MSU LIBRARIES ——. .7 RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES wiII be charged if book is returned after the date stamped below. Mode of Action of High Dietary Copper in Promoting Growth of Neanling Pigs. By Gerald Carlyle Shurson A Dissertation Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Animal Science 1986 ‘188—339/7 ABSTRACT Mode of Action of High Dietary Copper in Promoting Growth of Neanling Pigs. By Gerald Carlyle Shurson The mechanisms by which copper improves growth performance of growing swine have not been elucidated. Copper has antimicrobial properties and way affect the gut microflora to improve growth performance. The primary objective of the first experiment was to determine if feeding a diet containing 250 pun supplemental copper improves growth performance of germ-free pigs, as typically observed for conventional pigs. A second experiment was conducted to determine the effects of 250 ppm copper and 100 ppm chlortetracycline on microbial growth and fermentation patterns of gastrointestinal microflora of weanling pigs in vitro. In the first experiment, l0 germ-free and lO conventional weanling pi gs were fed one of two experimental diets containing either 16 ppm or 283 ppm copper fOr a three-week trial. Several criteria were measured including: growth performance, hematology, plasma and liver copper, zinc and iron, and plasma ceruloplasmin. In the second experiment, microbes present in gut contents and gut wall from five sections of the gastrointestinal tracts of 4 weanling pigs were used to inoculate culture tubes of anaerobic growth media containing one of four treatments (control, 250 ppm copper, 100 ppm chlortetracycline, 250 ppm copper + 100 ppm chlortetracycline). Culture tubes were incubated at 38'C for 24 hours and microbial growth, media pH. ammonia-nitrogen and organic acid production were determined. Results from the first experiment indicated that feeding the high copper diet improved growth rate of conventional pigs and reduced growth rate of germ-free pigs. However. this difference in growth rate was confounded by an apparent development of a moderate copper toxicity in germ-free pigs receiving the high copper diet. Germ-free pigs fed a high copper diet had a higher concentration of copper in liver and plasma and a lower concentration of liver iron compared to conventional pigs receiving the same diet, and lower hemoglobin, hematocrit and erythrocyte number compared to germ-free pigs fed the control diet. Microbial growth, ammonia-nitrogen and organic acid production were dramatically reduced in vitro when 250 ppm copper was present in the media compared to control and chlortetracycline treated microbes. However, the reduction in microbial growth confounded differences between treatments for ammonia-nitrogen and organic acid production. ACKNOWLEDGEMENTS In completion of my Ph.D. Degree. I am deeply grateful and fbrever indebted to my wife Laurie, who has made countless sacrifices and has given me a never ending source of encouragement, understanding. inspiration and love during the pursuit of this degree. I am also very proud and extremely grateful for our son Andrew, who without his knowing, has enriched our lives and has given so many hours of love and enjoyment even during the most trying times. I dedicate this dissertation to Laurie and Andrew. I am very honored to have had the opportunity to pursue my education at Michigan State University and to have been associated with many fine and outstanding pe0ple who have made these years at MSU a very enjoyable experience. I thank Dr. E.R. Miller for his interest in my education. advice and friendship. working with Dr. Miller has been an honor that I will forever cherish. Likewise, I thank Dr. M.G. Hogberg, Dr. D.R. Romsos, Dr. D.E. Ullrey and Dr. M.T. Yokoyama for their guidance and encouragement during the pursuit of this degree. I am very fbrtunate to have been guided by such a distinguished group. I also thank Dr. G.E. Haxler, Dr. Sheila Grimes and Mr. John Allen for the many hours of assistance they contributed in conducting the germ-free experiment contained in this dissertation. I especially thank Dr. P.K. Ku for his untiring assistance and advice in the laboratory and Ms. Nora Fisher and Mr. Dan Quackenbush for their assistance in conducting this research project. I am very grateful to have been associated with many of my fellow graduate students and thank them for their friendship, support, encouragement and assistance with many activities involved in conducting the experiments contained in this dissertation and other projects. Their friendship will always be remembered and cherished. I thank Ms. Judy Lentz for her friendship and for an exceptional job in typing and preparing this manuscript. Finally, I thank my parents for encouraging the pursuit of my goals and for instilling in me the philosophy that “you are never given a wish without also being given the power to make it true“. ii TABLE OF CONTENTS INTRODUCT I 0" O O O O O O O O O O O O O O 0 REVIEW OF LITERATURE . . . . . . . . . . . ESSENTIALITY OF COPPER. . . . . . II. III. EFFECTS OF IV. A. B. C. D. E. F. G. H. I. Introduction. . . . Copper Absorption and Excretion Bioavailability of Copper from Cop Interaction with Other Nutrients. Intermediary Metabolism of Copper Copper Storage Sites. . . . . . . Copper Requiring Enzymes. . . . . Physiological Functions of Copper l. Iron metabolism . . . . . . . 2. Cross-linking of connective tissue. 3. Neurotransmitters and brain function. 4. Lipid metabolism. . . . . . . . . . . 5. Pigmentation and keratinization "00] O O O O O O O O O O O 6. Intestinal integrity. . 7. Infertility . . . . . . 8. Immunology Copper Requirement of Swine Consequences of Feeding High Copper Growing Swine . . . . . . . . . . . DE 00000-30000 of hai 131 om0.0.0 . O . O m. 0 0 O O O . O o O o O c 0 g 0 o 0 m0 0 0 0 GROWTH PROMOTING EFFECTS OF HIGH COPPER DIETS A. B. C. 0.0.om0.0000000m000. O .fl.....1.........n.... FED 3 O o O o O Q 0 o O a. . O . 0.0.0Q000000000m000. TO SHINE Introduction. . . . . . . . . . . . . . . . . . . . . Effect of High Capper Feeding on Growth Performance . Proposed Mechanisms of Growth Promotion when High Copper Diets Are Fed to Growing Swine . . . . . . . . l. Bacteriostatic effects of copper. . . . . . . . . 2. Effects of high capper feeding on digestive and metabolic efficiency. . . . . . . . . . . . . a. Relationship of high copper and dietary protein source and level on protein digestion and utilization . . . . . . . . . . . . . . . b. Effect of high copper feeding on lipid mtabOI ism. O C O O O O O O O O O O O O O O O c. Mineral interrelationships relative to the effectiveness of high copper feeding. . . d. Other proposed mechanisms of growth promotion when high capper diets are fed. . . FEEDING HIGH LEVELS OF COPPER IN COMBINATION HITH ANTIBIOTICS ON GROWTH PERFORMANCE. . . . . . . . . . ANTIBIOTICS IN LIVESTOCK FEEDS AND THEIR MODE OF ACTION Metabolic Effect. . . . . . . . . . . . . . . . . . Nutrient Sparing Effect . . . . . . . . . . . . . . Disease-Control Effect. . . . . . . . . . . . . . . A. B. C. iii v D! O o 0 . 0000(1) (DNNNOSO‘OSUlm h-fih-fi . ._a 0 .S1. MN .14 MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF THE GERM’FREEANIMAL.0.0000000000000000... A. Survival, Germ-Free and Conventional Animal . . . . . . . . . . 1. 019 («ON 6. Organ weights of germ-free pigs . . . . . . . . . Systemic characteristics of the germ-free animal. Characteristics of the gastrointestinal tract of germ-free animals . . . . . . . . . . . . . . . . Digestive functions . . . . . . . . . . . . . . . Effect of germrfree environment on humoral and cellular defense mechanisms . . . . . . . . . . . Importance of the gut microflora to growth. . . . MATERIALS AND METHODS I. II. Growth and Reproduction of Germ-Free Animals. B. Differences in Organs. Tissues and Systems Between the EFFECT OF HIGH-COPPER FEEDING TO HEANLING PIGS IN GERM-FREE AND CONVENTIONAL ENVIRONMENTS ON GROWTH PERFORMANCE AND VARIOUS PHYSIOLOGICAL PARAMETERS. . . . . . . . . . . . A. Management of Germ-Free Pigs. . . . . . . . . . . . 8. Management of Conventional Pigs . . . . . . . . . . C. Collection of Data and Laboratory Analyses. . . . . l. 2. Determination of growth performance criteria. Collection and preparation of blood samples for laboratory analyses . . . . . . . . . . . . . . a. Hematological determinations. . . . . . . . b. Plasma trace element determinations c. Plasma ceruloplasmin determinations d. Plasma ammonia determinations . . . e. Plasma glucose determinations . . . f. Plasma amino acid determinations. . . Preparation of intestinal sections for histologia determinations. . . . . . . . . . . . . . . . . a. Intestinal histological determinations. . . . Body organ weights and preparation of organs and cecal contents for laboratory analyses. . . . . . a. Determination of ammonia and urea in cecal contents. . . . . . . . . . . . . . . . . . . C3 INFLUENCE OF COPPER AND CHLORTETRACYCLINE ON MICROBIAL FERMENTATION PATTERNS AND MICROBIAL POPULATION GROWTH IN FIVE SECTIONS OF THE GASTROINTESTINAL TRACT IN VITRO. . A. Preparation of Culture Tubes. . . . . . . . . . B. Preparation of Media. . . . . . . . . . . . I. 2. C. Inoculation of Culture Media. . . . . . . . . D. Data Collection and Laboratory Analyses . . . . RUN-P Anaerobic dilution media. . . . . . . . . . . Non-selective anaerobic growth media. . . . . Determination of microbial population growth. Determination of pH . . . . . . . . . . . . . Determination of ammonia nitrogen . . . . . . . . Determination of fatty acid butyl esters. . . . . iv Page RESULTS AND DISCUSSION 1. II. SUMMARY. APPENDIX 1. II. EFFECT OF HIGH COPPER FEEDING TO WEANLING PIGS IN GERM-FREE AND CONVENTIONAL ENVIRONMENTS ON GROWTH PERFORMANCE AND VARIOUS PHYSIOLOGICAL PARAMETERS. . . . . . . . . . . . . . 68 A. Growth Performance. . . . . . . . . . . . . . . . . . . 68 B. Hematology. . . . . . . . . . . . . . . . . . . . . . . 70 C. Plasma Copper, Zinc and Iron Concentrations . . . . . . 74 D. Plasma Ceruloplasmin Activity . . . . . . . . . . . . . 78 E. Liver Copper, Zinc and Iron Concentrations. . . . . . . 78 F. Plasma Ammonia Concentrations . . . . . . . . . . . . . 79 G. Concentration of Ammonia and Urea in Cecal Contents . . 80 H. Plasma Glucose Concentrations . . . . . . . . . . . . . 82 I. Plasma Amino Acid Concentrations. . . . . . . . . . . . 82 J. Organ Heights . . . . . . . . . . . . . . . . . . 85 K. IntestinalMorphology.................89 INFLUENCE OF COPPER AND CHLORTETRACYCLINE ON MICROBIAL FERMENTATION PATTERNS AND MICROBIAL GROWTH IN FIVE SECTIONS OF THE GASTROINTESTINAL TRACT IN VITRO. . . . . . . . . . . 94 A. Microbial Growth. . . . . . . . . . . . . . . . . . . . 94 B. Fermentation Changes in pH of Growth Media. . . . . . .104 C. Net Ammonia Nitrogen Changes. . . . . . . . . . . . . .105 D. Volatile and Non-Volatile Fatty Acid Production . . . .108 l. Comparison of the relative quantities of volatile and non-volatile fatty acids produced in the gastrointestinal tract. . . . . . . . . . . . . . .108 a. Volatile fatty acids. . . . . . . . . . . . . .108 b. Non-volatile fatty acids. . . . . . . . . . . .113 2. Total volatile fatty acid production. . . . . . . .117 3. Total non-volatile fatty acid production. . . . . .120 . . ............... . . . . . . . . . . . . .125 A C O O O O O O O O O O O O O O O O O O O O O I O O O I O .129 EFFECTIVENESS OF STERILIZATION OF CORN-SOYBEAN MEAL-DRIED WHEY STARTER DIETS BY AUTOCLAVING AND THE EFFECT OF AUTO- CLAVING DIETS, WITH OR WITHOUT VITAMIN SUPPLEMENTATION, ON GROWTH PERFORMANCE. . . . . . . . . . . . . . . . . . . . .129 A. Objectives. . . . . . . . . . . . . . . . . . . . . . .l3O B. Methodology . . . . . . . . . . . . . . . . . . . . . .130 C. Results . . . . . . . . . . . . . . . . . . . . . . . .133 l. Growthperformance.................l33 2. Effect of sterilization method and time on di t Steri‘lization O O O O O O O O 0 O O O O O O O O O .135 EFFECTIVENESS OF LYSINE AND METHIONINE SUPPLEMENTATION TO DIETS PRIOR TO AUTOCLAVING ON GROWTH PERFORMANCE. . . . . .136 A. Objective . . . . . . . . . . . . . . . . . . . . . . .136 B. Methodology . . . . . . . . . . . . . . . . . . . . . .136 C. Results . . . . . . . . . ..... . ....... . .137 Page 111.. BIOAVAILABILITY OF LYSINE IN AUTOCLAVED CORN-SOYBEAN MEAL- DRIED WHEY STARTER DIETS. . . . . . . . . . . . . . . . . .141 A. Objective . . . . . . . . . . . . . . . . . . . . . . .141 8. Methodology . . . . . . . . . . . . . . . . . . . . . .142 C. Results . . . . . . . . . . . . . . . . . . . . . . . .l42 IV.. EFFECTS OF AUTOCLAVING AND SUPPLEMENTATING DIETS WITH 250 PPM COPPER ON ENERGY, NITROGEN AND MINERAL BALANCE OF HEANLING PIGS O O O O O O O O O O O O O O O O O O 0 O O o .145 A. Objective . . . . . . . . . . . . . . . . . . . . . . .145 8. Methodology . . . . . . . . . . . . . . . . . . . . . .146 C. Results . . . . . . . . . . . ..... . . . . . . . .148 1. Energy balance. . . ..... . . . . . . . . . . .143 2. Nitrogen balance. . . . . . . . . . . . . . . . . .151 3. Mineral balance . . . . . . . . . . . . . . . . . .153 a. Calcium and phosphorus. . . . . . . . . . . . .153 b. Copper.....................153 c. Zinc. . . . . . . . . . . . . . . . . . . . . .156 d. Iron. . . . . . . . . . . . . . . . . . . . . .156 V. EFFECTS OF FEEDING A CORN-SOYBEAN MEAL STARTER DIET CONTAINING 250 PPM SUPPLEMENTAL COPPER ON ENERGY, NITROGEN AND MINERAL BALANCE . . . . . . . . . . . . . . . . . . . .156 A. Objective . . . . . . . . . . . . . . . . . . . . . . .156 8. Methodology . . . . ..... . . . . ....... . .157 C. Results . . . . . . . ...... . . . . ..... . .157 1. Energy balance. . . . . . . . . . . . . . . . . .157 2. Nitrogen balance. . . . . . . . . . . . . . . . . .l57 3. Mineral balance . . . . . . . . . . . . . . . . . .161 a. Copper.....................161 b. Zinc. . . . . . . . . . . . . . . . . . . . .161 . . . ........... . .161 c. Iron. . . . . . APPENDIX B I. PREPARATION OF REARING ISOLATORS FOR GERM-FREE RESEARCH Description of Rearing Isolators. . . . . Initial Cleaning of Isolators . . . . . . Installation of Gloves and Aluminum Rings Testing for Leaks in the Plastic Isolator Final Cleaning of Isolators . . . . . . . Preparation of Air Filters . . . . . . . Preparation of Sterilizing Cylinder . . . . Sterilization of Equipment for Use in Isolators . Final Preparation and Sterilization of the Isolators. .169 Transfer of Materials from Cylinder to Isolators. . . .171 0000000.. —l. at .h Goo—OIED‘HMUOW) C II. PREPARATION OF TRANSFER AND SURGICAL ISOLATORS FOR GERM- FREE RESEARCH . . . . . . . . . . . . . . . . . . . . . . .173 A. Description of Isolators. . . . . . . . . . . . . . . .173 1. Transfer isolator . . . . . . . . . . . . . . . . .173 2. Surgical isolator . . . . . . . . . . ..... . .173 vi Page B. Cleaning and Preparation of Isolators for Sterilization 174 1. Transfer isolator. . . . . . . . . . . . . . . . . .174 2. Surgical isolator. . . . . . . . . . . . . . . . . .174 C. Sterilization of Isolators and Preparation for Surgery .174 III. SURGICAL TECHNIQUE FOR OBTAINING GNOTOBIOTIC PIGS. . . . . .175 IV. PREPARATION AND TRANSFER OF MILK, WATER AND EXPERIMENTAL DIETS INTO REARING ISOLATORS . . . . . . . . . . . . . . . .177 A. Preparation and Transfer of Milk . . . . . . . . . . . .177 B. Preparation and Transfer of Water. . . . . . . . . . . .178 C. Preparation and Transfer of Experimental Diets . . . . .178 V. MONITORING ISOLATORS FOR GERM-FREE STATUS. . . . . . . . . .179 LIST OF REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . .181 vii TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE 10. 11. 12. 13. 14. 15. 16. LIST OF TABLES Nutrient Analysis of Evaporated Cow's Milk and Comparison to Nutrient Analysis of Sow's Milk. . . Diet Composition and Calculated Nutrient Analysis (Experiment])ooo00000000000000. Composition of Mineral Buffer Solutions Added to Dilution Media and Non-Selective Growth Media (g/l diStiIIed H20) 0 O O O O O O 0 0 O O O O O 0 O O 0 Composition of Anaerobic Dilution Media. . . . . . Composition of Non-Selective Anaerobic Growth Mdia. O O O O O O O O O O O O O O O I O O O O O 0 Diet Composition and Calculated Nutrient Analysis (Experiment 2) O O O I O O I O O O O O O 0 O O O 0 Effect of Diet and Environment on Growth Performance. I O O O O I O O O O O O O O I O I I O 0 Effect of Diet and Environment on Hematology . . . Effect of Diet and Environment on Plasma Capper, 2i "C and Iron. 0 O O O O O O O O O O O O O O O O 0 Effect of Diet and Environment on Portal and Peripheral Blood Plasma Ceruloplasmin. . . . . . . Effect of Diet and Environment on Liver Capper, Zinc and Iron (DM basis) . . . . . . . . . . . . . Effect of Diet and Environment on Portal and Peripheral Plasma Ammonia Concentration. . . . . . Effect of Diet and Environment on Urea and Ammonia-Nitrogen Concentrations in Cecal Contents (dry matter basis). . . . . . . . . . . . Effect of Diet and Environment on Portal and Peripheral Plasma Glucose. . . . . . . . . . . . . Effect of Diet and Environment on Peripheral Plasma Amino Acid (PPAA) Concentrations (uM) . . . Effect of Diet and Environment on Portal Plasma Amino Acid (PoPAA)Concentrations (uM). . . . . . . viii 59 59 61 63 69 71 76 76 76 81 81 82 83 84 Page TABLE 17. Effect of Diet and Environment on Mean Organ Weights (g/kgbOdyWETth)o00000000000000.0086 TABLE 18. Effect of Diet and Environment on Villus Height, Villus Width, Crypt Depth, Submucosa Depth and Total Intestinal Thickness of the Duodenum, Jejunum and Ileum (microns). . . . . . . . . . . . . . 90 TABLE 19. Comparison of Average Optical Density Readings (As a Measure of Microbial Growth) and Standard Deviations Within Microbe Source, Between Treatments, at 16 and 24 Hours of Incubation . . . . . 95 TABLE 20. Comparison of Average Optical Density Readings (As a Measure of Microbial Growth) and Standard Deviations at O, 16 and 24 Hours of Incubation Within Microbe Source, Within Treatments . . . . . . .101 TABLE 21. Comparison of Treatment Means for Change in Media pH After a 24-Hour Incubation Period, Within Microbe source 0 O O O O I O O O O O O O 0 O O O O O O O O O .107 TABLE 22. Comparison of Treatment Means for Net NH3-N Change (mg) After a 24 Hour Incubation Period, Within Microbe Source. . . . . . . . . . . . . . . . .108 TABLE 23A. Comparison of Volatile Fatty Acid (Formic and Acetic Acid) Production (umoles/ml) Between Microbe Sources and Treatments at 24 Hours of Incubation . . . . . . .110 TABLE 238. Comparison of Volatile Fatty Acid (Propionic and Isobutyric Acid) Production (umoles/ml) Between Microbe Source and Treatment at 24 Hours of Incubation . . . . . . . . . . . . . . . . . . . . . .111 TABLE 23C. Comparison of Volatile Fatty Acid (Butyric and Isovaleric Acid) Production (umoles/ml) Between Microbe Source and Treatment at 24 Hours of Incubation . . . . . . . . . . . . . . . . . . . . . .112 TABLE 230. Comparison of Volatile Fatty Acid (Valeric and Caproic Acid) Production (umoles/ml) Between Microbe Source and Treatment at 24 Hours of Incubation. . . . . . . .113 TABLE 24A. Comparison of Non-Volatile Fatty Acid (Lactic and Oxaloacetic Acid) Production (umoles/ml) Between Microbe Source and Treatment at 24 Hours of Incubation . . . . . . . . . . . . . . . . . . . . . .115 TABLE 248. Comparison of Non-Volatile Fatty Acid (Succinic Acid) Production (umoles/ml) Between Microbe Source and Treatment at 24 Hours of Incubation. . . . . . . . . .116 ix TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE TABLE 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. Page Comparison of Treatment Means for Total Volatile Fatty Acid Production (umoles/ml of media) After a 24-Hour Incubation Period, Within Microbe Source . . . . . . .119 Comparison of Tr atment Means for Non-Volatile Fatty Ac1d Production umoles/ml of media) After a 24 Hour Incubation Period Within Microbe Source. . . . . . . .122 Comgosition and Calculated Nutrient Analysis of Die 5 USEd in Trial 1. O O O O O O O O O O O O O O O .13] Amount of Vitamin and Trace Minerals Supplied Per Kg Of Diet in Trial 1. O O O O O O O O O O O O O O 0 .132 Effect of Autoclaving and Addition of Sugplemental Vitamin-Trace Minera Premix to Starter iets on GrOWth Performnce O O O O O O O O O O I O O O O O O .134 Composition and Calculated Nutrient Analysis of Diets used in Trial 20 O O O O O O O O O O O O O 0 O .138 Effect of Autoclaving and Addition of Supplemental Lys1ne and Meth1on1ne to Starter D1ets on Growth Performance 3 weeks) and the Effect of Addition of Non-Autoc aved Supplemental L-l sine HCl to Diets for an Additional 7-Days on rowth Perfomanceo O O O O I O O O O O O O O O O O O O I I .139 Composition and Calculated Nutrient Analysis of Diets ”sad in Trial 3. O O O O O O O O O O O O O O O O O O .143 Effect of Increasing Levels of Supplemental Lysine in Autoclaved Diets in Determining Ogtimal Lysine Supplementation as Measured by Growt Performance. . .144 Comgosition and Calculated Nutrient Analysis of Die 5 Used in Trial 4. . . . . . . .*. . . . . . . . .147 Effects of Autoclaving and Addition of 250 ppm Supplemental Capper to Diets on Energy Balance . . . .149 Effects of Autoclaving and Addition of 250 gpm Supplemental Capper to D1ets on Nitrogen Ba ance . . .152 Effects of Autoclaving and Addition of 250 ppm Supplemental Copper to Diets on Mineral Balance. . . .154 Composition and Calculated Nutrient Analysis of Diets used in Trial 5. O O O O O O C O O O O O O O O .158 Effects of Adding 250 ppm Supplemental Copper on Energy Balance . . . . . . . . . . . . . . . . . . . .159 Effects of Adding 250 ppm Supplemental Copper on Nitrogen Balance . . . . . . . . . . . . . . . . . . .l60 Effects of Adding 250 Bpm Supplemental Copper on Capper, Zinc and Iron alance. . . . . . . . . . . . .162 LIST OF FIGURES Page FIGURE 1. Influence of Capper and Chlortetracycline on Growth of Microflora from Stomach Contents and Wall During a 24 Hour Incubation Period. . . . . . . . . . . . . . . . . . .96 FIGURE 2. Influence of Copper and Chlortetracycline on Growth Microflora from Small Intestine Contents and Wall (lst Half) During a 24 Hour Incubation Period. . . . . . . . . .97 FIGURE 3. Influence of Copper and Chlortetracycline on Growth of Microflora form Small Intestine Contents and Wall (2nd half) During a 24 Hour Incubation Period. . . . . . . . . . . . . . . . . . . . . . . . . . .98 FIGURE 4. Influence of Copper and Chlortetracycline on Growth of Microflora from Cecal Contents and Wall During a 24 Hour InCUDation Peri 0d 0 O O O O I 0 O O O O O O O O O O O O O 099 FIGURE 5. Influence of Copper and Chlortetracycline on Growth of Microflora from Colon Contents and Wall During a 24 Hour IHCUbation Peri 0d 0 O O O O O O O O O O O O O O O O O O O 100 xi INTRODUCTION Addition of growth promoting levels of capper (125 to 250 ppm) above the copper requirement (4 to 6 ppm) to diets for growing swine is a generally accepted practice in today's swine industry. When copper is added to swine diets at levels up to 250 ppm, it is an equally effective and less expensive growth promoting agent compared to many broad spectrum antibiotics typically added to swine diets at subtherapeutic levels. The addition of high dietary levels of copper originated in 1955 when Barber and coworkers (1955a) demonstrated that inclusion of 250 ppm supplemental copper resulted in an improvement in growth performance of growing swine. Since that time, substantial evidence has been reported in the scientific literature to verify the growth promoting effect of copper but very little is known about how copper exerts its action to produce this beneficial response. At least three potential modes of action have been postulated for the growth promoting effects of subtherapeutic levels of antibiotics when included in diets for growing swine. These mechanisms could also apply to copper since copper has antimicrobial properties (Sollman, 1957). The growth promoting effects of antimicrobials may be mediated ' through 1) a metabolic effect, 2) a disease-control effect or 3) a nutrient-sparing effect (Hays, 1969). There is evidence to support each of these hypotheses, but most of the attention has focused on the influence of antimicrobials on the microbial population of the gastrointestinal tract in regard to a nutrient-sparing effect. It is known that antimicrobial agents (including c0pper) generally reduce the microbial population of the gut and also cause shifts in the microbial papulation. However, if these phenomena are to be nutritionally significant, it appears that the overall metabolism of gut microbes nust be altered in such a way as to elicit a growth promoting response. Studies have shown that when virginiamycin and spiramycin are added to diets for growing swine, utilization of glucose and amino acids as substrates for microbial metabolism is reduced compared to that of pigs fed diets containing no antimicrobial agents. As a result, the availability of these nutrients to the host appears to be a primary contributing factor to the total improvement of growth performance observed when these two antibiotics are added to diets for growing swine (Vervaeke et al., l979; Dierick et al., 1986a; 1986b). Perhaps copper also exerts its beneficial growth effects in this manner. Not only may the gut microbial population and metabolism be altered by antimicrobials to spare nutrients, but it may also be altered so that natural, endogenously produced,growth depressing bacterial toxins (e.g., ammonia) are reduced to provide an improved growth response. When antibiotics are fed to animals, portal ammonia concentration is reduced (Dintzis and Hastings, 1953; Silen et al. 1955) and when ammonia absorption is reduced, growth performance is improved (Holtzman and Visek, 1965). Since capper is an inhibitor of sulfhydryl enzymes such as urease, ammonia production may be significantly reduced to require less energy expenditure in the detoxification of absorbed amonia via the urea cycle. Due to the antimicrobial properties of capper and the limited data regarding the effects of c0pper on microbial papulations and metabolism in the gastrointestinal tract of the p19. this study was dESIQOEd t0 examine the relationship between high capper feeding and the gut microflora of the weanling pig. Several response criteria were measured to identify this relationship in an attempt to understand potential mechanisms by which c0pper exerts its growth promoting effects in growing pigs. REVIEW OF LITERATURE I. ESSENTIALITY OF COPPER A. Introduction Copper is an element unth atomic number 29, and an atomic weight of 63.546. Copper has two natural isotopes, capper-63 and copper-65 and occurs in nature as the metal in the +1 and +2 valence states. COpper is commonly found in the ecosystem associated with sulfide deposits, particularly in igneous rocks (NRC, 1977). Copper is one of fifteen naturally occurring trace elements known to be essential for animal life. However, capper, like all heavy metals, is also potentially toxic when ingested in sufficient quantities for an extended period of time. Biochemical mechanisms regulate the uptake and elimination of copper in order to maintain c0pper homeostasis during periods of dietary excess or deficiency so that adverse effects on the animal's health are avoided. B. Capper Absorption and Excretion Approximately 25 to 40 percent of c0pper consumed is absorbed depending upon dietary source and amount (O'Dell, 1984). Copper homeostasis is regulated by controlling the rate of absorption. Sulfur-rich proteins in the mucosal cells, which are identical or closely related to the thionine moiety of metallothionine, regulate the intestinal absorption of copper as well as zinc (Bremner and Davies, 1976). Capper is absorbed from the stomach and along all portions of the small intestine, especially in the upper small intestine (VanCampen and Mitchell, 1965). Primary route of capper excretion occurs via the bile and intestinal tract (O'Dell, 1984). C. Bioavailability of Copper from Capper Sources and Interaction with Other Nutrients Like most trace elements, the biological availability of capper depends to some extent upon its chemical form and its interaction with other dietary components. Capper carbonate and copper sulfate are more available forms of copper than capper sulfide, capper oxide and the copper in capper wire, which is largely unavailable (Chapman and Bell, 1963; Bowland et al., 1961). Copper assimilation is reduced by phytate which fbrms a very stable complex with capper (Vohra et al., 1965; Davis et al., 1962). Ascorbic acid, when fed at high levels, reduces copper retention by reducing intestinal absorption (Van Campen and Gross, 1968). Furthermore, several inorganic elements greatly influence capper absorption, retention and distribution in the body. These elements include: calcium, cadmium, zinc, iron, lead, silver, molybdenum and sulfur (Underwood, 1977). Although capper absorption mechanisms are poorly understood, these elements appear to compete with copper for protein-metal binding sites to inhibit capper absorption. D. Intermediary Metabolism of Copper When copper is absorbed from the intestine and enters the blood plasma, approximately 10 percent is loosely bound to serum albumin and amino acids and the remainder is bound tightly to ceruloplasmin fbr distribution to the various tissues of the body. Copper uptake by tissues tends to be greater from ceruloplasmin than from the cupric ion form (O'Dell, 1984). Ceruloplasmin serves as a direct donor of COPPer to metalloenzymes by entering the heart and to a lesser extent the kidney, brain and liver intact (Hsieh and Frieden, 1975; Owen, 1975; Linder and Moor, 1977). Copper also readily traverses into erythrocytes (Bush et al., 1956). E. Copper Storage Sites The main COpper storage organ in the body is the liver where capper is incorporated into the mitochondria, microsomes, nuclei and the soluble fractions of the parenchymal cells, depending on age, strain and copper status of the animal (Milne and Weswig, 1968; Thiers and Vallee, 1957). Capper may be stored in those sites or released for incorporation into erythrocuprein (synthesized in normoblasts of bone marrow; Bush et al., 1955), ceruloplasmin (synthesized in liver; Markowitz et al., 1955) or other cellular enzymes containing copper. Brain also contains appreciable concentrations of capper, while less copper is present in heart, spleen, kidneys and blood (NRC, 1977). F. flapper Requiring Enzymes At the cellular level, copper is primarily involved with capper proteins of which many are enzymes with oxidative functions. Copper is a very versatile cofactor in specific enzymatic reactions. Several copper containing enzymes include: tyrosinase, lactate dehydrogenase, ascorbic acid oxidase, uricase, monoamine oxidase, d0pamine-B-hydroxylase and cytochrome oxidase (Underwood, 1977). Cytochrome C oxidase and superoxide dismutase are good indicators of copper status (Phan and Mahler, 1976; Williams et al., 1975). G. Physiological Functions of Capper C0pper has a variety of physiological functions. Capper is involved in iron metabolism, cross-linking of connective tissue. neurotransmitters and brain function, lipid metabolism, pigmentation and keratinization of hair and wool, intestinal integrity, reproduction and immunology. 1. Iron metabolism Copper is vital in facilitating iron absorption and mobilization. In a copper deficiency, serum iron levels are generally low and iron levels in the intestinal mucosa and liver are higher than normal, resulting in a hypochromic anemia (Underwood, 1977). Since the oxidation rate of Fe (II) by oxygen limits the rate of iron mobilization, which is catalyzed by a ferroxidase, and since there are at least two plasma ferroxidases (ferroxidase I (ceruloplasmin) and ferroxidase II), copper plays a role in absorption and mobilization of iron through its function in ceruloplasmin (O'Dell, 1984). 2. Cross-linking of connective tissue Lysyl oxidase is a copper metalloenzyme which catalyzes the oxidation of specific lysyl and hydroxylysyl residues in soluble collagen and elastin (O'Dell, 1976). Without adequate copper, lysyl oxidase activity is low and the cross-links in collagen and elastin are not farmed. This results in fragile bone and other skeletal abnormalities (league and Carpenter, 1951). Furthermore, aortic rupture has been shown in capper deficient chicks (O'Dell et al., 1961) and in pigs (Carnes et al., 1961) due to reduced elastin content of the aortas of pigs (Weissmann et al., 1963) and chicks (Starcher et al., 4 1964) and less cross-linking groups due to depressed lysyl oxidase activity of the aorta. 3. Neurotransmitters and brain function Concentrations of at least two brain neurotransmitters, dopamine and norepinephrine, are reduced by capper deficiency (O'Dell, 1984). Copper deficiency appears to reduce the catalytic function of dopamine-s-hydroxylase in the adrenergic pathways and appears to adversely affect a structural component of the d0paminergic system through defective myelination (O'Dell, 1984). 4. Lipid metabolism Capper deficiency results in an increase of serum triglycerides, phospholipids and cholesterol; a decrease in the ratio of mono-unsaturated to saturated C15 and C13 fatty acids in subcutaneous adipose tissue; and a decrease in the C13. A9 desaturase enzyme (Wahle and Davies, 1975; Petering et al., 1977). The site of copper involvement in the desaturase reaction may be in the terminal component of the microsomal electron transport chain (Wahle and Davies, 1975). C0pper deficiency causes a rapid release of cholesterol fron the liver to the plasma due to a high zinc:copper ratio (Klevay, 1973). 5. Pigmentation and keratinization of hair and wool Failure of pigmentation of hair or wool (achromotrichia) among rats, rabbits, guinea pigs, cats, dogs, cattle and sheep, but not pigs, probably results in impaired conversion of tyrosine to melanin by c0pper-containing polyphenyloxidases in a copper deficiency (Underwood, 1977). Changes in hair or wool growth occur when these same Species are fed copper deficient diets. Copper deficiency adversely affects the typical physical characteristics of wool which are dependent on the disulfide groups to provide cross-linkages or bonding of keratin (Underwood, 1977). Inadequate copper also disrupts the alignment or orientation of the hair or wool keratin in the fiber (Underwood, 1977). Furthermore, a lack of copper can interfere with the arrangement of the polypeptide chain in keratin synthesis (Underwood, 1977). 6. Intestinal integrity In copper-deficient cattle, diarrhea (often called peat scours or teart scours) is usually a secondary symptom due to excess molybdenum intake (Miller, 1979). However, diarrhea is not a manifestation of capper-deficient swine (Miller et al., 1979). Copper-deficient cattle have reduced cytochrome oxidase activity in intestinal epithelium as well as villous atrOphy (Fell et al., 1975). 7. Infertility Copper deficiency results in reproductive failure due to fetal death and resorption in rats and guinea pigs, reduced egg production and hatchability in chickens, depressed estrus in cattle and abortion in sheep (Underwood, 1977). 8. Immunology Superoxide dismutase is a zinc, copper and manganese dependent enzyme and is important in the microbiocidal systems of phagocytes (Miller et al., 1979) . This enzyme is important when a bacterial stimulus causes normal phagocytes to undergo a respiratory burst which produces a very destructive superoxide radical. Superoxide dismutase converts the superoxide radical to less destructive hydrogen peroxide, which is subsequently converted to water via glutathione peroxidase. H. Copper Requirement of Swine The copper requirement for adequate growth in the pig is 4 to 6 ppn to 90 kg body weight (NRC, 1979). Work by Ullrey et a1. (1960) showed no treatment differences for growth rate and feed utilization efficiency when baby pigs were fed diets containing 6, 16 or 106 ppm of 10 copper. More definitive studies by Okonkwo et a1. (1979) and Hill et al. (1983) indicate that the copper requirement of the baby pig is about 5 ppm. Most swine diets contain much higher levels of copper than the requirement since the two major dietary ingredients include cereal grains and leguminous or oilseed meals (protein supplement) which contain 4-8 ppm copper and 15-30 pun copper, respectively (NRC, 1979). I. Consequences of Feeding High Copperl)iets to Growing Swine Although the copper requirement is 4 to 6 ppm, addition of supplemental copper up to 250 ppm in a normal swine ration results in an increase in rate of weight gain and feed utilization efficiency in growing pigs (Barber et al., 1955a, 1955b, 1955c). However, in some instances, addition of 250 ppm of capper can produce toxicosis. depending upon the dietary levels of zinc and iron. When copper toxicosis occurs, growth performance is often reduced (Bass et al., 1956). In copper toxicosis, anemia also develops but can be prevented by iron supplementation, (Ritchie et al., 1963; Wallace et al., 1960) and skin lesions may occur but are alleviated by zinc supplementation (Ritchie et al., 1963). Copper appears to be involved in the desaturase reaction of fatty acid metabolism because in copper-deficient rats there was a lower monounsaturated:saturated ratio and less desaturase activity in liver microsomes than in rats with adequate copper status (Wahle and Davies, 1975). It appears that the level of copper fed influences fatty acid composition and physical properties of backfat since higher concentrations of oleic acid and lower concentrations of stearic acid 11 in backfat lowered the mean melting point of whole backfat by lO’C (Moore et al., 1969). Taylor and Thomke (1964) fed 250 ppm copper to pigs and showed that high dietary levels of copper produced softer backfat with a lower melting point. Bowland and Castell (1964, 1965) also observed that high dietary copper levels softened backfat. Copper supplementation at 280 ppm caused a significant increase in the proportion of unsaturated fatty acids in the outer and inner layer of backfat, and in perinephric fat at 26, 47, and 70 kg body weight (Elliot and Bowland, 1968). In addition, a corresponding decrease in the proportion of saturated fatty acids was observed while no difference in fatty acid composition was found at 90 kg live weight. The increases in unsaturated fatty acids was due to increases in 16:1 and 18:1 fatty acids with decreases in 16:0 and 18:0 saturated fatty acids. Other workers have attributed the softness of depot fat arising from capper supplementation to changes to increases in major unsaturated fatty acids (Elliot and Bowland, 1968; 1969; 1970). Moore et al. (1969) reported that depot fat from pigs fed 250 ppm capper, had a higher concentration of oleic acid and a lower concentration of stearic acid. Castell et al. (1975) reported a lower amount of stearic acid, higher palmitoleic acid and a higher oleic:stearic acid ratio. Several researchers have shown that depot fat softness arising from high copper feeding was caused by an increase in the proportion of unsaturated fatty acids and a concomitant decrease in the amount of saturated fatty acids (Amer and Elliot, 1973; Myres and Bowland, 1973; Ho and Elliot, 1973, 1974; Ho et al.. 1975; Castell et al., 1975). Ho and Elliot (1973) suggested that the increase in '12 18:1 and the decrease in 18:0 fatty acids in depot fat of pigs fed high copper diets was related to an enhancing effect of copper on Specific activities of hepatic and adipose stearoyl-CoA desaturase systems but later implied the involvement of the entire fatty acyl desaturase system (No and Elliot, 1974). Later, these authors suggested that unsaturated fatty acid increases were caused by the role of copper in desaturation reactions as a component of some cuproprotein enzyme system or metalloprotein (Ho et al., 1975). II. GROWTH PROMOTING EFFECTS OF HIGH COPPER DIETS FED TO SWINE A. Introduction A considerable amount of research has been conducted in feeding high dietary levels of copper to stimulate growth and efficiency of gain in growing pigs. In 1955, Barber et a1. observed improved growth response from pigs fed a normal diet supplemented with 250 ppm of copper as copper sulfate. Since that time, British researchers have observed a consistent 81 improvement in daily weight gain and a 5.5% improvement in feed efficiency by feeding supplemental copper sulfate at the 250 ppm level. liowever, U.S. researchers have not observed the same consistency in growth promotion when high copper diets were fed, as noted in reviews by Braude (1967) and Wallace (1967). The mode of action of copper on improving growth perfbrmance is still not well understood (Underwood, 1977) and little research has been conducted in this area. B. Effect of High Copper Feeding on Growth Performance Numerous studies have shown that supplementing the diets of growing pigs with copper (in the form of copper sulfate) at levels up 13 to 250 ppm, results in improved growth rate and feed efficiency (Braude, 1945; Barber et al., 1955a, 1955b, 1955c; Bowler et al., 1955; Barber et al., 1960; Allen et al., 1961; Barber et al., 1961; Lucas at al., 1962a; Wallace, 1967; Braude et al.. 1970; Young and Jamieson, 1970; Braude and Ryder, 1973; Ho and Elliot, 1973). The beneficial effect from capper sulfate feeding has been shown to be due to the copper and not the sulfate radical (Barber et al., 1957; Bowland et al.. 1961; Hawbaker et al., 1959). Despite the reports of beneficial effects of high levels of copper feeding in the U.S. and Europe, other researchers observed no beneficial response (Lucas and Calder, 1957a; Teague and Grifo, 1966; Livingstone and Livingston, 1968; Parris and McDonald, 1969; Elliot and Bowland, 1970; Amer and Elliot, 1973). Gipp et al. (1973), reported that body weight gain was not consistently affected by feeding 250 ppm copper in corn-soy diets during the starter, grower, finisher stages or during the entire growth period. Ho et a1. (1975). observed that the presence of high dietary levels of copper reduced average daily gain and feed efficiency. Canadian workers have also shown inconsistent benefits, with most of the studies showing no improvement in growth rate or efficiency by feeding high levels of copper (Castell and -Bowland, 1968; Drouliscos et al.. 1970; Young et al.. 1970; Castell et al., 1975). Nevertheless, some Canadian workers reported beneficial effects with 250 ppm copper in starter diets of pigs up to 50 kg body weight (Beames and Lloyd, 1965; Young and Jamieson, 1970). Improvement in growth performance peaks and then plateaus when growing pigs fed high copper diets reach 50-60 kg in body weight, and a decline in 5. 23. human...» 14 response after this time is due to an accumulation of copper in the liver (Lucas and Calder, 1957b; Bunch et al., 1963; Bellis, 1961). Miller et a1. (1969) and the NCR-42 Committee on Swine Nutrition (1974) reported that growth rate during the growing phase was improved by feeding copper up to 250 ppm in the diet, but removing the copper after eight weeks did not affect subsequent growth performance. Elliot and Amer (1973) observed an improvement in growth rate in growing pigs fed high copper diets only up to 23, 46, or 69 kg body weight, which was not statistically significant, but upon removal of copper from the diet, growth was depressed. Withdrawal of copper from the diet when the [figs reached 46 or 57 kg in body weight did not affect performance but allowed liver copper concentration to return to normal levels by slaughter weight (Teague and Grifo, 1966). C. Proposed Mechanisms of Growth Promotion When High Copper Diets Are Fed to Growing Swine The mechanism of action relative to the effects of high c0pper feeding is not known but several suggestions have been made. 1. Bacteriostatic effects of copper The bacteriostatic properties of copper in regard to the gut microflora (Sollman, 1957) have been implicated due to the ”antibiotic-like" response observed when feeding high copper diets to swine. In other words, cooper may alter microbial populations so that growth of desirable organisms that synthesize vitamins or amino acids is stimulated, by depressing undesirable organisms which compete with the host for nutrients, or both. In one study, Hawbaker et a1. (1961) showed that lactobacilli, total aerobic, total anaerobic and ~15 streptococci fecal counts were reduced, and molds, yeasts and coliforms were increased when high copper (as CuS04) diets were fed. These authors noted that copper sulfate increased molds and yeasts much like that reported for broad spectrum antibiotics (Pappenfort and Schnall, 1951). Similar effects of high copper feeding (as capper sulfate) on fecal flora counts were observed by Bunch et al. (1961), where lactobacilli, total aerobic and total anaerobic counts were reduced but molds' and yeasts' counts were reduced. Miller et al. (1969) reported a lower total fecal bacterial count in feces from pigs fed diets containing 250 ppm copper than feces of pigs fed a combination of oxytetracycline and neomycin sulfate. Fuller et al. (1960) observed a reduction in the numbers of streptococci and a shift in the relative proportion of three lactobacilli species when pigs were fed high copper diets. However, Smith and Jones (1963) did not observe any differences in fecal flora counts when high c0pper diets were fed. Based on some of these reports, it is likely that copper, analogous to antibiotics, does alter the intestinal flora and this effect may be primarily responsible fbr the growth promoting effect observed when swine are fed diets containing high levels of copper. However, it must be realized that differences in fecal flora counts reported, represent only about 10% of the intestinal population. The gastrointestinal tract of man and animals has an oxidation-reduction potential of the contents below -250 mv which makes this an extremely anaerobic environment (Moore, 1969). Because of this extremely anaerobic environment, many of the bacteria present cannot be cultured with current anaerobic techniques. Therefore, approximately 902 of the l6 intestinal population of microbes has not been described and is essentially unknown (Moore, 1969). 2. Effects of high copper feeding on digestive and metabolic efficiency Several workers have reported that copper supplementation at high dietary levels exerts its pronounced effects on growing pigs by increasing digestive and/or metabolic efficiency (Lucas et al., 1962a; Bunch et al., 1963; Wallace, 1967; Drouliscos et al., 1970). Other researchers attribute the effect of high copper feeding to a stimulatory effect on feed consumption (Mitchell, 1953; Barber et al., 1962; Braude, 1965; Young et al., 1970). . a. Relationship of high copper and dietary protein source and level on protein digestion and utilizatigg By dosing young pigs with copper, an improvement in the digestibility of proteins has been observed and appears to be a result of an increase in cupric ion concentration, increasing pepsin activation and producing more peptic hydrolysis of proteins (Kirchgessner and Giessler, 1961; Kirchgessner et al., 1976). Protein level and source appear to influence the effectiveness of high level copper feeding in young growing pigs. British researchers have continually observed beneficial effects of feeding barley-fish meal diets containing 150 to 250 ppm copper as copper sulfate (Barber et al., 1955a, 1955b, 1955c, 1957; Bowler et al., 1955; Bellis, 1961; Braude and Ryder, 1973). Pigs fed corn-soybean meal basal diets. typically used in the U.S., have not produced consistent growth performance responses to copper supplementation and copper levels above 17 250 ppm have been shown to be toxic (Wallace et al., 1960). However, Bunch et a1. (1961) fed 22% protein corn-soybean meal diets containing capper levels up to 250 ppm and observed an improvement in growth performance but there was a linear decrease in hemoglobin levels. Canadian workers observed a 2.2 to 1.7% improvement in growth and a 2.0 to 2.2% improvement in feed efficiency from pigs when 125 and 250 ppm copper, respectively, were supplemented in these diets (Castell et al., 1975). Omole and Bowland (1974) fed pigs diets that were isonitrogenous and isocaloric, utilizing either soybean meal, low glucosinolate rapeseed meal or a combination of these two protein sources, along with 10, 135 and 210 ppm copper, and observed no differences on feed intake, gain, or feed efficiency. Pigs fed dried skim milk diets containing 250 ppm copper during both the growing and finishing phases, gained at the same rate as pigs fed a corn-soybean meal diet with or without copper supplementation, but pigs fed dried skim milk diets had carcasses with a higher fat content (Gipp et al., 1973). Edmonds and Baker (1984) have observed that when high levels of dried whey are added to a low-protein antibiotic fortified corn-soybean meal diet, and fed to weaning pigs, weight gain and feed efficiency are greatly improved. Edmonds and Baker (1984) showed that adding 250 ppm copper (from CuSO4-5H20) greatly improved rate and efficiency of gain, particularly during the first week postweaning, and an additive response for rate of weight gain was observed when 25% dried whey and 250 ppm copper were both added to a corn-soybean meal diet. There may be a unique relationship between the amount of copper and the amount of dried whey present in the diet to elicit such a response. b. Effect of high copper feeding on lipid metabolism Myres and Bowland (1973) measured plasma free fatty acids in both the fed and fasted state in pigs receiving 250 ppm or no supplemental copper. In the fasted state, pigs fed 250 ppm copper had lower levels of free fatty acids than pigs receiving no supplemental copper, with the opposite effects occurring in the fed state. c. Mineral interrelationships relative to the effectiveness of high copper feedipg Certainly, mineral interrelationships with copper would also determine the effectiveness of high level copper feeding. Zinc, iron, cadmium, molybdenum, sulfur, calcium, lead, silver and phytate either directly or indirectly influence the absorption and utilization of copper due to competition between metals for functional sites on enzymes and formation of metal complexes with protein, such as metallothionein (Underwood, 1977). In other words, an unusually high intake of one element can adversely affect the utilization of other elements. Ascorbic acid also chelates copper to reduce copper absorption (Underwood, 1977). Variations in growth response observed when pigs are fed high levels of copper may be due to the levels of iron and zinc in the basal diet (Underwood, 1977). Copper is known to compete with iron for absorption binding sites and with zinc for metal-binding protein. Zinc and iron have been shown to have a significant impact on copper absorption. When pigs were fed 250 ppm copper sulfate, they develOped skin lesions similar to parakeratosis (O'Hara et al., 1960). DeGoey et a1. (1971) observed a reduction in the incidence of parakeratosis in pigs fed a basal diet supplemented 1_ 9 with 100 ppm iron or basal plus 100 ppm zinc. Supplemental zinc has been shown to reduce liver copper accumulation from high dietary levels of copper (Hoefer et al., 1960; Wallace et al., 1960; Ritchie et al., 1963). Liver iron levels were lowered in pigs fed 250 ppm copper (Bunch et al., 1963). When toxic levels of capper are fed (750 ppm), high levels of zinc (500 ppm) reduce copper retention in the liver and reduce tissue damage. When 750 ppm iron was added to a 750 ppm copper diet. anemia was less severe than with only 750 ppm capper, less copper accumulated in the serum and tissue damage was reduced (Suttle and Mills, 1966). Several forms of c0pper such as copper sulfate, copper oxide, copper chloride, and copper carbonate are effective growth promoters (Wallace, 1967), while copper sulfide is not an effective growth promotant because the copper sulfide is insoluble and thus, copper is not available to perform its role as a growth promotant (Cromwell et al.. 1978). Molybdenum and sulfate in combination, prevent tissue buildup of copper in ruminants (Dick, 1953, 1954; Ammerman and Marcilese, 1968) but are ineffective in swine (Kline et al., 1971). Sulfide, however, appears to prevent excessive liver accumulation of c0pper in pigs fed high capper diets (Kline et al. 1973). Cromwell et a1. (1978) fed sodium sulfide to growing-finishing pigs and found a reduction of copper uptake in the liver, with no apparent interference with the ngth promoting effects of copper in pigs fed high capper diets. However, when these authors fed copper sulfide to pigs, no improvement 1“ Performance was noted, nor was there an increase in liver copper 20 levels. Sulfide, either in the form of sodium sulfide (Kline et al., 1973; Cromwell et al., 1978) or ferrous sulfide (Prince et al., 1979; Lima et al., 1981) are effective in preventing copper accumulation in the liver in pigs fed 250 ppm copper. A 2:1 ratio of sulfidezcopper reduced liver copper to approximately the same level as observed in control pigs, but didn't interfere with the growth promotion effects of copper. Elemental sulfur is only about half as effective as sulfide in reducing liver copper accumulation. Prince et a1. (1979) indicated that sulfide acts to prevent uptake of copper by liver tissue, apparently by reduced absorption, rather than by increasing mobilization of copper from the liver. This suggests that copper exerts its action in the digestive tract of the pig. Williams et al. 1981, suggested that a portion of the copper in the gut may be rendered insoluble by the inclusion of sulfide or sulfur in the diet, but this does not appear to be of sufficient magnitude to account for the decreased liver stores and does not explain the reason for sulfide interaction with the growth promoting effects of copper. d. Other proposed mechanisms of growth promotion when high copper diets are fed Braude (1967) suggested other possible modes of action as fungicidal action, anthelmintic activity, or a reaction of microbial-produced hydrogen sulfide in the intestine with copper to provide copper sulfide. Newton (1984) suggested that feeding high c“31313127: diets to young, growing swine may reduce the detrimental effects 0f naturally occurring phenol and polyphenol compounds present in plant Products which depress growth rate and feed efficiency. III. EFFECTS OF FEEDING HIGH LEVELS OF COPPER IN COMBINATION WITH ANTIBIOTICS ON GROWTH PERFORMANCE Since both high levels of copper and antibiotics frequently improve growth performance in the young pig, studies have been conducted to ascertain if a synergistic effect on growth stimulation could be obtained. Stahly et al. (1980) fed a diet supplemented with 250 ppm copper with or without 55 ppm chlortetracycline or 27.5 ppm of virginiamycin to early weaned pigs. When either copper or antibiotics were fed singly, growth rate increased by 17 to 22 percent and feed/gain improved by 5 to 9 percent but the combination of copper and antibiotics resulted in a 23 to 32 percent improvement in growth rate and 7 to 14 percent improvement in feed to gain ratios compared to pigs receiving the control diet. Similar additive responses were observed when copper was fed in combination with carbadox or with a combination of penicillin, chlortetracycine and sulfamethazine (Mahan, 1980). Beames and Lloyd (1965) reported an additive response in early weaned pigs fed a combination of copper and tylosin. It is less clear if there is a complementary effect from feeding cOPPer in combination with antibiotics to older, growing-finishing Pigs. An additive trend was reported by Barber et al. (1978) with coPPer and virginiamycin but Lima et a1. (1981) failed to show any additive response from the combination of capper and chlortetracycline or the combination of copper and virginiamycin compared to single additions of these antimicrobial agents. No differences in growth performance were observed when higher cOPPEr diets were fed in combination with tylosin (Lillie et al., 1977; Beames. 1969), zinc bacitracin or emtryl (Barber et al., 1965). Furthermore, when copper sulfate (CuSO4-5H20) was added at a level of 0.1% to diets containing dried skim milk, white fish meal or extracted soybean meal as protein sources, there was no synergistic effect with zinc b'aci tracin, tylosin, or formosulphathiazole (Livingstone and Livingston, 1968). However, feeding a combination of high copper and chlortetracycline improved growth performance compared to feeding chlortetracycline alone (Wallace, 1967). Similarly, Hawbaker et al. (1959) observed an additive response to weight gain when high copper was fed in combination with oleandomycin or oxytetracycline. In contrast, Braude (1967) observed no additive effect on growth performance when copper sulfate and oxytetracycline were fed in combination. Finally, Cromwell et a1. (1978) summarized Kentucky, Ohio, and Canadian studies where there was an additive effect in growth response from feeding high levels of copper sulfate along with antibiotics and suggested that copper may have a broader spectrum of antibacterial activity or that capper may have a different mode of action than the antibiotics studied. Because of the evidence that copper is “antibiotic-like" in its growth promoting effects and appears to influence the intestinal microflora, it is essential to understand the current knowledge of the mode of action of antibiotics to gain insight into possible modes of action of high copper feeding to swine. IV. ANTIBIOTICS IN LIVESTOCK FEEDS AND THEIR MODE OF ACTION Antibiotics have been widely used as feed additives in livestock feeds for the past 30 years because of their beneficial effects on 23 improving growth rate and feed conversion as well as reducing mortality and morbidity from clinical or subclinical infections. Many antibiotics have been used in livestock production and these feed additives vary in their effectiveness as growth promotants. Several commonly used antibiotics include: chlortetracycline, oxytetracycline, penicillin, sulfamethazine, bacitracin, tylosin and others. It is generally accepted that antibiotics have an ability to suppress or inhibit the growth of certain microorganisms in their growth promoting effects. Antibiotics differ in a variety of ways such as in their chemical composition. bacterial spectrum and absorption and excretion patterns (Hays, 1969). These characteristics certainly influence bactericidal and bacteriostatic properties and their effectiveness against systemic infections (Hays, 1969). When considering the mechanisms by which antibiotics improve growth performance, three general modes of action have been postulated: (l) a metabolic effect, (2) a nutrient-sparing effect and (3) a disease-control effect (Hays, 1969). A. Metabolic Effect The metabolic effect hypothesis suggests that antibiotics directly affect the rate or pattern of the metabolic processes in the host animal. which is also related to the disease-control effect hypothesis because the rate of metabolism may be related to systemic infections (Hays, 1969). Water and nitrogen excretion are affected by feeding chlortetracycline, (Braude and Johnson, 1953). Tetracycline inhibited f3tty acid oxidation by the mitochondria in rat liver homogenates 24 (Brody et.al., 1954). Phosphorylation and oxidation reactions requiring magnesium ions were inhibited by tetracycline (Weinberg, 1957), Tetracyclines have also been shown to inhibit protein synthesis (Hash et.aJ., 1964). Although antibiotics do appear to alter metabolic processes, the contribution of metabolic effects relative to other contributing factors probably does not account for a majority of the beneficial growth response typically observed because of the magnitude of animal responses relative to normal tissue levels of antibiotics when added to the diet at growth-promotant levels (Hays, 1969). 8. Nutrient Sparing Effect Hays (1969) noted that the nutrient-sparing effect hypothesis is based on the assumption that antibiotics may reduce the dietary requirement fbr certain nutrients by: 1) stimulating growth of desirable organisms that synthesize vitamins or amino acids. 2) depressing organisms that compete with the host for nutrients. 3) increasing the availability of nutrients by chelation mechanisms. 2 4) improving absorptive capacity of the intestinal tract. Certain intestinal organisms synthesize vitamins and amino acids which are needed by both the host animal and other intestinal microorganisms, and these microbes in turn, compete with the host animal for these nutrients (Hays, 1969). Visek (1978) proposed that it 15 not likely that antibacterials exert a vitamin-like action or Provide chemical moieties essential for metabolism. Streptomycin stimulated growth of some yeasts (Moore et al., 1946) and penicillin increases the number of coliforms (except E. coli) 25 (Anderson et al., 1952). Since these organisms are important for synthesizing dietary essential nutrients, the supplementation of diets with antibiotics would increase the synthesis of these nutrients. Since lactobacilli require amino acids in similar relative proportions as the pig, and since the level and source of protein supporting maximum growth in pigs is also near optimum for multiplication of lactobacilli in the intestinal tract (Kellogg et al., 1964), lactobacilli are considered to be an example of organisms which compete with the host animal for dietary needs. Certain antibiotics cause a reduction in the number of lactobacilli (March and Biely, 1952; Anderson et al., 1952; Johansson and Sarles, 1949). Furthermore, it appears that the most effective, routinely used growth promotants are those antibiotics that are the most effective in reducing the number of these intestinal organisms (Kellogg et al., 1966). Jukes (1955) and Taylor (1957) have indicated that the metabolism of intestinal microbes is changed rather than the relative number of each microbial species when antibiotics are fed. When an antibiotic is fed to pigs, the intestinal wall is thinner than the intestinal wall of pigs fed diets without antibiotics (Braude et al.. 1955). This same effect was observed in chicks (Eyssen and 'DeSomer, 1963a, 1963b; Pepper et al.. 1953) and when fed the intestinal contents of infected chicks, the intestinal wall was thicker (Coates, et al.. 1955). Hill et al. (1957) and Taylor and Harrington (1955) rePorted similar effects in chicks and pigs. A reduction in intestinal thickness implies a potential for improved absorption due to inhibition °f o"Slanisms which damage or produce toxins that damage intestinal tissue (Taylor, 1957). 26 Glucose absorption was increased when pigs were fed diets containing antibiotics which supports the basis for improved nutrient utilization from feeding antibiotics (Catron et al., 1953). Several researchers have also shown that less protein is required by pigs for maximum performance when pigs are fed diets containing antibiotics (Catron et al., 1952; Burnside et al., 1954; Beacom, 1959a, b). Several researchers have shown an association between diet and antibiotic response where the response to antibiotics is generally greater when antibiotics are added to the inadequate diet (Lucas and Calder, 1957b; Braude and Johnson, 1953; Stokstad, 1954; Burnside et al., 1949). Visek (1978) noted that the growth improvement observed when feeding diets containing antibiotics to swine is not well correlated with differences in bacterial counts in specific intestinal sections or for bacterial counts of the entire gut. This also appears to be true in pigs fed c0pper-supplemented diets (Smith and Jones, 1963; Miller et al., 1969; Hawbaker et al., 1961; Fuller et al., 1960; Bunch et al., 1961). As a result, Visek (1978) suggested that a change in bacterial metabolism may be nutritionally more important for the animal thania change in the relative number of microbial species. Vervaeke et al. (1979) studied the production rates of volatile fatty acids (VFA) and lactate when virginiamycin and spiramycin were added to incubated Stomach, ileal and cecal contents of pigs. These authors observed a decrease in organic acid production and a concommitant sparing of a measurable quantity of glucose upon addition of these two antibiotics to stomach and ileal contents. When virginiamycin was added to cecal 27 contents organic acid production was also reduced, but spiramycin increased organic acid production. Furthermore, these authors, using in vivo data, calculated that the sparing of carbohydrates by incorporating these two antibiotics in diets for swine resulted in more net energy available fer growth, and this estimated increase in net energy accounted for a majority of the growth improvement typically observed when these antibiotics are fed. This carbohydrate sparing action observed for these two antibiotics may also explain the improvements in growth of pigs fed other antimicrobials, particularly copper. For carbohydrates not digested and absorbed in the small intestine, these energy sources can be readily fermented to volatile fatty acids in the cecum and colon and contribute 5 to 28 percent of the pig's energy requirement (Friend et al., 1963a; 1963b; Farrall and Johnson, 1972; Kass et al., 1980). Imoto and Namioka (1978) measured the production of acetate, propionate and butyrate in the large intestine of the pig when either a high or low carbohydrate diet was fed. Mean total VFA absorption rates were 0.85 and 0.65 moles per day for the low carbohydrate and high carbohydrate diets, respectively. This amount of VFA absorption is equivalent to 230 and 190 kcal of energy for the high and low carbohydrate diets respectively, which correspond to 11.6 and 9.6% of the metabolizable energy for maintenance. Rerat (1978) indicated that digestion of carbohydrate in the Pig large intestine represents only 10 percent of digestible energy Intake. Thus, VFA production is important as an energy source as it is absorbed from the lower digestive tract. Perhaps copper increases VFA Production in the lower digestive tract to improve the efficiency of 28 energy utilization of carbohydrates not digested and absorbed in the upper intestinal tract. Several researchers have suggested that improved growth performance is due mainly to an increase in food intake because even though total nutrients retained is greater fbr animals fed antibiotics, the percentage retained between those animals fed antibiotics and those which are not, are similar (Meade and Forbes, 1956; Wallace et al.. 1954; Bush et al., 1959). C. Disease-Control Effect Finally, the third proposed mode of action for antibiotics is a disease-control effect by suppression of organisms causing clinical or subclinical disease (Hays, 1969). Ninety seven percent of the microbial population within the alimentary tract of man and animals is made up of obligate anaerobes (Moore and Holdeman, 1972) and these anaerobes are significant causes of disease (Keusch, 1974; Klainer and Beisel, 1969). Many studies lend support fbr the disease-control effect as a major factor in improved growth performance when animals are fed antibiotics. In general, the degree of response to antibiotics is inversely related to the well-being of animals. Antibacterial agents are generally more effective growth promotants in rearing conditions where hygiene is poor, compared to new or isolated , environments (Libby and Schaible, 1955), but not in every situation (Coates and Harrison, 1969). Healthy pigs fed well balanced diets and reared in a sanitary environment responded less to antibiotic supplementation then those pigs that were not (Speer et al., 1950; Hays and Speer, 1960). Visek (1978) postulated that antibiotics reduce microbial production of growth depressing toxins from bile acids and nitrogenous substances. In the absence of bacteria, portal blood ammonia concentrations are about 25% of portal ammonia concentrations in conventional animals (Warren and Newton, 1959) and are reduced in conventional animals fed antibiotics (Stahl, 1963; Warren and Newton, 1959). Urea and bile acid hydrolysis occur in the gastrointestinal lumen of conventional animals but not in germrfree animals and their hydrolytic products are reduced or abolished by antibiotics (Dintzis and Hastings, 1953; Kornberg and Davies, 1955; Levenson et al., 1959; Delluva et al., 1968; Norman and Widstrom, 1964; Walser and Bodenlos, 1959; Visek et al., 1959). It appears that the main source of ammonia in the body occurs when bacteria act on nitrogenous substances in the intestinal lumen (Folin and Denis, 1912; Phillips et al., 1952). Bacteria provide a major contribution to the portal ammonia concentration through deamination of ingested protein and urea hydrolysis as observed when antibiotics are fed (Dintzis and Hastings, 1953; Silen et al., 1955). Ammonia is recognized as a toxin in warm-blooded animal species (Phillips et al., 1952; Visek, 1964; 1972). Ammonia is one of the microbial products that increases the wet weight, alters nucleic acid synthesis and increases protein in the intestinal mucosa (Topping and Visek, 1977). Normal ammonia concentrations range between 8 to 10 mM (140 to 180 ppm) in the intestinal lumen (Prior et al., 1974; Wrong et al., 1965; Wilson et al., 1968a, b) which is several times the concentration required to destroy cells (Dang and Visek, 1968), alter the rate of nucleic acid 30 ’ synthesis by mammalian cells (Zimber and Visek, 1972) increase mycoplasma infections in controlled experiments in laboratory animals (Broderson et al., 1976) and to depress the immune response (Fridlyand, 1959). Copper is an inhibitor of sulfhydryl enzymes such as urease and a reduction of urease activity has been observed in the gastrointestinal lumen of birds fed copper-supplemented diets (Stokstad et al., 1953; Visek et al.. 1959). Products of hydrolysis produced from bile acids by bacterial enzymes also have detrimental consequences because they change micelle formation, lipid absorption, intestinal histology and calcium absorption (Combe et al.. 1976). They depress growth when fed in small amounts but there are no studies describing the effects of growth promoting concentrations of antibacterial agents upon bile acid metabolism. The microbial flora modify lipids and bile acids and has been described by Eyssen and DeSomer (1963a, 1963b, 1965) where transitory malabsorption of fats and carbohydrates in chicks fed casein-sucrose diets was observed along with decreased feed efficiency. Malabsorption of fats and carbohydrates was reduced and feed efficiency was improved by feeding 20 ppm of virginiamycin. It has been suggested that intestinal flora diminish the absorption of alimentary fat by modifying the intestinal mucosa (Sprinz, 1962) or by producing toxic unconjugated derivatives of bile acids (Dawson and Isselbacher, 1960). Feeding of 0.2% lithocholic acid to chicks caused malabsorption of fats, impaired efficiency of feed utilization and a profound increase In liver size with a proliferation of bile ductules (Leveille et al., 1962). The metabolites of bile acid metabolism, which are not produced .31 in germ-free animals, need further investigation in antibiotic supplemented conventional animals. According to Norman and Widstrom (1964), bile acids are excreted in a conjugated fbrm, with more than 90% of these bile acids being conjugated with taurine. In the small intestine, most of the bile acids are also conjugated, whereas the bile acids in the cecum, colon and feces are almost all unconjugated (Norman and Widstrom, 1964). Since bile acids in the feces of germ-free rats and rats treated with antibiotics are almost exclusively conjugated, the hydrolysis of taurine and glycine conjugates has been ascribed to the action of microbial enzymes. Combe and coworkers (1976) concluded that the presence of "non-pathogenic" bacterial flora in the digestive tract has to be considered as a factor having an unfavorable action on the digestion and absorption of dietary lipids and that action on the absorption of long and saturated fatty acids (palmitic and stearic acids) is particularly marked. These authors fUrther suggest that the microflora acts principally at this level through intermediary bile acid metabolism. V. MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF THE GERM-FREE ANIMAL It is essential to understand the morphological and physiological differences between germ-free and conventional animals in order to determine the association between the microflora and the systemic functions of the host. Early workers in germ-free research examined the ability of germ-free animals to survive, grow and reproduce (Pleasants, 1968). Most studies with germ-free animals involve laboratory rodents, primarily rats and mice, because of their acceptability as experimental models as well as the ease of managing these animals under germ-free conditions. Morphological and physiological data are lacking for the larger animal species (ruminants and pigs) because of size limitations, quantity of feed required and increased cost. Chickens have been used somewhat more extensively in germefree research. A. Survival, Growth and Reproduction of Germ-Free Animals Pleasants (1968) reviewed species differences on their abilty to survive, grow and reproduce in a germ-free environment. Germ-free rats and mice live longer than their conventional counterparts and also live longer than germ-free rabbits and guinea pigs. Mortality of germ-free guinea pigs and rabbits is lower than conventional animals in early life but is reversed for these animals later in life. Cause of mortality for all of these species is generally attributed to enlargement and distention of the cecum. This is particularly responsible for increased mortality among rabbits and guinea pigs due to the greater organ size for these species even in the conventional state. Dogs, cats and monkeys have been maintained for more than 10 months with no apparent problem. Lambs and kids have been maintained for 4 months but diet modifications were required to prevent altered mineral metabolism and kidney lesions. Pleasants (1968) classified the various species used in germ-free research based on relative growth rates compared to conventional animals of the same species. 33 Germ-Free Growth Equal to or Superior to Conventional Growth Chicken Japanese quail Monkey Pl9 Germ-Free Growth Similar to Conventional Growth Rat Turkey Mouse Calf Dog Burro Cat Germ-Free Growth Less Than Conventional Growth Guinea pig Kid Rabbit Lamb Most of the species that rely on microbial fermentation in their gastrointestinal tracts for nutritive sustenance, perform submaximally in a germ-free environment. However, germ-free ruminants were maintained only during the pre-ruminant period and were fed milk diets rather than dry feedstuffs. Landy and Ledbetter (1966) reported that conventionally reared miniature pigs weighed more at 3 weeks of age than their germ-free counterparts, but by 10 weeks, the germ-free pigs surpassed the conventional pigs in body weight. Waxler and Drees (1972) and Miller et al. (1982) observed a similar growth rate for germ-free and conventional pigs up to 3 weeks of age but no data were collected after 3 weeks. Miniats and Valli (1973) reported that conventional pigs grew about 50% faster than germ-free pigs up to 8 weeks of age but conventionally reared pigs were allowed to nurse the sow while germ-free pigs were fed a condensed cow's milk diet supplemented with Iron. 34 Pleasants (1968) noted that germ-free rats and mice have been maintained through 28 generations with no loss of reproductive capacity. Germ-free rabbits have been maintained through three generations but fertility declined with each successive generation and was finally lost due to the sensitivity of the species to inbreeding. Germ-free chickens and Japanese quail have been reproduced but not for extended periods of time. No data are available on reproductive efficiency for the larger animal species because of the physical limitations of rearing these animals through one generation. 8. Differences in Organs, Tissues and Systems Between the Germ-Free and Conventional Animal Since active defense against microbial action involves tissues, organs and systems which serve other functions, these may be altered in form or functional capacity of the germ-free animal. 1. Organ weights of germ-free pigs When organ weights of germ-free pigs were expressed as mg/lOO gm of body weight to compensate fbr differences in growth rates between germ-free and conventional pigs, the mandibular lymph nodes, adrenals, heart, spleen, kidneys, thyroid, stomach and small intestine of germ-free pigs weighed less than those organs from conventional pigs (Waxler and Drees, 1972). 2. Systemic characteristics of the germ-free animal Several systemic differences have been described when germ-free and conventional rats were compared (Pleasants, 1968; Gordon, 1968). The following characterisitcs were expressed on-the basis of percentage reduction of the germ-free level below the conventional level. 35 Basal metabolic rate 20% Iodine uptake by thyroid 50% Cardiac output (ml/min/kg BW) 32% Blood flow (mg/100 9 BW) 22% Arterial blood flow to liver 50% 3. Characteristics of the gastrointestinal tract of germ-free animals The gastrointestinal tract of germ-free rats is very different in its characteristics compared to conventional rats. Gordon (1968) reviewed several unique characteristics of the germ-free rat. The intestinal wall is thinner and the gastrointestinal tract is less well filled in the germ-free rat. The wet weight of the intestinal wall is reduced and the dry weight is increased when expressed per unit of body weight for rats in the germ-free state. Lamina propria is reduced in the germ-free rat and is probably the main cause of reduced weight of the small intestine. The pr0portion of intestinal epithelium is normal or slightly reduced. Mucosal surface area is reduced by about 30% and corresponds well to the reduction in dry matter per unit length of the intestinal wall. Renewal rates of the epithelium, lamina propria and Peyer's patches of the intestine are reduced in germ-free rats. A reduction in lamina propria may also be a cause for the reduced DNA content of the intestinal wall. An enlarged and distended cecum is usually observed and both wet and dry weight of the cecum wall is increased due to an increase in mucosa, submucosa and muscle tissue in the germ-free condition. Colon appearance is similar to conventional rOdents. Finally, alkaline phosphatase is elevated and acid Phosphatase is reduced in the epithelial lining of the cecum. 36 Germ-free rats generally consume 10% more feed and slightly more water than their conventional counterparts and cecal contents and feces are softer in consistency. The digestive tract of germ-free pigs differs from conventional pigs by its lower weight, reduced thickness and cellularity of lamina propria and muscularis, poorer development of the associated lymph tissue and by the liquid contents of the cecum and colon (Miniats and Valli, 1973). Most of these characteristics are similar to that described for germ-free rodents except that the cecum of the germ-free pig is not enlarged. 4. Digestive functions Since several characteristics of the gastrointestinal tract are different in a germ-free environment, and since gastrointestinal microflora play a role in digestive fonctions and in the nutrition of the animal, it is important to understand these differences when comparing germ-free and conventional animals. Gordon (1968) summarized research involving various factors related to digestive functions in the germ-free rat. The upper gastrointestinal tract pH of the germ-free rat is the same as the conventional rat but the lower tract of the germ-free rat is more alkaline. Osmolarity of the gastrointestinal tract of the germ-free rat is generally lower and the oxidation-reduction potential of the cecal contents is 250-300 mV more positive than conventional rats. In Gordon's (1968) review, he noted that germ-free rats excrete more fecal nitrogen which decreases the coefficient of nitrogen utilization. However, there is a 3 to 4 fold increase in nitrogen in 37 cecal contents. More specifically, the quantity of amino acids (primarily tyrosine, threonine and methionine which are characteristic of mucoproteins) are 50 to 100 times higher and there is also an increase in mucoproteins, hexosamines and urea, but histidine levels are lower in cecal contents of germ-free rats. More bilirubin is excreted in feces but no urobilin is found in urine or feces of germ-free rats. Gordon (1968) also described changes in lipid digestion and metabolism from reports by various researchers. Less fecal fatty acids were excreted in the germ-free rat and most of these fatty acids are unsaturated. Cyclical and branched chain fatty acids, which are characteristic of bacterial synthesis, were not found. Less total bile acids were excreted, and of those excreted, most was taurocholic acid which indicates a profound influence of the intestinal microflora on bile acid metabolism. Germ-free rats excreted more cholesterol while liver and serum cholesterol were increased, and systemic cholesterol metabolism was reduced. In regard to intestinal digestive enzymes, Gordon (1968) summarized research studies which indicated that feces of germ-free rats had higher levels of trypsin and invertase. Carbohydrate digestive enzymes were somewhat different in the germ-free rat. Disaccharidases (maltase, invertase. lactase, trehalase and cellobiase) were increased, and slightly more amylase was excreted in feces of the germ-free rat. In germ-free chicks, protease (trypsin fraction) and amylases were increased in the cecum, colon and cloaca while the reverse was observed for lipase. 38 Reddy et al. (1965a) observed that plasma iron and copper levels were lower, liver iron and copper concentrations were higher, while spleen, kidney and total body iron and copper levels were lower for germ-free rats compared to their conventional counterparts. Further evidence that capper and iron metabolism is influenced by the gut microflora was reported by Reddy et al. (1965b) with germ-free rabbits. Hypochromic anemia was observed in germrfree rabbits as evidenced by low hemoglobin and hematocrit levels. These rabbits had low plasma iron but normal copper levels and total iron binding capacity was increased. Iron and c0pper concentrations were lower in spleen and kidneys but higher in the liver of the germ-free rabbits. When these anemic rabbits were conventionalized, these symptoms were alleviated. Gordon (1968) reviewed evidence which indicates that the oxidation-reduction potential in cecal contents is more positive in germ-free compared to conventional animals. If this is also true for the intestinal contents, the availability of certain nutrients (such as iron) may be improved. When germ-free and conventional pigs were fed a sterile condensed milk diet from birth to 4 weeks of age, higher levels of hemoglobin and hematocrit were observed fbr germ-free pigs compared to conventional Pigs (Miller et al., 1982). Waxler and Drees (1972) noted the same relationship for hemoglobin and hematocrit in germ-free pigs receiving a condensed milk diet compared to conventionally reared baby pigs. Furthermore. mean corpuscular volume, mean corpuscular hemoglobin and serum iron tended to be greater for the germ-free pigs (Miller et al.. 1982). 39 Finally, Gordon (1968) indicated that calcium metabolism is somehow altered in the germ-free rat since a high incidence of urinary calculi has been observed. Tissue levels of manganese in germ-free rats are similar to those of conventional rats while the amount of sodium in the cecum is 6 to 10 times greater in germ-free rats due to the greatly enlarged cecum in the germ-free rats. However, the concentration of sodium in the cecum of germ-free rats was similar to conventional rats. Conversely, the total amount of potassium in germ-free and conventional rat ceca is comparable but its concentration is much lower in the germ-free rats. Germ-free rats require all known vitamins needed by their conventional counterparts. When deficiency experiments were conducted, no major differences in deficiency symptoms were observed between conventional or germ-free rats. However, vitamins such as thiamin, biotin, folic acid and vitamin K which are synthesized by gut microflora and contribute to meeting the animal's nutrient requirements in the conventional state, must be supplemented to a properly fortified diet to insure presence of adequate levels after autoclaving (Zimmerman and Wostmann, 1963; Wostmann et al., 1962). 5. Effect of germ-free environment on humoral and cellular defense mechanisms Wostmann (1968) reviewed research involving humoral defense mechanisms in the germ-free animals. In general, these animals have an a"t1b°dy production system that is quantitatively smaller, deficient in potential antibody forming cells and only partially accessible dePending upon dietary and environmental conditions. Sterilized diets 40 for germpfree animals which may have been heavily contaminated with microorganisms prior to sterilization can be antigenic. This system responds well to antigenic stimulation but prolonged stimulation may be required to overcome the original deficiency in size and cellularity. Bauer (1968) summarized the effects of rearing animals in a germefree environment on cellular defense mechanisms. MacrOphage morphology, distribution, phagocytic capacity or delivery of antigenic foreign material to the regional lymph nodes are not altered in germpfree animals. Conventional mice have a greater capacity for macrophages to process antigens due to continuous exposure to microbial flora even though these functions are equally develOped in germ-free mice. Absence of microflora stimulation causes a delayed onset of immune response in germ-free lymphatic tissue but slower digestion and release of immunogenic fragments results in a more sufficient response in conventional animals. Germ-free pigs have lower total leukocyte counts due to lower absolute lymphocyte counts compared to conventional pigs (Waxler and Drees, 1972). 6. Importance of gut microflora to growth When antibiotics are added to growing livestock diets, an improvement in growth performance is observed (Hays, 1969) . In attempts to understand the cause of this phenomenon, it was postulated that the microflora of the alimentary tract may interfere with an animal's ability to develop its full growth potential and that antibiotics (or any dietary constituent) capable of eliminating such °F9anisms, or discouraging their establishment, might have a beneficial effect on growth. 41 Several studies have demonstrated that dietary antibiotic supplementation had no effect on growth rate of germ-free chicks. Lev and Forbes (1959) introduced a strain of colstridium welchii to germ-free chicks and a growth depression was observed. However, when these monocontaminated chicks were given dietary procaine penicillin, growth depression was alleviated. No growth stimulation was observed when germ-free chicks and turkeys were fed antibiotics at a level of 50 mg/kg of diet (Luckey, 1952). Luckey et a1. (1955) later reported that low levels of antibiotics, 25 mg/kg of oxytetracycline and 11 mg/kg of procaine penicillin, produced a slight improvement in growth rate but these data are based upon a limited number of birds. A growth promoting effect in conventional but not germ-free turkeys was observed when 45 mg/kg of penicillin or 30 mg/kg or oleandomycin were added to the diet (Forbes et al., 1958). Germ-free chicks grew 15 to 25 percent faster than conventional chicks fed the same autoclaved casein-starch or soybean meal-corn diet containing 25 mg potassium penicillin or 45 mg procaine penicillin (Forbes and Park, 1959). When chlortetracycline, penicillin and streptomycin were injected into chick embryos under sterile conditions, there was no direct growth promoting action of the antibiotic on tissues of the host (Jukes and Williams, 1953; Stokstad, 1954). Furthermore, Whitehair and Thompson (1956) observed a lack of growth improvement when antibiotics were fed to pigs under germ-free conditions. MATERIALS AND METHODS 1. EFFECT OF HIGH-COPPER FEEDING TO WEANLING PIGS T0 GERM-FREE AND CONVENTIONAL ENVIRONMENTS ON GROWTH PERFORMANCE AND VARIOUS PHYSIOLOGICAL PARAMETERS. A comparative study of the effects of feeding a supra-nutritional level of dietary copper to weanling pigs reared in germ-free and conventional environments was conducted to examine potential differences in growth performance; portal and peripheral blood glucose, amino acids and ammonia levels; intestinal weight and morphology; organ weights; hematology; and tissue trace element concentrations. The study utilized two types of rearing environments, a germ-free environment and a conventional environment. Pigs were managed similarly in each environment and fed one of two autoclaved experimental diets. A. Management of Germ-Free Pigs One litter of 10 (3 gilts, 7 boars) crossbred pigs (Hampshire x Yorkshire-Duroc-Landrace) was delivered by caesarotomy on day 112 of gestation and placed in rearing isolators containing the necessary eQuipment for the 7-week germ-free portion of the trial. Refer to Appendix B for a detailed description of isolator preparation and procedures used in obtaining and successtlly rearing these germ-free pigs. Rearing isolators were marked and identified as I, II and III. Isolator I and III contained 3 pigs each (1 gilt, 2 boars) while isolator II contained 4 pigs (1 gilt, 3 boars). On day 1, all pigs were ear notched (#1 through #10) and needle teeth were clipped using Side cutters. 42 43 Pigs were bottle fed with evaporated milk (Carnation Company, Los Angeles, CA) three times daily at 6 am, 2 pm and 9 pm for 4 weeks. Nutrient analysis for the evaporated milk (Leveille et al., 1983) and a comparison to nutrient composition of sow's milk (Pond and Houpt, 1978) is shown in Table 1. Average daily milk consumption per pig fbr weeks 1 through 4 was as follows: Week 1 235 m1/pig/day Week 2 467 mllpig/day Week 3 617 mllpig/day Week 4 359 ml/pig/day ‘Total milk consumed per pig during the 28 day feeding period averaged 11.746 ml/pig which was approximately 54% of the amount consumed when pigs are nursing the sow (Hitchcock, 1973). Daily milk consumption was reduced during week 4 in order to encourage water and feed consumption before weaning, as well as to standardize pig weights with the conventionally reared pigs before imposing dietary treatments. Iron supplementation was provided by injecting 200 mg iron as iron dextran intramuscularly when pigs were 3 days of age. Rectal swabs were taken from one pig in each isolator on days 6, 21. 39 and 49 of the trial and monitored for anaerobe and aerobe bacterial growth in plated culture media. All isolators were germ-free uP to day 21 when Streptococcus faecalis was isolated from isolator III. Isolators I and 11 remained germ-free for the duration of the trial. On day 16, pigs were provided ad libitum access to water and creep feed (autoclaved control diet) and water. Composition and nutrient 44 mop om New cm a: .coaaou -1 -1 mo . m_ a: .o=_eo— mm.o o_.o m~.o m_.o me .coLH mo— om Na em as .s=.mm=moz cue om Nop— mom as .Eswmmmuoq amp on woe mc_ me .e=_eom New cop mus Now as .mscogqmoza mm__ o- moo— pom as .anopou m—mcocwz Nee— msm Pmm .uew as .ocwcpmc< New on onm ma as .cozaouaxgh wmpp o- mm__ son as .oc_=oocg» mm—_ o- wmm_ Npe as .m:_u=mpomH mum Nu new no me .o:_um»u «me mm mme .5. we .mcwcowguoz opmu oee omow saw as .ocwmxg mu_u< o=_e< o.¢ m~.o mm.o up.o m .ewuo upopo:_4 m.m o._ o.o m._ a .;m< mu o.m mm o_ m .muecexgonceu mm m.~ mm o.~ m .umm aw m.m om m.o m .=_muoc¢ can om. 2m 3: :8. .335 oo_ m_ co. mu m .Louues sue (a oopxcouuoz ago lmaloop\mp m< llwlmo—\cmuuez ago a oo—\ m_ m< u=m_eu=z nx—Pz m.:om o¥__z m.zou eouocoao>m . p xp.z n.3om we mpmh—~c< ucwwcuaz cu compounsou use x—wz m.zou umumgcnc>m ma m_mN—m=< acm—Luzz ._ m_nmh 45 .Awsmp .aqao: new ecoav xpwe m.:¢m we co_u_moasou acmwcuazn .Amma— .._e um m—_Po>mgv xpwe m.:ou uwumcoao>o Co eowu_moaeou acovcuszo as m_ N.“ m.p m5 .u_uo upncoum< p.~ Fe.o m.~ eo.o me .ewoo upcoguoucoa e.~ e._ .m o.m a: .e_oo_m mmou can Pm o.w a: .e_ue u__ou m.m ¢~.o m~.o m_.o me .=_uo¢z e~.o ¢—.o ~m.o up.o a: .mpm cpsoap> mo. ow map cm a: .ocwxou_cam a._ om.o ~.p —m.o me .cw>mpwoawm em.o no.o m_.o eo.o as .c_smwzh ¢~.o ep.o -.o o~.o 2H .u :_Emuw> mm a. com m“ 2H .o evacuw> m:_5ma_> o.~ om.c o.m -.o as .u=_N aloo_\oooooz so; waoo_\me m< a oop\ Looooz sea a oo_\ m_ m< oeoeooaz nx__x n.3om uxpwz m.zou umuucoao>m .xppz m.zom mo mvma~e=< acmwcuzz on cam_cmqeou use x__z n.3ou umpmeoa~>m yo mpmxfla=< u=m_cu=z ._ open» .46 analysis of the creep diet was the same as the experimental control diet which is shown in table 2. Pigs consumed approximately 31 g of creep feed/pig/day. Average environmental temperature was maintained at 33°C during week 1, 30‘C during week 2 and 3, 28’C during week 4 and was maintained at 27°C for the duration of the trial. Pigs were weaned on day 28 of age, weighed and fed their assigned experimental diets fOr 3 weeks. Each group of 3 pigs in isolator I and III were fed the control diet containing 16 ppm copper while the 4 pigs in isolator II were fed a high copper diet containing 250 ppm supplemental copper as copper sulfate (Table 2). Experimental diets were fbrmulated based on practical swine starter diets which are generally a corn-soybean meal-20% dried whey mixture. Furthermore, this type of diet tends to evoke a greater growth response from supplemental copper than other types of starter diets (Edmonds and Baker, 1984). However, sterilization of solid diets by steam results in partial loss of the nutritive value of proteins, particularly through its effects on lysine, methionine and cystine (Rice and Beuk, 1953). loss of certain B vitamins and also of Vitamins A and E (Zimmerman and Wostman, 1963). A series of preliminary 3-week growth performance trials with weanling pigs were conducted to compare the effect of vitamin and amino acid supplementation between autoclaved and non-autoclaved diets and to characterize the effects of autoclaving diets on growth performance and nutrient balance. The results of these trials are shown in Appendix A. Diets were formulated to meet or exceed NRC requirements (NRC, 1979) 47 and were fortified with 1% synthetic L-lysine HCl to elevate the lysine level to 1.82% in each diet before diets were autoclaved. Diet composition and analysis is shown in Table 2. Average initial weight of pigs receiving the control diet was 5.15 kg while the initial weight of high capper-fed pigs was 5.51 kg. Feed and water were provided ad libitum and each pre-weighed pan of autoclaved feed and sterilized water bottle were recorded when transferred into the isolators in order to calculate average daily feed and water consumption. Procedures used to sterilize and transfer feed and water and to transfer into isolators are discussed in detail in Appendix A. 8. Management of Conventional Pigs One litter of 5 (2 gilts, 3 boars) and one litter of 10 (7 gilts, 3 boars) crossbred Hampshire x Yorkshire-Landrace-Duroc pigs were allowed to nurse the sows for 3 days in order to obtain adequate colostrum before weaning from the sow and changing their diet to canned evaporated cow's milk. Prior to weaning, pigs were ear notched, needle teeth were removed and supplemental iron was provided by intramuscular injection of 200 mg iron dextran per pig at birth. When pigs were 3 days of age, they were removed from sows and placed in stainless steel rearing pens. Two pigs shared each pen measuring 0.75 1112 (except for one pen where 3 pigs were reared together) and were paired based on size and physical condition. The litter of 10 pigs developed diarrhea when nursing the sow and 4 out of the 10 pigs were somewhat smaller and dehydrated when weaned. P19'5 were bottle fed with evaporated milk 3 times daily at 7 am, 3 p11 and 10 pm analogous to the germ-free pigs. Average daily milk 48 consumption per pig for weeks 1 through 4 was as follows: Week 1 179 ml/pig/day Week 2 355 m1/pig/day Week 3 570 ml/pig/day Week 4 602 ml/pig/day Total milk consumed per pig during the 28 day feeding period averaged 11,941 m1/pig which was approximately the same as the germ-free pigs. During the 28 day period while pigs were receiving the evaporated milk diet, incidence and severity of diarrhea was monitored at each feeding. When diarrhea persisted among individual pigs, quantity of milk fed was slightly reduced and oral neomycin was administered to those pigs affected until diarrhea was under control. Because of the persistence of the diarrhea problem among individual pigs, a greater quantity of milk was fed to the conventional pigs during week 4 to allow them to compensate for their poorer condition, as a result of the diarrhea, in order to equalize initial weights of the conventional and germ-free pigs before imposing dietary treatments. All pigs were provided ad libitum access to water and creep feed (autoclaved control diet) identical to that provided for the germ-free pigs. Refer to Table 2 for diet composition and nutrient analysis. Pigs consumed 23 g/pig/day of the creep feed until weaning. Average room temperature was maintained at 31°C during week 1 and 29°C throughout the remainder of the trial. Supplemental heat was PPOVldEd by heat lamps located over each pen during the first 4 weeks of age which elevated the temperature in the pig's sleeping area to 49 Table 2. Diet Composition and Calculated Nutrient Analysis (Experiment 1). Dietary Treatment (%) Iggredient Int. Ref. No. Control _H1gh Copper Corn, ground shelled 4—02-935 50.90 50.80 Soybean meal, 44% 5-04-604 25.00 25.00 Dried whey 4-01-182 20.00 20.00 Mono-dicalcium phosphate 6-01-080 0.75 0.75 Calcium carbonate 6-01-632 1.00 1.00 Salt 6-04-152 0.35 0.35 Vitamin-trace mineral premixa A 0.50 0.50 Vitamin E-selenium premixa 0.50 0.50 L-lysine HCl 5-08-022 1.00 1.00 CuSO4'5H20 6-01-719 -- .10 Total 100.00 100.00 Calculated Analysis Metabolizable energy, kcal/kg 3103 3100 Crude protein, % 18.20 18.20 Lysine, % 1.83 1.83 Calcium, % 0.81 0.81 Phosphorus, % 0.62 0.62 aRefer to Appendix A (Table 28) for the amount of vitamins and trace minerals supplied per kg of diet. SO 35°C during this time period. Heat lamps were removed when pigs were weaned at 28 days of age and room temperature was maintained at 29°C throughout the duration of the trial. At weaning, pigs were weighed and the 10 most unifonn pigs were allotted by sex, litter and weight to one of the two experimental diets. Five pigs with an average initial weight of 5.97 kg were moved to a stainless steel pen measuring 1.50 m2 with a wire mesh floor and fed the autoclaved control diet, while another group of 5 pigs averaging 6.03 kg in initial weight were moved to another stainless steel pen (dimensions of 1.50 m2) and fed the autoclaved experimental diet containing 250 ppm supplemental capper. Both stainless steel pens were of the same dimensions and located in the same room where pigs . were previously fed the milk diet. Diet composition and nutrient analysis is shown in Table 2. Feed and water were provided ad libitum and the amount of feed fed was weighed and recorded before adding to the feeders in order to calculate average daily feed consumption. C. Collection of Data and Laboratory Analyses 1. Determination of growth performance criteria At the completion of the 7-week trial, pigs were weighed and individual weights were recorded. Any feed remaining in feeders was weighed and subtracted from total feed fed during the 3-week experimental period. Average daily weight gain, average daily feed consumption and feed utilization efficiency was calculated. Individual .average daily gain valueS‘were statistically analyzed using l=ederer-Zelen factorial analysis for unbalanced data (Gill, 1978). ‘Sinnce average daily feed consumption and feed utilization efficiency VE'IIlueS‘were pen values, and since there were no replications, these ‘Paxrameters could not be analyzed statistically. 51 2. Collection and preparation of blood sadlples for laboratory analyses Peripheral blood samples were taken by vena puncture from the anterior vena cava prior to anaesthetizing each pig. Before all remaining blood and tissue samples were collected, each pen or isolator of pigs was fed a meal 3 1/2 hours before anaesthetizing pigs to obtain blood and tissue samples. After 3 1/2 hours, pigs were anaesthetized by injecting iv approximately 6 cc of sodium pentobarbital (Sodium Pentobarbital Injectable, Butler Co., Columbus, OH) and state of anaesthesia was closely monitored to maintain a constant level of sedation. A vertical incision was made on the ventral side, approximately 25 cm from the posterior portion of the sternum with a scalpel. The portal vein was identified and approximately 20 ml of blood was drawn and placed in heparinized plastic tubes used to harvest plasma. An additional 10 m1 of blood was placed in tubes containing EDTA for hemotology determinations. After mixing blood with heparin (both portal and peripheral blood). the blood samples were placed in a centrifuge and spun for 15 minutes at 2000 x 9. Plasma was harvested and stored temporarily in coolers containing ice fbr subsequent laboratory analyses. Plasma samples for glucose, ammonia and ceruloplasmin determinations were analyzed immediately while plasma samples for amino acid and trace element determinations were frozen for later analyses. Hematology determinations were performed immediately. All data were analyzed statfistically using Federer-Zelen factorial analysis of variance for unbalanced data (Gill, 1978). 52 a. Hematological determinations Hemoglobin was determined by the cyanmethemoglobin method of Crosby et a1. (1954). Hematocrit was measured by the micro-method described by McGovern et al. (1955). Reticulocytes were enumerated per 1000 erythrocytes in blood smears stained with methylene blue on the hemocytometer. For leukocytes, blood was diluted using Turk's solution as a diluent and total leukocyte counts were determined with a hemocytometer. Differential leukocyte counts were determined by preparing a blood smear with Wright's stain and counting and differentiating 100 leukocytes. b. Plasma trace elements Plasma capper, iron and zinc concentrations were determined using atomic absorption spectrophotometry (Model 951, Instrument Laboratory, Inc., Lexington, MA). Capper and zinc concentrations were measured by diluting the plasma samples with appropriate amounts of distilled deionized water to obtain plasma zinc concentrations in the range of 0.1 to 1.0 ppm and copper concentrations in the range of 0.25 to 2.0 ppm. Plasma iron was determined by first deproteinizing the plasma with 20% tricarboxylic acid. After centrifugation the deproteinized supernatant was diluted and measured in the range of 1.0 to 5.0 ppm. Wavelengths used for maximum absorption were 213.9 nm, 324.7 nm and 248.3 nm fer zinc, copper and iron, respectively. c. Plasma ceruloplasmin determinations Plasma ceruloplasmin oxidase activity was measured with fresh samples according to the procedure of Smith and Wright (1974). 53 This procedure involves adding 2.4 ml of buffer (containing 0.8M sodium acetate AR which contained 3.19 EDTA disodium salt/liter and was adjusted with acetic acid to pH 6.5) to 0.5 m1 plasma. Samples were incubated at.37°C for 5 minutes and the substrate p-phenylenediamine dihydrochloride was added and samples were mixed. Change in absorbance was recorded at 540 nm wavelength on a spectrophotometer (Beckman-Gilford Spectrophotometer, Model 2400, Beckman Instruments, Inc., Fullerton, CA and Gilford Instrument Laboratories, Inc., Oberlin, ON). A blank sample was used to correct for substrate autooxidation in the absence of plasma. d. Plasma ammonia determinations Plasma ammonia concentrations were determined by using an ammonia diagnostic kit (Sigma Diagnostics, P.0. Box 14508, St. Louis, MO 63178). This procedure is based upon the reductive animation of 2- oxoglutarate using glutamate dehydrogenase and NADH. The decrease in absorbance at 340 nm on a spectrophotometer (Beckman-Gilford Spectrophotometer, Model 2400, Beckman Instruments, Inc., Fullerton, CA and Gilford Instrument Laboratories, Inc., Oberlin, OH), due to the oxidation of NADH. is proportional to the plasma ammonia concentration. e. Plasma glucose determinations Plasma glucose concentrations were determined using a glucose diagnostic kit (Sigma Diagnostics, P.O. Box 14508, St. Louis, MO 63178). This procedure is based upon the hexokinase method where gglucose is converted to glucose-G-phosphate by hexokinase. This reaction is coupled with the subsequent reduction of NADP to NADPH by 54 the action of glucose-6-phosphate dehydrogenase. In the presence of NADPH, phenazine methosulfate (PMS) is reduced to PMSH which is responsible fbr the reduction of iodonitrotetrazolium chloride (INT) to form INTH which is measured colorimetrically on a spectrophotometer (GilfOrd Instrument Laboratories, Inc., Oberlin, 0H) at 520 nm. The concentration of INTH is proportional to the plasma glucose concentration. f. Plasma amino acid determinations Two milliliters of plasma supernatant were added to each labelled Cortex centrifuge tube and 0.2 ml of’l mM norleucine-SB-(4 pyridyl:ethyl)-l cysteine was added as an internal standard to each tube and tubes were mixed on a vortex. Next, 0.2 m1 of 50% sulfosalicylic acid was added to each tube to deproteinize the plasma proteins. Tubes were then mixed and placed on ice for 30 minutes. Finally, samples were centrifuged at 20,000 x g for 20 minutes, supernatant was removed with a Pasteur pipet and placed in storage vials. Samples were frozen until amino acid analyses could be performed. When amino acid analyses could be performed, plasma samples were thawed and filtered through 0.5 um filter paper and were diluted 1:3 with 0.01 N HCl, mixed and put into vials. Individual plasma amino acids were determined by ion exchange chromatography using a lithium citrate buffer system and post column derivitization with ninhydrin. 3. Preparation of intestinal sections for histological determinations Three-5 cm intestinal sections were isolated by tying a piece (If suture material at each end of the intestinal section to be excised. 55 Once isolated, approximately 7 ml of a solution consisting of formalin-mercuric chloride was injected with a 23 gauge, 2.5 cm needle and syringe to uniformily distend each intestinal section. The formalin-mercuric chloride solution was used to quickly fix the living tissue before atrOphy could occur. Each of the three intestinal sections were excised and placed in a labelled beaker containing the fixing solution. Intestinal segments were removed starting with the duodenum near the pylorus and extending down the duodenum 5 cm, the most posterior portion of the ileum (adjacent to the ileal-cecal valve) and a section of jejunum approximately 155 cm cranially to the ileal-cecal valve. All samples were then taken to the laboratory for further preparation. Tissues were fixed for approximately 24 hours. Ends of the intestinal loops were then cut off, washed with tap water, trimmed and stored in 80% alcohol. Tissue buttons were then placed in labelled paraffin blocks, sliced and mounted on slides for examination under the microscope. a. Intestinal histological determinations Histologic differences due to dietary treatment and environment were determined by taking linear measurements of intestinal wall structures. Comparisons of three locations of the small intestine (duodenum, jejunum, ileum) were made by measuring villus height, villus *width, crypt depth, depth of the submucosa and total thickness using an ocular micrometer. Three representative samples from each intestinal section of each pig were examined, and 25 measurements for each parameter were made and then pooled. Mean values were calculated for each parameter for each pig and analyzed statistically using 56 Federer—Zelen factorial analysis of variance for unbalanced data (Gill, 1978). 4. Body organ weights and preparation of organs and cecal contents for laboratory analyses The pigs were then euthanized by giving an overdose of sodium pentobarbital. Various organs were then removed, weighed and put in plastic whirlpak bags and frozen for later laboratory analyses. Organs evaluated include: Liver Heart Lungs Kidneys Thyroid Gall bladder Spleen Thymus Stomach-empty Adrenals Pancreas Small intestine-empty Large intestine-empty Cecal contents were frozen in plastic whirl pak bags for ammonia and urea determinations. a. Determination of ammonia and urea in cecal contents Ammonia and urea nitrogen determinations were conducted for cecal contents by using diagnostic kits for ammonia and urea (Sigma Diagnostics, P.0. Box 14508, St. Louis, MO 63178). Frozen cecal content samples were thawed, weighed and diluted with water. After centrifugation, supernatant was deproteinized with trichloracetic acid (and change in absorbance (compared to blanks) was recorded after reagents were added to the sample. The change in absorbance was proportional to the ammonia or urea concentrations. The procedure was verified by comparing values obtained with those of Combe et a1. (1965) and were very similar. 57 II. INFLUENCE OF COPPER AND CHLORTETRACYCLINE 0N MICROBIAL ‘FERMENTATION PATTERNS AND MICROBIAL POPULATION GROWTH IN FIVE SECTIONS OF THE GASTROINTESTINAL TRACT IN VITRO. A. Preparation of Culture Tubes Prior to the start of the experiment, 1250 glass culture tubes (18 mm 0.0. x 150 mm long, 29 m1) obtained from (VWR Scientific Inc., Subsidiary of Univar, P.O. Box 3200, San Francisco, CA 94119) were sterilized by autoclaving at 121°C for 30 minutes. Rubber stappers were immediately placed in the glass culture tubes after sterilization to minimize contamination. B. Preparation of Media Two types of media were formulated and prepared to conduct this in vitro study: 1. anaerobic dilution medium. 2. non-selective anaerobic growth medium. Several reagents were prepared prior to mixing of the two types of media. First, two mineral buffer solutions were made and eventually added to the media. Chemical composition of these mineral buffer solutions is shown in Table 3. Next, an 8 percent NaZCO3 solution was prepared by weighing 24 g Na2C03 and dissolving in 300 ml distilled water. Sodium carbonate solution was then poured into a 1000 m1 round bottom flask and the flask was sealed with a cotton plug. The flask containing the IVa2C03 Solution was then autoclaved at 121°C for 30 minutes, cooled and pipetted into sterile test tubes in 10 m1 quantities after equilibrating with C02 gas fbr 30 minutes. 58 An ascorbic acid solution (2.38%)*was prepared by weighing 7.125 g of ascorbic acid and adding to 300 ml of distilled water. Ascorbic acid was used as the reducing agent for both the dilution and growth media even though it is a less powerful reducing agent than cysteine HC1 and other reducing agents containing sulfdryl groups, because it allowed the copper to remain in solution unlike these other reducing agents. After pouring the solution into a 1000 ml round bottom flask, the ascorbic acid solution was slowly boiled under C02 gas and a #7 rubber stopper was used to seal the flask as the C02 gas was removed. The rubber stopper was fastened in place with wire that was tightly twisted over the rubber stapper and around the neck of the flask to avoid the stopper from popping out during the autoclaved process (which would allow contamination by microbes and oxygen). After the ascorbic acid solution was sterilized by autoclaving at 121°C for 30 minutes, the flask was allowed to cool. The wire holding the rubber stopper in place and the rubber stapper were removed, and the contents of the flask were immediately pflaced under C02 gas. Ascorbic acid solution was then pipetted in 10 m1 quantities into sterile test tubes for later use. The pH was adjusted to neutrality by adding 10 N NaOH prior to boiling under C02 and sterilization. Once these solutions were made, the two types of media used in this experiment were prepared. 1. Anaerobic dilution media One thousand milliliters of anaerobic dilution medium was prepared in 500 m1 quantities by mixing ingredients shown in Table 4 with distilled water to provide a total volume of 465 ml. After adJUSting the pH to approximately 6.75 by adding a few draps of lON 59 Table 3. Composition of Mineral Buffer Solutions Added to Dilution Media and Non-Selective Growth Media (g/l distilled H20). Mineral Buffer 1 Mineral Buffer 2 Disti11ed H20 988.00 977.96 KzHPO4 6.00 -- KH2P04 6.00 6.00 NaCl -- 12.0 MgSO4'7H20 -- 2.45 CaClpfgflpo -- 1.59 Table 4. Composition of Anaerobic Dilution Media Compound (m1) Distilled H20 427.00 Mineral Buffer l 18.75 Mineral Buffer 2 18.75 Resazurin 0.50 Total 465.00 60 NaOH, media was added to 1000 m1 round bottom flask and boiled slowly under C02 gas (which was reduced by passing through a hot copper column) to remove the soluble oxygen present in the media. As the media cooled, a #7 rubber was used to seal the flask as the C02 gas was removed. Rubber stoppers were held in place by using wire to keep the flask sealed during sterilization (autoclaving at 121°C for 30 minutes) and during subsequent cooling prior to dispensing into sterile culture tubes. Once cooled, the rubber stoppers were removed and the media was placed under C02 gas. Previously prepared sodium carbonate and ascorbic acid solutions were added from sterile culture tubes to each flask containing 465 ml of dilution medium, (under C02 gas) in 25 and 10 ml quantities, respectively, to provide a final volume of 500 m1 of media in each flask. Carbon dioxide gas was then bubbled into the media through a glass pipette to mix the Na2C03 and ascorbic acid with the media for 10 minutes. Medium was then dispensed into sterile test tubes in 18 m1 quantities, under C02 gas, and tubes were tightly sealed with rubber stoppers. 2. Non-selective anaerobic growth media Composition of non-selective anaerobic growth media is shown in Table 5. A total of 5500 ml of anaerobic growth media was Prepared in 465 m1 quantities and the pH was adjusted to 6.75 with 10 N NaOH. After boiling growth media under C02 gas, flasks were sealed with rubber stoppers and autoclaved for 30 minutes at 121°C. Once the media was cooled to room temperature, 25 m1 of Na2C03 were added to each flask of media and mixed while under C02 gas. Media was then dispensed. in 8 m1 quantities, into 640 sterile culture tubes. Next, 0.2 m1 of previously prepared ascorbic acid was added to media 1" 93°" Table 5. Composition of Non-Selective Anaerobic Growth Media. 61 Inmedient (11) Distilled H20 91.48 Glucose 0.05 Starch 0.05 Cellobiose 0.05 Urea 0.10 Trypticase 0.50 Mineral buffer 1 3.75 Mineral buffer 2 3.75 Yeast extract 0.10 Resazurin 0.10 Na2C03 0.05 Ascorbic acid 0.02 Total 100.00 62 tube. Each of four treatment solutions (H20, CuSO4'5H20, chlortetracycline, CuSO4-5H20 + chlortetracycline) were filter sterilized through 0.45 pm pore filters (Nalge Co., Division of Sybron Corp., Rochester, NY 14602). Each treatment solution was added in 0.3 m1 quantities, to each of 160 tubes containing media to provide a total volume of 8.5 m1 of media in each of the 640 culture tubes. One hundred sixty culture tubes of each treatment contained either the control (distilled H20), 250 ppm capper, 100 ppm chlortetracycline, or 250 ppm copper and 100 ppm chlortetracycline. The addition of ascorbic acid and treatments to culture tubes was made at least 12 hours before the start of the experiment. All tubes were labelled according to treatment, source of inocula, replicate and pig number. An additional 8 culture tubes containing each of the treatments were. prepared to serve as blanks used for determining optical density readings, initial pH, ammonia, and organic acid profiles. C. Inoculation of Culture Media Four littermate crossbred pigs (2 gilts, 2 barrows) were weaned at 15 days of age and fed a typical corn-soybean meal-20% dried whey starter diet containing no supplemental copper or antibiotic (Table 6). These pigs did not receive creep feed or sow feed while nursing the sow so that they had not been exposed to antibiotics or high capper diets prior to the experiment. Two of the 4 pigs were fed the starter diet for 3 weeks while the other 2 pigs received the diet for 4 weeks. To obtain microbes from the gut contents and gut wall, 939" pair of pigs (1 gilt and l barrow) were euthanized by giving an overdose of sodium pentobarbital and were then exsanguinated before removing the digestive tract. Pigs were euthanized in pairs to allow 63 Table 6. Diet Composition and Calculated Nutrient Analysis (Experiment 2). ,Igggedient Int. Ref. No. (%) Corn, ground shelled 4-02-935 51.61 Soybean meal, 44% 5-04-604 25.00 Dried whey 4-01-182 20.00 Mono-dicalcium phosphate 6-01-080 0.75 Calcium carbonate 6-01-632 1.10 Salt 6-04-152 0.35 Vitamin-trace mineral premixa 0.50 Vitamin E-selenium premixa 0.50 L-lysine HCl 5-08-022 0.19 Total 100.00 Calculated Analysis Metabolizable energy, kcal/kg 3127 Crude protein, % 18.26 Lysine, % 1.20 Calcium, % . 0.85 Phosphorus, % 0.60 aRefer to Appendix A (Table 28) for the amount of vitamins and trace minerals supplied per kg of diet. 64 better management of inoculating tubes and collecting data. As a result, the first pair of pigs averaged 7.6 kg in body weight while the second pair of pigs, euthanized one-week later averaged 10.3 kg in body weight. Once the digestive tract was removed from each pig, each of five sections were removed by first removing the mesentery and stretching out the gastrointestinal tract, followed by using monofilament line to separate and tie each section from the other sections. The five sections studied and removed were: stomach, a 15 cm section of the first half of the small intestine obtained at 305 cm caudally from the pyloric valve, a 15 cm section caudally of the second half of the small intestine obtained at 245 om cranially from the ileal cecal valve, cecum, and a 15 cm section of the colon obtained 90 cm cranially from the rectum. Average gastrointestinal length was 1050 cm. Once the sections were removed, carcasses were disposed and gut sections were brought to the laboratory for further preparation. Two grams of gut contents from each gut section were weighed and immediately put into previously prepared culture tubes containing 18 ml of anaerobic dilution media while under C02 gas. Once the stappers were replaced, the inocula and medium were thoroughly mixed by shaking tubes after inoculation. A 2 x 2 cm section of each intestinal site was measured, cut and thoroughly washed with sterile physiological saline solution to remove any gut contents associated with the gut wall. Each piece of intestinal tissue was then placed into dilution thbes, while under C02. and mixed vigorously to inoculate the media. After the contents and tissues were placed in dilution tubes, and 65 mixed, tubes were allowed to stand for 1 hour to allow the particulate matter or tissues to settle. Each dilution tube was then reapened, placed under C02 gas and an additional 2 m1 of filter sterilized water (filtered through a 0.45 pm pore filter) was added and mixed to provide an additional 2 ml volume of fluid necessary to provide enough innocula to inoculate all culture tubes. Using a 1 m0 pipette, 1 ml of inocula from each microbe source was then added to one of sixteen treatment tubes (4 treatments of 4 replicates/treatment). D. Data Collection and Laboratory Analyses 1. Determination of microbial p0pu1ation growth Once all of the treatment tubes were inoculated from each pig, initial optical density readings were recorded on a spectrophotometer, Spectronic 70, (Bausch and Lamb Analytical Systems Division, 820 Linden Ave., Rochester, NY 14625) by zeroing the meter using a blank tube containing each of the four treatment media, but no inocula, to adjust for color differences. After initial readings were recorded, tubes were incubated at 38°C for 4 hours until the next reading was taken. This process continued in 4 hour intervals for a 24 hour period when the last reading was recorded. 2. Determination of pH At the end of the 24 hour incubation period, all treatment tubes were used to measure final pH with a pH meter Model PHM64a (The London Company. Westlake, OH). Each set of blank tubes, corresponding to each ‘treatment were also measured for pH, which served as a reference for the initial pH of the media before inoculation and incubation. After the pH was measured and recorded, all tubes were acidified by adding 66 0.25 ml of concentrated HCl to arrest microbial growth and fermentation. Samples were then immediately frozen until preparation and analyses could be performed for ammonia nitrogen and volatile and non-volatile organic acids. Changes in pH, after incubation, were statistically compared using a completely randomized design, 3-factor analysis of variance for balanced data, with 2 fixed factors (microbe source and treatment) and 1 random factor (pigs) (Gill, 1978). 3. Determination of ammonia nitrogen Ammonia nitrogen was measured according to the semi-automated method described in A.0.A.C. (1984) with an automatic analyzer where a NH3-salicylate complex is read in a flow cell at 660 nm wavelength. Samples were prepared for analysis by thawing and adding 0.5 m1 of 9N H2504 to 2.5 ml of fermented media. Samples were mixed and then 6 m1 of distilled, deionized water was added, mixed and centrifuged at 43,500 xg for 15 minutes (Sorvall Superspeed Centrifuge Model RCZ-B, Sorvall, Inc., Norwalk, CT 06852) in Corex glass centrifuge tubes. The supernatant was then transfered to disposable plastic tubes for analysis on the Technicon Auto-analyzer with appropriate standards. Data were statistically analyzed as previously described for pl changes. 4. Determination of fatty acid butyl esters Both volatile and non-volatile fatty acids were determined simultaneously by using the procedure of Salanitro and Muirhead (1975) which is a quantitative method for gas chromatographic analysis of short-chain monocarboxylic and dicarboxylic acids in fermentation 67 media. One ml of each fermented media sample was placed in culture tubes, frozen in an alcohol-dry ice bath, and placed in a continuous freeze drier for 12 hours. Chloroform (0.8 ml) and 0.2 m1 of 1-butanol, saturated with anhydrous HCl were added to the dry salts of acids. After mixing with a Vortex, tubes were tightly capped and heated at 80°C for 2 hours. Tubes were then cooled to room temperature and 0.2 ml of trifluoroacetic anhydride (TFA) was added to each tube and allowed to react for 1 hour. The TFA was used to react with hydroxy acids forming the trifluoroacetyl esters and to react with any excess butanol in the mixture. Samples were then washed twice with 1 ml aliquots of distilled water to remove excess TFA reagent, and the water layer was discarded. The 1 mfl chloroform layer, which contained the butyl esters, was placed in vials and sealed. Two microliters of each sample was injected and analyzed by gas chromatography (Hewlett Packard Model 5840A, Route 41, Avondale, PA 19311) by using a 183 cm x 0.32 cm custom packed column packed with Chromosorb W (80100 mesh, HP DMCS, AW) coated with 10% Dexsil 300 GC (Supelco Inc., Supelco Park, Belefonte, PA 16823). Data were statistically analyzed as previously described for pH changes. RESULTS AND DISCUSSION 1. EFFECT OF HIGH COPPER FEEDING TO WEANLING PIGS IN GERM-FREE AND CONVENTIONAL ENVIRONMENTS ON GROWTH PERFORMANCE AND VARIOUS PHYSIOLOGICAL PARAMETERS A. Growth Performance Germefree pigs grew at a faster rate (P<.001) than conventionally reared pigs (Table 7). Landy and Ledbetter (1966) reported that conventional miniature pigs were heavier at three weeks of age than their germ-free counterparts, but by 10 weeks of age, the conventional pigs weighed slightly more than the germrfree pigs. A diet by environment interaction (P<.O7) was observed for average daily gain (ADG) (Table 7) indicating that feeding the high capper diet improved ADG in conventionally reared pigs but decreased ADG in germ-free pigs. In other words, feeding 250 ppm supplemental copper appears to allow the conventional pig to grow at a rate more like that of the germrfree pig by reducing antagonistic effects of microbiota in the conventional environment. The reduction in ADG for germ-free pigs fed the high copper diet may have resulted from the development of a moderate copper toxicity (Sections 8, C, D, E). In regard to the development of a moderate copper toxicity state in the germ-free pigs receiving the copper supplemented diet, Ritchie et al. (1963) demonstrated that conventional pigs receiving diets containing supplemental copper in excess of 250 ppm, without iron or zinc supplementation, developed a copper toxicity state. Bunch et al. (1963) and Suttle and Mills (1966) rePorted similar results. In a copper toxicity, liver copper levels may'range between 800-2500 ppm on a dry matter basis (Wallace, 1968) 69 with a concommitant decrease in liver iron (Bunch er al., 1963; Suttle and Mills, 1966). Hemoglobin and hematocrit are reduced (Hoefer et al., 1960; Ritchie et al., 1963), serum copper levels are increased and feed intake and growth rate are depressed (Suttle and Mills, 1966). Table 7. Effect of Diet and Environment on Growth Performance. Treatment Combinationb Parametera GCo GCu CCo CCu MSEe Avg. initial wt., kg 5.27 5.51 ‘5.97 6.03 - Avg. final wt., kg 10.20 9.60 8.10 8.57 - ADG, gc.d 223 195 102 126 102 ADFI, g 520 500 320 340 - FIG 2019 2054 3012 2.82 - aADG=average daily gain. ADFI=average daily feed intake. F/G=feed/gain. bG=germ free, C=conventional, Co=control diet, Cu=250 ppm capper diet. cEnvironment effect (P<.001). dEnvironment x diet interaction (P<.O7). eMSE = mean square error. No statistical comparison could be made for average daily feed iratake (ADFI) and feed efficiency (F/G). However, germ-free pigs tended to consume more feed than conventionally reared pigs, and feeding the high c0pper diet tended to depress ADFI in germefree pigs but increase ADFI for conventional pigs (Table 7). Similarly, 7O germ-free pigs tended to be more efficient in converting feed to body weight gain than conventional pigs, and feeding the high copper diet tended to increase F/G in germ-free pigs but decrease F/G in conventional pigs (Table 7). During the course of the trial, the conventional pigs required an extended period of time fOllowing weaning, to adjust to the experimental diets, which may have confounded differences in ADG observed between germrfree and conventional pigs. 8. Hematology Hemoglobin and hematocrit values were higher (Table 8) for germ-free pigs than conventional pigs (P<.001), which is consistent with reports by Waxler and Drees (1972) and Miller et a1. (1982). Perhaps a lower blood volume, which has been observed in germ-free animals (Gordon et al., 1963). may be responsible fbr a higher concentration of hemoglobin and hematocrit observed in germ-free pigs in this study. No differences between germ-free and conventional pigs were noted for erythocyte number. Hematocrit (P<.01) and erythrocyte count (P<.06) were reduced and hemoglobin tended to be reduced by feeding the high copper diet (Table 8). Hemoglobin and hematocrit values are generally reduced by feeding high copper diets (Bunch et al., 1961; Wallace et al., 1960; Ritchie et al., 1963) but not in every case (Kline et al., 1972; Castell and Bowland. 1968). Gipp et a1. (1974) showed that the hypochromic microcyti c anemia induced by high dietary copper was due to an i mpairment of iron absorption. A decrease in hemoglobin and hematocrit and a reduction in plasma and liver iron concentrations (Section C and 51. may have resulted in a reduction in erythrocyte count due to an 71 .Aop.Aav peacomepe xpueou_ew:u_m accumz .mocou_e_cm_m .eu_um_aoum mo _o>m—uo=po> an .=o_uuocmu=_ um_eiu=mscoc_>:mnoxu .uumewm “open: .uuoemm acoscocp>couuu .Locco ocuacm ceosummzn .uopu cmaqou sag ommuau .um_e pocucouuou .peeowu=m>couuu .woLCIEmeuue moo. mz mz m_.o op.o oe.o m~.o oo.c a .moosoeeeoseo emcee—oez mz mz mz oe.o o~.o o~.o mm.o oe.o a .m_eeeemem mz mz «o. mm._ oo._ om.~ mm." oe.~ a .ePPeeee_mem .oo. .oo. .oo. m_.e oe.e o..e _.mp mm.m a .moesoeeez mz mz .o. m.me m.me e.mm e.we m.em a .mooxoeeeesa ea. mo. mz P~.e om.o oo._ mm.e o_.o a .e_eeeeee=oe eeem mz mz .co. m.om m.me m.em e.e~ “.mm a .e__eeeee=oe eoeeoemom mz mz .o. e.~_ e~e~_ oeem_ oe_m eoea mee\m__oo .oooaoeceos mz mo. mz __e wee m_m owe awe mee\eo_ x mppmu .mmuxuocguaLm mz .o. .oo. _e.. e._m e.~m e.em ~.em a .oeeoeeeeoz mz mz .oo. om.o o.op m.o_ _.__ e.__ _e\m .e_ee_meeoz axe a u now: sou ego sue ego cocoEeeee cospo> a oooeeoee_euem .eo_om_eeem o:o_uocpnsau aeoEHemcp .mwopouueoz co acmeeoc_>:m use even mo gummeu .m opnmh 72 antagonistic effect of the high copper diet on normal iron utilization. In other words, feeding the high copper diet to pigs may have resulted in a reduction of iron absorption which could reduce iron incorporation into reticulocytes resulting in a reduction of the number of mature erythrocytes present. Germ-free pigs had fewer leukocytes (P<.0l) than conventional pigs (Table 8) which is in agreement with previous reports (Waxler and Drees, 1972; Miller et al., 1982). Since leukocytes are involved in the immune system, it would be expected that total leukocyte number would be lower in germ-free pigs because of the lack of infectious agents in the environment. Feeding a high dietary level of copper also tended to reduce total leukocyte number. Differentation of leukocytes is used in diagnosis of immunological status of the body. Leukocytes are typically differentiated into the various constituent classes which include: l) segmented (mature) neutrophils, 2) band (immature) neutOphils, 3) lymphocytes, 4) monocytes, 5) eosinophils, 6) baSOphils. Germ-free pigs had a lower proportion of mature (segmented) neutrophils (P<.001) than conventional pigs which is reflected by the lack of antigenic stimulation of pigs reared in a germ-free environment (Table 8). NeutrOphils (microphages) participate in the initial stages of inflammation and destroy invading agents by phagocytosis (Schalm, 1955). In severe infections, immature (band) neutrophils appear in the Peripheral blood but are not capable of participating in phagocytosis of antigens. Waxler and Drees (1972) observed an increase in the number of neutrophils but had no explanation for this occurence. 73 Feeding high c0pper diets to pigs in the germpfree environment increased the percentage of immature (band) neutrOphils but decreased the percentage of these cells in the conventional pig (P<.04). In conditions of stress, there is an increase in ACTH which causes a decrease in eosinophils and lymphocytes but an increase in neutrOphils (Gordon, 1955). Perhaps during the last few days of the trial, germ-free, high capper fed pigs were too crowded in their isolator so that stress (increased ACTH) stimulated a rapid rate of neutrophil production which had not matured before blood samples were obtained. It is unclear why the percentage of lymphocytes was increased (P<.Ol) for germ-free pigs compared to conventional pigs as observed in this study (Table 8). Lymphocytes are involved in antibody formation and decrease during the initial phases of infection, but return to normal levels during convalescence (Schalm, l965). Lymphocytes originate from the thymus, spleen and lymph nodes and are widely distributed throughout the body (Schalm, 1965). Naxler and Drees (1972) indicated that lymph nodes of pigs raised in the presence of microbial flora are much more active than in germpfree pigs and that the degree of activity of the lymph nodes is generally correlated with the lymphocyte counts of the peripheral blood. These researchers consequently observed a lower percentage of lymphocytes in germ-free pigs compared to conventional pigs. Percentage of monocytes (Table 8) was dramatically increased for QBFM-free pigs fed the high copper diet (P<.00l), and this resulted in germ-free pigs having a higher percentage of monocytes (P<.001) Compared to conventional pigs. There was also a diet by environment interaction (P<.001) indicating that feeding the high copper diet 74 increased the percentage of monocytes in germ-free pigs but decreased the percentage of these cells in conventional pigs fed the same diet (Table 8). As indicated in Sections C, D, and E, a moderate copper toxicity developed in germ-free pigs fed the high capper diet. Copper poisoning has been shown to result in the development of a hemolytic crisis when liver copper concentrations are high (Schalm, 1965). In this situation, when pigs in this condition are stressed, copper is released from the liver into the blood resulting in rapid hemolysis of erythrocytes (Gordon and Luke, l957). As previously mentioned the germ-free pigs receiving the high copper diet may have been subjected to stress during the last few days of the trial and this, coupled with a high concentration of copper in the liver may have resulted in a hemolytic crisis. As a result, more monocytes may have been produced. to remove the erythrocyte remnants resulting in the response observed in this trial. C. Plasma Copper, Zinc and Iron Concentrations Germ-free pigs had higher peripheral and portal plasma concentrations of copper, zinc and iron (P<.00l) than their conventional counterparts (Table 9). Reddy et al. (l965b) observed a reduction in hemoglobin, hematocrit and plasma iron and an increase in Plasma total iron binding capacity in germ-free rabbits fed either of two steam sterilized diets which proved adequate when fed to conventional rabbits. When these germ-free animals were conventionalized, these parameters returned to normal. These authors concluded that iron availability was reduced in the absence of gut microflora. In rats, however, there were only slight indications that 75 iron is less available in the absence of gut flora (Reddy et al., 1965a). These researchers reported that germ-free rats had lower Spleen and kidney iron concentrations but liver iron levels were increased compared to conventional rats receiving the same diet. Hemosiderin and ferritin iron concentrations were similar for rats reared in both environments. Copper levels followed the same pattern as for iron. Furthermore, Geever and Levenson (l964) observed dense foci of an iron containing pigment in the tunica propria of conventional but not germrfree pigs, suggesting that gut microflora may influence iron metabolism. More copper, zinc and iron appeared to be absorbed by germpfree pigs (P<.001) than by conventional pigs as indicated by portal element concentration (Table 9) and liver copper, zinc and iron concentrations (Table ll). Wostmann and Bruckner-Kardoss (l966) and Phillips et al. (1959) showed that the cecal contents of germ-free rats and guinea pigs have a more positive oxidation-reduction potential than their conventional counterparts. If this is also true for the intestinal contents of germ-free animals, then the availability of certain nutrients (such as copper, zinc and iron) may be affected. Perhaps a more positive oxidation-reduction potential in the intestinal contents Of germ-free pigs allowed greater copper, zinc and iron absorption observed in this study. Feeding the high copper diet increased peripheral plasma copper (P<-001) (Table 9). In general, plasma copper appears to be a poor criterion from which to assess copper status in the pig since Buntain (1951) observed no differences in plasma copper levels between nonmal Pigs and those dying from copper toxicity. Other reports substantiate 76 Table 9. Effect of Diet and Environment on Plasma Copper, Zinc and Iron. Treatment Combinationa Statistical Significancec P Valued Parameter GCo GCu CCO CCu nssb E 0 ,:g50 Peripheral plasma Cu. ug/dl 184 270 119 149 861 .001 .001 .06 Zn. ug/dl 84 90 62 63 141 .001 NS NS Fe. ug/dl 159 83 131 156 576 .06 .03 .001 Portal plasma Cu. u9/d1 187 252 126 167 763 .001 .001 NS Zn. u9/dl 106 113 66 67 348 .001 NS NS Fe,‘gg[d1 167 98 133 145 590 NS .02 .003 aG=germ-free, C=conventiona1, Co=control diet, Cu=250 ppm c0pper diet. bMSE=mean square error. cE=environment effect, D=diet effect, ExD=environment-diet interaction. dP value=level of statistical significance, NS=not significantly different (P>.10). Table 10. Effect of Diet and Environment on Portal and Peripheral Blood Plasma Ceruloplasmin. Treatment Combinationa Statistical Significancec P valued Parameter GCO GCu CCo CCu MSEb E 0 ExD Ceruloplasmin, AA/min/ml Portal plasma .463 .606 .338 .264 .014 .001 NS .07 Peripheral plasma .478 .560 .339 .300 .020 .001 .29 .1] aG=germ~free, C=conventional, Co=control diet, Cu=250 ppm copper diet. bMSE=mean square error. cE=environment effect, D=diet effect, ExD=environment-diet interaction. dP va1ue=1eve1 of statistical significance, NS=not significantly different (P>.10). Table 11. Effect of Diet and Environment on Liver Capper, Zinc and Iron (DM basis). Treatment Combinationa Statistical Significancec P Va1uea Parameter GCO GCu CCo CCu useb E 0 ExD Cu. 90m 63 1911 15 537 7436 .001 .001 .001 Zn. Ppm 436 969 105 104 13124 .001 .001 .001 .Eg. ppm 155 93 126 112 773 NS .01 .01 aGfgerm-free, Csconventional, Co=control diet, Cu=250 ppm copper diet. MSE=mean square error. cE-environment effect, D=diet effect, ExD=environment-diet interaction. E value=1evel of statistical significance, NS=not significantly different P<.10). 77 the inadequacy of plasma copper levels as an index of copper status (Castell and Bowland, 1968; Parris and McDonald, 1969; Kline et al., 1972). However, Suttle and Mills (1966) showed that feeding dietary levels of 425 to 750 ppm markedly increased serum copper levels. Furthermore, Underwood (1977) indicated that copper intakes of 100 ppm copper or more are necessary to produce a significant elevation of plasma copper. Portal plasma copper levels were higher (P<.001) for pigs fed the diet containing 250 ppm supplemental c0pper (Table 9). Copper in the form of water-soluble salts (Cu504) is more available than copper in copper oxides or copper wire (Underwood, 1977). COpper absorption appears to be regulated in accordance with body copper need at low dietary capper levels (Crampton et al., 1965; Gitlin et al., 1960), but at high dietary copper levels, absorption appears to parallel the dose until a tissue saturation point is reached at which additional copper intake results in no further absorption. High dietary levels of calcium, zinc and iron have been demonstrated to reduce c0pper absorption. Since copper in the form of copper sulfate is a highly available source of copper and since copper absorption increases when P195 are fed a high dietary level of copper, it would be expected that portal copper concentrations would be greater than that of pigs fed the control diet. The dietary levels of calcium, zinc and iron used in this study (Table 2), were not high enough to interfere with copper absorption. Finally, an environment x diet interaction (P<.06) was observed for Peripheral and portal plasma copper indicating that feeding the hi9h capper diet increased peripheral and portal c0pper concentrations 78 more dramatically in the germ-free pigs than in conventional pigs (Table 9). This relationship suggests that the presence of gut microflora alters copper metabolism as previously suggested by Reddy et al. (1965a). 0. Plasma Ceruloplasmin Activity Ceruloplasmin, measured as change in absorbance/minute/ml of plasma was higher (P<.001) in portal and peripheral plasma of germ-free pigs than their conventional counterparts (Table 10). Since there was an apparent increase in copper absorption creating a moderate copper toxicity in germ-free pigs (discussed in Sections C and E), there appeared to be a concommitant increase in plasma ceruloplasmin in order to accomodate the increased copper load entering the plasma. Germ-free pigs fed the high c0pper diet had higher levels of portal plasma ceru10plasmin but when conventional pigs were fed this same diet, portal ceruloplasmin decreased (P<.07). It appears that the gut microflora greatly influences the absorption and metabolism of copper and other trace elements. E. Liver Capper, Zinc and Iron Concentrations In this study, germ-free pigs had higher liver copper and zinc concentrations than conventional pigs (P<.001), while liver iron levels were not different (Table 11). Since the liver is the primary storage organ for c0pper and zinc fellowing absorption (Underwood, 1977). and since it appears that germ-free pigs absorbed more copper and zinc, as indicated by increased portal concentrations of these elements (Table 9), then a higher concentration of liver copper and zinc would be expected. 79 Feeding the high copper diet increased liver copper and zinc (P<.001) and increased the concentration of these elements more dramatically in germ-free pigs than their conventional counterparts (P<.001) (Table 11). In contrast, liver iron concentrations were reduced by feeding the high capper diet (P<.Ol) and liver iron was reduced more dramatically in germ-free than in conventional pigs (P<.Ol). Feeding a high copper diet typically results in increased copper levels in the liver (Wallace, 1967). Copper is known to compete with iron for absorption binding sites and with zinc for metal-binding protein (Miller et al., 1979). Therefore, an abnormally high intake of one element can adversely affect utilization of other elements. Hhen high dietary levels of copper have been fed, zinc deficiency symptoms have been alleviated (Hallace et al., 1960; Hoefer et al., 1960; Ritchie et al., 1963). Perhaps copper is less inhibitory to zinc absorption in the germ-free pig than in the conventional pig due to the absence of gut microflora. Bunch et al. (1963) observed that high copper feeding decreased liver iron concentrations which could be alleviated by liberal supplementation of these diets with iron (Suttle and Mills, 1966). Again, as fer copper and zinc, the decrease in liver iron was not as dramatic in the presence of the gut microflora, which "my suggest that absorption and metabolism of these trace elements are markedly affected by the gut environment. F. Plasma Ammonia Concentrations Peripheral plasma ammonia concentrations were reduced (P<.07) and tended to be reduced in the portal plasma of pigs fed the high COPPer diet (Table 12). An inhibitory effect of copper on urease me am Or. '80 activity would support the decrease in plasma ammonia concentration in the conventionally reared pigs, but since germrfree animals (pigs) would not have any bacterial urease activity (Kornberg and Davies, 1955; Levenson et al., 1959), copper might be acting in another capacity to elicit the response observed. Germ-free pigs had lower levels of portal ammonia (P<.001) than conventional animals apparently due to a lack of bacterial urease activity (Table 12). G. Concentration of Ammonia and Urea in Cecal Contents In this study (Table 13), germ-free pigs had a higher concentration of urea (P<.001) and a lower concentration of ammonia-nitrogen (P<.001) in cecal contents compared to conventionally reared pigs. these values are consistent with those reported by Combe et al. (1965) when comparing these two response variables between gernhfree and conventional rats. Cecal contents of germ-free rats contain a greater amount of non-precipitable nitrogen and very little amonia compared to conventional rats (Combe and Sacquet, 1966; Levenson and Tennant, 1963). It appears that nitrogenous compounds are degraded to a lesser extent in germ-free animals than in conventional .animals. Germ-free rats excrete more fecal nitrogen in their feces than their conventional counterparts (Levenson and Tennant, 1963; Evrard et al., 1964) and urea accounted for more than 25 percent of the rritrogen in feces of germrfree rats, while conventional rats had no measurable urea in feces. Feeding the high c0pper diet had no effect on urea or amonia-nitrogen concentrations in cecal contents of either germ-free or conventional pigs (Table 13). Chachulowa (1964) observed a 81 Table 12. Effect of Diet and Environment on Portal and Peripheral Plasma Ammonia Concentration. Treatment Combinationa Statistical Significancec P Valued Parameter GCO GCu CCo CCu nsgg E 0 ExD Peripheral NH3. ug/ml 1.26 1.01 1.39 1.14 0.08 NS .07 NS Portal NH2.9ug/m1 3.48 3.27 8.69 7.29 4.20 .001 NS NS aG=germ free, C=conventiona1, Co=control diet, Cu=250 ppm copper diet. bMSE=mean square error. cE=environment effect, D=diet effect, ExD=environment-diet interaction. dP value=level of statistical significance, NS=not significantly different (P>.10). Table 13. Effect of Diet and Environment on Urea and Ammonia Nitrogen Concentrations in Cecal Contents#(dry matter basis). Treatment Combinationa Statistical Significancec P Va1ued Parameter GCo GCu CCO CCu MSEb E 0 ExD lJrea in cecal contents, 109/9 0.99 1.16 0.20 0.15 0.03 .001 NS NS Awnmonia in cecal contents,gqu 0.30 0.31 0.57 0.56 0.03 .005 NS NS aG=germ free, C=conventional, Co=control diet, Cu=250 ppm c0pper diet. bNISE=mean square error. cE=environment effect, D=diet effect, ExD=environment-diet interaction. ‘0’ value=level of statistical significance, NS=not significantly different (P>.10). 82 reduction in cecal ammonia when c0pper (as c0pper sulfate) or chlortetracycline was included in the diet but this effect was not observed in this study. H. Plasma Glucose Concentrations Peripheral glucose levels (P<.08) and portal glucose levels (P<.Ol) were higher for germ-free pigs compared to conventional pigs (Table 14). Portal plasma glucose levels were also increased (P<.07) by supplementing the diets with 250 ppm c0pper. Table 14. Effect of Diet and Environment on Portal and Peripheral Plasma Glucose. Treatment Combinationa Parameter GCo GCu CCo CCu H§§E_ Peripheral glucose, mg/dib 91.8 88.4 70.2 85.8 199 Portal_glucose, mgldlC-dl48 226 84.5 109 32.6 aG=germ-free, C=conventional, Co=control diet, Cu=250 ppm capper diet. bEnvironment effect (P<.08). cEnvironment effect (P<.Ol). dDiet effect (P<.07). eMSE = mean square error. 1. Plasma Amino Acid Concentrations Germ-free pigs had lower peripheral amino acid concentrations (P<-05) with the exception of methionine and isoleucine, which tended to be lower, and higher cystine (P<.001), histidine (P<.02) and a"Qinine (P<.003) concentrations, compared to peripheral amino acid 19V81Es conventional pigs (Table 15). This same relationship existed. 1:114:10 1:4 Lee: to «004M m om~ m. K) .01 l 83 .Ao—.on ucwgoweou »_ucouowocmoo accumz .mooooowcmoo _ououo_uouo mo pa>wpum=po> ow .coouuoeoaeo coon x acmscoeozoouoxu .uuooew “ozone .uumemm acmscoco>conmo .eoecm mtooco comsuumzo .umou cmoooo son ammuao .umoo Poeucouuou .pocopu:o>couuu .omee snowman oo. No. ooo. ooo. oe oo oo, oo. oeoeoouo. oz oz No. No. N_ o_ oo oe oeooooooz ooo. oz oo. None ooo No_ No o_~ aeoooo oz oo. Foo. oNo. No. Now oo oo. eeoeozopzeoza oo. ooo. _oo. ooo. oPN ooN oo _o_ oeoooezw oz oz oo. oooo ooo ppo oo_ .om aeoooeo oz oz oz oooo No. No. o_o oe— oe_ooo.ooo oz oz oz so, oo oo .o oe ae_eo_zooz oz oz Poo. oo. me No oo Po aeooozo oz oz _oo. .oooo ~oo o_~ oo. ooo aeopez oo. oz. ooo. ooooo ooo ooo _oo _o~ aeoeozo ooo. oo. eoo. oozwo Noo_ ooo_ ANN oooz oe_oopo oz oz poo. Nooe own z_o ~o_ zoo aeoeooo_o oz oz woo. _o__~ ooo .ze oNN oow monsooopo oz oz ,o. oe~o_ _oo ooz .oo ooo oeoeao ooo. oz eoo. oozoe ooo ooo ~_o ooo ae_eooeeo oz oz No. «Koo so. oo. oo ea aoeoeeooo oxo o o oooz ooo ooo ooo ooo «oae um: —o> a mmco 5w: 2:59 20:500.; oaoeeooooeo_o _eooooooaoo .Azao oeooooeoeaoeoo Aezaoo oooo oeoeo aeoe_o .oeozooeoa eo oeoeeoeo>eo oee oo_o co oooceo .op ozone 84 ..o..xa. oeueo...o o.oeoo...eo.o ooeuoz .ooeoo...eo.o .oo.oo.oooo co .asa. u oo.o> oo .oo.»uoemuo. um.o x newscoeo>couoxu .uuommm goon»: .uuocom u:o§:oe.>=muuo .eoecm mcoaao cowsuumzo .uu.u emooou eon omuuou .uw.u .oeucoouou ..o:o.u=m>=oouu .uucpnsgamuoo oz .oo. oz oNoo ~.~ ..o No. ooo ae.e.oum oz ooo. .oo. ~.o oo oo. oo .o ae.o.oo.z oo. oo. oo. ooo.. oo. oo. o.. ooo oe.ooo oz oz oz ooo oo. oe. oo. oo. ae.ee.o.oooza ooo. .oo. oo. ooo oo. ooo ... .oN ae.ooeo. oo. oz oz Nooo o.o oeo ooo ooo ae.ooao .o. oz oz o.o. ~o~ ooo ... ooo ae.ooe.oo. oo. No. oz .o oe oz oo .o ae.eo.zoaz oz oz No. oo. oo. oo .o. oo. ae.oooo eo. oz oz oooo o.o woo woo ooo ae..e> .o. oo. oz ~.oo. o... oo.. o.o ooo. ae.ee.< oo. .oo. No. .oooo Now. .oo. oz. .oo. ae.oo.o oo. oz oz eooo ooo ooN o.. oo~ ae.eeoo.o oz oz .oo. .oeo .oo .oo o.o ..o aoeeeoo.o oz oz oz .ooo. oNo ooo o.o ooe ae.eoo oo. oz No. Noooo .oo ooo ooo ooo ae.eoaez. oz oz .oo. oom oo. om. o. oo aoooeoooo oxo o o oooz ooo ooo ooo oo < a. maze run: 3500 ucguoogh oooeoo.e.eo.w .oo.oo.oooo ..zeo oeo.ooeoeaoeoo .<eo oeo oo.o co Homeco .o. e.oe. £15 but to a lesser extent for portal plasma amino acids (Table 16). Germ-free pigs had lower aspartate (P<.001), threonine (P<.02), glutamate (P<.001), glycine (P<.02) and histidine (P<.007), and tended to have less tyrosine in portal blood plasma than conventional pigs. However, germ-free pigs had higher cystine (P<.02) and lysine (P<.05) concentrations than conventional pigs. Herskovic et al. (1967) showed that germ-free mice absorb amfino acids more efficiently than conventional animals, and as a result, it would be expected that portal amino acid concentrations would be elevated in the germ-free animal after a meal feeding. Feeding the high c0pper diet decreased, and in some cases tended to decrease portal, amino acid levels in the germ-free pig but increased or tended to increase plasma amino acid levels in the conventional pigs fed the same diet (Table 16). Glycine and tyrosine however, were reduced by feeding the high copper diet to pigs in both environments (P<.001). High copper feeding also tended to reduce portal serine, histidine and arginine levels in pigs in both environments. This same relationship existed for peripheral amino acid concentrations with the exception of aspartate, leucine, tyrosine, phenylalanine and arginine which tended to be lower for pigs fed the high copper diet in both environments (Table 15). J. Organ Heights In this experiment, germ-free pigs had a smaller thyroid (P<.001), liver (P<.001) stomach (P<.Ol), small intestine (P<.001) and large intestine (P<.001) but larger spleen (P<.003), lungs (P<.03), heart (P<.Ol) and gall bladder (P<.02) than conventional pigs (Table 17). No differences were noted for thymus, adrenals, kidneys or 86 .Ao..Aoo u:ocooo.u ».o:ou.o.=m.o uocuoz .muoou.o.co.o .ououo.uoao mo .o>o.uw=.o> on .oo.uooemao. woooluomsooeo>oouoxm .uoooow amoouo .uoowoo pauseoeo>omumo .Loeem aeoaao oomsuuozo .umou coooou zoo ommuzo .amou .oeucouuoo ..o=o.u=m>=ouuo move» Ecmouoo mz mz poo. n~.o~ m.m~ ¢.¢~ o.op ¢.N— acouomacu magma mz mz poo. om.~m o.mm o.om n.om ~.om mcpumouco pposm mz mz soo. wom.o m~.o mo.o No.o up.o gumsoum mz mz No. oNo.o sm.o on.o o¢.o mo.o Levon—o ppmo mz mz mz uno.o op.~ ¢—.~ oo.— no.— mumgucma mz mz poo. emo.m o.mm e.¢m N.o~ ¢.¢N Lm>vo mz mz mz —~o.o um.o pm.o —~.~ mo.o mhmcowx mz mz ooo. —mp.o m—.m ¢~.m oo.m oo.m «cum: mz mz mo. ~¢~.o o.o— ~.o~ ¢.—— o.~p moczo mz mz moo. ooo.o oe.— ¢¢.P m~.— mo.~ cmmpqm mz mz poo. .oo.o oo.o o_.o oo.o so.o upogxgp eo. mz mz —oo.o ¢F.o sp.o op.o mp.o m—mcmgo< mz Noo. mz pom.o o~.P Nm.~ po.p oo.~ masxzh mxo o u pom: zoo coo :oo coo oneo um:.o> o eaoeoo...eo.o .oo.oo.oooo ocoouocoosoo unusuooe» .Ausm.m: Noon mx\mo ougaom: cameo com: co «coscogo>ou uco pmoo oo powwow .n— o—nop pa Dr 1110; hal 50 the hac ger sma Hax' difi M1 n1' tree for Gem were EXpl. EXpeg 201 c 87 pancreas weights between germ-free and conventional pigs. -Hax1er and Drees (1972) examined differences in organ weights between germ-free, monocontaminated and conventional pigs and noted that germ-free pigs had a smaller thyroid and spleen but larger adrenals, heart, kidney, stomach and small intestine than conventional pigs when expressed on the basis of mg/100 g body weight. Pigs monocontaminated with §;.£Qli had larger adrenals, thyroid, stomach and small intestine than germ-free pigs but a smaller spleen, and a larger kidney, stomach and small intestine than conventional pigs (Haxler and Drees, 1972). Waxler and Drees (1973) and Kruml et al. (1969) did not observe differences in thymus weight of germ-free and conventional pigs. Miniats and Valli (1978) noted that the weight of the gastrointestinal tract, as a percentage of body weight, for germ-free pigs was less than for conventional pigs. However, in both reports, conventional pigs grew faster and weighed more at the termination of these experiments, which is contrary to the results observed in this experiment (see discussion on growth performance in Section A). As a result, even though organ weights were expressed on a body weight basis, large differences in body weight of pigs, along with small changes in organ weights, could explain some discrepancies between organ weights 1 observed in this study compared to those reported by other workers. Germ-free pigs were heavier in this experiment while conventional pigs were heavier in the other two studies previously discussed which could explain these conflicting findings. A smaller thyroid gland as observed in this study might be expected since basal metabolic rate in germ-free rats is approximately 20% of that in conventional rats (Deplaces et al., 1963; Levenson et al., 1 approx Since may 31 smalle envir gastr pigs. studi other the r CODVG lntes rEdur (lab COPP diff 9160 as a 1r01 adre 11‘01 4er 99n1 .88 al., 1966) and iodine uptake by the thyroid of germ-free rats is approximately 50% of that in conventional rats (Levenson et al., 1966). Since basal metabolic rate is reduced in germrfree rats, germ-free pigs may also have a lower basal metabolic rate which could explain the smaller thyroid observed for germ-free pigs in this study. The gastrointestinal tract is one organ in direct contact with environmental microbes and wet weight of the germ-free pig gastrointestinal tract was reduced compared to that of conventional pigs. This relationship is in agreement with other germ-free pig studies (Haxler and Drees, 1972; Miniats and Valli, 1973) and with other germ-free animal studies (Gordon, 1968) except for the cecum of the rat and mouse which is greatly enlarged relative to their conventional counterparts (Gordon et al., 1966). This reduction in intestinal weight of germ-free pigs appears to be primarily due to a reduction in lamina propria (Gordon and Bruckner-Kardoss, 1959; 1961b). Thymus was the only organ affected by feeding the high copper diet (Table 17). Thymus weights were reduced (P<.002) by feeding the high copper diet in both environments. Although no other significant differences due to diet were observed, liver, kidney, heart and adrenal glands tended to increase in germ-free pigs fed the high copper diet, as a percentage of body weight. Miller et al. (1967) observed that iron-deficient pigs had larger livers, kidneys, hearts, spleen and adrenal glands, as a percentage of body weight, than pigs receiving an iron adequate diets. A diet by environment interaction (Table 17) was observed for adrenal gland weights (P<.04). The heavier adrenal weights of the germefree pigs receiving the high c0pper diet may have been due to str tri hig 1119 V11 the long exar obse ente norn inte corr mOre Slnc smal time them that rep] time germ. COrri 99ml. aSSoc 89 stress from overcrowding in that isolator during the last week of the trial, but adrenal weights were reduced for conventional pigs fed the high copper diet. K. Intestinal Morphology Results from this study (Table 18) indicate that germ-free pigs had longer ileal villi (P<.05), tended to have longer duodenal villi (400 um vs. 346 um), but had shorter jejunal villi (P<.02) than their conventional counterparts. Miniats and Valli (1973) observed longer jejunal villi in the intestine of germ-free pigs but did not examine duodenum or ileun sites. Measurements of villus height and observations on villus shape provide a reasonable estimate of villus enterocyte numbers (Hampson, 1986). Wright (1982) reported that in normal, finger-like villi, (analogous to villi in the germ-free intestine) villus height and cell population are highly correlated, but correlations between villus height and cell population are lower for more complex shaped villi relative to cell population (Creamer, 1964). Since there is a slower turnover of epithelial cells in the germ-free small intestine, there may be a greater number of mature cells at any time, and thus, potentially higher amounts of enzymes associated with them (Coates and Fuller, 1977). Therefore, even though it may seen 'that the villi should be shorter for the germ-free pig, a slower cell replacement rate allowing more mature cells to be present at any given time, would explain the longer ileal and duodenal villi, observed in germ-free pigs in this experiment (assuming that villi height is highly correlated with cell number). If there are more mature cells in the germ-free small intestine, resulting in higher amounts of enzymes associated with them, then the increase in absorption of xylose and 90 ..o..xoo oeoeooo.o o.oeeo.o.eo.o ooeuoz .ooeoo...eo.o .oo.oo.oooo co .o>o..oo.o> oo .coouuoemuco um.ouu=m§coeo>:muoxm .uomoom «movno .uomoom pomscoeo>cmumu .coecm meoaoo cooEummzo .uooo emooou sou ommuoo .umou .oepcounoo ..oooou:m>couuo .omeo Eemouoo oo. oz oz oooo. o.o ooo ooo ..o oooezo.zo .ooo. oo. oz oz oo.o oo. .o. ow. oo. zooao oooooEooo wo. oz .oo. woe oo. o.. ... oo. ooooo gooeo .o. oz oz oo ow. ow. ow. wo. zoo.z oo...> oz oz oo. oooo ooo w.o ooo ooo ozo.oz oo...> :50: oo. oo. wo. owoo owo ooo ooo w.o oooexo.zo .ooo. oz oz oz ooo. oo. oo. oo. oo. zoooo oooooEooo wo. oo. .oo. ooo oo. ... oo. .o. zoooo oooeo oo. .o. oz ow. ww. ow. .o. ow. zoo.z oo...> eoo. oz wo. ooo. ooo o.o ooo ooo ozo.ez oo...> Eocanmo oz oz .o. owoo ooo o.o ow. woo ooaexo.o. .ooo. oz oz oo. ooww .ow oow oo. oo. zooao oooooeooo oz oz .oo. ooo. o.o .oo ... ww. zoooo oooeo .o. oz .oo. ..w o.. oo. .w. oo. zoo.z oo...> oo. .o. oz owoo owo woo eoo ooo oooooz oo...> Eocwuooo oxo o o oooz ooo ooo ooo ooo eooeeoeoo vmzpm> a oooeoo.o.eo.o .oo.oo.oooo o:o.uoooosoo acmsuomeh .Aocoeuoso Eom—. new Eocawmol«E=:muo:o us» we ooocxuozh .ocouomaco .ooo. oeo zoooo oooooEooo .zoooo oooeo .zoo.z oo...> .ozo.oz oo...> eo oeoeeoe.>eo oeo oo.o co ooaooo .o. o.oo. amin germ 0059 more appar lncre rate. (P<.o Digs Pigs to al ileal highl I“mini raduc( Vl'Hi prOdu: '91 amino acids observed by Heneghan (1963) and Herkovic et al. (1967) in germ-free rats, and the increase in portal glucose concentration observed in this study may be partially explained. Kenworthy and Allen (1966) showed that when germ-free pigs were exposed to the "bacterial environment”, villi became stunted, fused and clubbed. Width of villi (measured at the base of the villi) was reduced in the duodenum (P<.001), tended to be reduced in the jejunum (115 um vs. 123 um) but was not different in the ileum of germ-free pigs compared to conventional pigs (Table 18). Since the conventional pigs in this study had shorter villi (duodenal and ileal), there was also a likely reduction in enterocyte number even though villi were more irregular than that observed for the germ-free pigs. This apparent reduction in enterocyte number could either be a result of an increased rate of cell loss or a reduction in crypt cell production rate. Feeding high copper diets reduced villus height in the duodenum (P<.Ol) and to a greater extent in the germ-free pigs (P<.05) (Table pigs when fed the high copper diet, but increased when conventional pigs were fed this same diet (P<.004). This same relationship tended to also exist for the ileal site. Apparent increases in jejunal and ileal villus height (enterocyte number) of conventional pigs fed the high copper diet may have been a result of local alterations in the luminal environment caused by copper. In other words, c0pper may have reduced the accumulation of toxic bacterial metabolites shown to cause villi shortening (Kenworthy, 1976). Crypt depth gives a general indication of the rate of crypt cell production (Hampson, 1986). In other words, crypt cell production 92 increases as additional cells are recruited to the crypt epithelium to cause it to elongate. As crypt cells migrate upward, they become villus enterocytes and this cycle takes 2 to 4 days to occur in three-week old, unweaned conventional pigs (Moon, 1971). During their migration, these cells become increasingly differentiated and their absorptive capacity develops (Kinter and Nilson, 1965). Measurement of crypt depth therefore gives a general indication of the likely maturity and functional capacity of enterocytes of the villi. In this experiment, crypt depth was decreased (P<.001) in germ-free pigs at each site along the small intestine (Table 18) indicating a slower rate of enterocyte production which is consistent with work by Abrams et al. (1963) and Lesher et al. (1964). The slower turnover of epithelial cells in the small intestine of germ-free pigs implies that there is a greater number of mature cells present at any time, and thus, potentially higher amounts of enzymes associated with them. When high copper diets were fed, crypt depth decreased in the germ-free pig but increased in the conventional pig in the jejunum and ileum (P<.02). The small intestinal mucosa is the most rapidly regenerating tissue in the body (LeBlond and Walker, 1956). Any tissue undergoing such a rapid turnover needs a readily available supply of nutrients. Perhaps high copper feeding improves the amount of readily available nutrients so that cell turnover is reduced in the germ-free pig but microflora in the conventional pig utilize some of these nutrients and cell turnover is increased. Depth of the submucosa was less in the duodenum (P<.03) and tended to be reduced in the jejunum (159 um vs. 167 um) and ileum (141 um vs. t1 1'r Fu th the drag for thlc redt diet the thicl Since 5“901 thick 93 166 pm) of germ-free pigs compared to their conventional counterparts (Table 16). The submucosa (containing the larger blood vessels, lymphatics and neural complexes) is a loose connective arrangement paralleling that of the lamina pr0pria and accomodates contractions of the adjacent muscularis mucosa. Since germrfree animals have a thinner intestinal wall due to a reduction in connective tissue (Coates and Fuller, 1977; Gordon, 1968; Minats and Valli, 1973) then a reduction in thickness of the submucosa would also be expected for the intestinal tract of germ-free pigs in this experiment because of its connective tissue constituents. Total intestinal thickness of the duodenum (P<.Ol) and jejunum (P<.02) was reduced in the germrfree pigs and tended to be reduced . (785 um vs. 874 um) in the ileum (Table 18). This is in agreement with other reports when germ-free and conventional animals were compared (Coates and Fuller, 1977; Gordon, 1968; Miniats and Valli, 1973). Feeding the high copper diet reduced total intestinal thickness of the jejunum of both germ-free and conventional pigs (P<.04) but more dramatically in the germ-free pigs (P<.06). This same trend existed for the duodenal site. High capper feeding tended to increase total thickness of the ileum of conventionally reared pigs but tended to be reduced in germ-free pigs (P<.09). In general, feeding high copper diets to germ-free pigs reduced total intestinal thickness but not to the extent of that in the conventional pigs. This implies that gut thickness may not play a role in the mechanism of action of copper since conventional pigs appeared to grow at a faster rate when supplemental copper was included in the diet even though total gut wall thickness appeared to be not appreciably different. 94 II. INFLUENCE OF COPPER AND CHLORTETRACYCLINE 0N MICROBIAL FERMENTATION PATTERNS AND MICROBIAL GROWTH IN FIVE SECTIONS OF THE GASTROINTESTINAL TRACT IN VITRO. A. Microbial Growth Microbial growth, measured by change in optical density of the inoculated media per unit time, was greater for the control (Co) and 100 ppm chlortetracycline (CTC) treated microbes, with each microbe source, compared to either the 250 ppm c0pper (Cu) or 250 ppm c0pper and 100 ppm chlortetracycline (Cu + CTC) treated microbes at 16 hours (P<.Ol) and 24 hours (P<.Ol) of incubation (Table 19 and Figures 1-5). Microbial growth was not different between Co and CTC treated microbes within microbe source except for cecal contents at 16 (P<.Ol) and 24 hours (P<.Ol) of incubation (Table 19 and Figures 1-5). Similarly, no difference was noted for microbial growth within microbe source when the Cu treatment was compared to the Cu + CTC treatment of 16 and 24 hours of incubation (Table 19 and Figures 1—5). Within each microbe source (except for 1st half small intestine contents and cecal contents) there appeared to be a general tendency for CTC to slightly reduce growth compared to growth of Co treated microbes and fer the Cu + CTC treated microbes to grow at a slightly reduced rate compared to microbes incubated with the Cu treatment (Figures 1-5). Within the Co and CTC treatments, microbial growth was increased at 16 hours (P<.001) and at 24 hours (P<.001) of incubation compared to the initial optical density readings at 0 hours (Table 20). However, microbial growth within these two treatments was not different between 16 hours and 24 hours of incubation (Table 20). Within the Cu treatment, microbial growth occurred, and peaked at 16 hours of 95 48.x: accent... ....ucouzocmoo 8:. an.» .3333..— 55... 3.58 39.2.... 5.3... 33.8833 accent... .3... ocoozud... .o....u»u~..aofo...u 5.. 8. + .258 IE com-o5 + ...o .oo.—uxuoeuoto...u ...... oo.-o8 .338 .o.... own-3 ..oeucouuooa ewo. u wo.o ...o. u oo.w oo.. o wo.o ...o. ... oo.w ooo. u ...o ...o. u .o.. a... .o ...o oow. .o. oo.. ...... 5.8 u... u So .8. .o oo.. eoo. ..n .w.o ooo. u oo.. oo.. .... .o.o ...o. u oo.w uoo. ... wo.o ...o. .... oo.. 352.8 .88 u.o. ... 85 .oo. o oo.. o... u so o... n oo.w goo. u ...o ooo. ... .... oo.. u ...o ...w. u oo.. :2. .38 eon. H 86 nmo. H on.— umc. 1+. ww.o 36. H ...o.— usw. H on.o ...o. H oo.. ucm. H on... a8. H 8.. 3:35.... .33 u.o. n so .8. u oo.. oo.. n so oo.. u oo.. goo. ... ow.o oz. ... o... oo.. u ...o oow. n oo.. .w. ...... .oo. ....o ooo. q. ...o oo.. u wo.. 88. u ...o oo.. o oo.. ooo. u :5 .3. u o... uoo. u .3 oww. u oo.. .w. 35.28 .oo. ....o 88. ..o wo.o oow. ... oo.. owo. u so ...o. ... oo.w ooo. ... .w.o o.w. u oo.. ooo. ... ww.o ooo. ... oo.. ... ...... J... .3 uoo. q. o..o oow. u oo.. uoo. n ...o ...o. .w o... oo.. n so owo. ... .... uoo. n 35 ooo. ... oo.. .3 3.6.28 .oo. ...o oo.. o wo.o oow. o oo.. ooo. ... ooo oow. o oo.. oo.. n owo ...o. ... 8.. “...o. ... ow.o oow. ... ww.. ...... 5283 aoo. ... owo oo.. .o o... uoo. u ww.o ooo. ... oo.. aoo. u so ooo. u oo.. o... n :o ooo. u .... 35:8 zusaoo "eeeooo ooaeo.z o ._.o + no oh. so co o8 + 3 ob so 3 $5.53..» 9.3.. cu 3.6.. o. as.» .5333... 22.53 :5 .58.... ...—afoot mo 9:58: a o5 3.5.3.. 33...... .333 $9.22 .6 Sotogo .8533... we «use: cu ...... o. a. .3553.» .3253 69.3...” 39.2: 55:. 23325.. .2 oz: 96 E 5:: :3: '7' E 2’ 8 m :3 53 2! (oo.)oo - a .. / E -a 5.33.3 & D-O-Og E ‘ -.~e E o 8 3. g ...... or) ‘ '° 1 . , —o a4. 61 no ' I l I (oo.) oo . Figure 1. Influence of Copper and Chlortetracycline on Growth of Microflora from Stomach Contents and Hall During a 24 Hour Incubation Period. 97 24 SMALL meme WALL (lsi half) —' IIIUWI1III . :1 a :2 :2 z :1! a a a a a 8 (oo.)ao ‘ -."~ --8 SMALL INTESTINE CONTENTS (151 half) r- a - v I I I I I I I r I I - ° :1 :3 =3 :3 :5 =3 :3 :3 =3 :3 :7 :3 (oo.) co Figure 2. Influence of Copper and Chlortetracycline on Growth of Microflora from Small Intestine Contents and Hall (lst half) During a 24 Hour Incubation Period. 98 «'1'. h ‘5 g {:2 -... -'= .3153 'D t: n+‘ov- iiflL - :2 <1: 3:: BE: “24 ~== -.._- E m 1...: E: "‘ A 2‘ 2 (f) "" I U I l j I I I fl I "9 :3 :3 =3 :2 :1 :2 E! :3 :3 :5 :1 :3 (oo.)oo ‘ ‘ a -8 r2 SMALL INTESTINE CONTENTS (2nd half) [VIII] -9 :2 2 s a e a: a I use 154 (oo.) co Figure 3. Influence of Copper and Chlortetracycline on Growth of Microflora from Small Intestine Contents and Hall (2nd half) During a 24 Hour Incubation Period. 99 «c: EB: _.l 2'5 Lu Q (co->00 ‘ * a “-fi i838 g—‘Q 9.4-0. {—2 2 Lu b 2 8 -... Z _..l < E 0 Lu 0 ~ —- d. L..- I l a I . I — 53 :3 :3 :3 :3 :3 1.2-J :: a 2': 2':- 52 (900) no Figure 4. Influence of Copper and Chlortetracycline on Growth of Microflora from Cecal Contents and Hall During a 24 Hour Incubation Period. 100 COLON WALL 2.2 20 Lb Li“ H- 1.2- LO" ”4 16‘ M“ 0.2 0.0 (can) no 0 lerd + Cl 9 CE I CniCl C COLON CONTENTS .2 MM '$l111fi "'9 V :1 a :2 .- .- 2 s a a z. a s (oo.)ao Figure 5. Influence of Copper and Chlortetracycline on Growth of Microflora from Colon Contents and Hall During a 24 Hour Incubation Period. .86... acacmwm.c x—ucou.».:m.m men acmEuamgu =.=p.z mugzom unocu.s :_;».3 mua.cumcoa=m a:mgomw.v gu.z «cauzu.n .m:..u.u~u»maco.su Eng oo—uUPU ..ocucouuouu lOl 9.... H .....N a... H ...... ...... H 3... 0.... H .....N as. H ...... ...... H 3... ...... 5.8 on... H ...... u.... H 8.~ ...... H a... um... H ...... 0.... H 2.... ...... H a... 3528 5.8 um... H ...... 08. H .... ...... H 8... u... H ...: um. H ...... .2... H 8... :2. .88 um... H ...... ...... H ...... .2... H ...... um... H ...... ...... H 3.. ...... H ...... $328 .88 as. H ...... 93. H ..... ...... H 8... 0.2. H 8.. um. H ...... ...... H 2... a. ..e. .2. ....m a... H N... 0:. H m... .2... H S... a... H a... as. H a... ..8. H 2... .3 3528 .2. ....m 08. H ...... as. H 8.. ...... H 8... as. H .....N can. H 2... as. H 3... ... ..e. .2. ....m as. H ...... 08. H .... ...... H S... 0.... H 2.. own. H 2.. ...... H ...... ... 3528 .2. ....m ”.8. H 8.. as. H 8.. ...... H ...... as... H 2.. 08. H 2.. ...... H 8... ...... €252 um¢..H m... gem..u mm.— n.o..H ww.o umo..H mm._ oom..fl .... 9....“ ww.o mucmucou gumeoum .mmucsom wnocu.z ..N o. .. a ... .. .25 S... 5.222. who am mucwsuauch .mucmEummLp c.:u.3 .mugacm mnogu.z 2.5».3 :o.uan:u=. we mgso: cu can a. .o um mco.uo.>ma unaucaum vco Agpzoco .m.nocu.z we mesmooz a m< we com.gaaeoo .oN upon» (t LIF‘IILF 102 ...o.va. acute...c ...:au...=a.m mum acmEuomcu c.gu.z oo.:om anogu.s gu.: mua.cumgmn=m u:mgowm.u gu.: mcuwzu.n .m:..u»ducuuuco—gu sag oop+cmaaoo Eng omuuohu+au .cmanou an: ammuauo n~o..H ~o.c umo..H w..c neo..H ec.o um...H wo.o 2....H ...o 2....H mc.o ..a: =o.oo .....H wo.° ao...H .¢.o anc..H om.o awe..H .~.o neo..H ~¢.o n.o..H w~.o mpcaucou cc.oo n.o..H mo.c occ..H ...o nwo..H eo.o .....H .o.o am...H ...o ne...H wo.o ..a: .20». n»...H wo.o n.~..H om.o nom..H m..o ame..H_-.o aem..H mm.o n.o..H ...o mucaucou .auau a.o..H wo.o umo..H c~.o nwo..H mo.o ac...H_.o.o aw...H ...o gm...H wo.o .N. ..a: .22. .2. =mc..H ...c umo..H em.o neo..H ~..o nmo..H ...o umo..H..m.o nmo..H ...o .N. mucaucou .92. .sm n.o..H ~o.o um:..H .~.o n.o..H mo.o n~o..H co.o ueo..H -.o amo..H.eo.o ... ..a: ..c. .5. ne¢..H ...o um...H.o¢.o a.o..H ...o a.o..H ...o umo..H em.o nmo..H m..c ... mucapcou ..g. .5. am...H we.c ne...H m~.o am...H ma.o nmo..H wo.o ueo..H m~.o n~o..H wo.o ..22 gonzoum nmo..H m~.o umo..H om.o neo..H m~.o nmo..H -.c 0....H ee.o 2.0..H .~.o m.=m.=cu someoum .mmugaom unocu.z cu m. o e~ . m. o .m.:. as.. cc..un=u=. 9.9 + au so oucwEuamgh mco.ua.>mo venucaum vcm Aguxoco .c.aocu.z mo ogzmowz a m< we com..uasou .o~ «pack 103 incubation compared to initial Optical density readings (P<.Ol) for microbes from the stomach contents (SC), stomach wall (SH), small intestine contents (lst half) (Slic), small intestine wall (lst half) ($11“) and small intestine contents (2nd half) (SIZC), but not for microbes associated with the small intestine wall (2nd half) (SIZH), cecal contents (CeC), cecal wall (Cew), colon contents (COC) or colon wall (CON) as shown in Table 20. However, after 16 hours of incubation, Optical density readings declined to initial levels (P<.0l) at 24 hours of incubation for microbes from SC, SN, SI]C, 51]“ and SIZC sources, and tended to decline for microbes from $12", CeC, Cew, COC and COM (Table 20). For each microbe source incubated in Cu media, there was an 8 hour lag phase where little, if any, growth occured, which may have been due to an attempted adaptation of microbes to the adverse effects of Cu in the media. This lag phase appeared to be particularly marked for the microorganisms associated with the CeC and COC sources (Figures 4 and 5). Similarly, when optical density readings were determined at 0, l6 and 24 hours, within the Cu + CTC treatment for each microbial source, growth did occur and peaked at l6 hours of incubation for microbes from the sc, SIIC, 51]”, SIZC, SIZW, CeN and Cow, (P<.0l) and growth tended to occur for SW, CeC and COC microbe sources (Table 20). It appears that after maximum growth was obtained at l6 hours of incubation, the attempts of the microbes to compensate for the apparent adverse environment created by OOpper, were not adequate, and bacterial cell lysis occurred, since Optical density readings at 24 hours of incubation were approximately the same as the initial optical density readings (Table 20). Changes in optical density readings during 104 incubation may be due to either an increase in bacterial cell number, caused by multiplication, or an increase in cell size. Studies have been conducted to examine the influence of high levels of capper on specific bacterial species in digesta and feces of pigs. Kellogg et al. (l966) observed that when pigs were fed 250 ppm supplemental copper, growth rate was improved, compared to pigs fed a control diet and associated with this growth improvement, they observed a lower number Of lactobacilli, total aerobes and total anaerobes. When Fuller et al. (1960) fed diets supplemented with 250 ppm capper, they observed no difference in the number of lactobacilli, and coli- aerogenes compared to microbes of pigs fed a control diet, but they did Observe a decrease in the total number of streptococci and a shift in the species Of lactobacilli (decrease in L. acidophilus and a corresponding increase in L;.EE§!1§ and L;_cellobiosus). Smith and Jones (l963) studied the effects of 250 ppm and l000 ppm supplemental copper on the bacterial flora of the alimentary tract of the pig and observed no differences in g; 9911, lactobacilli, streptococci, Ql;__ welchii, bacteroides or yeasts compared to control pigs. However, the minimum inhibitory concentration (MIC) for microbes grown in reinforced clostridial medium (RCM) was as follows: MIC of copper (ppm)a Test medium Lactobacilli Streptococci E. coli Cl welchii RCM agar lo 200 200 19 RCM broth 100 200 200 50 Aqueous suspension of pigvfeed (30% wt./vol.) 400 400 -400 100 aSmith and Jones (1963). 105 As these workers Observed, a higher concentration of cOpper was required to inhibit growth of these organisms in liquid than on solid media. These workers suggested that the bactericidal effectiveness Of cOpper sulfate is decreased in the alimentary tract by other factors that are not present in an aqueous suspension of pig feed (e.g., digestive secretions), which would explain their results. Therefore, the detrimental effects of capper on in vitro microbial growth observed in this study, may not be nearly as dramatic in vivo, but capper does appear tO affect microbial population growth at all sites along the digestive tract in the absence of factors in the gut that reduce the bactericidal effectiveness Of copper. Based on these data, it appears that l00 ppm chlortetracycline reduced microbial growth only in cecal contents as compared to the control treated microbes and the presence of 250 ppm copper, singly, or in combination with 100 ppm CTC, dramatically reduced growth of microbes when Optical density readings were compared with those Of the control treated microbes at 16 and 24 hours Of incubation. Apparently, the level of 250 ppm capper, which consistently promotes growth when included in diets for the young pig, is toxic to the gastrointestinal microflora in both the gut contents and the gut wall along all sections of the digestive tract. Bacterial growth is inhibited in vitro, but based on work reported by Smith and Jones (l963), the presence Of other factors in the gut environment may reduce the bactericidal effectiveness of copper in vivo, as Observed in this study. B. Fermentation Changes in pH of Growth Media Since the buffer systen Of the media used in this study was designed to prevent large changes in pH which would severely affect , 106 microbe viability, a large change in acid or base production would have to occur from the microbial fermentation process before changes in pH could be Observed. In other words, a large increase in amine production caused by the fermentative action of bacterial amino acid decarboxylases on amino acids or a release Of these highly basic amines from bacterial cells upon lysis could cause a small increase in the pH of the growth medium (Prins, l977). Similarly, a large increase in fatty acid production from microbial fermentation of carbohydrate sources could result in a small reduction in media pH. An initial pH of 6.54 was measured in the anaerobic growth media and when microbes from each source were incubated in media containing either the Co or CTC treatments, pH was generally reduced (P<.05) by 0.03 and 0.05 pH units, respectively, compared to an increase in pH (P<.05) by 0.09 and 0.06 units for Cu and Cu+CTC treatments, respectively, regardless of microbe source (Table Zl). Perhaps an increase in total organic acid production for microbes incubated in the Cu and CTC media (Tables 23 and 24) was sufficient to cause this small increase in pH over the initial level, and an apparent bacterial cell lysis after l6 hours Of incubation (which may explain the reduction in Optical density readings shown in Table 20) when microbes were incubated in Cu and Cu+CTC media, may have liberated highly basic amines tO cause the small increase in pH observed in this study. C. Net Ammonia Nitrogen Changes In this study, net ammonia nitrogen change after 24 hours of incubation was generally increased for CO and CTC treated microbes, within microbe source, compared to a net reduction in ammonia nitrogen for Cu and Cu+CTC treated microbes (P<.05) (Table 22). 107 Table 2l. Comparison Of Treatment Means for Change in Media pH After a 24-Hour Incubation Period, Hithin Microbe Source. Treatmenta Microbe Source CO Cu CTC Cu+CTC Stomach contents -O.Osb +0.09d -O.07b +0.05c Stomach wall -o.03b +O.lod -O.Osb +0.06c Small intestine contents (lst half) -o.07b +0.09d -O.O7b +0.06c Small intestine wall (lst half) -O.Osb +O.lld -O.OSb +0.07c Small intestine contents (2nd half) -O.O4b +0.10d -o.O7b +0.06c Small intestine wall (2nd half) -O.O4b +o.lld -O.O7b +0.07c Cecal contents +O.Olb +O.lOd +0.00b +0.05C Cecal wall -O.03b +0.09c -o.o4b +0.06c Colon contents +0.02b +0.08d +O.Olb +0.05c Colon wall -O.Oab +0.08c -O.O7b +0.06c ' Overall -O.O3b +0.09d -o.osb +0.06c aCo=control, Cu=250 ppm capper as CuSO4°5H20, CTC=l00 ppm chlortetracycline, Cu+CTC=250 ppm capper as CuSO4°5H20 and 100 ppm chlortetracycline. bacadMean changes between treatments, within microbe source, with different superscripts are significantly different (P<.05), Std. Error = 0.02. 108 Table 22. After a 24 Hour Incubation Period, Hithin Microbe Source. Comparison Of Treatment Means for Net NH3-N Change (mg) Treatmenta Microbe Source CO Cu CTC Cu+CTC Stomach contents +0.73b -O.25c -o.17c -o.23c Stomach wall +0.66b -O.27c +0.30b -O.26c Small intestine contents (lst half) +0.65b -O.22c +0.54b -o.28c Small intestine wall (lst half) +0.6lb -o.23c +0.74b -o.29c Small intestine contents (2nd half) +l.l6b -0.20c +0.88b -0.30c Small intestine wall (2nd half) +0.90b -O.l2cd +0.17c -o.33d Cecal contents +2.59b -o.l3c +1.95d -O.z4c Cecal wall +0.82b -O.l4c +l.l3b -O.23C Colon contents +3.35b -O.07c +2.50d -o.zoc Colon wall +0.66b -0.l3¢ +0.77b -o.23c Overall +l.2lb -o.l7c +0.38b -o.26C aCo=control, Cu=250 ppm COpper, CTC=l00 ppm chlortetracycline, Cu+CTC=250 ppm COpper + 100 ppm chlortetracycline. bacidiieans within microbe source, among treatments with different superscripts are significantly different (P<.06), Std. Error = 0.27. 109 In the large intestine of the pig, there is considerable bacterial deamination of amino acids (Michel, 1966; Fauconneau and Michel, 1970). Urea also diffuses from the body tissues into the intestinal lumen and is a readily available source of nitrogen for those bacteria having urease activity (Ratcliffe, l985). Microflora have an important role in the recycling of endogenous nitrogen secreted into the gut as urea (Richards, 1972), but hydrolysis of endogenous urea alters turnover rate of intestinal cells and ammonia production in the animal has a deleterious effect Visek et al. (1972). Alvares et al. (1964a, 1964b) and Harbers et al. (1963) with chicks, and Visek et al. (1959) with rats, showed that l00 ppm of chlortetracycline or 3 ppm COpper inhibit urease activity in vitro. Chachulowa (1964) observed a reduction in cecal ammonia production in pigs fed diets containing COpper (as copper sulfate) or chlortetracyline. 0. Volatile and Non-Volatile Fatty Acid Production 1. Comparison of the relative quantities of volatile and non-volatile fatty acids produced in the gastrointestinal tract. a. Volatile fatty acids The relative proportions Of VFA's found in fermentation media are shown in Tables 23A, 238, 23C and 230. Of the organic acids measured, acetic acid and formic acid contributed the greatest proportion of the total, followed by lower amounts Of valeric, butyric and caproic acid. PrOpionic, isobutyric and isovaleric acid were found primarily in the cecum and colon sites. Isocaproic acid was not found in any Of the incubated media samples. For microbes incubated in Cu and Cu+CTC media, acetic acid was the primary VFA produced with smaller quantitites of butyric, isovaleric, valeric and H0 .oe..u»eetueuce.gu Egg on. + Lengeu Eng emu-ewe + :u .ue..e»uesueute_ge Egg oo.nuwu .tengeo Eng omwueu ..ecueeuneue 8. H 8.. ...~ H m... ... H 8.... ~.~ H m... e... H o... ... H a... e... H ...e m... H ...... ...... 5.8. 8. H 8... ... H ...: 8.. H 8.. ... H ...... .... H e... .... H ...... e... H e... .... H 8... 3528 .38 .... H 8.. .... H N... ... H ...... .... H .... e... H e... ...o. H m... e... H e... ... H..... ....e .88 ...~ H 8.. .... H .... ... H 8.. ... H 2... .... H e... .... H m... e... H e... .... H a... 3528 .88 .... H n... 8. H 8... 8. H 8.. .... H ...... e... H ...e ... H ...: e... H ...e .... H ...... ... ..e. .2. .8 ... H 84 ..~ H ..e. .o. H 8.. ...... H a... e... H ...e ... H H... o... H e... .... H .... .... .3528 .2. ...... .... H ...... ... H 8.... 8. H 8.... m... H ...... e... H e... ... H e... e... H e... .... H .... ... ..e. .2. .e. ... H 84 N. H a... 8. H 2... ... H m... e... H ...e ... H ...: e... H e... ... H a... ... .3528 .2. 2. .... H m... e.. H 8.. ... H .... .... H 8.8 e... H ...e ... H ...... ...e H ...e .... H ..e. ..a: 528m .... H 8... .... H ...: .e. H 82 e... H e... e... H e... e... H e... e... H e... ...o H e... 3528 5.52 ”ouceem unecuw: “E + 8 e5 8 8 e... + 8 e5 8 8 .2222. ...o. 222 22 2.28 ...o. .22 2.2.2. ecu neuceem oeecu.z cooxuem A.E\me—eE:V =e.uueeegg Ae.u< uwuou< ecu quLen. v.u< muueg o..ue.e> we :em.tegEeu .ce.ueeeuc. we acne: cw an maceEueme . we eem.cegEeu .mnw e.eew 112 .ocw—uauecuete.5 Egg 8. + ..engeu Eng omNnuwu + 8 .o....uauebete—gu Eng 87qu ...eggee Egg ammueu ..etueeuweue .... H 88 ... H ...e .... H ....e .... H ...... N... H ...... ... H 8... 8. H 8... .n. H 8... ...... 5.8 .... H 8... 8. H 8.. .... H 8... .... H .... 8. H 2... .... H ...N 8. H ...... N. H ...... 3528 .88 .... H ...... ... H .... .... H 8... ~... H ...... .... H ...... ... H 3... .... H ...... 8.. H ...... ...... .88 .... H 8... 8. H ...~ .... H 8... ... H 8.. ~.. H 8... .... H 8... N... H ...... ...... H ...... 3528 .88 N... H ...... e... H ...e .... H 8... ...e H ..... .... H ...... .... H ...... .... H ...... ... H 8... .... ...: .2. .8 .... H ...... ...e H ...e .... H 8... ...e H ...e .... H ...... 8. H ....e 8. H ...... 8. H ..... ... 3528 .2. .8 .... H 8... ...e H ...e N... H ...... e... H ...e .... H ...... 8. H ...... .... H ...... .... H 8... ... ...... .2. .5 .o..H wo.o o.o.H e.e .o..H wo.o e.e.H e.e .o..H «e.e .c..H «e.e .o..H wo.o ....H m..o ... mueuueeu .ue. .Em .... H 8... e... H ...e N... H ...... ...e H e... .... H ...... .... H 8... 8. H 8... .... H 8... ...... 5.52. .e. H e.e e.e H e.e .o. H 8... e.e H e.e .o. H No... 2. H 2... .e. H 8... .e. H 8... 352.8 58.3.. “eugeem maetew: US + 3 98 a... eu uh. + 3 oh. 3 en $5.53.... 85.88. 2.58 ...o. .38 2.2.3. .ee.uee:ue. we acne: «N we HeeEueocw .2.. 3.58 395.: 52.25 253.83 552.8,; .32 eta—gem. ecu 3.328 Sec Due“. 2.522. we eemtegEeu .umN e.e... 113 .oe.—u»oosueuLe—:e Eng oo. + Leggeu Eng omwwuwu + :u .oe..u»uesueuce.gu Eng oo.huwu .cegneu Eng omwwnu ..ecueeu-eue .... H ...e .... H ...... .... H 8.... 8. H ...... ... H 8... 8. H .... ... H 8... 8. H 8... ...... 5.8 8. H 8... 8. H ...... 8. H 8... .... H 2... 8. H 8... .... H 8.. 8. H 8... .... H 8.... 3528 .88 8. H ...e .... H ...e e.. H 8... 8. H ...... 8. H 8... 8.. H 8.. e.. H 8... .... H ...e :8 .88 .... H 2... .... H ...... N... H 8.... ~... H ...e ...... H ...e .... H ...... ...... H 2... .... H ....N 3528 .88 ~... H ...... .... H a... N... H ...... 8. H 8.... 8. H 8... ... H ...... .... H 8... 8.. H 8... .~. :2. .2. .8 8. H N... N... H ...e 8. H ...e N... H 2... 8. H 8... 8. H 8... ... H 8... .m. H .o.. ... 3528 .2. .8 8. H 2... 8. H ...e 8. H ...... 8. H ...... ... H 8... .... H 8... ... H 8... H... H 8... ... ...... .2. .8 N... H ..... .... H ...... 8. H ..... .... H 8... 8. H ...... 8. H 8... ... H 8... 8. H .... ... 3528 .2. .8 .... H ...... .... H 8... 8. H 8... 8. H 8... ... H 8... .... H 8... .~. H ...e ... H ...... ...: 5.58 8. H ...e .... H ...... .... H ...e 8. H ...... 8. H ...... 8. H 8.. ... H 8.. .~. H 8.. 35.8 5.58. "meteem eeetu.x e.e . 8 e.e 8 8 e.e . 8 e.e 8 8 - .2222. e.ecgeu u.co.e> e.e< Huang e..u~.e> .Ee.ueeeue. we mgee: «N as HeeEueeLw one oegeem eeege.z eeexeem ..E\me.eE=. :e.ue=eegg .e.u< e.etgeu ecu e.ce.e>. e.e< ...e. o..ue.e> we eem.cegEeu .nmw e.new 114 caproic acid. No formic or propionic acid were produced, and isobutyric acid was produced only in small quantities in the cecum and colon contents. b. Non—volatile fatty acids For the non-volatile fatty acids, lactic, oxaloacetic and succinic acids were the only acids produced (Tables 24A and 24B). No pyruvic, oxalic, methyl malonic, malonic or fumaric acids were detected. Of these organic acids, lactic acid was the most predominant and high concentrations were found in media containing microbes from the stomach and small intestine. Oxalacetic and succinic acids were produced in greater amounts in the stomach and small intestine compared to concentrations of the acids in the lower gastrointestinal tract. Friend et al. (1963a,b) found that the total organic acids in the stomach amounted to approximately 30-92 meq/l in early weaned pigs. Of this, lactic acid comprised over 90 percent of the molar concentration when pigs were fed a whey diet, but this proportion declined to 50 percent when pigs were fed primarily cereal diets. Propionic acid comprised approximately half of the remaining organic acids with lesser concentrations of butyric and valeric acids. These authors also noted that in older pigs on cereal diets, formic acid comprised 3.5-4.5 percent of organic acids, which was greater than the amount of butyric acid production pigs fed a cereal diet and greater than the amount of propionic acid produced in pigs fed a whey diet. However, when diets contain more digestive or fermentable carbohydrate, there is considerably more fermentation in the stomach and less in the large intestine (Mason and Just, l976). 115 .oc—puauuguoauopzu Egg oo. + Loggoo Egg cmwuupu + no .oc._u»uoguougopgu Egg oo.wupu .Loggou Egg omwnsu ..ogucouuouu 8.. H :8 8. H 88 8.. H 88 ... H :8 8. H 8.8 8. H 88 8. H 88 ... H 98 :2. 8.8 8.. H 8.. ... H 88 2. H 88 8. H 88 8. H 88 8.. H 88 8. H 88 8. H 88 383:8 .88 8. H 88 8. H 8.. 8. H 3.. ... H 88 8. H 8.8 8. H 88 8. H 88 8. H 88 ...... .88 8. H 88 8. H 88 8. H 88 8. H 88 .8. H 88 8. H 88 8. H 88 8. H 88 3888 .88 8. H 88 8. H .88 8. H 88 8.. H 38 8. H 88 8.. H 88 8. H ....8 8. H 88 8. ...... ...: .8 8. H 8.. ... H 8.8 8.. H 88 8.. H 38 8. H 88 8.. H 88. 8. H 88 8.. H 8..... .8 3888 .8. .8 8. H 88 8. H :8 m... H 88 8. H 8.8 8. H 8.8 ... H 88 8. H 8.8 8.. H 88 E ...... .8. .8 ... H 8.8 8. H :8 8. H 8.. 8. H 8.8 8. H 8.8 .8 H 88. 8. H 88 8.. H .8. .3 3:388 8... .8 8. H 88 8. H 8.8 8. H 8... 8. H 8.8 8.. H 88 8.. H :8 8. H 88 8.. H 88 :2. 5888 8.. H 88 8. H 8.8 8.. H 8.. 8.. H 88 8. H 88 ...8 H 88. 8. H 8.8 8.8 H 8.8. 3588 5288 "oucaom unocu.z u»u + su uhu gu ou u»u + nu upu su cu aucuEuou.» uwu< o.uoumo.~xo ku< u8uuaa v_u< xuuog up.ua_o>u:oz .co.uon=uc_ mo mcao: em an acoEuuoL» new ouc=Om uaogu.z cuoxuoo ..E\ma_oE=v cg.uo=uogg .v.u< u.uouao.uxo ecu u.uungv u.u< swung u..uo.o>.egz 8o cgm..ogEou .<¢~ o.nu» 116 Table 248. Comparison of Non—Volati1e Fatty Acid (Succinic Acid) Production (umoles/ml) Between Microbe Sources and Treatments at 24 Hours Incubation. Non-Volatile Fatty Acid Succinic Acid Treatmenta Co Cu CTC Cu+CTC Microbe Source: Stomach contents 1.01 1 .18 0.23 1 .06 0.52 1 .08 0.16 1 .06 Stomach wall 2.12 1 .03 0.26 1 .08 1.05 1 .25 0.25 1 .10 Sm. int. contents (1) 1.41 1 .22 0.24 1 .07 1.24 1 .16 0.24 1 .12 $111. int. wall (1) 1.15 1 .13 0.23 1 .10 1.26 1 .20 0.23 1 .15 Sm. int. contents (2) 1.25 1 .l3 0.25 1 .07 1.27 1 .23 0.22 1 .l3 Sm. int. wall (2) 0.69 1 .10 0.20 1 .06 0.90 1 .25 0.18 1 .09 Cecal contents 0.11 1 .02 0.21 1 .06 0.09 1 .03 0.16 1 .07 Cecal wall 0.08 1 .03 0.22 1 .05 0.07 1 .04 0.15 1 .04 Colon contents 0.24 1 .10 0.20 1 .05 0.08 1 .02 0.35 1 .20 Colon wall 0.13 1 .09 0.211 .04 0.04 1 .03 0.20 1 .15 aCo=control, Cu=250 ppm cOpper, CTC=100 ppm chlortetracycline, Cu + CTC=250 ppm c0pper + 100 ppm chlortetracycline. 11 7 In the small intestine, Friend et al. (1963a,b) reported that the total organic acid concentration was lower in the small intestine than in any other part of the alimentary tract. These authors consistently found formic acid in the small intestine which was not present in the large intestine, and only present in the stomach of older pigs. Considerable quantitites of lactic acid were found in the small intestine but lactic acid contributed a smaller proportion of the total organic acids than in the stomach. 0f the remaining organic acids, acetic acid was found along with some pr0pionic acid. Friend et al. (l963a,b) found levels of total organic acids to be up to 300 meq/l in the cecum and colon which is the highest of any site of the gastrointestinal tract. 0f the total amount of organic acids, acetic acid was present in the greatest quantity, followed by propionic acid, butyric acid, lactic acid and valeric acid. These authors observed little difference in levels or pr0portions of organic acids between the cecum and colon. Argenzio and Southworth (1974) observed similar levels (150-250 meq/l) of total VFA's. Clemens et a1. (1975) observed low VFA production in stomach and small intestine but a dramatic increase in VFA production in cecum and colon. Argenzio and Southworth (1974) measured VFA transport across the intestinal mucosa and observed a high capacity for VFA transport in the cecum and colon but relatively low transport of VFA's in other types of gut mucosa. However, Barcroft et al. (1944) and Elsden et a1. (1946) showed that VFA's are present in the stomach and small intestine, as well as the large intestine, and these VFA's are absorbed. Imoto and Namioka (1978) observed that 31 percent of the acetate was absorbed from the large intestine of the pig when a low 118 carbohydrate diet was fed while 43 percent was absorbed when a high carbohydrate diet was fed. Only 1 to 3 percent of propionic and butyric acid was absorbed. Hhen Imoto and Namioka (1983) added acetate to diets of swine at 0, 5 and 10 percent of metabolizable energy intake, the efficiency of acetate utilization was calculated to be between 56 and 59 percent. Since acetate was the predominant VFA measured in this study and since its energetic efficiency is relatively high, it could be a contributing factor to any improvement in energy utilization from feeding a high cOpper diet to growing swine. 2. Total volatile fatty acid production Total volatile fatty acid (VFA) production between treatments, within microbe source was compared by determining the sum of the eight volatile fatty acids detected and measured after 24 hours of incubation in fermentation media inoculated with microbes from each of ten microbe sources. These microbe sources include the stomach contents (SC), stomach wall (SN), small intestine contents (lst half) (5110), small intestine wall (lst half) (511“), small intestine contents (2nd half) (SI1C), small intestine wall (2nd half) ($12”), cecal contents (CeC), cecal wall (CeN), colon contents (CoC) and colon wall (Cow). In general, the control (Co) and 100 ppm chlortetracycline (CTC) treatments resulted in a greater production of VFA's (P<.05) compared to that of microbes incubated in the 250 ppm c0pper (Cu) and the copper-chlortetracycline (Cu+CTC) treated media (Table 25). This relationship was evident for microbes obtained from $11", SIZC, SIZN, Cew and Cow. For SN, 5110 and 000 microbe sources, total VFA production was reduced for the CTC treatment compared to that for the 119 8.0. u .0... .008 ..88.v.. 000.0...0 ..0000...08.m 8.0 mug.gum.wg=m acmgmmeU gu.z mucmEuoo.» mcoEu .oo.:om one.u.E e.e».x mcamza.u.u .m:..u»oa.umu.o.nu Egg oo. + .mggoo Egg ommuopo+=u .mEP—oaumgumugopgu Egg oo.uu.u ..oggoo Egg ammugu ..ogpcoououa .mougzom maogu.E c. we zoom Eon. menogo.E o:.c.0u=oo 0.0mE mo .E .mg .m0.oe o.ocg00 wee u..m.0> .o..m.0>om. .o..»pgn .o..»pgaom. .o.:opgogg .uwumua .u.ELow mo AmoFOEgv xu.u=0=a ecu mo we Esm «ea 0 :o.uu=uo.g v.00 have. m..u0.o> pouopo vm~.m um.g~ u—m.n om.~m m ppmsw>o 8.8. 008.8 08.88 000.0 00..m ..02 00.00 8.88 000.8 0...0 088.8 08.88 00000000 00.00 0.8. 000.8 00.88 00.8. 0...m ..03 .0008 8.08 088.8 08.00 088.. 0..88 00:00:00 .0000 8.0. 080.0 08..8 088.0 00.88 ...0. 008. ..0: .00. ..008 8.8. 088.8 08.88 088.0 08... ...0; 0:8. 80:00:00 .00. ..0Em 8.8. 0.0.0 08.88 000.. 08.88 ...0; 00.. ..0: .00. ..008 8.8. 080.8 08.08 008.. 08.88 ...0; 00.. 00000000 .00. ..008 8.8. 0...0 08.88 088.8 0..m0 ..03 0000008 88.0 088.8 08.8. 000... 088.. 00000000 30000.8 m :95:. 8.0.50 0.8 :0 cu mug—Sm ~30..on nucmEuoaLE .oo.:om onogopz cpsuwz .vo..mg co.u0n=uc. Lao: 0~ 0 emuw< AuwcwE .0 .E.0.0E0V 0:0..0000.. 0.0< 8.00. a...0.0> .088. .8..m=0oz 880E800.» .8 =ow..0gsou .mN 0.80. 12.0 C0 treatment (P<.05) and increased for microbes of the SC and CeC microbes incubated in CTC treated media compared to the control treatment (P<.05). However, for all microbe sources, Cu and Cu+CTC treated microbes produced less VFA's compared to Co and CTC treated microbes (P<.05). This apparent reduction in VFA production was confounded with a severe depression in microbial growth (Table l9 and Figures l-S) when microbes were incubated for 24 hours in the Cu and Cu+CTC treated media. When microbe sources were compared within treatments, no differences were noted between microbes obtained from contents and wall at the same intestinal site, between microbes from gut contents, or gut wall at each intestinal site for the Cu and Cu+CTC treatments. For the Co and CTC treatments, total VFA production was greater for microbes of contents from the cecum and colon (and 511C of the control treatment) than for microbes of the wall at these intestinal sites (P<.05). However, for the Co and CTC treatments, more VFA's were produced by the SH microbes than by SC microbes (P<.05). Nhen microbes from gut contents of each intestinal site were compared, within treatments, the lowest amount of total VFA production occurred from SC microbes compared to other microbe sources exposed to the Co and CTC media (P<.05). For the Co treatment, CoC microbes produced the highest amount of VFA's (P<.05) compared to cecum and small intestine content sources. These results are consistent with work reported by Friend et al. (l963a,b). This same relationship was also true for CTC treated microbes except that total VFA production from CeC microbes was as great as that for CoC microbes (P<.05). 121 For microbes associated with the gut wall at each intestinal site, were compared within treatment, total VFA production was the greatest for SH microbes (P<.05), compared to that by Cew and COM microbes, which produced more total VFA's than 51]" and $12“ microbes (P<.05) within the Co and CTC treatments. In general, microbes of the stomach are relatively low in number and produce a limited amount of VFA's (Friend et al., l963a; l963b) because the highly acidic environment is prohibitive to the establishment of a large bacterial population and as a result the bacterial species that are capable of tolerating this environment are highly specific (Clarke, 1977b). Species of Lactobacillus, Bifidobacterium and Streptococcus are the primary microflora of the stomach (Smith and Jones, l963) and lactic acid is the primary organic acid produced (Friend et al., 1963a; l963b). Perhaps the relatively high amount of fermentable carbohydrate present in the growth media and the higher pH of the fermentation media (6.7) allowed more microbial fermentation and VFA production than typically observed in vivo. 3. Total non-volatile fatty acid production The sum of the quantity of non-volatile acids (lactic, oxaloacetic and succinic acids) per ml of media are presented in Table 26. In general, total non-volatile fatty acid production (NVFA) was greater for microbes incubated in Co and CTC treated media compared to that for Cu and Cu+CTC treated microbes (P<.05). As for the total VFA production, these differences in NVFA production were confounded by the dramatic reduction in microbial growth when microbes were incubated in Cu and Cu+CTC media (Table l9 and Figures l-5). No differences in NVFA production were noted between treatments within CeC, CeH, CoC and Cow 122 88.8 0 88888 .8.8 ..88.88V .8080.8.8 8..800.8.88.8 080 80g.8u880g=8 0808088.8 8..: 8.:0E8008. acoE0 .008888 080808E 8.8.8: 880020.8.u .m:..uau0cumuco.go Egg oo. +_80ggou Egg omuuoho+=u .0:..88808.0.80.gu Egg oo.u888 .8oggou Egg 888088 ..8cucouuoon .8088808 088888E o. 80 8000 8588 8080888E 80.080.800 .0880E 80 .E 80g 80.00 8.0.0888 8:0 u.uuu0o.0xo .8.880— 80 .80.oE:v 8.8808: 0:. 80 E88 0;. n :o..o=8o8g 8.80 8.808 0.8.0.o>ucc= .0uoh0 888.. 088.. 8.8.. 088.8 m ..080>8 8... 088.. 088.. 088.. 088.. ..0.8 88.88 8... 088.. 088.8 088.. 08..8 8.80.880 88.88 mm.. 888.. 888.. 888.. 088.. .80: .0088 .8.8 0.... 088.8 088.. 0.8.8 8.80.880 .0008 .8.. 088.. 8.8.8 088.. 0.8.. .8.08 8888 ..0: ..8. ..088 ..8. 08... 08..8 8.8.. 08.8. .8.08 0888 8.80.880 ..8. ..088 8..8 8.8.. 08..8 888.. 08..8 ...08 .8.8 ..08 ..8. ..088 8.8. 888.8 0..88 88... 08.8. .8.08 .8.8 8.80.880 ..8. ..088 88.8 888.. 0.8.8 888.. 088.8 ..08 80088.8 om.w nmp.— mo.mp ume.— an.ep mucmucou someoum m ..08088 8.8+08 8.8 08 88 .I 008088 08880.: nucoEu0oLE .mucsom unocu.z 08:88:.408880g :08008800. 8:0: 88 0 80.8< 80.00E we .E\80.oqu 008.088088g 8.u< 8.008 o...0.o>-:oz .0888 808 8:08: .cwEu0088 88 888880gEou .88 0.808 123 microbe sources, which is contrary to that observed for VFA production (Table 25). Microbes from SC and SIZC produced more NVFA's when incubated in CTC media than that produced from those microbes in the Co media (P<.05). However, the inverse was true for Co and CTC treatments within the SW microbe source (P<.05). When microbes of gut wall and contents were compared for each intestinal site, microbes from SC, SIIC and SIZC produced more NVFAs than their respective SW, SIIW and SIZW microbe sources within the Co and CTC treatments (P<.05). Only the microbes of CeW produced more NVFA's than microbes of the CeC within the CTC treatment (P<.05). No differences between gut wall and gut content microbe sources were noted within either the Cu or Cu+CTC treatments. When total NVFA production of microbes from gut contents of each intestinal site were compared, within treatment, microbes of SC, SI]C and SIZC produced more NVFA's than CeC and CoC microbes within the Co and CTC treatments (P<.05). This is likely due to the greater lactic acid production typically observed in the upper digestive tract compared to more total VFA production in the lower digestive tract. No differences in total NVFA production were noted between gut content sources within the Cu treatment but for the Cu+CTC treatment, more total NVFA's were produced from SIIC microbes than for SC and CeC microbes (P<.05). As for gut contents, microbes of SW, 51]" and $12" produced more total NVFA's than for CeW and CoW sources within the CTC treatment (P<.05). Microbes from SW produced more total NVFA's than $11" microbes which produced more total NVFA's than SIZH, CeW and COW microbes within the Co treatment (P<.05). No differences for NVFA 124 production were noted between gut wall sources within the Cu and Cu+CTC treatments. In general, more VFA's are produced in the lower digestive tract than in the upper digestive tract and the reverse is true for NVFA production. Volatile fatty acid and NVFA production were reduced by adding Cu and Cu+CTC to fermentation media, but this reduction in VFA production was confounded with lower microbial growth and mass which greatly influence the quantity of substrate fermented. Copper may inhibit organic acid production and thus spare carbohydrate as observed for the antibiotics Virginiamycin and Spiramycin (Vervaeke et al., 1979) but further studies are required to test this hypothesis. A. 125 SUMMARY Feeding a diet containing 250 ppm supplemental copper increased growth rate of conventional ptgs but decreased growth rate of germ-free pigs compared to pigs receiving a control diet in each environment. In the absence of microflora, the effects of feeding a high capper diet on copper, iron and zinc metabolism in the pig were greatly exaggerated compared to that observed for pigs in the conventional environment. In other words, high copper feeding increased copper concentrations in liver and plasma, increased plasma ceruloplasmin, decreased liver and plasma iron concentrations, and hematocrit and erythrocyte count were reduced more dramatically for germ-free pigs fed the high copper diet compared to conventional pigs fed the same diet. Furthermore, these effects in the germ-free pigs receiving the high capper diet, were of the magnitude typically observed when pigs develop a moderate copper toxicity and may explain the apparent reduction in growth rate of these pigs compared to germ-free pigs receiving the control diet. These results suggest that gut microflora have a significant impact on absorption and retention of copper, iron and zinc in the pig. Germpfree pigs had a lower percentage of leukocytes and mature (segmented) neutrOphils than conventional pigs due to a lack of antigenic stimulation of pigs reared in the germ-free environment. Feeding the high copper diet increased the percentage of immature (band) neutrophils in germ-free pigs but decreased the percentage of these cells in conventional pigs fed the high capper diet. E. 126 Germ-free pigs had a higher percentage of lymphocytes than conventional pigs. The percentage of monocytes was dramatically increased. by feeding the high c0pper diet to germ-free pigs but was reduced in conventional pigs fed the same diet. Peripheral plasma ammonia concentrations were reduced, and portal plasma ammonia concentrations tended to be reduced in pigs fed the high copper diet. Germrfree pigs had lower levels of ammonia than conventional animals due to a lack of bacterial urease. Similarly, germ-free pigs had higher concentrations of urea, and lower concentrations of ammonia in cecal contents of germ-free pigs compared to cecal contents of conventional pigs. However, no effect of copper was noted on urea or amnonia-nitrogen concentrations. Germ-free pigs had a smaller thyroid, liver, stomach, small intestine and large intestine but a larger spleen, lungs, heart and gall bladder than conventional pigs. No differences were noted for thymus, adrenals, kidneys or pancreas weights between germ-free and conventionally reared pigs. Feeding the high copper diet reduced thymus weight. Total thickness of the wall of the duodenum and jejunum was reduced for pigs in the germ-free environment compared to conventional pigs. Feeding the high copper diet reduced total intestinal thickness of the jejunum, tended to reduce total thickness of the duodenum, and tended to reduce thickness of the ileum of germ-free pigs but increased total thickness of the ileum of conventional pigs. G. I. _127 When microflora from gut contents and gut wall from five gut sections were incubated in an anaerobic growth media, the presence of 250 ppm capper dramatically reduced nficrobial growth and, for microbes obtained from the lower digestive tract, inhibited growth, compared to microbes from these same sources grown in the control or 100 ppn chlortetracycline medium. After l6 hours of incubation, microbes appeared to die when incubated in media containing 250 ppn copper which apparently was too toxic to support microbial growth. These results verify that copper, at the 250 ppm level, does affect microbial growth in vitro, but in vivo, the bactericidal effectiveness of copper may be reduced by other factors present in the gut which were not present in the growth media. Furthermore, a level of 250 ppm capper appears to reduce or inhibit microbial growth along all gastrointestinal sites. A small reduction in pH of the growth media after 24 hours of incubation (compared to initial pH) of microbes fron all microbe sources, within the control and l00 ppm chlortetracycline treated microbes, may have been due to acid production during carbohydrate fermentation. When 250 ppm copper and the COpper-chlortetracycline combination were present in growth media, pH increased compared to initial pH. Ammonia-nitrogen production of microbes was greatest in the control treated media, tended to be less when microbes were incubated in loo ppm chlortetracycline media, but both the control and chlortetracycline treated microbes produced significantly higher amounts of amnonia nitrogen than microbes treated with 250 128 ppm copper or the copper-chlortetracycline combination. Most ammonia-nitrogen production occurred in the lower sections of the digestive tract. Differences between treatments in regard to ammonia production were confounded with the mass of microbes present at 24 hours of incubation but a negative net ammonia change was noted for the media containing capper which may indicate that bacterial urease activity was inhibited. Total volatile and total non-volatile fatty acid production was greatest for microbes incubated for 24 hours in media containing the control and lOO ppm chlortetracycline treatments. When 250 ppm copper was present in media, volatile and non-volatile fatty acid production was reduced but this reduction was confounded by a reduction in microbial growth and less microbial mass. Microbes incubated in media containing 250 ppm cOpper or the COpper-chlortetracycline combination treatments, produced no formic or propionic acid which were produced by microbes incubated in control and chlortetracycline media. 0f the volatile fatty acids measured, acetic and formic acid were produced in the greatest quantities particularly by the cecum and colon microbes. Lactic acid was the predominant non-volatile fatty acid produced and was produced in the greatest quantities in the stomach and small intestine. APPENDIX A APPENDIX A A series of five preliminary trials were conducted to determine the effect of autoclaving a typical corn-soybean meal-20% dried whey starter diet on growth performance and nutrient balance. Effectiveness of sterilization was also evaluated to determine the minimum time needed for complete sterilization. Sterilization of solid diets by steam results in loss of nutritive value of proteins through its effects on lysine, methionine and cysteine (Rice and Beuk, 1953), loss of certain B vitamins and vitamins A and E (Zimmerman and Wostmann, l963), formation of potential antagonists to important nutrients or metabolites (Reyniers et al., l946) and the formation of potentially antigenic products. Theoretically, some loss could also occur by leaching, but a comparison of iron and copper contents of diets before and after autoclaving showed no demonstrable loss by leaching (Reddy et al., 1965a, l965b). Nutrient losses resulting from steam sterilization of diets can be minimized by careful control of temperature and sterilization time. Furthermore, nutrient losses from sterilization can be estimated and compensated for by supplementation of the original diet. The results of these five studies presented in this appendix helped provide the basis for formulating diets for autoclaving and use in the germ-free experiment contained in this dissertation. I. EFFECTIVENESS 0F STERILIZATION 0F CORN-SOYBEAN MEAL-DRIED WHEY STARTER DIETS BY AUTOCLAVING AND THE EFFECT OF AUTOCLAVING DIETS, WITH OR WITHOUT VITAMIN SUPPLEMENTATION, ON GROWTH PERFORMANCE OF WEANLING PIGS. 129 130' A. Objective The primary objectives of this trial were to l) determine if processing a practical swine starter diet by autoclaving affects nutrient composition of the diet as measured by growth performance of weanling pigs, 2) determine if supplementing these autoclaved diets with a vitamin-trace mineral premix improves growth performance, 3) determine the effectiveness of sterilization by two methods and 4) determine the minimum time needed to provide adequate sterilization. 8. Methodology Thirty-two crossbred weanling pigs averaging 7.5 kg in initial body weight were assigned to one of four experimental diets for a 3-week growth perfOrmance trial. Experimental diets consisted of a typical corn-soybean meal-202 dried whey starter diet formulated as shown in Table 27. Diet l was a non-autoclaved conventional diet which served as a control for comparison of growth performance of pigs fed autoclaved diets 2, 3 and 4. Diets l and 2 were identical in nutrient composition and were formulated to meet or exceed NRC (1979) recommendations far starter pigs. Diet 3 contained 150% of the vitamin-trace mineral premix and vitamin E-selenium premix while diet 4 contained 200% of each of these two ingredients compared to the levels added in diets l and 2. Refer to Table 28 for the amount of vitamins and trace minerals supplied per kg of diet. Pigs were weighed weekly and feed consumption was measured. Average daily gain was analyzed statistically using a one-way analysis of variance (Gill, 1978). In order to determine the effectiveness of the autoclave procedure on sterilization of these diets, two types of containers were evaluated as well as the minimum length of time necessary for complete sterilization so that unnecessary nutrient loss could be avoided. F1 lullnr 131 ..x.Eoeg .0.0:.E ou0..-c.E0u.> uoom + .o.»:ou 80>0.uou:0. 8 wo.o ..x.E88g .080:.E 080..-:.E0p.> now. + .oeucou 80>0.8ou=0. m 80.8 ..ogucou 80>0.oo.:0. 8 80.8 ...ogucou um>0.uou=0n:o=. . wo.o0 o8.o om.o omno o8.o a .83888g888g 88.8 88.8 88.8 88.8 8 .88.0.08 8... o~.. o~.. o~.. a .8:.8»8 ...8. 88.8. 88.8. 88.8. 8 .8.0.888.00088 8888 8..8 .8.8 .8.8 88..008 .88 8.88.0:< 880.8982 8880.88.08 88.88. 88.88. 88.88. 88.88. 8..8 8..8 8..8 8..8 888-88-8 .88 08.88.-. 88.. 8..8 88.8 88.8 8.8088 08-8 8.80... 88.. 8..8 88.8 88.8 8.8088 8.-8.80..> 88.8 88.8 88.8 88.8 88.-88-8 ..08 8... 8... 8... 8... 888-.8-8 0.0888800 88.0.08 8..8 8..8 8..8 8..8 888-.8-8 0.0888888 88...00.8-8888 88.88 88.88 88.88 88.88 88.-.8-8 8088 80.88 88.88 88.88 88.88 88.88 888-88-8 888 ..008 8008.88 .8.88 ....8 .8..8 .8..8 888-88-8 8880 80..088 880888 8 8 8 . .82 ..08 ..8. .8. 8.80.80888. 0.0.8 .0.808..80888 .. .0.8. 8. 8088 8.0.8 .8 8.8..088 .80...8z 80.0.88.08 880 88...888888 ..8 0.80. 1. 32 Tab1e 28. Amount of Vitamin and Trace Minera1s Supp1ied Per Kg of Diet in Tria1 '1. Experimenta1 Diet 0.50% 1eve1 0.752 1eve1 1.002 1eve1 Nutrient 1 and 2 3 4 Vitamin A. IU 3300 4950 6600 Vitamin D, 10 660 990 1320 Vitamin E. 1U 16.5 24.8 33.0 Menadione sodium bisu1fite. mg 2.20 3.30 4.40 Ribof1avin, mg 3.30' 4.95 6.60 Niacin. mg 17.6 26.4 35.2 D-pantothenic acid, mg 13.2 19.8 26.4 Cho1ine, mg 110 165 220 Vitamin B12. "9 19.8 29.7 39,5 Zinc, mg 75.0 113 150 Iron, mg 60.0 90.0 120 Manganese, mg 37.0 55.5 74,0 Copper, mg 10.0 15.0 20.0 Iodine, mg 0.20 0.30 0,40 Se1enium. mg .10 .15 .20 133 Diets were either p1aced in paper bags in 2.5 kg quantities or in a1uminum pans in 2.5 kg quantities. Feed was even1y distributed to provide approximate1y a 4 cm thickness in either container and paper bags were sea1ed with autoc1ave tape. Before autoc1aving feed, Ki1it ampu1eS- (BBL Mi cr0b10109y Systems. P.o. Box 243, Cockeysvi11e, MD 21030) containing Baci11us stearothermophi1us spores, were inserted into the center of the feed samp1es and feed samp1es were autoc1aved at 121'C for 10. 15, 20, 25 or 30 minutes. Upon comp1etion of the steri1ization procedure. ampu1es were removed and incubated at 60'C for 48 hours to determine the effectiveness of sterilization by monitoring the ampu1es for co1or change. c. Resu1ts 1. Growth performance Growth performance of pigs fed these experimenta1 diets is shown in Tab1e 29. Average dai1y gain for the 3-week period was reduced by approximate1y 542 when the contro1 diet was autoc1aved, and tended to be reduced even further upon addition of higher 1eve1s of vitamin-trace minera1 premix. A1though statistica1 ana1ysis of average dai1y feed intake and feed efficiency cou1d not be performed because of 1ack of rep1ication, feed intake appeared to be reduced when the contro1 diet was autoc1aved (in comparison to pigs fed the non-autoc1aved contro1 diet), but not as great1y when the vitamin-trace minera1 premix was added at higher 1eve1s. The apparent reduction in feed intake was probab1y caused by a reduction in paiatabi1ity of diets after autoc1aving. Feed efficiency tended to mirror the response observed for average dai1y gain observed. Because of the rapid 134 Table 29. Effect of Autoclaving and Addition of Supplemental Vitamin-Trace Mineral Premix to Starter Diets on Growth Performance. Dietary Trea tmenta Growth Parameterb l 2 3 4 Avg. initial wt., kg 7.2 7.3 7.9 7.5 Avg. fina1 wt., kg 10.8 9.2 9.3 8.9 ADG, g 2'54c 136d sad 98d ADFI. g 407 271 365 314 FIG 1.60 1.99 3.72 3.20 aDiet l (non-autoc1aved control). Diet 2 (autoc1aved control). Diet 3 (autoclaved control + 150% vitamin-trace mineral premix). Diet 4 (autoc1aved control + 200% vitamin-trace mineral premix). bADG=average daily gain. ADFI=average daily feed intake. F/G=feed/gain. c'dMean values fbr ADG with different superscripts are significantly different (P<.Ol). MSE = 1108. 135 reduction in growth observed by fEEdl'DQ the autoclaved diets, regardless of the level of vitamin-trace mineral premix present in the diet. a significant amount of protein (amino acid) destruction was su5pected as being a greater contributing factor, when diets were subjected to steam sterilization. A reduction in growth response due to vitamin defi ciency is generally a long-term effect and was not improved by providing a higher level of premix for the 3-week period. A lack of available amino acids generally produces an immediate growth depression analogous to that observed in this trial. As a result, a second trial was conducted to determine the effects of autoc1aving diets fortified with supplemental lysine andimethionine. 2. Effect of sterilization method and time on diet sterilization. In regard to the effectiveness of sterilization of diets using two different (methods) containers for different lengths of time, the paper bag method proved undesirable. The paper bag method required a greater amount of time to package the diets and made it difficult to maintain a uniform thickness of feed, which consequently resulted in a lack of uniformity of heating, and ineffective steri1ization, even with 35 minutes of autoclaving. This lack of uniformity of heated feed was undesirable not only from a sterility standpoint but also in regard to consistency of product quality. Furthermore, after autoclaving was completed and the feed cooled in the bags, the feed became very hard, adhered to the walls of the paper bags and was difficult to remove. In contrast, the use of aluminum pans was much more desirable because of ease of packaging and maintenance of uniformity of thickness, which resulted in uniformity of heating and effective 136- sterilization. It was also much easier to remove the feed from the pan after the feed cooled from the autoclave process. Minimum effective sterilization was obtained by autoclaving feed for 25 minutes at 121’C. Shorter autoc1aving times of 10. 15 or 20 minutes did not consistently provide complete steri1ization as determined with the Kilit ampu1es, while autoc1aving for greater than 25 minutes was unnecessary and avoided to minimize nutrient loss by autoclaving. II. EFFECTIVENESS OF LYSINE AND METHIONINE SUPPLEMENTATION T0 DIETS PRIOR TO AUTOCLAVING ON GROWTH PERFORMANCE. A. Objective As mentioned in the previous trial, supplementation of diets with vitamin-trace mineral premix did not alleviate the depression in growth performance when autoclaved diets were fed to starter pigs. Because of an immediate growth depression, it was suspected that amino acid destruction was the major contributing factor for this growth depression. Lysine and methionine were considered because lysine is the first limiting and methionine is the third limiting amino acid in a corn-soybean meal diet and because these two amino acids are markedly affected by autoclaving (Rice and Beuk, 1953). Therefore, the objective Of this trial was to determine if supplementation of starter diets with 0.38% L-lysine'H01 and 0.102 D,L-methionine alleviates growth depression of pigs fed autoc1aved diets. 8. Methodology Sixteen crossbred weanling pigs from 2 litters averaging 8.1 kg in initial body weight were allotted by sex and weight to one of two dietary treatments for a three-week growth performance trial and housed 137 in two slotted- floor nursery pens. Die‘t composition and calculated nutrient analyses are shown in Table 30. Diets 5 and 6 were identical in nutrient composition but diet 6 was autoclaved. Individual pig weights were recorded on a weekly basis and feed consumption was measured by recording feed added to feeders. Average daily gain, average daily feed intake and feed efficiency were calculated and analyzed statistically using one-way analysis of variance (Gill, 1978). After obtaining the results from the three-week growth performance trial (described in the following discussion). diet 6 was modified by adding non-autoclaved 0.382 L-lysine HCl to that already present in this autoc1aved diet and is indicated as diet 8 in Table 30. Diet 7 served as a non-autoclaved control and had the same level of ingredients and calculated nutrient analyses as in diet 5 which was used in the previous three-week growth performance trial. Diets 7 and 8 were fed to the same groups of pigs as for diets 5 and 6, respectively. for an additional week to ascertain if lysine destruction is the primary nutrient affected by autoclaving to cause the poor growth performance observed when autoclaved diets are fed to starter pigs. Average daily gain, average daily feed intake and feed efficiency were calculated and analyzed statistically using one-way analysis of variance (Gill, 1978). C. Results Nhen 0.38% L-lysine HCL and 0.10% D,L-methionine were added to the diets, autoc1aving appeared to be 9.0% less deleterious to average daily gain and 5.262 less deleterious to feed/gain (Table 31) compared to growth performance of pigs fed autoclaved diets containing 0.19% _138 .88: 08.8>.-8 .8080.0o080ucoc 888.8 + 8 00.8 80>0.0o080. 8 00.8 ..8 00.8 8080.08080-888. . 00.8 .88080.08.808 8 .0.8 ...880880 8080.08080-8888 m 00.80 88.8 88.8 88.8 88.8 8 .8880888088 88.8 88.8 88.8 88.8 8 .88.0.08 88.. 88.. 88.. 88.. 8 .08.888 88.8. 8~.8. 88.8. 8~.8. 8 .8.00088 08888 88.8 ...m 8..8 ...8 88\.008 .8: 8.88.08< 080.8082 8000.80.08 88.88. 88.88. 88.88. 88.88. 8..8 8..8 8..8 8..8 888-88-8 08.88.8008-8.8 88.8 - - - 888-88-8 .08 08.8..-8 8080.08.80-88: 88.8 88.8 88.8 88.8 ~88-88-m .8: 08.8>.-8 88.8 88.8 88.8 88.8 8.8088 08-8 8.80... 88.8 88.8 88.8 88.8 8.5088 2.18.500.> mm.8 88.8 88.8 88.8 ~8.-88-8 0.08 8... 8... 8... 8... ~88-.8-8 000808800 888.0.08 8..8 8..8 8..8 8..8 888-.8-8 000888888 88.0.00.8-0coz 88.88 88.88 88.88 88.88 ~8.-.8-8 8082 80.88 88.88 88.88 88.88 88.88 888-88-8 888 ..008 8008888 88.88 .8..8 .8..8 .8..8 888-88-. 8880 80..088 888880 8 8 8 8 .oz ..08 .08. 8080.80888. 000.8 .00808.80888 .022.:0383:8:88288828283232.20888858.202: Tab‘ Grow F/G “Die' Die' Die' Die' non- *- ”ADG: ADF] F/G= t-d (P<. eMean 139 Table 31. Effect of Autoclaving and Addition of Supplemental Lysine and Methionine to Starter Diets on Growth Performance (3 weeks) and the Effect of Addition of Non-Autoclaved Supplemental L-Lysine HCl to Diets for an Additional 7-Days on Growth Performance. Dietary Treatmenta 3-week Growth Performance 4th Week Growth Performance 5 6 7 8 Growth Parameterb NAC+Lys+Met AC+Lys+Met NAC+Lys+Met AC+Lys Avg. initial wt., kg 8.3 8.5 9.6 9,2 Avg. final wt.. kg 9.6 9.2 9,9 9.5 mg, g 267c 155d 435e 4516 ADFI. g 639 4ll llBD 939 FIG 2.39 2.65 2.71 2.08 aDiet 5 (non-autoclaved + 0.38% L-lysine HCl + 0.lOZ D,L methionine). Diet 6 (autoclaved + 0.38% L-lysine HCl + 0.10% D,L methionine). Diet 7 (non-autoclaved + 0.38% L-lysine HCl + 0.102, D,L methionine). Diet 8 (autoclaved + 0.38% L-lysine HCl + 0.l0% D.L methionine + 0.38% non-autoclaved L-lysine HCl). bADG=average daily gain. ADFI=average daily feed intake. F/G=feed/gain. c"‘Mean values fOr ADG with different superscripts are significantly different (P<.03). MSE = 5609. ‘eMean values fOr ADG are not significantly different (P>.l0). MSE = 20,614. 140 supplemental L-lysine HCl in the previous trial (Table 29). Average daily feed intake was depressed (P<.03), but to a lesser extent when 0.38% supplemental L-lysine HCl and 0.10%, D,L-methionine was included in the autoclaved diet compared to results in the previous trial. These results suggested that growth performance tended to improve by supplementing diets with lysine and methionine before autoclaving but these levels of lysine and methionine were not high enough to give growth performance similar to pigs fed the non-autoclaved control diet. Since lysine is the first limiting amino acid in these diets, and because methionine can be toxic when added at higher levels, and to verify that the depression in growth response was due to amino acids (lysine), the autoclaved diet (Diet 6) was reformulated and fortified with an additional 0.38% L-lysine HCl after autoclaving to provide a total of 1.65% lysine in the diet (Diet 8) at the end of the three week trial. It was estimated that 35% of the lysine in the diet was destroyed by autoclaving. Non-autoclaved L-lysine HCl was added at the level of 0.38% (0.30% actual lysine) in addition to that already present when the diet was originally mixed. If 35% of the lysine was destroyed by autoclaving, diet 6 would have an available lysine level of approximately 0.88% after autoclaving which would explain the poor growth performance observed in this trial. Consequently, the addition of non-autoclaved 0.382 L-lysine HCl to diet 6 would raise the level of available lysine to 1.18% (diet 8). The reformulated diet was fed along with the original diet 5 (indicated in Table 30 as diet 7) to the same pigs for an additional week to ascertain if the destruction of lysine was the major factor for poor growth with the autoclaved diet. 141 At the end of the 7-day feeding period, average daily gain for pigs fed diet 7 was 435 g/day while it was 451 g/day for pigs fed diet 8 containing 1.65% lysine (1.18% estimated available lysine) which would be similar to that present in the non-autoclaved diet. Feed efficiency was 2.71 and 2.02 kg feed/kg weight gain while average daily feed intake was 1.18 kg and 0.91 kg feed consumed per day for pigs fed diet 7 and 8, respectively. Although this feeding period was of short duration, it reconfirmed that the majority of growth depression observed was due to a reduction in availability of lysine. and that diets could be fortified with L-lysine HCl prior to autoclaving to give similar growth responses for a 3-week growth performance trial when autoclaved diets were fed to germ-free and conventional pigs in the germ-free experiment. Although a level of 1.65% lysine in the pre-autoclaved diet appeared to alleviate growth depression from the reduction of lysine availability during the last week of this trial, a third trial was conducted to determine the optimum level of supplemental L-lysine HCl needed in autoc1aved diets to produce a growth response analogous to the non-autoc1aved original diet used in this trial. III. BIOAVAILABILITY 0F LYSINE IN AUTOCLAVED CORN-SOYBEAN MEAL-DRIED HHEY STARTER DIETS. A. Objective In order to determine the optimum level of synthetic L-lysine HCl to add to pre-autoclaved diets to obtain growth performance similar to that of pigs fed a non-autoc1aved diet containing approximately 1.35% lysine (Trial 2), this experiment was designed to examine the growth performance responses of pigs fed diets containing increasing levels of L-lysine HCl when diets were autoclaved for 25 minutes at 121'C. 142 8. Methodology Four litters of crossbred weanling pigs (28) averaging 7.8 kg in initial body weight were allotted in pairs,based on sex, weight and litter, to one of 7 experimental diets for a three-week growth performance trial. Experimental diet composition and calculated nutrient analyses are shown in table 32. Diets were autoclaved for 25 minutes at 121°C. Weight gain was recorded for each pig and feed consumption was measured and recorded on a weekly basis in order to calculate average daily gain, feed efficiency and average daily feed intake. Data were analyzed statistically using orthogonal polynomial contrasts (Gill, 1978) to characterize the form of response and to estimate the level of supplemental lysine needed in autoclaved diets to obtain maximum growth rate. C. Results Cumulative 3-week growth performance data are shown in Table 33. No significant differences for average daily gain (ADG) were noted for first, second or third degree nonlinearity but the response was strongest (P<.40) for second degree (quadratic) nonlinearity. As a result, a prediCtion equation was deve10ped for second degree nonlinearity which is Y=83.49 + 102.16xi -26.88x12. The stationary point (x5), where an estimate of the level of lysine at which maximum growth is expected to occur is xS=-§]/2§2=-102.16/2(-26.88)=1.90% lysine. Therefore, a level of approximately 1.9% total lysine was added to eXperimental diets before autoc1aving for use in the subsequent germ-free experiment. It appears that at levels higher than 2.0% total lysine, growth rate and feed consumption decline, perhaps due to an amino acid imbalance. Because 143 amen paucmswcquu ~e.e Ne.o Ne.c ~e.o wo.o ~e.o ~e.o a .maeeeemeea .m.o _m.o .m.o Fm.o .w.o _m.o .w.o a .e=_epeu m~.~ om.~ m~.~ oc.~ m~._ om._ m~.F a .ee_m»4 oo.w. ~_.wp m..mp m_.w_ _~.w_ e~.w_ N~.mp a .e_eeeee eeeeu neon eeom mmom meow Napm ~_.m m~_m m¥\_eex .mz mwmxflm=< u:m_eu=z umump:u_ou oo.oo_ oo.ocp oo.oo_ co.oo_ oo.oo_ oo.oo. oo.co_ _eeep ~_.~ mm._ mm._ _~._ ww.o ~m.o m~.o Pu: ee_m»_-4 om.o om.o cm.o om.o om.o om.o om.o x_Eeea em-w =_Eeee> om.o om.o om.o om.o om.o om.o om.c x_eeee-zh c.5ee_> mm.o mm.o ww.o mm.o mm.o ww.o mm.o e.em oc._ co.. oo.. oo._ oo.. oc._ oo.. eeeeeeeee e=,e_eo m~.o m~.o m~.c m~.o m~.o m~.o m~.o eeeeemeee e=_e_ee_e-eeez oo.o~ oo.c~ oo.o~ oo.o~ oo.o~ oo.o~ oo.o~ see: ee_eo oo.m~ .cc.m~ oo.m~ oo.m~ oo.ww oo.m~ oo.m~ nee ._eee eeeeaem m~.me mo.om sm.cm ae.om .o._m mm._m me._m eeee ee__eem eeaeeu m. e, m_ N_ ,_ o. a dag eee_eeemmw m»_ nom.F m». um~._ m». so._ a». am“. a». gem. map amN. ee_e ... 33m + pommm ... Emma + pummm + pmmum + memm Emma tm>o _.uou:< um>m puouz< uo>o —uou=< vo>m —uoa=< um>w —uou:< v96 puoa3< Uw>m puouz< .m E: 5 32. ”38.5 B 23:22 23.532 3:328 25 5:53.89 .3 m3: 144 .mopp n um: .Aoe.vav »amgmmcwpcoc “owuaeumzav cognac vacuum .>p_gmo:__co: mogmou Aupnsuv ugpgu Lo Aopumgvesav vacuum .Acmmcwpv umcwm gee manguemcu xcepm_v coozuon acocmeemu apueeuwmvcmpm no: me: even xpwmn masco>au~mo< .cpmm xpwoo mamgm>uuuoapuous< c. cava»; Poucmsopgqsm ea apo>mb mcpmemeucH we uumeeu .mm open» ‘145 growth rate appeared to be affected more dramatically than feed consumption, feed efficiency is also worsened. Average daily feed intake (ADFI) tended to follow the same pattern as for average daily gain while the feed efficiency (F/G) response appeared to be third degree (cubic) in nonlinearity (Table 33). Statistical analyses were performed only for average daily gain because it provides a more reliable estimate of lysine bioavailability than ADFI or F/G and because there were more observations for ADG (4) compared to ADFI and F/G (2) per treatment. When comparing ADG, ADFI and FIG values for pigs receiving the autoclaved diet containing 2.0% lysine versus the non-autoclaved diet containing l.35% lysine (Table 30 and 31), ADG appeared to be approximately 85%, ADFI was 76% and FIG was lll% of the growth response of pigs fed the control non-autoclaved diet. Perhaps, some of the reduction in growth performance of pigs fed the autoclaved diets was due to a reduction in palatability which appears to be the case in all of the preliminary trials. Nevertheless, it appears that a major proportion of the growth depression of‘ autoclaved swine diets is due to the apparent destruction or lowered bioavailability of lysine which can be corrected by proper ‘fortification of diets with synthetic-lysine HCl prior to autoclaving. It also appears that approximately 35% of the lysine in the diet is destroyed by autoclaving which supports our findings in Trial 2. Iv. EFFECTS OF AUTOCLAVING AND SUPPLEMENTING DIETS NITH 250 PPM COPPER 0N ENERGY, NITROGEN AND MINERAL BALANCE OF WEANLING PIGS. A. Objective Our previous studies, as well as work by Rice and Beuk (l953), have indicated that the bioavailability of certain amino acids (lysine, 146 methionine and cysteine is reduced by autoclaving. Furthermore, Zimmerman and Wostmann (l963) showed that certain B vitamins and A and E are reduced upon sterilizing conventional diets with steam even though our initial studies showed no benefit in growth performance from adding additional vitamin-trace mineral permix to autoclaved diets fed to pigs for a 3-week growth performance trial. Reddy et al. (1965b) compared the copper and iron content of diets before and after autoclaving and observed no demonstrable loss from leaching which may theoretically occur. Although the effects of autoclaving diets on' energy utilization have not been studied, energy utilization is not likely to be affected and may be improved by heating. Because of the lack of knowledge of the specific effects of autoclaving conventional starter diets, this study was designed to quantitatively examine differences in energy, nitrogen and mineral utilization of autoclaved and non-autoclaved corn-soybean meal-dried whey starter diets wfith or without high levels of supplemental copper, when feed intakes are standardized. B. Methodology Sixteen crossbred pigs, 4 weeks of age, obtained from two litters and averaging 7 kg of initial body weight, were allotted by sex, litter and initial body weight to one of the four experhnental diets (Table 34) in a 2 x 2 factorial arrangement of treatments for a lD-day balance trial. Pigs were housed in individual collection cages and fed twice daily an amount of their respective experimental diets equivalent to 2% of their average initial body weight. Pigs were removed from their collection cages at each feeding and fed a gruel mixture in individual feeding cages to alleviate problems associated 147 umpu _uu:mswcmnxu ww.o No.0 Ne.o No.0 a .mseoeemeee .m.o .m.o _w.o _m.o a .e=_u_eu mm._ mm._ mm._ mm._ a .eeemas o~.w_ o~.m_ o~.w_ o~.m_ a .e_eueee eueeu oo_m mo_m oo_m mo.m a¥\_eex .m: m .—m>.—a:< ”:5 75:2 cava ~30 Eu o.oo_ o.oo_ o.oo_ o.oo_ .eeoe c_.o -- o_.o -- m_~-_o-e ouzm.eom=u oo.. oo.. oo.. oo.. -o-mo-m .9: eer»_-s cm.o om.o om.o om.o x_eeee em-“ esseu_> om.o om.o om.o om.o x_eeeq ze-e_2ee_> ww.o mm.o mm.o mm.c ~m_-eo-e e.em oo._ oo., oo.P oo.. mme-_o-e eueeoeeeu e=_e_eu m~.c m~.c m~.o m~.o omo-po-e eueeemeee e=_e.eupe-eeez oo.o~ oo.c~ oo.o~ oo.o~ ~m_-_o-e see: emcee oo.m~ oo.m~ oo.m~ oo.m~ eoe-eo-m gee ..eee eeeexem cm.om om.om om.om oa.om mm¢-~o-e eeee ee__eem eeeeeu a. w_ A. e. .ez .eez .ues any eee_eeemmw omxm cameo + .eeeeoe o~=m eomau + Peeeeoe um>m_uou=< um>mpoopa< uo>opuou=aicoz um>o_uou=micoz .e Fe_eh =_ new: meewo ea m_mxaee¢ u=e_eu=z emee_=u_eu wee eo_s_moeeeu .em e.eee 148 with contaminating the collected feces. After a 5-day adjustment period, used to acclimate pigs to their respective diets, feeding schedule and environment, collection cages were cleaned to start the 5-day collection period. Concentrated hydrochloric acid was added to urine containers to eliminate microbial growth in the collected urine. At the end of the collection period, urine volumes were measured and a 100 ml sample was retained for subsequent laboratory analyses. Collected feces were dried, weighed and taken to the laboratory for analysis along with feed samples of each diet. Gross energy of feed feces and freeze dried urine was determined by using an adiabatic bomb calorimeter (Parr Instrument Company, Moline, IL) standarized with benzoic acid. Nitrogen content of feed, feces and urine was determined by a semi-micro Kjeldahl method (AOAC, 1984). Calcium, iron, zinc and copper concentrations were determined by atomic absorption spectrophotometry (Model 95l, Instrument Laboratory, Inc., Lexington, MA) while phosphorus determinations were conducted using colorimetric spectrometery (Gomori, 1942). Concentrations of these nutrients were then used to calculate daily intake and excretion of nutrients to determine daily nutrient balance. Data were analyzed using Federer-Zelen factorial one-way analysis of variance (Gill, l978). C. Results l. Energy balance Although daily feed intake was standardized across all treatments, some pigs would not consume all of their meal, particularly those pigs receiving the autoclaved diets because of apparent palatability differences. As a result, daily feed intake was different .comuuocuucm ammo i acmuom: u axxm .uuomom ammo .Amouooms mm com ue—~_ no acm>opuouzov powwow acmuamg .Ao—.on accomomcamm ac: u m: .mucoumwmcomm .oomummuoum mo szmp u oopo> on .eoccm oeosom come u mmxu .mz umpumcgou :mmccumc u 2m: .Amom— ..po um mmomav z a\_oox oo.w x Amv mucopoo z u comuumLLou umcomou .om o_omm eoee coco—mo z m__mo II II I D 0 '0- zmcocm wmao~mponaume u m: .mmcmco mmnmumommc n ma .mmcmem mmocm u man .Aouzm.comau mm cmooou monom5m_ooam sag emu + um>o_uou=uv ow ammo .A_oemooo oozmzoooomo o. bozo .Acwzm.¢om=u mm Loqoou .ouco5o_aa:m Eng omN + um>omoou=oioocv w— Home .Apocucou um>opuou=mi=o=v up woman 149 moo. mz _oo. mo.~ o.mm m.om o.mm _._m moo_ x oo\zmm moo. mz No. moo. m_.m om.m om.m o~.m o\_moz .zoz -- -- -- -- ~.o~ o.~_ o.om m.m~ _moz .oomoeoeeoe ozeo_mo mz moo. _oo. .oo. oo.w oo.. mm.o No.m o .ooommmo z m_.mo moo. mz moo. moo. o~.m om.m oo.m _m.m o\_moz .oz -- -- -- -- ~.o_ m._~ o.m~ ~.m~ .moz .oo oemeo m_ooo No. mz poo. mo.~ o.~o z.mm m.oo o.oo moo_ x mo\oo .o. mz moo. moo. ~m.m oo.m om.m mo.m o\_moz .oo mz mz mz Nmmm m._oo m.~om m._mo m._mo o .ezmoom mo m_mmo mz oo. mz omm o.mm~ _.mo~ o.oo~ o.om~ o .ozoooo come m__mo -- -- -- -- mo.o mo.o oo.m mo.m o\_ooz .oo x a“: a 2 2 2 ozooozoé oo.w '1 ll .1! mung—5&9; beau _. D K 303 PO> n— ooooo_ oo. mm «1. Tiiiiiiiqiiimflmmw iwowwopwwowwomamflfloflafl 5.5 cm oo 5:52 ogmzoé mo Sootw mm '130 across dietary treatments which confounds the true effects of nutrient balance. The non-autoclaved diets were higher in digestible energy ( DE) (P<.003), metabolizable energy (ME) (P<.002) and metabolizable e nergy corrected for nitrogen balance (MEN) (P<.02) compared to a utoclaved diets (Table 35). However, pigs receiving these I". (in-autoclaved diets tended to consume more gross energy although there was no statistical difference in gross energy intake. If intakes were 5 ‘tandardized to 250 g of feed intake per day, and assuming that DE, ME a nd MEN are proportional to the amount of GE consumed, DE values would be 3.6l, 3.63, 4.l0 and 3.48 kcal/g of feed consumed which would i ndi cate that energy utilization of autoclaved diets is improved as mi ght. be expected, since heating of grain diets (i.e.. as in a pelleting process) does improve energy utilization by the pig (Erickson et al., 1980; Seerley et al., l962). Nevertheless, comparing numerical d‘i fferences between the non-autoclaved and autoclaved control diet for DE, NE and MEN. they are almost identical even though the pigs consumed less energy when the diets were autoclaved. There were no differences due to copper supplementation between the two diets, but there was a heating x diet interaction for DE, ME and MEN (Table 35). I h each case, adding copper to the non-autoclaved diet increased DE, ME and MEN but decreased these parameters when COpper was added to the autoclaved diets. This general relationship exists even when these parameters are adjusted to the same energy intake and is supported by ef'lergy balance data of non-autoclaved diets from preliminary experiment 5 which will be described in the next section. It appears that autoclaving these diets has no effect on, or may ’1 rIcrease, DE, ME and MEN. and that copper supplementation increases 151 E)E, ME and MEN When included in non-autoclaved diets. However, these energy parameters are reduced when pigs are fed autoclaved diets containing supplemental copper. 2. Nitrogen balance As for energy balance, differences in feed (nitrogen) intake ( P<.09) tended to confound the true picture of nitrogen utilization in pigs fed these diets (Table 36). Daily nitrogen absorbed, apparent d igestibility of nitrogen, daily nitrogen balance, net protein 1.1 tilization and biological value of protein were higher (P<.00l) when n on-autoclaved diets were fed compared to pigs fed the autoclaved diets ( Table 36). However, this same relationship would also exist if pigs consumed a standardized amount of nitrogen. These data support our previous observations in regard to improvements in growth performance of pigs when diets were supplemented with L-lysine HCl prior to autoclaving, and the reduction of growth performance when autoclaved diets were not fortified with lysine, compared to pig performance when non-autoclaved diets were fed. Copper supplementation increased daily n itrogen absorption (P<.03). net protein utilization (P<.02) and b1'ological value of protein (P<.05) when both autoclaved and hon-autoclaved diets were fed (Table 36). This same relationship 1 exists when daily nitrogen intakes are standardized, which suggests that feeding high copper diets improves nitrogen utilization, perhaps by increasing peptic hydrolysis of proteins as shown by Kirchgessner et a] - (l976) or by reducing the amount of bacterial urease activity and thus reducing the absorption of ammonia and production or urea, (Visek e1; al., l959) which would produce such effects. This relationship 1fielnded to exist in a preliminary experiment (discussed in the next 152 ..moooeoe om tom o.m~m om oomzmmoomom. mooemo oomoeeo .oommomeemem Hemoioomooez u oxzo .ouoomo ammo II II I D a) ‘0- .Aom.xov acoumwmcmmm no: u m2 .mocoummmcmmm poumpmmampm mo mo>om u o=—o> on .coccm ocozcm come u umzo .Aomzm.¢omso mo goooou poucmsopoozm sag omm + uo>opuoa=o. ow ammo ...oemooo oezmmoomooo om memo ..omzm.¢omzu mo zoaoou popcoEomoosm son omN + uo>omuopzoicocv mp ammo .Apogucou um>opuou=oicocv so woman .Auoncomno z « mucopoo 2V com u ammuozo mo msmo> Foommopomm .zozmoom z « ooom_mo zo oom n m .oommmNmmmeo omoooeo mozo op. mo. moo. o.oo o.om m..o m.mo m.mo omomoea mo oomez .mo_m mz No. moo. o.om o.oo m.om m.mm m.mm m ...mmo .omoooeo moz moo. mz moo. moo. oo.w oo.. mm.o ~m.m o .ooommmo z zmmoo -- -- -- -- mo.~ mo.~ om.~ m..~ o .z oomeo mpmoo mz om. moo. oo.o o.mm o.mm m.mm m.mo m .z oo .omo .ooz mz mo. .oo. .oo. mo.m oo.o .m.o oo.o o .z oooeomoo zmomo -- -- -- -- oo.m oo.. om.o mm.” o .z Poem: zmmmo mz mo. oo. mmmo .o.m om.o oo.m m_.m o .ezome. z z_.mo ooxz oo oz ommz om mm mm mm meoooEmema ooooemmz mozfim> a nozozomozz zzmoooo I11 ll\ oozoomm_ow_m .oo_mm_moon (iiiiiiiii .ooommmm oooozmzz .5 33o 3 zooooo 355333 23 cm N we 55:84 can 957060954 we macaw“; .cm 020». 153 section) when daily feed intakes were identical, even though dried whey was not included in the diet. A heat treatment x copper interaction ( P<.003) existed for daily nitrogen balance indicating that copper was much more effective (50% more effective) in improving daily nitrogen Da lance for pigs fed the autoclaved diets compared to the pigs fed the n on-autoclaved diets. This could be possible since there is poorer a. mi no acid availability of autoclaved diets due to amino acid de struction during the autoclave process which consequently results in 'I e 55 amino acids being absorbed. As a result, more amino acids are available to the gut microflora for deamination and urea production which may be inhibited by COpper. 3. Mineral balance a. Calcium and phosphorus Effects of autoclaving and copper supplementation on mi neral balance are shown in Table 37. Daily calcium retention was not a ffected by autoclaving or copper supplementation. Phosphorus "e tention was also not affected by autoclaving but was improved (P<.04) by adding 250 ppm supplemental copper to these diets. b. Leena: Copper retention was reduced in pigs fed autoclaved diets (P<.00l) a"<1 a heating x copper interaction was noted (P<.002) since high copper feeding increased copper retention of the non-autoclaved diet but decreased copper retention when present in autoclaved diets fed to the se pigs (Table 37). A portion of the reduction in copper retention f0? pigs fed the autoclaved high-copper diets was no doubt due to the 1 Ower copper intake relative to fecal copper excretion compared to the "On-autoclaved high-copper diet. Perhaps another cause for this reduction in copper retention is that autoclaving diets affects the a"hilability of copper. 154 Noo. mz poo. o.o~ mo.o mo.om oo.o~ mo.o. oo_meoooe m moo. poo. poo. mm.m mm.m mo.o mo.om mo.o oe .ooemmmo zmmoo oo. .oo. mz omo. mo.o mo.o mo.o mo.o oe .oemez zmmmo .o. moo. No. No.m mo.om om.~ oo.oo No.m oe .pmoem zmmmo moo. moo. moo. omo. mo.om mm.m _o.om mm.m oe .ozomo. z_moo ummmmw mz oo. mz ~.- o.om m.om o.mm m.~m oo_oooooz m mz oo. om. ooo. mz.o mo.o mo.o mo.o o .eoommmo mmmmo No. ooo. moo. moo. oo.o mo.o mo.o mo.o o .eemeo mzmoo mz mz mz omo. om.o om.o om.o mo.o o ..mooo zmmoo mz mz mz omo. om._ o~.. om._ om.. o .ozmmom zmmoo msgogmmcgo mz mz mz o.mm m.om ~.mo m.po o.oo ooooooooz m mz mz .oo. o_o. oo., oo.o o... oo., o .oooommo zmmoo mz mz .o. ooo. mo.o mo.o mo.o. mo.o o .oomeo z_mmo oo. mz mz omo. mo.o mo.o oo.o mo.o o ..moeo mmmmo No. mz ooo. omo. eo._ mm._ No.m m_.~ o .ezome. zmmmo Esmupou oxz o z oomz om mm mm mm moeeomz ooze, z mucosa: ago; 3st.. a 22:53 ll). ,1 co 32o 3 .538 3:22.53 :3 SN 1.1.42.2 Es; we co'uwcc4 cc“ acw>opuopz¢ we muuwwwm .fim apps». 155 .comuuogmucm ummuimcpaoozuo x :* .uumowo name a no .Amouocme mm so; u.p~p um m=m>o—uou:o. uumwwm mcmuomz u I “U .Aom.xov acoummmcmmm accumz .ooooumomcmmm poomummuoum we mo>mm u osmo> ou .goszo ocosam some n umzn .mowzm.¢om:o mo cmooou monomsomoosm zoo omm + ou>opoou:o. o~ puma .mpoeucou uozopuouso. mp ammo ..om:m.¢om=o mo cocoon moucmsmpoosm Ego omm + om>opuou=oicocv.m— ammo _ .Amozueou um>omuou=oicocv mp woman oo. mo. mz m.~o mo.o oo.o~ No._~ oo.- ooomooooe m oo. oo. mo. ~.oo oo.om ~.m~ m.m~ o.o~ oe .ooommoo zmmeo mz mz mz mo.o o~.~ oo., oo.. mo.o oe .oomeo mpomo oo. mz mz om. m.oo m.om o.mo o.om me .mmeeo mmmmo mz mz mz mum o.mom m.mom m.oom m.mmm oe .ozmmem mmmoo :9: mo. mz oo. omo m.oo- ..m. o..~ o.om commeomez m oo. mz mo. m.om o...- mo.o oo.m mo.m oe .ooommmo mmmmo .oo. mz moo. oo.o mo.~ mm.. mo.o eo.o os .oomeo mmmmo oo. mz mz o.mm m..m o.om m.m_ ~.m~ oe .mooem zmomo mo. mz moo. mo.m o.- ~.- o.m~ o.o~ me .ozooo. zpmmo ucpN ox: o z oumz om o. o. No .meee_z 2;“ Egg Eta iii (1:. (iiiiiiii. vacuumwmcmmm poomummuopm I! co 35 3 .533 Psogoaoom :3 SN .f .9 $325 .22; oo oo'owooz coo mowzmmoopzz oo mooomzu .mm opomp 156 c. Zinc ~Zinc retention was reduced by autoclaving (P<.04) and a heating x diet interaction was noted (P<.03). where zinc retention ‘i’ ncreased in the non-autoclaved-high-copper diet but decreased when the same diet was autoclaved (Table 37). The bioavailability of zinc may have been reduced when the high capper diet was autoclaved but the reason for a loss of zinc from the pig (as indicated by the negative Zn retention of pigs fed diet 20) is not very apparent and may be a result 0 f contamination of fecal samples. d-M Iron balance (P<.02) and iron retention (P<.03) was reduced (Table 37) by feeding the high copper diets and were reduced to a greater extent when diets were autoclaved (P<.06, P<.09, respectively). Reductions in iron balance and retention (and zinc balance and retention of pigs fed diet 20) from feeding the high copper diets may be expected, since copper, zinc, iron and other divalent fi‘letals are believed to compete for binding sites in the intestinal mucosa (Underwood, l981). Thus, a high copper diet would reduce zinc and iron absorption by inhibiting the uptake of these elements to a greater extent than when a low copper diet (diet l7 and 19) are fed. V. EFFECTS OF FEEDING A CORN-SOYBEAN MEAL STARTER DIET CONTAINING 250 PPM SUPPLEMENTAL COPPER ON ENERGY, NITROGEN AND MINERAL BALANCE . A. Objective In order to determine the influence of feeding high copper diets to weanling pigs on energy and nitrogen utilization when feed "ntake is standardized, a balance trial was conducted to partition 157 digestive and metabolic effects of energy and nitrogen as influenced by high capper feeding. The effects of a high copper diet on copper, iron and zinc balance were also determined. 8. Methodology Twelve crossbred pigs, 50 days of age and averaging 20.4 kg in initial body weight, were assigned to one of two experimental diets (Table 38) for a lO-day balance trial. All pigs were fed 375 g of their respective experimental diets, twice daily for a 5-day adjustment period and during the subsequent S-day collection period. Procedures used in conducting this trial are identical to those described for trial IV in this Appendix. Data were analyzed statistically by using one-way analysis of variance (Gill, l978). C. Results l. Energy balance No differences were noted for digestible energy (DE), metabolizable energy (ME). or metabolizable energy corrected far nitrogen (MEN) when a diet containing 250 ppm supplemental copper was fed at the same daily intake level as the control diet (Table 39). There was a slight tendency for DE, ME and MEN to be improved by high copper feeding as observed in Trial IV when pigs were fed the non-autoclaved diets. 2. Nitrogen balance More nitrogen was absorbed (P<.Ol) when diet 22 was fed, which subsequently resulted in an increase in nitrogen balance (P<.0l) and was not affected by the slight increase in daily nitrogen intake since net protein utilization was also improved (P<.0l) compared to pigs fed the control diet 2l (Table 40). This increase in nitrogen .158 Table 38. Composition and Calculated Nutrient MIGJJS“ 01' Diets U599 1" "'1“ 5- Experimental Diet Control + 250 ppm Cu ingredients (%) 2) 22 Ground shelled corn 4-02-935 68.67 68.57 Soybean meal, 44% 5-04-604 27.40 27.40 Mono-dicalcium phosphate 6-0l-080 l.13 l.l3 Calcium carbonate 6-0l-632 l.45 l.45 Sal t 6-04-152 0. 35 0.35 Vitamin-TM premix 0.50 0.50 Vitamin E-Se premix 0.50 0.50 CuSOg' H20 6-0l-7l9 -- 0.10 Total 100.00 100.00 Calculated Nutrient Analysis ME. kcal/k9 3130 3127 Crude protein, 1 18.1 lB.l Lysine. 1 0.97 0.97 Cal c1 um. 1 0.87 0.87 ghosphorus. 1 0.60 0.60 159 Table 39. Effects of Adding 250 ppm Supplemental Copper on Energy Balance. Statistical Dietary Treatment Significance Control +250 ppm Cu Energy Parametera . 2l 22 MSEb P valuec GE, kcal/kg 4.02 4.02 -- -- Daily feed intake, g 750 750 -- -- Daily GE intake, g 3015 3015 -- -- Daily fecal GE, kcal 427.5 394.3 1980 us DE. kcal/k9 3.45 3.50 0.001 NS ME/GE x 1002 85.8 35.9 2.18 NS Daily urine GE, kcal 58.8 74.9 48.67 NS ME. kcal/9 3.35 3.39 0.001 us Daily N balance. 9 10.5 11.5 0.07' .01 Caloric correction, kcal 70.0 76.8 -- -- MEN . kcal/9 3.27 3.29 0.001 NS ME x 100% 81.2 81.9 1.71 NS aggsgross energy. og=digestible energy. Mas-metabolizable energy. Daily N balance from Table 40. Caioric correction = N balance (9) x 6.77 kcal/g N (Diggs et al., 1965). M5N=ni trogen corrected ME. szfismean square error. Cp value = level of statistical significance, NS: not significant (P>.l0). 160 Table 40. Effects of Adding 250 ppm Supplemental Copper on Nitrogen Balance. Statistical Dietary Treatment Significance Control +250 ppm Cu Nitrogen Parametera 21 22 MSEb P valuec Daily N intake, 9 19.80 20.55 -- -- Daily fecal N, g 3.72 3.57 0.14 NS Daily absorbed N, g 16.1 17.0 0.14 .01 Apparent dig. of N, % 81.2 82.6 3.34 NS Daily urine N, g 5.51 5.46 0.15 NS Daily N balance, 9 10.6 11.5 0.07 .01 Net protein util., % 53.4 56.1 1.61 .01 Biol. value of protein 65.8 67.9 3.46 NS aNet protein utilization, % = 100 (N balance 2 N intake). Biological value of protein = 100 (N balance 2 N absorbed). bMSE=mean square error. cP vahlue=1eve1 of statistical significance, NS=not significant (P>.10). 161 utilization could be a result of increased peptic hydrolysis of proteins during digestion when in the presence of high cupric ion concentrations (Kirchgessner et al., 1976). There was also a tendency for copper supplementation to improve the biological value of protein. 3. Mineral balance a. 22222 Daily copper intake was greater and daily fecal and urinary copper excretion was greater (P<.Ol) when pigs were fed diet 22 (Table 41). Although copper excretion increased by feeding the high copper diet, the amount of copper retained by the pig, relative to daily copper intake, was also increased (P<.05). These results are consistent with other reports (Underwood, 1977) where copper absorption increases as dietary copper increases until copper storage organs are saturated and homeostatic mechanisms then reduce the amount of copper absorbed relative to intake. b. 2135 Fecal zinc excretion was increased (P<.05) by feeding the high copper diet (diet 22) but zinc retention was not affected (Table 41). c. 1323 No differences were observed fer iron balance or retention when the copper supplemented diet was fed as compared to pigs fed the control diet (Table 41). 162 Table 41. Effects of Adding 250 ppm Supplemental COpper on Copper, Zinc and Iron Balance. Dietary Treatment Statistical Significance Mineral 21 22 MSE P value 92222 Daily intake, mg 14.3 214 -- -- Daily fecal, mg 12.5 168 110 .01 Daily urine, mg 0.22 2.08 0.10 .01 Daily balance, mg 1.64 43.4 110 .01 % retention 11.4 20.3 32.2 .05 Zinc Daily intake, mg 198 91.3 -- -- Daily fecal, mg 72.6 84.4 56.0 .05 Daily urine, mg 4.83 4.59 4.68 NS Daily balance, mg 13.9 19.0 66.2 NS % retention 15.2 17.6 62.2 NS 193 Daily intake, mg 212 210 -- -- Daily fecal, mg 173 171 57.8 NS Daily urine, mg 3.01 2.15 0.54 NS Daily balance, mg 37.1 37.8 59.9 NS % retention 17.5 18.0 13.4 NS aMSE=mean square error. bP value=1eve1 of significance, NS=not significant (P>.10). APPENDIX B APPENDIX 8 Prior to the start of the gnotobiotic portion of the experiment, a great amount of preparation was required. The following discussion describes in detail, the various steps involved in preparing and sterilizing the isolators and equipment necessary to obtain and successfully rear one litter of germ-free pigs for the seven-week trial. In addition, the surgical procedure used to obtain the germ-free pigs by Caesarian-section, steri1ization and transfer of milk, feed and water into isolators, and microbial culture procedures used to verify germ-free status are described. I. PREPARATION OF REARING ISOLATORS FOR GERM-FREE RESEARCH A. Description of Rearing Isolators Three small ruminant stainless steel isolator units measuring approximately 122 x 60 x 91 cm and enclosed with vinyl film were used to provide adequate floor space, durability and ease of rearing during the seven-week trial. Two isolators housed three pigs providing 0.25 m2 of floor space per pig while the third isolator accomodated four pigs and provided 0.19 m2 of floor space per pig. 8. Initial Cleaning of Isolators Isolator preparation was begun by washing the vinyl film of the isolator with warm water containing a quaternary ammonia detergent (Diversey Chemicals, Division of the Diversey Corp., Des Plaines, IL), placed along with dry towels inside the deflated isolator. Once the inside cap was placed on the outside of the entry port and the nipples of the vinyl film of the isolator were plugged with rubber stoppers, the isolators were inflated by attaching an air hose to the air intake 163 154 nipple on the plastic portion of the isolator. Both inside and outside of the vinyl film portion of the isolator were thoroughly washed, rinsed and dried with cloth towels to prevent accumulation of detergent residue. Any tape or soil remaining after initial washing was removed by applying Super Takeoff (Diversey Chemicals, Division of the Diversey Corportation, Des Plaines, IL)_to a dry cloth towel and rubbing the soiled spot until clean. Once the vinyl film was cleaned satisfactorily, a11 cleaning materials were taken out of the isolators by removing the cap which sealed the outside of the entry port. C. Installation of Gloves and Aluminum Rings Yellow vinyl tape, C320 (Standard Safety Equipment Company, Palatine, IL) and fiberglass tape, #898 (Scotch Brand, Minnesota Mining and Manufacturing Co., St. Paul, MN), holding each of the six rubber- gloves to the aluminum rings on each isolator was removed. Each of the six rings on each isolator was then removed from the preformed plastic grooves and were cleaned to remove all soil and corrosion. These aluminum rings were then replaced and new rubber gloves were properly oriented and attached to the rings. Rubber gloves were then secured to the rings by several wraps of the fiberglass tape followed by yellow vinyl tape to protect and insure a tight seal between the rib at the -end of the rubber glove and the adjacent ring groove. 0. Testing for Leaks in the Plastic Isolator The vinyl film portion of the isolator was tested fer leaks by placing the inside cap on the outside of the entry port, inserting rubber stoppers in nipples of the plastic, and inflating the vinyl film with air from the air hose until it was approximately 75 percent 165 inflated. Vinyl film was further inflated With freon gas from a compressed gas cylinder until the gloves were fully extended. A halogen leak detector (Type: H-l- Obtained from General Electric Co., Medical Systems Division, Milwaukee, HI 53201) was used to examine the entire vinyl portion of the isolator to identify small holes, cracks and leaks needing repair. Particular attention was given to the seams, rubber gloves, seal around compression rings (where gloves were attached to the isolator) and the base of the vinyl attached to the stainless steel frame of the isolator cage. Hhen air leaks were identified, they were marked and later repaired when the vinyl portion was removed from the stainless steel frame. A11 holes and cracks identified with the halogen leak detector were repaired by first cleaning the vinyl surface with Poly Kleen (Schwartz Chemical Company, Inc., 50-01 Second St., L.I.C., NY) followed by sealing a small piece of vinyl over the hole or crack. E. Final Cleaning of Isolators Once all repairs were made to the vinyl portion of the isolator, both the inside and outside surfaces of the vinyl were cleaned by applying Poly Kleen to a dry cloth towel and polishing the surfaces to remove any finger prints, soil and detergent residue. All cleaning was done using rubber gloves to avoid getting oil from fingers during handling on the vinyl surface. Before the vinyl could be reattached to the stainless steel portion of the isolator, the entire frane of the stainless steel isolator was disassembled and thoroughly cleaned with detergent. rinsed, and all rusted bolts, nuts and screws were replaced. The wire mesh floor material was wrapped in paper, autoclaved and placed at the 165 bottom of the isolator tank while two aluminum storage trays were attached with wire ties to the aluminum sheet located at the 1201’ 0f the stainless steel frame. When all components of the isolator were cleaned, dried and reassembled, the vinyl portion of the isolator was reattached to the stainless steel frame of the isolator by wrapping yellow vinyl tape several times (overlapping each previous wrap) to properly seal the vinyl to the metal frame. Steel support bars lined with adhesive weather stripping (3M Company, Minneapolis, MN) were used to give further support to the vinyl-metal attachment and to hold the yellow vinyl tape in place. Following the attachment of vinyl to the metal frame, the isolators were reinflated with 75 percent air and 25 percent freon gas. A final check for leaks was conducted, as previously described, to 1 identify any holes or cracks not identified in the initial test. If leaks were identified at this time. they were repaired by placing a piece of yellow vinyl tape over the location in question until no leaks were identified using halogen leak detector. Once all isolator leaks were repaired, the isolator units were ready to be sterilized. F. Preparation of Air Filters For the air intake filter, a 43 x 137 cm section of 1.27 cm thick filter media (FM 004 Glass Fiber Media obtained from Owens Corning) was cut and wrapped tightly around the air filter frame. wire was then wrapped around the air filter fiber media to hold it in place. A wire screen was then wrapped around the filter media to further secure the filter media to the filter frame and also to provide protection from potential damage to the glass fiber media. Mylar tape 167 was wrapped around both the top and bottom edges of the filter media and yellow vinyl tape was wrapped over the mylar tape to seal any leaks that may have been present between the filter media and the filter frame. The inletand outlet filter connections were then covered with mylar film to prevent contamination before attachment to the sterilized isolator. All intake air filters were then sterilized in a dry heating oven f0r 4 hours at lSO'C. Oil bath exhaust filters were cleaned, checked for leaks and approximately 500 ml of mineral oil was added so that 50 percent of the filter was filled with oil. G. Preparation of Sterilizing Cylinder A 51 x 457 cm section of filter media (sufficient to provide four compete wraps around the sterilizing cylinder) was cut and applied in the same manner as previously described for the air intake filter. Filter media was held in place by two M-196 clamps at each end of the cylinder. A piece of wire screen measuring 112 x 51 cm was cut to cover the filter and overlap it by 3 cm on each end of the cylinder. This screen was then secured at each end of the cylinder with two M-ZOO clamps. 1 H. Sterilization of Equipment for Use in Isolators Each of three wire baskets were lined with cloth towels and filled with the following equipment needed to accomplish various procedures within each isolator: - 2 can openers 10 screw cap vials containing rectal swabs - 1 ear notcher - 1 side cutter 168 2 (Sec) syringes 4 (20 gauge, 3.8 can needles 10 pieces of 40 cm nylon cord 1 suction bulb and one 30 cm length piece of plastic tubing 1 feeder (Starcraft, Star Manufacturing Co., Oklahoma City, OK) 1 plastic waterer (Staley Livestock Products, A.E. Staley Mfg. Co., Decatur, IL) 4 (3.8 liter) plastic bottles with plastic caps These baskets, plus 12 folded cloth towels, 2 pair of cloth gloves, 5 (300 m1) plastic milk bottles and 5 rubber nipples were placed inside of two sterilizing cylinders on a 22 x 61 cm perforated stainless steel sheet located at the bottom of the cylinder. This stainless steel sheet allowed circulation of steam in the bottom of the cylinder for maximum steam penetration and provided a flat surface to place equipment. All items were carefully packed within the cylinders to provide adequate steam circulation and glass screw cap vials were placed upside down to prevent steam from condensing. Nhen all materials were properly placed in the cylinders, the open end of the cylinder was covered with a sheet of .001"-1 mil mylar film (Standard Safety Equipment Company, Palatine, IL) and held in place with an “O" ring p1aced approximately one inch behind the retaining edge of the cylinder. After the mylar film was pulled tight and wrinkles were removed, the mylar film was attached to the cylinder using two wraps of mylar tape on the side of the "O“ ring toward the open end of the cylinder. The first wrap completely sealed the edge of the mylar film to the edge of the cylinder while the second wrap 169 overlapped the first wrap by one half toward the "O" ring. Excess mylar film was then trimmed as close to the ”0" ring as possible, away from the open end of the cylinder. The “O" ring was then removed and three additional wraps of mylar tape were applied, followed by four wraps of yellow vinyl tape to completely cover the mylar so that none was exposed. In order to insure that no creases, wrinkles and bubbles developed during tape application, tape was applied with a strong, even tension at all times. The sealed cylinders, loaded with the previously described equipment, were placed in the autoclave (American Sterilizer Co., Erie, PA) and were ready to be sterilized. The autoclave door was closed and sealed tightly and a vacuum of 65 cm of mercury was imposed for 20 minutes. After the expired 20 minutes, the vacuum pump was turned off and the vacuum valve was closed prior to opening the steam valve. All materials located within the cylinders were sterilized at 121'C fer at least one hour. At the completion of the steri1ization process, steam was exhausted from the autoclave and another vacuum of 65 cm of mercury was imposed for 20 minutes to dry the contents of the cylinders. Finally, the vacuum was turned off and the autoc1ave door was opened, upon reaching neutral pressure, to allow the cylinders to cool before preparing to transfer cylinder contents into the isolators. After cooling. the mylar film on the cylinder should remain tight. If it iwere not tight, there likely was a leak in the mylar film or a leak in the tape attaching mylar film to the cylinder, and the autoclaving process was repeated after replacing the mylar film and tape. 1. Final Preparation and Sterilization of the Isolators Plastic covers were placed on the presterilized air intake filters and secured with M-52 clamps fellowed by wrapping with yellow 170 vinyl tape. Both air intake and exhaust filters were attached to the isolator base with screws, and both filters on each isolator were attached to the isolator nipples and secured with M-16 clamps. Rubber stoppers were tightly inserted in the bottom of the stainless steel tank of each isolator and sealed with mylar film, held in place with yellow vinyl tape on the outside bottom of the tank. Next, all items which could be sterilized with peracetic acid (i.e. any item with a smooth surface which could be completely exposed to peractic acid) were placed in the isolator. These items included: 2 inside plastic caps 2 rubber bands preautoclaved wire mesh floor 2 aluminum storage pans 1 long, blunt nosed forceps Using a graduated cylinder, 20 ml of concentrated peracetic acid (40% aqueous peracetic acid obtained from F.M.C., Speciality Chemicals Division, Buffalo, NY) was added to 380 ml distilled water and a few flakes (approximately 0.1%) of Nacconal detergent (triethanolamine alky arye sulfonate) were added as a wetting agent. This 2% peracetic acid solution was then poured into a plastic spray bottle, mixed and (attached to the spraying apparatus linked to a compressed nitrogen gas cylinder with a plastic hose. Compressed nitrogen gas was used because of its inert properties and as a source of pressure for applying the peracetic acid solution. The spray bottle containing peracetic acid was placed inside of the isolator and the flexible plastic tube connecting the gas cylinder to the spraying apparatus was inserted through a vinyl stopper located in one of the nipples on the outer cap 171 of the entry port. The outer cap was sealed to the entry port by five overlapping wraps of yellow vinyl tape. Two operators entered their arms into the rubber gloves, one on each side of the isolator. One operator sprayed the gloves and equipment on the opposite side of the isolator while the other operator manipulated the gloves and equipment to insure adequate coverage by the acid. When the entire inside of the isolator and its contents were saturated with acid and the vinyl film was fully inflated, the spray bottle was placed in the entry port for removal from the isolator. The inside cap was then placed on the inside of the entry port and sealed with a rubber band. Next, the outside cap was removed and the spray bottle was detached from the plastic tubing, and reattached when the plastic tubing was pulled through the nipple of the outside cap. Finally, the entry port was sprayed and resealed by pulling the outside cap over the outside of the entry port, wrapped with yellow vinyl tape and nipples were plugged with vinyl stoppers after the entry port was sprayed and inflated. Isolators remained sealed for at least one hour. In the meantime, the blower hose from the air pump was attached to the nipple on the air inlet filter with an M-52 clamp. After the required time had elapsed, one operator entered the rubber gloves and punctured the mylar on the air inlet and outlet filters. The operator then removed his hands from the gloves, started the blower and adjusted the air intake and exhaust flow clamps. Isolators were dried by the circulating air and were ready to receive animals at the end of 48 hours. J. Transfer of Materials from Cylinder to Isolators In order to transfer the required equipment into the isolator has well as transferring autoclaved feed and any other equipment needed 172 later in the trial) the outer cap of the GOtry port was removed and the entry port was wiped dry with a paper towel to remove any debris from the previous transfer. A vinyl transfer sleeve was then attached to the entry port of the isolator and over the taped section of the cylinder by wrapping yellow vinyl tape around each component three times. Four hundred milliliters of the previously described 2% peracetic acid solution was mixed and some of the solution was sprayed through a nipple located in the center of each side of the vinyl sleeve until the sleeve was fUlly inflated and the entire inside of the exchange area was thoroughly saturated with peracetic acid. Vinyl stoppers were then inserted into the nipples and taped with yellow vinyl tape to hold stoppers in place when the inside cap was removed during the transfer process. After a 30 minute steri1ization period“ following spraying of the exchange area, the operator entered the gloves of the isolator and removed the inside cap, while the second operator pushed the cylinder up to the entry port of the isolator. The operator working inside of the isolator then punctured the mylar film at the mouth of the cylinder with a pair of blunt end forceps. Materials present in the cylinder were then transferred into the isolator through the vinyl sleeve and entry port. When all of the cylinder contents were transferred, the inner cap was replaced and a rubber band was stretched around the inner cap to secure it. The transfer sleeve was then removed, the entry port was resprayed with peracetic acid and the outer cap was replaced and taped as previously described. Finally, peracetic acid was sprayed into the nipple of the (Miter cap until the inside cap was inflated. Vinyl steppers were then inserted into the nipples and sealed with yellow vinyl tape. 173 Vlhen all of these preparations were completed, these isolators were ready to receive the baby pigs. 11. PREPARATION OF TRANSFER AND SURGICAL ISOLATORS FOR GERM-FREE RESEARCH 1 The comprehensive discussion of the steps involved in preparing the rearing isolators previously described also applied to preparation of the transfer and surgical isolators with a few of the following exceptions. A. Description of Isolators 1. Transfer Isolator The transfer isolator measured approximately 152 x 102 x 102 cm and was constructed entirely of flexible plastic with a 46 cm diameter entry port on one end. The vinyl isolator was placed on a paper covered, 1.2 cm thick plywood sheet located on top of a steel cart with wheels for ease of manipulation. The stainless steel entry port was attached to the plywood sheet by a bracket to give stability to the isolator. Air intake and exhaust nipples were located in the rear of the isolator for attachment to air filters when needed. 2. Surgical isolator A vinyl surgical isolator measuring approximately 150 x 102 x 102 cm was attached to a steel rod frame which gave stability and was used to suspend the isolator with rope and pully from the ceiling of the surgical room. A 46 cm diameter entry port attached to the suspended frame was located at one end so that once pigs were removed from the uterus, they could be readily passed into the transfer 150131101”- A nipple was located above and behind the entry port and contained electrical wires embedded in a vinyl stOPPe" fur 174 electrocautery use during surgery. In addition, a 30 cm port containing a fiber glass ring with rubber bands attached and covered with a vinyl cap was located at the bottom of the isolator, which was eventually attached to the sow's skin for the surgical operation. Air intake and exhaust nipples were located in the rear of the vinyl isolator for attachment to air filter and outlet trap when needed. 8. Cleaning and Preparation of Isolators for Sterilization Both transfer and surgical isolators were cleaned and checked for leaks in the same manner as described for the rearing isolators. 1. Transfer isolator Four stainless steel cages were cleaned, covered with brown wrapping paper and sealed with autoclave tape for sterilization. These cages were autoc1aved, as previously described, for at least one hour at 250'C. After cages were cooled, they were placed inside of the transfer isolator on a plastic mat and later the paper was removed before spraying with peracetic acid. These cages were later used to sort and hold the baby pigs when being transferred from the surgical isolator to the rearing isolators. 2. Surgical isolator The 30 cm cap at the bottom of the surgical isolator was put in pl ace after the fiberglass ring containing rubber bands was put in place for the surgery. The surgical isolator was ready for steri1iza tion when a plastic mat and long, blunt end forceps were Placed in the isolator. C. Sterilization of Isolators and Preparation for Surgery B0th the transfer and surgical isolators were Sprayed with 2% peracetic acid with the sane procedure as described previously 10" the 175 rearing isolators. Sterilized, dry cloth towels and all surgical equipment were then transferred from cylinders to the surgical isolator. Next, the transfer isolator was attached and sealed to the surgical isolator by a vinyl transfer sleeve and the glove was sterilized with peracetic acid. III. SURGICAL TECHNIQUE FOR OBTAINING GNOTOBIOTIC PIGS A pregnant sow was anesthetized on the 112th day of gestation by injecting 15 m1 of lidocaine hydrochloride (Lid-o-cain 2%, Butler Co., Columbus, OH) in the epidural space at the lumbosacral articulation, using an 18 gauge, 15.25 cm needle. This was followed by an intramuscular injection of 5 ml of promazine hydrochloride (Sparine, Hyeth Laboratories, Inc., Philadelphia, PA). The sow was secured on the surgical table in lateral recumbency with the left side uppermost. The left flank area was then prepared for surgery. The hair was clipped and shaved, and the skin was scrubbed with Betadine Surgical Scrub (Purdue Frederick Co., Norwalk, CT). Betadine Solution (Purdue Frederick Co., Norwalk, CT) was then applied to the site, followed by 70% ethyl alcohol. Chloroform was next applied to the skin, and the area was dried with sterile towels. The skin of the surgical site and the vinyl film covering the lZ-inch port in the floor of the surgical isolator were sprayed with Vi-Drape Adhesive (Parke-Davis Co., Detroit, MI). When the adhesive was drY. the vinyl film of the isolator was placed over the surgical site, and the lZ-inch port was secured in place with three cords. Each cord was tied to one side of the port and extended around the abdomen of the saw before being tied to the other side of the port, Working through shoulder-length rubber gloves in ei ther side of 176 the surgical isolator, the operators node a 9 inch incision through the vinyl film covering the lZ-inch port located at the bottom of the surgical isolator and the skin of the sow with a thermocautery unit (Thermo Cautery, National Electric Instruments Div., Englehard Hanovia, Inc., Elmhurst, L.I., N.Y.). As the incision was made, the cut edges of the vinyl film and the skin and the handles of the forceps were secured to rubber bands extending through holes in the circumference of the 12-inch port. The forceps were placed approximately 1 inch apart throughout the length of the incision. The remainder of the abdominal well (muscle, fascia, and peritoneum) was incised with scissors. A loop of the gravid uterus containing a fetus was pulled through the incision into the interior of the isolator. The uterine wall over the dorsal aspect of the head and neck of the fetus was incised with scissors, and the pig was pulled into the interior of the isolator. An umbilical clamp (Hollister Double-grip Disposable Cord-Clamp, Hollister, Inc., Chicago, IL) was placed on the umbilical cord near the bed)! wall, and the cord was cut approximately one inch below the clamp. The pig was then passed into an attached transport isolator where it was dried and placed in a cage. The loop of the uterus was then passed back into the abdominal cavity of the sow, another loop was brought through the incision, and the process was repeated. When all the pigs had been removed, the sow was euthanatized by the intravenous injection of an overdose of sodium pentobarbital (Sodium Pentobarbi ta1 Injectable, Butler Co., Columbus, OH). Following delivery of all the pigs, the inside cap was placed on the 9'1th port of the transport isolator, and the sleeve connecting the 177 surgical and transport isolators was removed. The outside cap was placed on the entry port of the transport isolator, and the space between the two caps was sprayed with a solution of 2% peracetic acid. The transfer isolator containing the baby pigs was moved to the room containing the rearing isolators and attached to each individual isolator by a plastic sleeve. The space within the plastic sleeve between the two isolators was sprayed with 2% peracetic acid for each transfer. Pigs were removed from cages and placed in rearing isolators after removing the inside cap. Pigs were evenly distributed between the rearing isolators based on sex, size and number. When all pigs were placed in their respective isolators, the outside caps were replaced and sealed after the entry ports were sprayed with 2% peracetic acid solution. IV. PREPARATION AND TRANSFER OF MILK, HATER AND EXPERIMENTAL DIETS INTO REARING ISOLATORS A. Preparation and Transfer of Milk Twenty-354 ml cans of evaporated milk (Carnation Company, Los Angeles, CA) were prepared for transfer into each isolator by first removing all labels, glue and rust. Damaged cans were not used because of potential contamination. Each can was then rolled in 70% ethanol solution and placed under a fume hood to dry. Hhen needed, cans were thoroughly sprayed with 2% peracetic acid solution and stacked on a stainless steel tray in the entry ports of the isolators after removing the outside cap. When the outer cap was replaced and sealed, the entry Port was resprayed with 2% peracetic acid solution. All cans were traPSfEN‘Ed into isolators following a 30 minute time PEPiod after spraying. Any empty cans were then placed into the entry port for removal from the isolator. 178 B. Preparation and Transfer of Hater Six-2000 ml glass Pyrex flasks (American Sterilizer Company, Erie, PA) were filled with tap water and sealed with rubber disposable closures (American Sterilizer Company, Erie, PA). All sealed flasks were placed in the liquid autoclave (Hilmot Castle Co., Rochester, NY) and sterilized for at least one hour at 121°C. Flasks were then cooled and wiped with a paper towel saturated with 70% ethanol to remove any oil or debris. Two flasks were then thoroughly sprayed with 2% peracetic acid solution and placed in the entry port of each isolator. Entry ports were capped and sealed as previously described. After a 30 minute time period allowed for steri1ization, the flasks were transferred into the isolator by removing the inside cap and water was poured into plastic storage containers for later use. Empty water flasks were then placed into the entry port and ready to be removed from the entry port after replacing the inside cap. These flasks were filled with water and autoclaved to repeat this process as needed. C. Preparation and Transfer of Experimental Diets An amount of experimental diet, in meal form, was placed in a labeled aluminum pan and weighed to measure 2.5 kg. This amount of diet measured 2.54 cm thick in the bottom of the pan which allowed satisfactory sterilization of the diet which was previously determined by incubating Kilit ampu1es (American Scientific Products, Industrial Division, McGaw Park, IL 60085) containing Bacillus sterolhermophilus spores, subjected to this sterilization procedure. Prior- to putting the diet in the pans, small holes (2 cm apart) were drilied in the P°tt°m 01’ the pens so that adequate steam penetration COuid be accomplished without the diet sifting out of the pan. Two 2.5 cm diameter mean pipes were attached to the top of one pan with 113110“ 179 cord to allow a 2.5 cm space between the bottom and top pan in each cylinder for adequate steam circulation. Pans were positioned in the center of the sterilizing cylinder. Cylinders were then sealed with mylar film, as previously described, and placed in the autoc1ave (American Sterilizer Co., Erie, PA). After a 20 minute, 65 cm of mercury vacuum, diets were sterilized by steam for 25 minutes at 121°C. Another 20 minute vacuum was then imposed to dry the diets. Cylinders were allowed to cool before transfer of diets into the isolators. Once diets were transferred into the isolators, the dried cooked slab of feed was broken into smaller pieces and reground using a mortar and pestle before putting into feeders. V. MONITORING ISOLATORS FOR GERM-FREE STATUS Standard aerobic and anaerobic microbial culture procedures were used to verify germ-free status of the pigs in the rearing isolators on day 6, 21, 39 and 49 of the experiment. Swabs contained in presterilized screw cap vials located in each isolator were used to sample the feces from the rectal area of one pig in each isolator and the feeding equipment within each isolator. Once the swabs were inoculated with feces and feed, they were replaced inside of the screw cap vials, which were sealed tightly, and passed out of the isolator through the entry port. The entry port was sprayed with 2% peracetic acid to resterilize the entry port following removal of the vials as previously described. These vials containing the swabs were then taken to the laboratory and media were inoculated with the swabs, Three types of media were used to assess the presence of aerobic and anaEPObic microbial growth. Thioglycolate broth was used to assess the Presence of anaerobic organisms and plates were lncubated at three 180 temperatures (25’, 27° and 56'C) when inoculated with a swab from an isolator. Plates were examined daily for 5 days and weekly thereafter for 3 weeks for presence of anaerobic bacterial growth. Blood agar plates were inoculated with swabs to determine the presence or absence of anaerobic and aerobic bacterial growth when plates were incubated at 25', 37' and.56'C. Both aerobic and anaerobic plates were checked daily f0r 3 days and were discarded if negative after this time period. If there was microbial growth present in these plates, organisms were allowed to grow and were then identified. Mycoplasma broth was the third culture media used to assess the presence or absence of mycoplasma organisms when plates were inoculated with swabs and incubated at 37'C for 3 days. After 3 days, 0.1 m1 of mycoplasma broth was transferred to plates containing mycoplasma agar and were incubated in jars at 37'C for 1 week. Plates were examined microscopically for QVOWth and were discarded if negative. LIST OF REFERENCES A.0.A.C. 1984. Official Methods of Analysis (14th Ed.), Assoc. of Official Anal. Chem., Washington, D.C. Abrams, G.D. , H. Bauer and H. Sprinz. 1963. Influence of the normal flora on mucosal cell morphology and cellular renewal. Lab. Invest. 12:355. Allen, M.S., R.S. Barber, R. Braude and K.G. Mitchell. 1961. Further studies on various aspects of the use of high-copper supplements for growing pigs. Br. J. Nutr. 15:507. Alvares, A.P., R.F. Hendrickson and W.J. Visek. 1964a. Effects of isoniazid, chlortetracycline, and copper upon growth and gastrointestinal urease activity in chicks. J. Nutr. 83:79. Alvares, A.P., L.H. Harbers and W.J. Visek. 1964b. Effect of barbituric acid, chlortetracycline and carbohydrates upon growth and gastrointestinal urease activity of chicks. J. Nutr. 82:93. Amer, M.A. and J.A. Elliot. 1973. Influence of supplementary dietary copper and vitamin E on the oxidative stability of porcine depot fat. J. Anim. SCiO 37:87. Amnerman, C.B. and N.A. Marcilese. 1968. Nutritional interrelationships of copper, molybdenum and sulfate in ruminants. Proceedings NFIA Annual Convention. Osage Beach, MD. Anderson, G.W., 0.0. Cunningham and S.J. Slinger. 1952. Effect of protein level and penicillin on growth and intestinal flora of chickens. J. Nutr. 47:175. Argenzio, R.A. and M. Southworth. 1974. Sites of organic acid production and absorption in the gastrointestinal tract of the pig. Am. J. Physiol. 228:454. Barber, R.S., R. Braude and K.G. Mitchell. 1955a. Antibiotic and copper supplements for fattening pigs. Brit. J. Nutr. 9:378. Barber, R.S., R. Braude and K.G. Mitchell. 1955b. Effect of adding copper in the diet of suckling pigs on creep meal consumption and live weight gain. Chem. Ind. 21:1954. Barber, R.S., R. Braude, K.G. Mitchell and J. Cassidy. 1955c. High capper minera1 mixture for fattening pigs. Chem. Ind. 21:601. Barber, 11.5,, R. Braude, K.G. Mitchell, and J.A.F. Rook. 1957. Further studies on antibiotic and copper supplementation for fattening pigs. Brit. J. Nutr. 11:70. Barber. R.S., R. Braude and K.G. Mitchell. 1950. Further studies on anti biotic copper and zinc supplements for growing - 5, Br. J. Nutr. 14:499. 9‘9 181 182 Barber, R.S., J.P. Bowland, R. Braude, K.G. Mitchell and J.W.G. Porter. 1961. Copper sulfate and copper sulfide as supplements for growing pigs. Br. J. Nutr. 15:189. Barber, R.S., R. Braude and K.G. Mitchell. 1962. Copper sulfate and molasses distillers dried solubles as dietary supplements for growing pigs. Anim. Prod. 4:233. Barber, R.S., R. Braude and K.G. Mitchell. 1965. Studies on various potential growth stimulants for growing pigs with particular reference to their activity in supplementing that of copper sulfate. Br. J. Nutr. 19:575. Barber, R.S., R. Braude, K.G. Mitchell and R.J. Pittman. 1978. The value of virginiamycin (Eskalin) as a feed additive for growing pigs in diets with or without a high cOpper supplement. Anim. Prod. 26:151. Barcroft, J., R.A. McAnally and A.T. Phillipson. 1944. Absorption of volatile acids from the alimentary tract of sheep and other animals. J. Exp. Biol. 20:120. Bass, 8., J.T. McCall, H.D. Wallace, G. Combs, A.Z. Palmer and J.E. Carpenter. 1956. High level c0pper feeding of growing-finishing swine. J. Anim. Sci. 15:1230 (Abstr.). Bauer, H. 1968. Defense mechanisms in the germ-free animals. Part 11. Cellular defense mechanisms. In: The Germ-Free Animal in Research, M.E. Coates ed. Academic Press, Inc.; New York and London. Beacom, S.E. 1959a. Chlortetracycline and protein level in rations for market hogs. I. Effect on rate of gain and efficiency of feed utilization. Can. J. Anim. Sci. 39:71. Beacom, S.E. 1959b. Chlortetracycline and protein level in rations for market hogs. II. Effect on carcass quality. Can. J. Anim. Sci. 39:79. Beames, R.M. and L.E. Lloyd. 1965. Response of pigs and rats to rations supplemented with tylosin and high levels of copper. J. Anim. Sci. 23:1020. Beames, R.M. 1969. Further trials on the response of early-weaned pigs and rats on diets supplemented with tylosin and high levels of copper. J. Anim. Sci. 29:573. Bellis, D.G. 1961. Supplementation of bacon pig rations by aureomycin and two levels of copper sulfate. Anim. Prod. 3:89. Bowland, J.P., R. Braude, A.G. Chamberlain, R.F. Glascock and K.G. Mitchell. 1961. The absorption, distribution and excretion of labelled copper in young pigs given different quantities, as sglggate or suplphide, orally or intravenously. Br. J. Nutr. 183 Bowland, J.P. and A.G. Castell. 1964. Supplemental copper for market pigs fed rations varying in source and level of protein. 43rd Annual Feeders' Day Report. Dept. of Anim. Sci., University of Alberta, Edmonton. Bowland, J.P. and A.G. Castell. 1965. Supplemental copper for market pigs. 44th Annual Feeders' Day Report. Dept. of Anim. Sci., University of Alberta, Edmonton. Bowler, R.J., R. Braude, R.C. Campbell, J.N. Caddock-Turnbull, H.F. Fieldsend, E.K. Griffith, I.A.M. Lucas, K.G. Mitchell, N.J.D. Nickalls, and J.H. Taylor. 1955. High-copper mineral mixture for fattening pigs. Brit. J. Nutr. 9:358. Braude, R. 1945. Some observations on the need for copper in the diet of fattening pigs. J. Agri. Sci. 35:163. Braude, R. and B.C. Johnson. 1953. Effect of aureomycin on nitrogen and water metabolism in growing pigs. J. Nutr. 49:505. Braude, R., M.F. Coates, M.K. Davies, G.F. Harrison and K.G. Mitchell. 1955. The effect of aureomycin on the gut of the pig. Brit. J. Nutr. 9:363. Braude, R. 1965. COpper as a growth stimulant in pigs (cuprum pro pecunia). .Ig Cuprwn pro vita (Transactions of a Symposium). COpper Development Assn., London, p. 55. Braude, R. 1967. Copper as a stimulant in pig feeding. World Review of Animal Production 3, No. 11:69. Braude, R., K.G. Mitchell, M.J. Newport and R.J. Pittman. 1970. Response to different levels of supplementation with copper sulfate)of diets for growing pigs. Proc. Nutr. Soc. 29:1OA Abstr. . Braude, R. and K. Ryder. 1973. Copper levels in diets of growing pigs. J. Agric. Sci., (Cambridge) 80:489. Bremner, I. and N.T. Davies. 1976. Studies on the appearance of a hepatic COpper-binding protein in normal and zinc deficient rats. Br. J. Nutr. 36:101. Broderson, J.R., J.R. Lindsey and J.E. Crawford. 1976. Role of environmental ammonia in respiratory mycoplasmosis of rats. Amer. J. Path. 84:115. Brody, T.M., R. Hurwitz and J.A. Bain. 1954. Magnesium and the effect of the tetracycline antibiotics on oxidative processes in mitochondria. Antibiot. Chemother. 4:864. Bunch, R.J., V.C. Speer, V.W. Hays, J.N. Hawbaker and D.V. Catron. 1961. Effects of copper sulfate, copper oxide and chlortetracycline on baby pig performance. J. Anim. Sci. 20:723. 184 Bunch, R.J., V.C. Speer, V.W. Hays and J.TL McCall. 1963. Effects of high levels of copper and chlortetracycline on performance of pigs. J. Anim. Sci. 22:56. Buntain, D. 1961. Deaths in pigs on a high copper diet. Vet. Rec. 73:707. Burnside, J.E., T.J. Cunha, A.M. Pearson, R.S. Glasscock and A.L. Shealy. 1949. Effect of APF supplement on pigs fed different protein supplements. Arch. Biochem. Biophys. 23:328. Burnside, J.E., R.H. Grummer, P.H. Phillips and G. Bohstedt. 1954. The influence of crystalline aureomycin in vitamin B 2 0n the protein utilization of growing-fattening swine. J. nim. Sci. 13:184. Bush, J.A., J.P. Mahoney, H. Markowitz, C.J. Gubler, G.E. Cartwright and M.M. Wintrobe. 1955. Studies on capper metabolism. XVI. Radioactive copper studies in normal subjects and in patients with hepatolenticular degeneration. J. Clin. Invest. 34:1766. Bush, J.A., W.N. Jensen, J.W. Athens, H. Ashenbrucker, G.E. Cartwright and M.M. Wintrobe. 1956. Studies on copper metabolism. XIX. The kinetics of iron metabolism and erythrocyte life-span in copper deficient swine. J. Exp. Med. 103:701. Bush, L.J., R.S. Allen and N.L. Jacobson. 1959. Effect of chlortetracycline on nutrient utilization by dairy calves. J. Dairy Sci. 42:671. Carnes, W.H., G.S. Shields, G.E. Cartwright and M.M. Wintrobe. 1961. Vascular lesions in copper deficient swine. Fed. Proc. Fed. Am. Soc. Exp. Biol. 20:118. Castell, A.G. and J.P. Bowland. 1968. Supplemental copper for swine: Growth, digestibility and carcass measurements. Can. J. Anim. Sci. 48:403. Castell, A.G., R.D. Allen, R.M. Beames, J.M. Bell, R. Belfile, J.P. Bowland, J.I. Elliot, M. Ihnat, E. Larmond, T.M. Mallard, D.T. Spurr, S.C. Stothers, S.B. Wilton, and L.G. Young. 1975. Copper supplementation of Canadian diets for growing-finishing pigs. Can. J. Anim. Sci. 55:113. Catron, D.V., A.M. Jensen, P.G. Homeyer, H.M. Maddock and G.C. Ashton. 1952. Re-evaluation of protein requirements of growing-fattening swine as influenced by feeding an antibiotic. J. Anim. Sci. 11:221. Catron, D.V., M.D. Lane, L.V. Quinn, G.C. Ashton and H.M. Maddock. 1953. Mode of action of antibiotics in swine nutrition. Antibiot. Chemother. 3:571. 185 Chachulowa, J. 1964. Wpylw niektorych dodatkow do paszy na poziom ammoniaku w jelicie swin. l. Zawartosc ammoniaku w jelicie slepym swin przy roznym dodatku celulozy do paszy, chlortetracykliny i siarczanu miedzi. Roczn. Nauk Roln. 84-B-lz83. Chapman, H.L., Jr. and M.C. Bell. 1963. Relative absorption and excretion by beef cattle of copper from various sources. J. Anim. Sci. 22:82. Clemens, E.T., C.E. Stevens and M. Southworth. 1975. Sites of organic acid production and pattern of digesta movement in the gastrointestinal tract of swine. J. Nutr. 105:759. Clarke, R.T.J. 1977a. Methods for studying gut microbes. In: Microbial Ecology of the Gut, R.T.J. Clarke and T. Bauch0p ed., Academic Press, New York, NY. Clarke, R.T.J. l977b. The gut and its microorganisms. In: Microbial Ecology of the Gut, R.T.J. Clarke and T. Bauchop ed., Academic Press, New York, NY. Coates, M.E. 1968. Nutrition and metabolism. In: M.E. Coates, H.A. Gordon and 8.5. Wostmann, Ed. Part III. The Germ-Free Animal in Research. Academic Press, New York. Coates, M.E., M.K. Davies and S.K. Kon. 1955. The effect of antibiotics on the intestine of the chick. Brit. J. Nutr. 9:110. Coates, M.E. and G.F. Harrison. 1969. Observations on the growth-promoting effects of procaine penicillin and zinc gacitracin in chicks in different environments. J. Sci. Food Agr. 0: 83. Coates, M.E. and R. Fuller. 1977. The gnotobiotic animal in the study of gut microbiology. In: Microbial Ecology of the Gut, R.T.J. Clarke and T. Bauchop,Ed., Academic Press, New York, NY. Combe, E., E. Penot, H. Cahlier and E. Sacquet. 1965. Metabolisme du rat "germ-free" Teneurs des contenus digestifs en certains composes azotes, en sodium et an potassium. Teneurs de gullques tissues en acides nucleiques. Ann. Biol. Anim. Biochem. Biophys. 5: 89. Combe, E. and E. Sacquet. 1966. Influence de 1'etat axenique surs divers compses azote contenus dans le caecwm de rats albinos recevant des quantities variables de proteins. Compt. Rend. Acad. Sci. 262:685. Combe, E., Y. Demarne, L. Gueguen, O. Ivorec-Szylit, J.C. Meslin and E. Sacquet. 1976. Some aspects of the relationships between gastrointestinal flora and host nutrition. World Rev. Nutr. Diet. 4:1. 186 Crampton, R.F., D.M. Matthews and R. Poisner. 1965. Observations on the mechanism of absorption of copper by the small intestine. J. Phys. (London) 178:111. Creamer, B. 1964. Variations in small-intestinal villous shape and mucosal dynamics. Brit. Med. J., Part 2, pp. 1371-1373. Cromwell, G.L., V.W. Hays and T.L. Clark. 1978. Effects of copper sulfate, copper sulfide and sodium sulfide on performance and copper stores of pigs. J. Anim. Sci. 46:692. Crosby, W.H., J.I. Munn and F.W. Furth. 1954. Standardizing a method for clinical hemoglobinometry. U.S. Armed Forces Med. J. 5:693. Dang, H.C. and W.J. Visek. 1968. Some characteristics of blood in normal and immune rabbits after urease injection. Amer. J. Physiol. 215:502. Davis, P.N., L.C. Norris and F.H. Kratzer. 1962. Interference of soybean proteins with the utilization of trace minerals. J. Nutr. 77:217. Dawson, A.M. and K.J. Isselbacher. 1960. Studies on lipid metabolism in the small intestine with observations on the role of bile salts. J. Clin. Invest. 39:730. DeGoey, L.W., R.C. Wahlstrom and R.J. Emerick. 1971. Studies of high levels of copper supplementation to rations for growing swine. J. Anim. Sci. 33:52. Delluva, A.M., M. Markley and R.E. Davies. 1968. The absence of gastric urease in germ-free animals. Biochim. Biophys. Acta. 51:646. Desplaces, A., D. Zagury and E. Saquet. 1963. Etude de la fonction thyodienne du rat prive de bacteries (germ-free). C.R. Acad. Sci. (Paris) 257:756. Dick, A.T. 1953. The control of copper storage in the liver of sheep by inorganic sulphate and molybdenum. Australian Vet. J. 29:233. Dick, A.T. 1954. Preliminary observations on the effect of high intakes of molybdenum and of inorganic sulphate on blood copper and on fleece character in crossbred sheep. Australian Vet. J. 30:196. Dierick, M.A., I.J. Vervaeke, J.A. Decuypere and H.K. Hendrickx. 1986a. Influence of the gut flora and of some growth-promoting feed additives on nitrogen metabolism in pigs. 1. Studies in vitro. Livestock Prod. Sci. 14:161. Dierick, N.A., I.J. Vervaeke, J.A. Decuypere and H.K. Hendrickx. 1986b. Influence of the gut flora and of some growth-promoting feed additives on nitrogen metabolism in pigs. II. Studies in vivo. Livestock Prod. Sci. 14:177. 187 Diggs, B.G., D.E. Becker, A.H. Jensen and H.W. Norton. 1965. Energy values of various feeds for the young pig. J. Anim. Sci. 24:555. Dintzis, R.Z. and A.B. Hastings. 1953. The effect of antibiotics on urea breakdown in mice. Proc. Natl. Acad. Sci. 39:571. Drouliscos, N.J., J.P. Bowland and J.I. Elliot. 1970. Influence of supplemental dietary copper on copper concentration of pig blood, selected tissues, and digestive tract contents. Can. J. Anim. Sci. 50:113. Edmonds, M.S. and D.H. Baker. 1984. Efficacy of supplemental copper and dried whey for newly weaned pigs. I11. Swine Res. Rep., Univ. of 111., Urbana-Champaign, p. 1-5. Elliot, J.I. and M.A. Amer. 1973. Influence of level of copper supplement and removal of supplemental copper from the diet on the performance of growing-finishing pigs and accumulation of copper in the liver. Can. J. Anim. Sci. 53:133. Elliot, J.I. and J.P. Bowland. 1968. Effects of dietary copper sulfate on the fatty acid composition of porcine depot fats. J. Anim. Sci. 27:956. Elliot, J.I. and J.P. Bowland. 1969. Correlation of melting point with the sum of the unsaturated fatty acids in samples of porcine depot fat. Can. J. Anim. Sci. 49:397. Elliot, J.I. and J.P. Bowland. 1970. Effects of dietary c0pper sulfate and protein on the fatty acid composition of porcine depot fat. J. Anim. Sci. 30:923. Elsden, S.R., M.W.S. Hitchcock, R.A. Marshall and A.T. Phillipson. 1946. Volatile acids in the digesta of ruminants and other animals. J. Exp. Biol. 22:191. Erickson, J.P., E.R. Miller, G.M. Hill, J.R. Black, D.M. Bebiak and P.K. Ku. 1980. Wheat versus corn in pelleted and meal swine diets. J. Anim. Sci. 51:1065. Evrard, E., P.P. Hoet, H. Eyssen, H. Charlier and E. Sacquet. 1954. Fecal lipids in genn-free and conventional rats. Brit. J. Exp. Path. 45:409. Eyssen, 1i. and P. DeSomer. l963a. Effect of antibiotics on growth and nuti~ient absorption of chicks. Poul. Sci. 42:1373. Eyssen. ii. and P. DeSomer. 1963b. The mode of action of antibiotics in stimulating growth of chicks. J. Exp. Med. 117:127. Eyssen, H. and P. DeSomer. 1965. Studies on gnotobiotic chicks: Effects of controlled intestinal flora's on growth and nutrient absorption. Ernahrungsforschung 10:262. 188 Farrell, D.J. and K.A. Johnson. 1972. Utilization of cellulose by pigs and its effect on the cecal function. Anim. Prod. 14:209. Fauconneau, G. and M.C. Michel. 1970. The role of the gastrointestinal tract in the regulation of protein metabolism. In: Mammalian Protein Metabolism, H.M. Munro Ed. Academic Press, New York, NY 4:481. Fell, B.F., D. Dinsdale and C.F. Mills. 1975. Changes in enterocyte mitochondria associated with deficiency of copper in cattle. Res. Vet. Sci. 18:274. Folin, O. and W. Denis. 1912. Protein metabolism from the standpoint of blood and tissue analysis. Second paper. The origin and significance of the ammonia in the portal blood. J. Biol. Chem. 11:161. Forbes, M. and J.T. Park. 1959. Growth of germ-free and conventional chicks: Effect of diet, dietary penicillin and bacterial environment. J. Nutr. 67:69. Forbes, M., H.C. Supplee and G.F. Combs. 1958. Response of germpfree and conventionally reared turkey poults to dietary supplementation with penicillin and oleandomycin. Proc. Soc. Exp. Biol. Med. 99:110. Fridlyand, I.G. 1959. The problem of the effect of industrial poisons on the immunobiological state of the organism. Gigiena Sanitariia 24:55. Friend, D.W., H.M. Cunningham and J.W.G. Nicholson. l963a. Volatile fatty acids and lactic acid in sections of the alimentary tract of the young pig. Can. J. Anim. Sci. 43:174. Friend, D.W., H.M. Cunningham and J.W.G. Nicholson. l963b. Production of organic acids in the pig. II. The effect of diet on the levels of volatile fatty acids and lactic acid in sections of the alimentary tract. Can. J. Anim. Sci. 43:156. Fuller, R.L., G.M. Newland, C.A.E. Briggs, R. Braude, and K.G. Mitchell. 1960. The normal intestinal flora of the pig. IV. The effect of dietary supplement, of penicillin, chloretetracycline or COpper sulfate on faecal flora. J. Appl. Bacteriol. 23:195. Geever, E.F} and S.M. Levenson. 1964. Iron pigmentation of the gut of gerflhrfree and conventionalized guinea pigs. Experentia 20:391. Gil1. J.l.. 1978. Design and Analysis of Experiments. Volume 1. 2 and 3. The Iowa State University Press, Ames, IA. Gipp. H.F.. 11.6. Pond and E.F. Walker, Jr. 1973. Influence of diet composition and mode of copper administration on the response of gEowing-fin1shing swine to supplemental copper. J. Anim. Sci. :91. 189 Gipp, H.F., W.G. Pond, F.A. Kallfelz, J.B. Tasker, D.R. Van Campen, L. Krook and W.J. Visek. 1974. Effect of dietary copper, iron and ascorbic acid levels on hemagglogy, bggod and tissue copper, iron and zinc concentrations and Cu and Fe metaoblism in young pigs. J. Nutr. 104:532. Gitlin, D., W.L. Hughes and C.A. Janeway. 1960. Absorption and excretion of copper in mice. Nature 188:50. Gomori, G. 1942. A modification of the colorimetric phosphorus determination for use with the photoelectric colorimeter. J. Lab. Clin. Med. 27:955. Gordon, A.S. 1955. Some aspects of hormonal influences upon the leukocytes. Ann. NY Acad. Sci. 59:907. Gordon, H.A. 1959. Morphological and physiological characterization of germ-free life. Ann. NY Acad. Sci. 78:208. Gordon, W.A.M. and 0. Luke. 1957. Copper poisoning in the pig. Vet. Rec. 69:37. Gordon, H.A. and E. Bruckner-Kardoss. 1959. The distribution of reticulo-endothelial elements in the intestinal mucosa and submucosa of germ-free, monocontaminated and conventional chickens orally tested with penicillin. Antibiotics Annual Medical Encyclopedia, New York. p. 1012. Gordon, H.A. and B.S. Wostmann. 1960. Morphological studies on the germ-free albino rat. Anat. Rec. 137:65. Gordon, H.A. and E. Bruckner-Kardoss. 1961a. Effect of normal microbial flora on intestinal surface area. Amer. J. Physiol. 201:175. Gordon, H.A. and E. Bruckner-Kardoss. 19610. Effects of the normal microbial flora on various tissue elements of the small intestine. Acta Anat. 44:210. Gordon, H.A., B.S. Wostmann and E. Bruckner-Kardoss. 1963. Effects of microbial flora on cardiac output and other elements of blood circulation. Proc. Soc. Exp. Biol. Med. 114:301. Gordon, H.A., E. Bruckner-Kardoss and B.S. Wostmann. 1966. Aging in germfree mice: Life tables and lesions observed at natural death. J. Gerontol. 21:380. Gordon. H.A. 1968. Is the germ-free animal normal?: A review of its anomalies in young and old age. In: The Germ-Free Animal in Eesgarch, M.E. Coates ed. Academic Press, Inc., New York and on on. Ha"IPSOH. D.J. 1986. Alterations in piglet small intestinal structure at weaning. Res. Vet. Sci. 40:32. 190 Harbers, L.H., A.P. Alvares, A.I. Jacobson and W.J. Visek. 1963. Effect of barbituric acid and chlortetracycline upon growth, ammonia concentration, and urease activity in the gastrointestinal tract of chicks. J. Nutr. 80:75. Hash, J.H., M. Wishnick and P.A. Miller. 1964. On the mode of action of the tetracycline antibiotics in Staphylococcus aureus. J. i Biol. Chem. 239:2070. Hawbaker, J.H., V.C. Speer, J.D. Jones, V.W. Hays and D.V. Catron. 1959. Effect of copper sulfate and antibiotics on growth rate, feed conversion and fecal flora of growing pigs. J. Anim. Sci. 18:1505 (Abstr.). Hawbaker, J.A., V.C. Speer, V.W. Hays and D.V. Catron. 1961. Effect of copper sulfate and other chemotherapeutics in growing swine rations. J. Anim. Sci. 20:163. Hays, V.W. 1969. Biological basis for the use of antibiotics in livestock production. The Use of Drugs in Animal Feeds. National Academy of Sciences, Pub. 1697. Hays, V.W. and V.C. Speer. 1960. Effect of spiramycin on growth and feed utilization of young pigs. J. Anim. Sci. 19:938. Hays, V.W. and R.D. Kline. 1969. Copper-molybdenum-sulfate interrelationships in growing pigs. Feedstuffs 41:44:18. Heneghan, J.B. 1963. Influence of microbial flora on xylose absorption in rats and mice. Amer. J. Physiol. 205:417. Herskovic, T., J. Katz, M.M. Floch, R.E. Spencer and H.M. Spiro. 1967. Small intestinal absorption and morphology in germ-free, ggnpcontaminated and conventional mice. Gastroenterology : 36. H111. C.H., A.0. Keeling and J.W. Kelley. 1957. Studies on the effect 2; antibiotics on the intestinal weights of chicks. J. Nutr. : 55. Hill, G.M., P.K. Ku, E.R. Miller, D.E. Ullrey, T.A. Losty and B.L. 0 Dell. 1983. A capper deficiency in neonatal pigs induced by a high zinc maternal diet. J. Nutr. 113:867. HitChCOCk. J.P. 1973. Thyroprotein for lactating sows. Report of Swine Res.. Michigan State University, East Lansing, MI, 232:19. Hoefer. J.A., E.R. Miller, 0.5. Ullrey, H.D. Ritchie and R.W. Luecke. 1960. Interrelationships between calcium, zinc and copper in swine feeding. J. Anim. Sci. 19:249. H0. S.K. and J.I. Elliot. 1973. Supplemental dietary copper and the desaturation of l-l4C-steroyl-coenzyme A by porcine hepatic and adipose microsomes. Can. J. Anim. Sci. 53:537. 191 Ho, S.K. and J.I. Elliot. 1974. Fatty acid composition of porcine depot fat as related to the effect of supplemental dietary copper on the specific activities of fatty acyl desaturase systems. Can. J. Anim. Sci. 54:23. Ho, S.K., J.I. Elliot and G.M. Jones. 1975. Effiects of copper on performance. fatty acid composition of depot fat and fatty and desaturase activities in pigs fed a diet with or without supplemental copper. Can. J. Anim. Sci. 55:587. Holtzman, J.L. and W.J. Visek. 1965. Growth of rats fed chlortetracycline or an ion exchange resin. J. Nutr. 87:101. Hsieh, H.W. and E. Frieden. 1975. Evidence fer ceruloplasmin as a copper transport protein. Biochem. Bi0phys. Res. Commun. 67:1326. Imoto, S. and S. Namioka. 1978. VFA production in the pig large intestine. J. Anim. Sci. 47:467. Imoto, S. and S. Namioka. 1983. Nutritive value of acetate in growing pigs. J. Anim. Sci. 56:858. Johansson, K.R. and W.B. Sarles. 1949. Some considerations of the biological importance of intestinal microorganisms. Bacteriol. Rev. 13:25. Jukes, T.H. 1955. Antibiotics of Nutrition. Monograph, Medical Encyclopedia, Inc., New York. Jukes, T.H. and W.L. Williams. 1953. Nutritional effects of antibiotics. Pharmacol. Rev. 5:381. Kass, M.L., P.J. VanSoest and W.G. Pond. 1980. Utilization of dietary fiber from alfalfa by growing swine. II. Volatile fatty acid concentrations in and disappearance from the gastrointestial tract. J. Anim. Sci. 50:192. Kellogg. T.F., V.W. Hays, D.V. Catron, L.Y. Quinn and V.C. Speer. 1964. Effect of level and source of dietary protein on performance and cecal flora of baby pigs. J. Anim. Sci. 23:1089. Kellogg, T.F., V.W. Hays, D.V. Catron, L.Y. Quinn and V.C. Speer. 1966. Effect of dietary chemotherapeutics on the performance and cecal flora of pigs. J. Anim. Sci. 25:1102. Kenworthy, R. and W.D. Allen. 1966. Influence of diet and bacteria on small intestinal morphology, with special reference to early weaning and Escherichia Coli, studies with germfree and gnotobiotic pigs.TTJ. Comp. Path. 76:291. Kenworthy, R. 1976. Observations of the effects of weaning in the young pig. Clinical and histopathological studies of intestinal function and morphology. Res. in Vet Sci. 21:69. Keuseh, G.T. 1974. Opportunistic infections in colon carcinoma. Amer. J. Clin. Nutr. 27:1481. 192- Kinter, W.B. and H. Wilson. 1965. Autoradiographic study of sugar and amino acid absorption by everted sacs of hamster intestine. J. Cell Biol. 25:19. Kirchgessner, M. and H. Giessler. 1961. Activation of pepsin (EC 3.4.4.1) by heavy-metal ions including a contribution to the mode of action of copper sulphate in pig nutrition. Z. Tierphysiol., Tierernaehr. Fittermittlkd. 16:297. Kirchgessner, M., M.G. Beyer and H. Steinhart. 1976. Activation of pepsin (EC 3.4.4.1) by heavy-metal ions including a contribution to the mode of action of copper sulphate in pig nutrition. Br. J. Nutr. 36:15. Klainer, A.S. and W.R. Beisel. 1969. Opportunistic infection: A review. Amer. J. Med. Sci. 258:431. Klevay, L.M. 1973. Hypercholesterolemia in rats produced by an increase in the ratio to zinc in c0pper ingested. Am. J. Clin. Nutr. 26:1060. Kline, R.D., V.W. Hays and G.L. Cromwell. 1971. Effects of copper, molybdenum, and sulfate on performance, hematology. and copper stores of pigs and lambs. J. Anim. Sci. 33:771. Kline, R.D., V.W. Hays and G.L. Cromwell. 1972. Related effects of copper, zinc, and iron on performance, hematology and copper-stores of pigs. J. Anim. Sci. 34:393. Kline, R.D., M.A. Corzo, V.W. Hays and G.L. Cromwell. 1973. Related effects of copper, molybdenum and sulfide on performance, hematology, and capper stores of growing pigs. J. Anim. Sci. 37:936. Kornbgrg, H.L. and R.E. Davies. 1955. Gastric urease. Physiol. Rev. 5:169. Kruml, J., J. Ludvik, I. Trebichavsky, L. Mandel and F. Kovaru. 1969. Morphology of germ-free piglets. Folia Microbiol. 14:441. l.andy, J.J. and R.K. Ledbetter. 1966. Delivery and maintenance of the germ-free pig. In: Swine in Biomedical Research. Richland, Washington:Pacific Northwest Laboratory, pp. 619-632. LeBlond. C.P. and B.E. Walker. 1956. Renewal of cell populations. Physiol. Rev. 35:255. Lesher, S., M.E. Wallbury, Jr. and G.A. Sacher. 1964. Generation cycle in the duodenal crypt cells of germ-free and conventional mice. Nature 202:884. Lev, M. and M. Forbes. 1959. Growth response to dietary protein of germ-freelchicks and chicks with a defined intestinal flora. Br. . Nutr. 3:78. 193 Leveille, G.A., H.E. Sauberlich and R. Hunt. 1962. Effect of dietary lithocholic acid on liver size of the chick. Poul. Sci. 41:1991. Leveille, G.A., M.E. Zabik and K.J. Morgan. 1983. Nutrients in Foods. The Nutrition Guild, Cambridge, MA. Levenson, S.M., L.V. Crowley, R.E. Horowitz and 0.J. Malm. 1959. The metabolism of carbon-labeled urea in the germ-free rat. J. Biol. Chem. 234:2061. Levenson, S.M., D. Kon, M. Lev and F.S. Daft. 1966. Oxygen consumption, carbon dioxide production and body temperature in germ-free rats. Fed. Proc. 25:482 (Abstr). Levenson, S.M. and Tennant. 1963. Contributions of intestinal microflora to the nutrition and the host animal. Some metabolic and nutritional studies with germ-free animals. Fed. Proc. 22:109. Libby, D.A. and P.J. Schaible. 1955. Observations on growth response to antibiotics and arsonic acids in poultry feeds. Science 121:733. Lillie, R.J.. L.T. Frobish, M.C. Steele and G. Graber. 1977. Effect of dietary copper and tylosin and subsequent withdrawal on growth, hematology and tissue residues of growing-finishing pigs. J. Anim. Sci. 45:100. Lima, F. Ribeiro, T.S. Stahly and G.L. Cromwell. 1981. Effects of copper, with and without ferrous sulfide, and antibiotics on performance of pigs. J. Anim. Sci. 52:241. Linder, M.C. and J.R. Moor. 1977. Plasma ceruloplasmin. Evidence for its presence in and uptake by heart and other organs of the rat. Biochem. Biophys. Acta 499:329. Livingstone, R.M. and D.M.S. Livingston. 1968. Copper sulphate and antibiotics as feed additives for early weaned pigs. J. Agric. Sci. Camb. 71:419. Lucas, I.A.M. and A.F.C. Calder. 1957a. A comparison of five levels of copper sulfate in rations for growing pigs. Proc. Nutr. Soc. 16:1 (Abstr.). Lucas, I.A.M. and A.F.C. Calder. 1957b. Antibiotics and a high level 2; popper sulfate in rations for growing bacon pigs. J. Agr. Sci. : 84. Lucas. I.A.M.. R.M. Livingstone, A.W. Boyne and I. McDonald. 1962a. The early weaning of pigs. VIII. COpper sulfate as a growth stimulant. J. Agr. Sci. 58:201. Lucas, I.A.M., R.M. Livingstone and A.W. Boyne. 1962b. Copper sulfate as a growth stimulant f0r pigszEffect of composition of diet and level of protein. Anim. Prod. 4:177. 194 Luckey, T.0. 1952. Effect of'feeding antibiotics upon the growth rate of germ-free birds. Colloquium held at the University of Notre Dame, Notre Dame, Ind. Luckey, 1.0., J.R. Pleasants, M. Wagner, H.A. Gordon and J.A. Reyniers. 1955. Some observations on vitamin metabolism in germ-free rats. J. Nutr. 57:169. Mahan, D.C. 1980. Effectiveness of antibacterial compounds and copper for weanling pigs. Ohio Swine Res. and Ind. Rep. 80-2:1. Mason, V.C. and A. Just. 1976. Bacterial activity in the hind-gut of pigs. I. Its influence on the apparent digestibility of dietary energy and fat. 2. Tierphysiol. Tierernahr. Futtermittelk., 36:301. March, 8. and J. Biely. 1952. The effect of feeding aureomycin on the bacterial content of chick feces. Poultry Sci. 31:177. Markowitz, H.. C.J. Gubler, J.P. Mahoney, G.E. Cartwright and M.M Wintrobe. 1955. Studies on copper metabolism. XIV. Copper, ceruloplasmin and oxidase activity in sera of normal human subjects, pregnant women, and patients with infection, hepatolenticular degeneration and the nephrotic syndrome. J. Clin. Invest. 34:1498. McGovern, J.J., A.R. Jones and A.G. Steinberg. 1955. The hematocrit of capillary blood. New Eng. J. Med. 253:308. Meade, R.J. and R.M. Forbes. 1956. The influence of chlortetracycline and vitamin 812 alone and in combination on nitrogen utilization by growing swine. J. Nutr. 59:459. Michel, M.C. 1966. Metabolisme de la flore intestinale du porc. Degradation de fermes L et 0 des acides amines. Private Coll. Soc. Org. Biol. Chem., Paris, Cahier 6:307. Miller. E.R., H.0. Stowe, P.K. Ku and G.M. Hill. 1979. Copper and zinc in swine nutrition. In: Copper and Zinc in Animal Nutrition. National Feed Ingredients Association, West Des Moines, IA. Miller, E.R.. D.E. Ullrey, D.J. Ellis, B.L. Shoepke and J.A. Hoefer. 1969. Comparison of copper sulfate and a selected antibiotic for growing-finishing swine. J. Anim. Sci. 29:140. Miller, E.R., D.E. Ullrey, D.A. Schmidt, R.W. Luecke and J.A. Hoefer. 1967. Effects of nutrient deficiencies upon organ weights of the baby pig. J. Anim. Sci. 26:1046. Miller. E.R., G.L. Waxler, 9.x. Ku, 0.5. Ullrey and 0.1:. Whitehair. 1982. Iron requirements of baby pigs reared in germ-free or ggnventional environments on a condensed milk diet. J. Anim. Sci. : O6. 195 Miller, W.J. 1979. Copper and zinc in ruminant nutrition. In: Copper and Zinc in Animal Nutrition. National Feed Ingredients Association, West Des Moines, IA. Milne, 0.8. and P.H. Weswig. 1968. Effect of supplementary copper on blood and liver copper-containing fractions in rats. J. Nutr. 95:429. Miniats, 0.P. and V.E. Valli. 1973. The gastrointestinal tract of gnotobiotic of pigs. In: Germfree Research. Biological Effect of Gnotobiotic Environments, J.B Henegan, ed., Academic Press Inc., New York and London. Mitchell, K.G. 1953. Observations on the effect of adding copper to the diet of suckling piglets. Chem. and Ind. p. 871. Moon, H.W. 1971. Epithelial cell migration in the alimentary mucosa of the suckling pig. Proc. Exp. Biol. Med. 137:151. Moore, J.H., W. Christie, R. Braude and K.G. Mitchell. 1969. The effect of dietary copper on the fatty acid composition and physical properties of pig adipose tissue. Br. J. Nutr. 23:281. Moore, P.R., A. Evenson, T.D. Luckey, E. McCoy, C.A. Elvehjem and E.B. Hart. 1946. Use of sulfasuxidine, streptothricin, and streptomycin in nutritional studies with the chick. J. Biol. Chem. 165:437. Moore, W.E.C. 1969. Current research on the anaerobic flora of the gastrointestinal tract. In: The Use of Drugs in Animal Feeds. Proceedings of a Symposium, National Academy of Sciences, Washington, D.C. p. 107. Moore, W.E.C. and L.V. Holdeman. 1972. Identification of anaerobic bacteria. Amer. J. Clin. Nutr. 25:1314. 1 Myres, A.W. and J.P. Bowland. 1973. Effects of environmental temperature and dietary copper on growth and lipid metabolism in pigs. III. Concentration and composition of plasma free fatty acids. Can. J. Anim. Sci. 53:127. NCRr42 Committee on Swine Nutrition. 1974. Cooperative regional studies with growing swine: effects of vitamin E and levels of supplementary capper during the growing-finishing period on gain, gged conversion and tissue copper storage in swine. J. Anim. Sci. :512. NRC. 1977. Copper : Medical and Biologic Effects of Environmental Pollutants. National Academy of Sciences, Washington, D.C. NRC. 1979. Nutrient requirements of domestic animals. Nutrient requirements of swine. National Academy of Science - National Research Council, Washington, D.C. 196 Newton, G.L. 1984. Copper as a feed additive: Possible interaction with polyphenol compounds as a mode of action. Animal Science Division Report, Univ. of Georgia, Athens, p. 305. Norman, A. and O.A. Widstrom. 1964. Hydrolysis of conjugated bile acids by extracellular enzymes present in rat intestinal contents. Proc. Soc. Exp. Biol. Med. 117:442. O'Dell, B.L. 1976. Biochemistry of copper. Med. Clin. North Am. 60:687. O‘Dell, B.L. 1984. Copper : Present Knowledge of Nutrition. Nutrition Review's, 5th Ed.. The Nutrition Foundation, Inc., Washington, D.C. O'Dell, B.L., B.C. Hardwick, G. Reynolds and J.E. Savage. 1961. Connective tissue defect in the chick resulting from copper deficiency. Proc. Soc. Exp. Biol. Med. 108:402. O'Hara, P.J., A.P. Newman and R. Jackson. 1960. Parakeratosis and copper poisoning in pigs fed a copper supplement. Australian Vet. J. 36:225. Okonkwo, A.C., P.K. Ku, E.R. Miller, K.K. Keahy and D.E. Ullrey. 1979. Copper requirement of baby pigs fed purified diets. J. Nutr. ' 109:939. Omole, T.A. and J.P. Bowland. 1974. Copper, Fe, Mn supplementation of pig diets containing either soybean meal or low glucosinolate rapeseed meal. Can. J. Anim. Sci. 54:481. Owen, C.A., Jr. 1975. Uptake of 67cu by ceruloplasmin in vitro. Proc. Soc. Exp. Biol. Med. 149:681. ' Pappenfort, R.B. and E.S. Schnall. 1951. Clinical and laboratory evidence that aureomycin stimulates the growth of Candida albicans. Arch. Int. Med. 88:729. Parris, E.C.C. and B.E. McDonald. 1969. Effect on dietary protein source on copper toxicity in early weaned pigs. Can. J. Anim. Sci. 49:215. Pepper, H.F., S.J. Slinger and I. Motzok. 1953. Effect of aureomycin on the niacin and manganese requirements of chicks. Poul. Sci. 32:656. Petering, M.G., L. Murthy and E. O'Flaherty. 1977. Influence of dietary copper and zinc on rat lipid metabolism. J. Agr. Food Chem. 25:1105. Phan, S.H. and H.R. Mahler. 1976. Studies on cytochrome oxidase. Partial resolution of enzymes containing seven or six subunits. from yeast and heart, respectively. J. Biol. Chem. 251:257. 197 Phillips, G.B., R. Schwartz, G.L. Gabuzda and C.S. Davidson. 1952. The syndrome of impending hepatic coma in patients with cirrhosis of the liver given certain nitrogenous substances. N. Engl. J. Med. 247:239. Phillips, B.P., P.A. Wolfe and H.A. Gordon. 1959. Studies on rearing the guinea pig germfree. Ann. N.Y. Acad. Sci. 78:183. Pleasants, J.R. 1968. Characteristics of the germ-free animal. In: The Germ-Free Animal in Research, M.E. Coates., ed. Academic Press Inc., New York and London. Pond, W.G. and K.A. Houpt. 1978. The Biology of the Pig. Cornell University Press, Ithaca, NY. Prince, T.J., V.W. Hays and G.L. Cromwell. 1979. Effects of copper sulfate and ferrous sulfide on performance and liver cOpper and iron stores of pigs. J. Anim. Sci. 49:507. Prins, R.A. 1977. Biochemical activities of gut micro-organisms. In: Microbial Ecology of the Gut, R.T.J. Clarke and T. Bauchop ed., Academic Press, New York, NY. Prior, R.L., D.C. Topping and W.J. Visek. 1974. Metabolism of isolated chick small intestinal cells. Effects of ammonia and various salts. Biochemistry 13:178. Ratcliffe, B. 1985. The influence of the gut micorflora on the digestive processes. In: Proceedings of the 3rd International Seminar on Digestive Phys. in the Pig, Copenhagen, Denmark, A. Just, H. Jorgenson and J. A. Fernandez, Ed. Beretning fra Statens Husdyrbrugsforsog 580: 245. Reddy, 8. 5., B. S. Wostmann and J. R. Pleasants. 1965a. Iron, copper and manganese in germ-free and conventional rats. J. Nutr. 86: 159. Reddy, B.S., J.R. Pleasants, D.R. Zimmerman and B.S. Wostmann. 1965b. Iron and copper utilization in rabbits as affected by diet and germ-free status. J. Nutr. 87:189. Rerat, A. 1978. Digestion and absorption of carbohydrates and nitrogenous matters in the hindgut of the omnivorous nonruminant animal. J. Anim. Sci. 46:1808. Reyniers. J.A., P.C. Trexler and R.F. Ervin. 1946. Rearing germfree albino rats. Lobund Reports No. 1, Notre Dame, Indiana, University Press, p. 1-84. Rice. E.E. and J.F. Beuk. 1953. The effects of heat upon the nutritive value of protein. In: Advances in Food Research Vol. IV, E.M. Mrak and G.F. Stewart, Ed., Academic Press Inc., New ork, NY. 198 Richards, P. 1972. Nutritional potential of nitrogen recycling in man. Amer. J. Clin. Nutr. 25:615. Richardson, L.R., L.G. Blaylock and C.M. Lyman. 1953. Influence of dietary amino acids on the free amino acids in the blood plasma in chicks. J. Nutr. 51:515. Ritchie, H.0., R.W. Luecke, B.V. Baltzer, E.R. Miller, D.E. Ullrey and J.A. Hoefer. 1963. Copper and zinc interrelationships in the pig. J. Nutr. 79:117. Salanitro, J.P. and P.A. Muirhead. 1975. Quantitative method fer the gas chromatographic analysis of short-chain monocarboxylic and dicarboxylic acids in fermentation media. App. Microbiol. 29:374. Schalm, O.W. 1965. Veterinary Hematology. Lea and Febiger, Philadelphia, PA. Seerley, R.W., E.R. Miller and J.A. Hoefer. 1962. Growth, energy and nitrogen studies on pigs fed meal or pellets. J. Anim. Sci. 21:829. Silen, W., H.A. Harper, D.L. Mawdsley and W.L. Weirich. 1955. Effiect of antibacterial agents on ammonia production within the intestine. Proc. Soc. Exp. Biol. Med. 88:138. Smith, B.S.W. and N. Wright. 1974. Improved manual and automated procedures fer estimation of ceruloplasmin oxidase activity. Clin. Chim. Acta 50:359. Smith, H.W. and J.E.T. Jones. 1963. The effect of the addition of copper sulphate to the diet on the bacterial flora of the alimentary tract of the pig. J. Appl. Bacteriol. 26:262. Sollman, T. 1957. A Manual of Pharmacology. 8th Ed., W.B. Saunders and Co., Philadelphia, PA. Speer, V.C., R.L. Vohs, D.V. Catron, H.M. Maddock and C.C. Culbertson. 1950. Effect of aureomycin and animal protein factor on healthy pigs. Arch. Biochem. 29:452. Sprinz. H. 1962. Morphological response of intestinal mucosa to enteric bacteria and its implication fer sprue and asiatic cholera. Fed. Proc. 21:57. Stahl, J.G. 1963. Studies of blood ammonia in liver disease. Ann. Intern. Med. 58:1. Stahly, T.S., G.L. Cromwell and H.J. Monegue. 1980. Effects of the dietary inclusion of copper and(or) antibiotics on the performance of weanling pigs. J. Anim. Sci. 51:1347. .199 Starcher, B.C.. C.H. Hill and G. Matrone. 1964. Importance of dietary copper in the formation of aortic elastin. J. Nutr. 82:318. Stokstad, E.L.R., T.H. Jukes and W.L. Williams. 1953. The growth promoting effect of aureomycin on various types of diets. Poul. Sci. 32:1054. Stokstad, E.L.R. 1954. Antibiotics in animal nutrition. Physiol. Rev. 34:25. Suttle, N.F. and C.F. Mills. 1966. Studies of the toxicity of copper to pigs. 1. Effects of oral supplements of zinc and iron salts on the development of copper toxicosis. Brit. J. Nutr. 20:135. Taylor, J.H. 1957. The mode of action of antibiotics in promoting animal growth. Vet. Rec. 69:278. Taylor, J.N. and F. Harrington. 1955. Influence of dietary antibiotic supplements on visceral weights of pigs. Nature 175:643. Taylor, M. and S. Thomke. 1964. Effect of high-copper on the depot fiat of bacon pigs. Nature 201:1246. Teague, M.S. and L.E. Carpenter. 1951. The demonstration of a copper deficiency in young growing pigs. J. Nutr. 43:389. Teague, H.S. and A.P. Grifo, Jr. 1966. The continuous or intermittent supplmentation of copper in feed and water. Ohio Swine Res. Summ. l3 Thiers, R.E. and B.L. Vallee. 1957. Distribution of metals in subcellular fractions in rat liver. J. Biol. Chem. 226:911. Topping, D.C. and W.J. Visek. 1977. Synthesis of macromolecules by intestinal cells incubated with anmonia. Amer. J. Physiol. 233:E341-E347. Ullrey, D.E., E.R. Miller, O.A. Thompson, C.L. Zutaut, D.A. Schmidt, H.D. Ritchie, J.A. Hoefer and R.W. Luecke. 1960. Studies of copper utilization by the baby pig. J. Anim. Sci. 19:1298 (Abstr.). Underwood, E.J. 1977. Trace Elements in Human and Animal Nutrition, 4th Ed. Academic Press, New York. Underwood, E.J. 1981. The Mineral Nutrition of Livestock, 2nd Ed. Commonwealth Agricultural Bureau, London. Van Campen, D. and E. Gross. 1968. Influence of ascorbic acid on the absorption of copper by rats. J. Nutr. 95:617. Van Camggn, 0 and E.AS Mitchell. 1965. Absorption of Cu54, Zn , Mo99 and Fe 9 from ligated segments of the rat gastrointestinal tract. J. Nutr. 86:120. rs..- ..r—_. 200 Vervaeke, I.J., J.A. Decuypere, M.A. Dierick and H.K. Hendrickx. 1979. Quantitative in vitro evaluation of the energy metabolism influenced by Virginiamycin and Spiramycin used as growth promoters in pig nutrition. J. Anim. Sci. 49:846. Visek, W.J., J.M. Baron and D.M. Switz. 1959. Urea metabolism and intestinal ureolytic activity of rats fed antimicrobial agents. J. Pharm. Exp. Ther. 126:359. Visek, W.J. 1964. Urease immunity in liver disease: A new approach. Gastroenterology 46:326. Visek, W.J. 1972. Effects of urea hydrolysis on cell lifespan and metabolism. Fed. Proc. 31:1178. Visek, W.J., G.M. Kolodny and P.R. Gross. 1972. Ammonia effects in cultures of normal and transformed 3T3 cells. J. Cell Physiol. 80:373. Visek, W.J. 1978. The mode of growth promotion by antibiotics. J. Anim. Sci. 46:1447. Visek, W.J., J.M. Baron and D.M. Switz. 1959. Urea metabolism and intestinal ureolytic activity of rats fed antimicrobial agents. J. Pharm. Exp. Ther. 126:359. Vohra, P., G.A. Gray and F.H. Kratzer. 1965. Phytic acid-metal complexes. Proc. Soc. Exp. Biol. Med. 120:447. Wahle, K.W.J. and N.T. Davies. 1975. Effect of dietary copper deficiency in the rat on fatty acid composition of adipose tissue and desaturase activity of liver microsomes. Brit. J. Nutr. 34:105. Wallace, H.0. 1967. High level copper in swine feeding. International Copper Research Association, Inc., New York. Wallace, H.D. 1968. Effects of high level copper on performance of growing pigs. Feedstuffs 40:22. Wallace, H.0., M. Milicevic, A.M. Pearson, T.J. Cunha and M. Koger. 1954. The influence of aureomycin on the protein requirement and carcass characteristics of swine. J. Anim. Sci. 13:177. Wallace, H.0., J.T. McCall, 8. Bass and G.E. Combs. 1960. High level copper for growing-finishing swine. J. Anim. Sci. 19:1153. Walser, M. and L.J. Bodenlos. 1959. Urea metabolism in man. J. Clin. Invest. 38:1617. Warren, K.S. and W.L. Newton. 1959. Portal and peripheral blood ammonia concentrations in germ-free and conventional guinea pigs. Amer. J. Physiol. 197:717. 201 Waxler, G.L. and D.T. Drees. 1972. Comparison of body weights, organ weights and histological features of selected organs of gnotobiotic, conventional and isolator-reared contaminated pigs. Can. J. Comp. Med. 36:265. Weinberg, E.D. 1957. The mutual effects of antimicrobial compounds and metallic cations. Bacteriol. Rev. 21:46. Weissman, M., G.S. Shields and W.H. Carnes. 1963. Cardiovascular studies on COpper-deficient swine. IV. Content and solubility of the aortic elastin, collagen and hexosamine. J. Biol. Chem. 238:3115. Whitehair, C.K. and G.M. Thompson. 1956. Observations on raising "disease-free" swine. J. Amer. Vet. Med. Assoc. 122:94. Williams, D.M., R.E. Lynch, G.R. Lee and G.E. Cartwright. 1975. Superoxide dismutase activity in copper deficient swine. Proc. Soc. Exp. Biol. Med. 149:534. Williams, H.0., G.L. Cromwell and T.S. Stahly. 1981. Effects of elemental sulfur and ferrous sulfide on capper solubility in various sections of the digestive tract of pigs fed high copper diets. J. Anim. Sci. 53 (Suppl. 1). Wilson, D.R., T.S. Ing, A. Metcalfe-Gibson and D.M. Wrong. 1968a. In vivo dialysis of faeces as a method of stool analysis. II. The effect of intestinal antibiotics. Clin. Sci. 34:211. Wilson, D.R., T.S. Ing, A. Metcalfe-Gibson and O.M. Wrong. 1968b. The chemical composition of faeces as revealed by in vivo faecal dialysis. Clin. Sci. 35:197. Wostmann, 8.5. 1968. Defense mechanisms in germ-free animals. Part I. Humoral defense mechanisms. In: The Germ-Free Animal in Research, M.E. Coates ed. Academic Press, Inc., New York and London. Wostmann, B.S., P.L. Knight and D.F. Kan. 1962. Thiamine in germrfree and conventional animals: Effect of the intestinal microflora on thiamine metabolism of the rat. Ann. New York Acad. Sci. 98:516. Wostmann, B.S. and E. Bruckner-Kardoss. 1966. Oxidation-reduction protentials in cecal contents of germ-free and conventional rats. Proc. Soc. Exp. Biol. Med. 121:1111. Wright, N.A. 1982. The experimental analysis of changes in proliferative and morphological status in studies on the intestine. Scand. J. of Gastroenterology 17: Supp. 74:3. Wrong, 0., A. Metcalfe-Gibson, R.B.I. Morrison, S.T. Ing and A.V. Howard. 1965. In vivo dialysis of faeces as a method of stool analysis. 1. Technique and results in normal subjects. Clin. Sci. 28:257. 202 Young, L.G., R.G. Brown, G.C. Ashton and G.C. Smith. 1970. Effect of copper on the utilization of raw soybeans by market pigs. Can. J. Anim. Sci. 50:717. Young, L.G. and J.D. Jamieson. 1970. Protein and copper supplementation of corn-soybean meal diets for young pigs. Can. J. Anim. Sci. 50:727. Zimber, A. and W.J. Visek. 1972. Efect of urease injections on DNA synthesis in mice. Amer. J. Physiol. 223:1004. Zimmerman, D.R. and B.S. Wostmann. 1963. Vitamin stability in diets sterilized for germ-free animals. J. Nutr. 79:318. l11111111111111111l