UUA U'.CILIZATION STUDIES with RUMI~TS By
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UUA U'.CILIZATION STUDIES WITH RUMI~TS By ANDQW JOSEPH CLIFFORD !\ Bachelor of Science University College Dublin Dublin, Ireland 1962 Master of Science University College Dubli~ · Dublin, Ireland 1963 Sub"1i(ted to the faculty of the Graduate College of · the Oklahoma State Univer~ity in partial fulfillment of the requirements for the degree of· DOCTOR OF PHILOSOPHY July, 1967 OKLAHOMA STATE UNIVERSITY LIBRARY ,' JAN 9 l968 ,. ~ UREA UTILIZATION STUDIES WITH RUMINANTS .~. ,··-·~ . -•,. --.... Thesis Approved: ~/ - .d~Th" Adviser Dean of the Graduate School 858411 ii ACKNOWLEDGEMENTS The author expresses his appreciation to Dr. A. D. Tillman, Pro fessor of Animd Science, during the course of this study and in the preparation of this thesis. Appreciation is also extended to Dr. I. T. Omtvedt, Associate Pro fessor of Animal Science, Dr. L. J. Bush, Associate Professor of Dairy Science, Dr. D. D. Goetsch, Professor of Physiology and Pharmacology, and Dr. E. c. Nelson, Assistant Professor of Biochemistry, for their suggestions in the preparation of this thesis. Further acknowledgement is due to Dr. E. I. Williams, Assistant Professor of Veterinary Medicine and Surgery; Mrs. Jane Witte, Labora tory Techni:chn, and John R. Bourdette, Undergraduate Assistant, for their assistance during this study. Special recognition is extended to the wife and family of Dr. Allen D. Till~an for the many courtesies durin~ the author's stay in O~lahoma and to Carolyn K. Harman, Instructor, Poultry Science Depart ment, who assisted in preparing and typing this thesis. iii TABLE OF CONTENTS Chapter Page I. INTRODUCTION. • . 1 II. LITERATURE REVIEW . 2 Properties of Urease. • • • • • • • • • • • 2 Fermentation of Amino Acids ••••••••••• • • 4 Ammonia Utilization. • • • • • ••• . 5 Adaptation to Urea • • • • ••• 9 Control of Urease Activity. • • • . 10 Ill. UREA AND ISOLATED SOY PROTEIN IN SHEEP PURIFIED DIETS. 13 Introduction •••• . 13 Experimental Procedure. .. 13 Results and Discussion. ~ . 16 Summary ••• . 25 IV. AMINO ACID SUPPLEMENTATION OF UREA RICH DIETS FOR LAMBS. 26 Introduction • • • • · . 26 Experimental Procedure. 26 Results and Discussion 28 Summary. • • 34 v. STUDIES ON RUMINAL UREASE ACTIVITY. 36 Introduction. • • . 36 Experimental Procedure. 36 Results and Discussion. • • 39 Summary. • • • •• . 47 VI. EFFECTS OF UREASE INHIBITORS ON SHEEi> FED UREA RICH DIETS 48 Introduction •••••••••• • • • . 48 Experimental Procedure. 48 Trial 1 • • • . 48 Trial 2. 50 Trial 3 • • • • ••• . 50 Trial 4. • • . • • . '. 51 Trial 5. • • • • • . 51 Trial 6 • • • • • • • • • • • • • • . 52 Chemical and Statistical Analyses •••• . 52 iv Chapter Pj;ige VI. CONTINUED • • 53 Results and Discussion. • 53 Sununary. 72 Vil. GENERAL DISCUSSION. 74 Vlll. GENERAL SUMMAR~ • • • 77 LllERA?URE C~TED. ., • • 79 V LIST OF TABLES Table Page :r. Percentage Composition of Diets. • • • • . 14 II. Effects of Urea, Soy Protein and Various Combinations of UJ,"ea .. soy Upon Daily Gains and CaJ;'cass Measurements • • • • • • • • • • • • • • • • • • • • 17 III. Effect of Urea or Soy Protein Alone and Urea-Soy Combinations on Fecal and Urinary Excretions and on Nitrogen Ba 1 ance. • • • • • • • . • • • . 18 IV. Effect of Time Upon Percentage of Nitrogen Retained. • 19 V. Effects of. Urea, Soy Protein and Various Combinations of Urea-Soy on Ruminal Fluid PH and Fatty Acid Concentrations. • • • • • • • • • • • • • • • • • • 20 VI. Effect of Urea, Soy Protein, and Various Urea-Soy Combinations on Ruminal Fluid Fatty Acid Concentrations •••••• . 21 VII. Effect of Urea, Soy Protein, and Vario~s Urea-Soy Combinations on Nitrogen Fractions in Rumen Fluid. • 23 VIII. Effect of Urea~ Soy on Free Plasma Amino Acids Concentrations. • • • • • • • • • • • • •• . 24 Percentage Composition of the Diets •• . 27 x. Effects of Amino Acid Supplementation of Urea Containing Purified Diets on the Growth Performance of Lambs • • , • • . • • • • • • • • • • • 29 XI. Effects of Amino Acid Supplementation of Urea Containing Purified Diets on Nitrogen Fractions ;30 In Blood • • • • • • • • • • • • • • • • • • • XII. Effects of Amino Acid Supplementation of Urea Containing Purified Diets on Plasma Volatile Fat:ty Acid Concentrations •••••••••• • • • • 31 XIII. Effects of Amino Acid Supplementation of Urea Containing Purified Diets on Nitrogen Components and PH of Rumen Fluid, ••••••••••••• . 32 vi Table Page XIV. Effects of Amino Acid Supplementation of Urea Containing Purified Diets on Ruminal Volatile Fatty Acid Concentrations. • • • • • • • • • • • • • 33 XV. Percentage Composition of the Diets. • • • • • • • • • 38 XVI. Effect of Fasting!! Feeding of Mixed Diet on Rumen Urease Activity •••••••• • • • • • • • • 39 XVII. Effect of Fasting~ Feeding on Ruminal Volatile Fatty Acid Concentrations. • • • • • • • • • • • • • 41 XVIII. Effect of Mixed!! Cottonseed Hulls Diets on Ruminal Urease Activity •••••••••• . 42 XIX. Effect of Mixed!! Cottonseed Hulls Diets on Rumen Volatile Fatty Acids •••••••• • • • • • 43 XX. Effect of Alfalfa!! Prairie Hay Diets on Ruminal Urease Activity •• . • • • . • • 44 XXI, Effect of Alfalfa!! Prairie Hay on Rumen Volatile Fatty Acid Concentration •••••••••••• • • 45 XXII. Effect of Mixed and Cottonseed Hulls Diets, and Fasting on Rumen PH. .. • • • • • 46 XXIII. Percentage Composition of Control Diets Used in Each Trial •••••••••••••••••• • • • 49 ·xxiv. Effect of Urease Inhibition on Sheep Performance on Urea-Containing All-Concentrate Diets (Trials l, 2, and 3) ••••••••••••• • • • 54 xxv. Effect of Barbituric Acid or Nitrates on the Performance of Sheep Fed High Roughage Diets (Trial 4) ••••••••••••••••••• • • • 56 XXVI. Effects of Dietary Copper, Barbituric Acid, and Nitrates on Ruminal PH, Urease, Amylase Activities, . Nitrogen Balance and Cellulose Digestibility (Trial 5)~ • • • • • • • • • • • • • • • • • • • • • 57 XXVII. Effect of Days on Feed and Urease Inhibitors on Ruminal Urease Activity ••••• , •••••• • • • 60 XXVlll. Effects of Days on Feed and Time After Feeding on Ruminal Urease Activity ••••••••••••• . 61 XXIX •. Main Effects of Urease Inhibitors on Ruminal .PH •• 62 vii Table Page XXX. Ji;ffects qf Days on Feed and Time After Feeding on Ruminal PH • • • • • • • • • • • . ' . 63 XXXI. Effects of Days on Feed and Urease Inhibition on Ruminal Urea-Nitrogen Levels • • • • • • • • • • • • 64 XXXII. Effects of Time After Feeding on Ruminal Urea- Nitrogen Concentrations. • • • • • • • • • • • • 65 XXXIII. Main Effects of Urease Inhibitors on Ruminal Anunonia-Nitrogen Concentrations •••••• . • • 66 XXXIV. Effects of Days on Feed and Time After Feeding on Ruminal Anunonia-Nitrogen Levels. • • • • • • • • • • 67 XXXV, Main Effects of Urease Inhibitors on Ruminal Nitrate- Nitrogen Concentrations. • • • • • • • • • • • • • • 68 XXXVI. Effects of Days on Feed and Time After Feeding on Ruminal Nitrate-Nitrogen Levels. • • • • • • • • • • 69 XXXVII. Main Effects of Urease Inhibitors on Total-Nitrogen Concentrations in Rumen Fluid. • • • • • • • • • • • 70 XXXVUI. Effects of Urease Inhibitors and Time After Feeding on Total Nitrogen Concentrations in Rumen Fluid. • • 71 viii CHAPTER l INTRODUCTION· 'It is well established that at least one-third of the preformed · protein in ruminant diets can be replaced by non-protein nitrogen such as urea.'; lndeed, many investigators h.;ive used urea as the sole source of dietary nitrogen and obtained ver-:, promising growth performance and milk production. However, when urea served as the sole source of nitrogen growth performance and milk production were approximately 70 percent as great as when preformed proteins were fed. Possible explanations for this reduced performance concern (a) the inability of rumen microbes to synthesize adequate quantities of branched-chain fatty acids for maximum microbial protein synthesis when urea is fed, and .(b) the rapidity with which urea is hydrolyzed to ammoJ;lia in the rumen relative to the rate of microbial incorporation of ammonia into protein. Since urea is destined to play a major role in $Upplying dietary nitrogen for ruminant animals, it is important to learn if diets containing urea as the sole source of . nitrogen could promote g;owth and milk production eq1,1al to, or greater than, that obtained with preformed protein. This report describes .attempts to investigate the posijibilities (a) t,:hatbranched-chain fatty acids are deficient when urea-containing diets are fed, and (b) of re ducing the rate.of urea q.ydrolysis to a level commensurate with microb ial ability to incorporate the ammonia released into protein. l CHAPTER II LITERATURE REVIEW Properties of Urease Urea amidohydrolase (urease) which catalyses the hydrolyds of urea to ammonia and co2 was isolated from the jackbean by Sumner (1926) and has since been found to occur in bacteria, yeast, fungi and many highet' plants (Sumner and SolUers, 1953). Urease, a 19.6 S globulin (Sumner and Somers, 1953; Gorin an,d Chin, 1967); has a molecular weight of 483,000 (Sumner!! al., 1938); an isoelectric point at pH 5.0 (Hand, 1939); and optimum pl{ of 7.0 (Sumner and Somers, 1953). The urease molecule call be dissociated into 6 subunits of 83,000 molecular weight which are held together by covalent bonds (Creeth and Nichol, 1960; Reithd il !!.•, 1964). Hand (1939) reported the dissociation of urease into 16 subunits of 17;000 molecular weight which may have ureolytic activity. Gorin and Chin (1967) dissociated urease into two biologically active subunits using O.lM phosphate buffer at pH 3.5. Each subunit was a.9.8 S protein and