepest appreciation is extended to my parents, Jonathan 0. Ankrah and Grace K. Simpson for their love, care, support and en couragement throughout my education. Dad, I wish you had lived to see your dream come true. You saw in me what I did not think possible. H i Finally, I am extremely grateful to my wife Juliana and two sons, Anthony and Peter for their love, constant support, patience, understanding and enduring the rough times with me. iv VITA April 22, 1954 . Born - Acora, Ghana. 1978 ............. B.Sc. (Hons.), Agriculture, Animal Science, University of Ghana, Ghana. 1979 -.. 1980...... Teaching Assistant, Department of Animal Sci ence, University of Ghana. 1983 ..... • ...... M.Sc., Agriculture, Animal Science, Universi ty of Sydney, Australia. 1983 - 1984...... Agricultural Projects Officer, Ghana Invest ment Center, Ghana. 1984 - 1985...... Graduate Teaching Assistant, Department of Animal Science, The Ohio State University, Columbus, Ohio. 1986 - 1988...... Graduate Research Associate, Department of Animal Science, The Ohio State University, Wooster, Ohio. Publications Williams, G. E. S. and P. Ankrah. 1979. Evaluation of heat-treated full-fat soybean seeds as protein source for broiler chicks. Proceedings of the Tenth Animal Science Symposium, Ghana Animal Science Association 10:49-51. Ankrah, P., S. C. Loerch and B. A. Dehority. 1987. Occurrence of 2- aminoethylphosphonic acid in feeds, rumen bacteria and duodenal digesta from defaunated sheep. Proceedings of the Nineteenth Biennial Conference on Rumen Function, Chicago, IL., Nov. 17-19, 19i38. (Abstr.). Ankrah, P., S. C. Loerch and B. A. Dehority. 1988. Occurrence of 2- aminoethylphosphonic acid in feeds, rumen bacteria and duodenal digesta from defaunated sheep. J. Anim. Sci. (In press).. v Ankrah, P., S. C. Loerch and B. A. Dehority. 1988. Effects of di etary protein source and defaunation on lamb performance. J. Anim. Sci. 66(Suppl. l):497-498. Ankrah, P., S. C. Loerch, B. A. Dehority and K. A. Kampraan. 1988. Effects of defaunation on lamb performance and ruminal nutrient digestion in steers, tin preparation for submission to J. Anim. Sci.). FIELDS OF STUDY Major Field: Animal Science - Ruminant Nutrition vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ...................................... ill VITA ............................................. v LIST OF TABLES....................... x LIST OF FIGURES....................... xii CHAPTER PAGE I- 1 INTRODUCTION.............................. 1 LITERATURE REVIEW ............................. 3 Rumen Microbial Markers ............... 3 Alkyl-phosphonates and their Derivatives. 4 Physical and Chemical Properties of AEP .... 5 Synthesis and Metabolism of Alkyl-phosphonates. 6 Distribution of AEP in Biological Material. 9 Laboratory Methods for Analyzing AEP..... 10 Defaunation Methods ........................ 12 Effects of Defaunation on: Animal Performance . .................... 15 Nutrient Digestion ...................... 17 Rumen and Blood Metabolites.......... 19 Rumen Bacterial Population........... 22 Escape of Protozoal Protein from the Rumen. 25 LITERATURE CITED........................... 31 II. OCCURRENCE OF 2-AMIN0ETHYLPH0SPH0NIC ACID IN FEEDS, RUMINAL BACTERIA AND DUODENAL DIGESTA FROM DEFAUNATED SHEEP........................... 44 INTRODUCTION............................... 44 EXPERIMENTAL PROCEDURE..................... 45 Apparatus............................. 45 HPLC Procedures ................ ...... 45 Reagents............................... 46 vii Linearity, Precision and 2-Aminoethylphosphonic Acid Recovery............................. 47 Acid Hydrolysis Procedures.................. 48 Organic Phosphorus Analysis ......... .... 48 Isolation of Mixed Ruminal Bacteria and Protozoa................................... 50 Pure Cultures of RumenBacteria .............. 51 In Vitro AEP Fermentation.................. 51 RESULTS AND DISCUSSION......................... 53 LITERATURE CITED............................... 68 III. EFFECTS OF DIETARY PROTEIN SOURCE AND DEFAUNATION ON LAMB PERFORMANCE........................... 72 INTRODUCTION................................... 72 MATERIALS AND METHODS .......................... 74 Experimental Animals andDiets ............... 74 Defaunation and Refaunation Procedures....... 76 Microbial Assays........................... 78 Experimental Design ........................ 78 RESULTS AND DISCUSSION......................... 79 LITERATURE CITED............................... 89 IV. DERIVATION OF A SELECTIVE CONTAINER TO STUDY THE PHENOMENA OF PROTOZOAL SEQUESTRATION, MIGRATION AND LYSIS IN THE RUMEN............................. 93 INTRODUCTION................................... 93 MATERIALS AND METHODS .......................... 94 Study A: Evaluation of the Selective Container Design..................... 97 Study B: Investigation of the Phenomena of Protozoa Sequestration, Migration and Lysis in the Rumen................................. 100 Experiment 1............................... 100 Experiments 2 - 6 ........................... 101 Experimental Design ........................ 101 vlli RESULTS AND DISCUSSION.......................... 101 Experiment 1.......... ................. 101 Experiment 2. ........................... 105 Experiment 3............................... 108 Experiment 4............................... 112 Experiment 5............................... 113 Experiment 6...... ............. .... 119 LITERATURE CITED............................... 124 V. SUMMARY...................................... 127 APPENDICES.................................... 129 A. Data relative to Chapter.4 .............. 130 B. Data relative to Chapter 4.............. 131 C. Data relative to Chapter 4............ 132 BIBLIOGRAPHY................................... 134 ix LIST OF TABLES Table Page 1 Recovery of AEP added to ruminal fluid ............... 56 2 Extent of in vitro fermentation of AEP by mixed ruminal bacteria.......................................... 57 3 Concentration of AEP in pure strains of ruminal bacteria and in mixed bacteria and protozoa isolated from sheep . 61 4 Concentration