CGPPER AND MANGANESE REQUlREMENTS OF THE BABY PlG Dissertation for the Degree of Ph. D. MICHBGAN STATE UNIVERSITY ANTHONY CHUKWUEMEKA OKONKWO I. 9 76 . __ 1132.412)! Michaan Sam .5 ’ Univcm'ty This is to certify that the thesis entitled Copper and Manganese Requirements of the Baby Pig presented by Anthony Chukwuemeka Okonkwo has been accepted towards fulfillment of the requirements for Ph. D. . Animal Husbandry degree in ‘”;':.M..t 8 Z ((1/th Major professor [hue Octoher 26, 1976 0-7639 cinema WAG & SUNS' 300K HINDI" W). I Ian DV .IHn'ID. Mum Scam ty LIBRARY ‘Unival' ( . . . tn... 5 35.5 l l ~.~ snazanznnuszzz mmmaammwmmmmmm own-ansnow-eanoa—aau manaanon.auuwxmwaamunnmmmwrammmw .— r- 5 .— FF55_-----~FF---_p---~F-thh-w‘__.- amwaauouumuaacamummmmammumuuueauuammmuaoaumwoam.umomao nnnmmwmmmmmmmmnmnwmmnrmmnmnMflmmmnmnmnnnmmnmmmnmnmmmmmm uvquvwvvvov‘~.vfiv.¢ev.w...#.v¢.~VV¢~vqm‘e..—~.rwv'~.. nnnnmnnnnnnnnnnnnnnnrnmnnnnmnnmnannnnnfinnnnnnnnnmm“van NNNNNfiNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN--~NNN~N _______________—_____—______—_______________r_______r_ x==::==n=:==a::=::=:a=:::az==s=:=::r::=::==:::::=====F aaaaaaeaaaaaeeaaauaaaaeaaaeaaeaaaauoaaaaaaaaarraeaaaaa r HccpIa Lu ABSTRACT COPPER AND MANGANESE REQUIREMENTS OF THE BABY PIG BY Anthony Chukwuemeka Okonkwo Three experiments involving 52 baby pigs were conducted to deter- mine the minimum copper requirement of baby pigs on semi-purified diets. In the first experiment 16 week-old baby pigs were fed semi- purified diets supplemented with anhydrous copper sulfate resulting in the following dietary copper levels: diet 1 (basal), 1.3 ppm; diet 2, 3.2 ppm; diet 3, 5.6 ppm; diet 4, 9.3 ppm. Initial weights of pigs were taken and blood parameters including plasma copper were determined at this time and at subsequent 2-week intervals. After 6 weeks, a copper balance trial was conducted. The pigs were killed at the conclusion of the trial and organs and tissues were taken and kept at -20°C until used for analysis. Ceruloplasmin activity, plasma copper levels, copper balance and hemoglobin were useful indicators of copper status, with all but hemoglo- bin significantly (P<0.05 or P<0.01) depressed by the basal diet (1.3 ppm Cu). Hemoglobin, while not significantly influenced by treatments, was consistently depressed by the baSal diet, suggesting a sub-clinical anemia as a result of copper deficiency. From this study it appeared that the minimum.copper requirement of the baby pig is lower than 5.6 ppm. Anthony C. Okonkwo In the second experiment, 15 three-day old baby pigs were main- tained on three semi-purified diets which analyzed 0.6 ppm, 1.9 ppm and 2.8 ppm copper. The casein used in the basal diet was treated with NaZEDTA to remove as much capper as possible. The protocol used here was similar to that used in Experiment 1. Hemoglobin was depressed con- siderably by the basal diet (0.6 ppm Cu) but the effect was not statisti- cally significant. Ceruloplasmin activity and plasma copper showed sig- nificant treatment differences (P<0.05 or P<0.01) particularly between the basal (0.6 ppm) and the other diets (1.9 ppm and 2.8 ppm Cu). Kidney copper concentration and physical measurements of the left femur, with the exception of weight and elasticity showed significant (P<0.05 or P<0.0l) difference due to treatments. Results of the copper balance trial showed that absolute copper retention was significantly (P<0.05 or P<0.0l) influenced by dietary copper levels. In the third experiment, 21 week-old baby pigs were assigned to four dietary treatments composed of distilled deionized water and semi- purified diets analyzing 0.9 ppm, 2.0 ppm, 4.0 ppm and 4.9 ppm copper. The experimental design was similar to that used in Experiments 1 and 2. Hemoglobin values on weeks 4 and 8 showed significant (P<0.05 and P<0.01, respectively) differences between diets, with the basal diet inducing a very low hemoglobin value of 7.3 g/100 ml on week 8. The corresponding mean corpuscular hemoglobin concentration (MCHC) was likewise depressed (23.72). These hemoglobin and MCHC values are indicative of a marginal anemia due to inadequate dietary copper (0.9 ppm). Ceruloplasmin activity was considerably depressed by the basal diet until week 8, after which it rose slightly. There were significant (P<0.05 or P<0.01) differences in ceruloplasmin activity due to treatments in weeks 4 through 10. After week 2, plasma copper values were consistently and significantly (P<0.05 Anthony C. Okonkwo or P<0.0l) affected by dietary copper levels. Pigs on the basal diet had plasma copper values (5.9 and 5.2 meg/100 ml) on weeks 8 and 10, respec- tively, which were much below the critical hemopoietic level of 20 mcg/ 100 m1. Data from the above experiments indicate that although anemia and copper deficiency were not grossly evident, the consistent depression of hemoglobin and plasma copper levels by diets low in copper (0.6 ppm, 0.9 ppm and 1.3 ppm) showed that copper deficiency and anemia were biochemi- cally manifested. Such a sub—clinical hypocuprosis was also indicated by the significant depression of ceruloplasmin activity, an observation strongly suggesting that on low dietary copper, ceruloplasmin activity cannot be sustained. Based on these parameters the minimum copper requirement for the baby pig is very low and is probably between 3.0 and 4.0 ppm on an as-fed basis, or between 3.4 and 4.6 ppm on a dry basis. As a follow-up to the study previously conducted by Kayongo-Male (1974), a trial was designed to determine the manganese requirement of baby pigs on semi-purified diets. Sixteen 8-day old baby pigs from sows on loWbmanganese diets (13.9 ppm Mn) were fed diets analyzing 0.9 ppm, 2.2 ppm, 3.8 ppm and 7.4 ppm Mn. Blood samples were taken at the begin- ning of the trial and subsequently on days 7, 14, 28 and 42 for determi- nation of hemoglobin, hematocrit, alkaline phosphatase activity and serum manganese levels. A manganese balance trial was conducted at the end of the growth trial period. The pigs were killed thereafter and tissues and organs kept at -20°C until used for analysis. Manganese balance, serum manganese concentration and serum alkaline phosphatase activity were indicators of manganese status. Anthony C. Okonkwo Hemoglobin on day 42 was significantly (P<0.05) higher on diet 4 than on diets l and 3. Mean corpuscular hemoglobin concentration (MCHC) of pigs on diet 4 was significantly (P<0.05) higher than the correspond— ing values of pigs on diets l and 2, while MCHC value for diet 3 was sig— nificantly (P<0.01) lower than that for diet 4. Initial serum alkaline phosphatase activity was high on all diets, but decreased dramatically throughout the trial; there were, however, no statistically significant treatment differences. Serum manganese concentrations merely fluctuated without showing any statistically significant response to dietary levels of manganese. Manganese intake, manganese retention (absolute and as percent of intake), and fecal and urinary manganese.excretion were significantly (P<0.01) influenced by dietary manganese levels. Absolute fecal manganese excretion was significantly (P<0.05) different between diets 1 and 2, and between diets 1, 3 and 4 (P<0.01). There was a negative absolute manga- nese retention induced by diets l and 2 (-0.05 and -0.004 mg/day, respec- tively) resulting in negative retention as percent of intake (-l6.4 and -0.242, respectively); thus manganese excretion via urine and feces was greater than manganese absorption on diets 1 and 2. Heart, thyroid and spleen weights as percent of final body weight were significantly (P<0.05 or P<0.01) affected by dietary treatments as were liver and kidney manganese concentrations. Manganese intake was significantly (P<0.01) correlated with fecal manganese, manganese reten- tion, and negatively but significantly (P<0.05 or P<0.01) correlated with manganese excretion as percent of intake. Based on all parameters examined, the dietary manganese require- ment for the baby pig is approximately 3.8 ppm (as-fed basis) or 4.3 ppm (dry basis). COPPER AND MANGANESE REQUIREMENTS OF THE BABY PIG By Anthony Chukwuemeka Okonkwo A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 1976 Dedicated to My Beloved Mother ii ACKNOWLEDGEMENTS The author is grateful to Dr. D. E. Ullrey and Dr. E. R. Miller for their constructive advice, able instruction, guidance and supervision throughout his doctoral program, and for their critical appreciation and evaluation of this manuscript. The expertise, thoroughness and experi- ence of Dr. E. R. Miller in the principles and practices of swine husban- dry and nutrition provided the author with a series of rich learning exercises which kept him constantly on his toes and which deserve special mention. The writer is also grateful to Dr. R. M. Cook and Dr. K. K. Keahey, the other members of his guidance committee, for their encourage- ment and helpful suggestions during the course of this study. The assistance of Dr. K. K. Keahey with the histopathological examination is greatly appreciated. Sincere gratitude is expressed to Dr. R. H. Nelson, chairman of the Department of Animal Husbandry, and his staff for providing the financial support and facilities which made this research possible and my academic sojourn in this country fruitful. I wish to thank Dr. W. T. Magee, Mr. Charles McPeake and Dr. J. L. Gill whose help with the statistical analyses is greatly appreciated. I also wish to express gratitude to fellow graduate students and labora- tory personnel whose unreserved assistance was noteworthy, particularly Dr. P. K. Ku who gave me close guidance, advice and instructions regard- ing laboratory procedures and who directly assisted in some of the iii analyses, Ms. Phyllis Whetter, Mr. Paul Brady and Ms. Mona Shaft who willingly helped with some analytical procedures. The author is deeply grateful to Miss Liesel M. Heil who, despite her academic load, devoted many hours to the skillful typing, arrange- ment and constructive proofreading of the first, second and final drafts of this manuscript. I also wish to express sincere appreciation and gratitude to Mrs. Comfort C. Nwabara whose support and encouragement, particularly during my moments of depression and despair, motivated me to forge ahead. Most importantly, I am indebted to my dear mother, Cecilia, who has been, to me, a tremendous pillar of inspiration, encouragement, patience and support throughout my entire academic venture, and who persuaded me to return to this country to complete my studies; and to my sisters, Maria and Anne, for their understanding, support and love. iv ANTHONY C. OKONKWO CANDIDATE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DISSERTATION: COPPER AND MANGANESE REQUIREMENTS OF THE BABY PIG OUTLINE OF STUDIES: Main Area: Animal Nutrition and Husbandry Department of Animal Husbandry Minor Areas: Biochemistry Statistics Education BIOGRAPHICAL ITEMS: Born: April 12, 1944, Nkpor, Nigeria Undergraduate Studies: Michigan State University, 1965-1969 Graduate Studies: Central Michigan University, 1970-1971 Michigan State University, Jan. 1972—July 1972 Sept. l973-Dec. 1976 Experience: Graduate Teaching Assistant Central Michigan University, 1970-1971 Graduate Research Assistant Michigan State University, Jan. 1972-July 1972 Sept. l973-Dec. 1976 Student Representative to the Institute of Nutrition Michigan State University, 1974-1975 MEMBERSHIPS: Institute of Nutrition, Michigan State University American Society of Animal Science TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . ix LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . xiii INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . 4 A. Copper as an Essential Trace Element . . . . . . . . . 4 B. Tissue Distribution of Copper . . . . . . . . . . . . . 7 C. Metabolism of Copper . . . . . . . . . . . ._. . . . . . 9 1 0 Absorption O O O O I O O O O O O O O O O O O O O O O 9 2. Transport and Tissue Storage . . . . . . . . . . . ll 3. Excretion C O O O O O O O I O O O O O O O O O O O O 11 D. Metabolic Roles of Copper . . . . . . . . . . . . . . . 12 l. Hematopoiesis . . . . . . . . . . . . . . . . . . . 12 2. Cardiovascular Integrity . . . . . . . . . . . . . 15 3. Bone and Connective Tissue Metabolism . 2 . . . . . 17 a. Ceruloplasmin . . . . . . . . . . . . . . . . . 19 b. Superoxide Dismutase . . . . . . . . . . . . . 21 c. Cytochrome Oxidase . . . . . . . . . . . . . . 22 d. Other Enzymes . . . . . . . . . . . . . . . . . 23 5. Nervous System.Myelination . . . . . . . . . . . . 25 6. Fatty Acid Metabolism in Depot Fat . . . . . . . . 28 7. Reproduction . . . . . . . . . . . . . . . . . . . 30 E. Interaction of Copper with other Minerals . . . . . . . 31 l 0 Iron 0 O O O O O O O O 0 O O O O O O O O O O O O O 31 2. Molybdenum and Sulfate . . . . . . . . . . . . . . 33 3. Calcium and Zinc . . . . . . . . . . . . . . . . . 35 4. Other Minerals . . . . . . . . . . . . . . . . . . 37 F. High Level Copper Feeding . . . . . . . . . . . . . . . 37 G. Copper Requirements . . . . . . . . . . . . . . . . . . 41 vi Page 1. Pigs O O O O C O O C O O O O O O O O Q Q Q Q Q Q Q 41 2. Sheep and Cattle . . . . . . . . . . . . . . . . . 42 3. Other Species . . . . . . . . . . . . . . . . . . . 43 EXPERIMENTAL PROCEDURES . . . . . . . . . . . . . . . . . . . . . 44 A. IntrOduction O O O O O O O O O C O O O O I O O O O O O 44 B. Experiments . . . . . . . . . . . . . . . . . . . . . . 44 1. Experiment 1 . . . . . . . . . . . . . . . . . . . 44 2. Experiment 2 . . . . . . . . . . . . . . . . . . . 47 3 0 Experiment 3 O O O O O O O O O O O O O O O O O O O 47 C. Analytical Methods . . . . . . . . . . . . . . . . . . 50 l. Hematology . . . . . . . . . . . . . . . . . . . . SO 2. Physical Determinations . . . . . . . . . . . . . . 51 3. Chemical Analyses . . . . . . . . . . . . . . . . . 54 4. Enzymology . . . . . . . . . . . . . . . . . . . . 57 D. Statistical Analyses . . . . . . . . . . . . . . . . . 59 RESUIOTS AND DISCUSSION 0 O O O O O O O O O O O O O O O O O O O O O 61 A. Experiment 1: Copper requirement of baby pigs on semi-purified diets supplemented with varying levels of copper to yield upon analysis: 1.3 ppm, 3.2 ppm, 5.6 ppm and 9.3 ppm copper . . . . . . . . . . . . . . 61 l. Histopathology . . . . . . . . . . . . . . . . . . 82 B. Experiment 2: Copper requirement of baby pigs on semi-purified diets (the casein in the basal diet was treated with NazEDTA) supplemented with copper sulfate to yield upon analysis: 0.6 ppm, 1.9 ppm and 2.8 ppm copper . . . . . . . . . . . . . . . . . . . . . . . . 83 l. Histopathology . . . . . . . . . . . . . . . . . . 100 C. Experiment 3: Copper requirement of baby pigs maintained on distilled deionized water and fed semi- purified diets supplemented with copper sulfate to yield upon analysis: 0.9 ppm, 2.0 ppm, 4.0 ppm and 4.9 ppm copper . . . . . . . . . . . . . . . . . . . . 102 l. Histopathology . . . . . . . . . . . . . . . . . . 125 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 vii Page MANGANESE INTRODUCTION 0 O O O O O O O O O I O O O 0 O O O O O O O O O C I 1- 31 EXPERIMENTAL PROCEDURES . . . . . . . . . . . . . . . . . . . . . 133 A. Introduction . . . . . . . . . . . . . . . . . . . . . 133 B. Experiment 1 (Mn) . . . . . . . . . . . . . . . . . . 133 C. Analytical Methods . . . . . . . . . . . . . . . . . . 135 1. Bone Minerals . . . . . . . . . . . . . . . . . . 135 2. Serum Manganese . . . . . . . . . . . . . . . . . 136 3. Serum Alkaline Phosphatase . . . . . . . . . . . . 137 D. Statistical Analyses . . . . . . . . . . . . . . . . . 139 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . 140 A. Experiment 1: Manganese requirement of baby pigs on semi-purified diets supplemented with manganese sulfate to yield upon analysis: 0.9 ppm, 2.2 ppm, 3.8 ppm and 7.4 ppm manganese . . . . . . . . . . . . 140 1 0 H18 topathOIOgy O O O O O O O O O O O O O O O O I O 164 CONCLUS IONS O O O O O O O O O O I O O O O O O O O O O O O O I O O 1 66 BIBLIWRAPHY O O I O O O O O O O O O O O O O O O O O O O O O O O 16 7 APPENDIX A O O O O O O O O O O O O O O O O 0 O O O O 0 O O O O O 194 APPENDIX B O O C O O O O O O O I O O O O O O O O O O O O O O O O 202 viii Table 10 11 11a £12 £13 .14 LIST OF TABLES COMPOSITION OF DIETS USED IN EXPERIMENT 1 . . . . . . . COMPOSITION OF DIETS USED IN EXPERIMENT 2 . . . . . . . COMPOSITION OF DIETS USED IN EXPERIMENT 3 . . . . . . AVERAGE COPPER CONTENT OF TREATED AND UNTREATED CASEIN AND DIETS (EHT O 2) I O I O I I O O O O O O O I O O C C THE EFFECT OF DIETARY COPPER LEVELS ON GROWTH (EXPT. 1) THE EFFECT OF DIETARY COPPER LEVELS ON HEMATOCRIT, HEMOGLOBIN AND MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (EHT. 1) O O O I O O O O O O C O O O O O O O O O O C 0 THE EFFECT OF DIETARY COPPER LEVELS ON CERULOPLASMIN AND PLASMA COPPER CONCENTRATION (EXPT. 1) . . . . . . . . . THE EFFECT OF DIETARY COPPER LEVELS 0N TISSUE COPPER CONCENTRATIONS AND BRAIN SUPEROXIDE DISMUTASE ACTIVITY (EXPT. 1) . . . . . . . . . . . . . . . . . . . . . . . . THE EFFECT OF DIETARY COPPER LEVELS ON COPPER BALANCE (EXPTO l) o o O o o o o o o o o o o o o o o o o o o 0 THE EFFECT OF DIETARY COPPER LEVELS ON PHYSICAL AND CHEMICAL CHARACTERISTICS OF THE LEFT FEMUR (EXPT. 1) . . CORRELATIONS BETWEEN COPPER BALANCE, PLASMA COPPER, CERULOPLASMIN AND AVERAGE DAILY GAIN (EXPT. 1) . . . . . CORRELATIONS BETWEEN LIVER AND KIDNEY COPPER AND CERULOPLASMIN AND PLASMA COPPER (EXPT. 1) . . . . . . . THE EFFECT OF DIETARY COPPER LEVELS ON GROWTH AND TISSUE COPPER CONCENTRATIONS (EXPT. 2) . . . . . . . . . . . . . THE EFFECT OF DIETARY COPPER LEVELS 0N HEMATOCRIT, HEMDGLOBIN AND MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (EDT. 2) C O O 0 . C O O O O I O O C O O O O O O O O C THE EFFECT OF DIETARY COPPER LEVELS 0N CERULOPLASMIN ACTIVITY AND PLASMA COPPER CONCENTRATION (EXPT. 2) . . . ix Page . 45 . 48 49 . 53 62 63 69 75 77 78 80 81 84 89 89 Table Page 15 THE EFFECT OF DIETARY COPPER LEVELS ON COPPER BALANCE (WT. 2) I I I I I I I I I I I I I I I I I I I I I I I I I 96 16 THE EFFECT OF DIETARY COPPER LEVELS ON PHYSICAL AND CHEMICAL CHARACTERISTICS OF THE LEFT FEMUR (EXPT. 2) . . . 98 17 CORRELATIONS BETWEEN COPPER BALANCE, PLASMA COPPER, CERULOPLASMIN AND AVERAGE DAILY GAIN (EXPT. 2) . . . . . . 99 17a CORRELATIONS BETWEEN LIVER AND KIDNEY COPPER AND CERULOPLASMIN AND PLASMA COPPER (EXPT. 2) . . . . . . . . 101 18 THE EFFECT OF DIETARY COPPER LEVELS ON GROWTH AND ORGAN WEIGHTS AS PERCENTAGE OF FINAL BODY WEIGHT (EXPT. 3) . . . 103 19 THE EFFECT OF DIETARY COPPER LEVELS 0N HEMATOCRIT, HEMOGLOBIN AND MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (EHTI 3) I I I I I I I I I I I I I I I I I I I I I I I I 105 19a ACTIVITY OF ERYTHROCYTE SUPEROXIDE DISMUTASE (SOD) IN RESPONSE TO DIETARY COPPER LEVELS (EXPT. 3) . . . . . . . 113 20 THE EFFECT OF DIETARY COPPER LEVELS ON CERULOPLASMIN ACTIVITY AND PLASMA COPPER CONCENTRATION (EXPT. 3) . . . . 114 21 THE EFFECT OF DIETARY COPPER LEVELS ON TISSUE COPPER CONCENTRATIONS (EHT I 3) I I I I I I I I I I I I I I I I I 1 1 7 22 THE EFFECT OF DIETARY COPPER LEVELS ON COPPER BALANCE (EDT. 3) o o o o o o o o o o I o O o o o I o O o o o o o 122 23 THE EFFECT OF DIETARY COPPER LEVELS ON PHYSICAL AND CHEMICAL CHARACTERISTICS OF THE LEFT FEMUR (EXPT. 3) . . . 123 24 CORRELATIONS BETWEEN COPPER BALANCE, PLASMA COPPER, CERULOPLASMIN AND AVERAGE DAILY GAIN (EXPT. 3) . . . . . . 124 24a CORRELATIONS BETWEEN TISSUE COPPER CONCENTRATIONS, PLASMA COPPER AND CERULOPLASMIN ACTIVITY (EXPT. 3) . . . . . . . 126 25 COMPOSITION OF DIETS USED IN EXPERIMENT 1 (Mn) . . . . . . 134 26 THE EFFECT OF DIETARY MANGANESE LEVELS ON GROWTH (WT. 1) I I I I I I I I I I I I I I I I I I I I I I I I 141 27 THE EFFECT OF DIETARY MANGANESE LEVELS ON HEMATOCRIT, HEMOGLOBIN AND MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (mTI 1) I I I I I I I I I I I I I I I I I I I I I I I I 142 28 THE EFFECT OF DIETARY MANGANESE LEVELS ON SERUM ALKALINE PHOSPHATASE ACTIVITY AND SERUM MANGANESE CONCENTRATION (EHTI 1) o o o o o o o o o o o o o o o o o o o o o o o o 149 Table 29 30 31 32 33 34 Page THE EFFECT OF DIETARY MANGANESE LEVELS ON MANGANESE BALANCE, FECAL AND URINARY ZINC, COPPER AND IRON (mTI 1) I I I I I I I I I I I I I I I I I I I I I I I I 153 INFLUENCE OF DIETARY MANGANESE LEVELS ON ORGAN WEIGHTS AS PERCENTAGE OF FINAL BODY WEIGHT (EXPT. 1) . . . . . . . 155 THE EFFECT OF DIETARY MANGANESE LEVELS ON LIVER, KIDNEY AND MUSCLE MANGANESE, ZINC, COPPER AND IRON CONCENTRATIONS (IN PPM) (EXPT. 1) o o o o o o o o o o o o o o o o o o o o 157 THE EFFECT OF DIETARY MANGANESE LEVELS ON PHYSICAL AND CHEMICAL CHARACTERISTICS OF THE LEFT FEMUR (EXPT. 1) . . . 159 CORRELATIONS BETWEEN MANGANESE BALANCE, SERUM MANGANESE, SERUM ALKALINE PHOSPHATASE AND AVERAGE DAILY GAIN (EXPT. 1) I I I I I I I I I I I I I I I I I I I I I I I I 162 CORRELATIONS WITHIN MANGANESE BALANCE PARAMETERS (WT. 1) I I I I I I I I I I I I I I I I I I I I I I I I 163 x1 LIST OF APPENDIX TABLES APPENDIX A Table Page A91 LOW-COPPER VITAMIN-TRACE MINERAL PREMIX USED IN GESTATION AND LACTATION RATIONS . . . . . . . . . . . . . 194 A92 LOW-COPPER GESTATION AND LACTATION RATIONS . . . . . . . . 195 A93 LOW9COPPER MINERAL PREMIX USED IN SEMI-PURIFIED DIETS . . 196 A94 VITAMIN MIXTURE USED IN COPPER AND MANGANESE SEMI- PURIFIED DIETS . . . . . . . . . . . . . . . . . . . . . . 197 A-5 IL 453 ATOMIC ABSORPTION SPECTROPHOTOMETER SPECIFICATIONS FOR COPPER MYS Is I I I I I I I I I I I I I I I I I I I 1 9 8 A96 ANALYSIS OF VARIANCE FOR EFFECTS OF DIETARY COPPER LEVELS ON COPPER BALANCE, TISSUE COPPER CONCENTRATIONS AND GROW (mTI 1) I I I I I I I I I I I I I I I I I I I 199 A97 ANALYSIS OF VARIANCE FOR EFFECTS OF DIETARY COPPER LEVELS ON COPPER BALANCE, TISSUE COPPER CONCENTRATIONS AND GRWTH (EDTI 2) I I I I I I I I I I I I I I I I I I I 200 A98 ANALYSIS OF VARIANCE FOR EFFECTS OF DIETARY COPPER LEVELS ON COPPER BALANCE, TISSUE COPPER CONCENTRATIONS AND GROW (mT I 3) I I I I I I I I I I I I I I I I I I I 201 APPENDIX B Table Page B91 LOW9MANGANESE MINERAL PREMIX USED IN SEMI-PURIFIED DIETS . 202 392 LOW9MANGANESE GESTATION AND LACTATION RATIONS . . . . . . 203 B93 LOW9MANGANESE VITAMHN TRACE MINERAL PREMIX USED IN GESTATION AND LACTATION DIETS . . . . . . . . . . . . . . 204 B94 IL 453 ATOMIC ABSORPTION/EMISSION SPECTROPHOTOMETER - IL mDEL 455 muss I I I I I I I I I I I I I I I I I I 205 B95 ANALYSIS OF VARIANCE FOR EFFECTS OF DIETARY MANGANESE LEVELS ON MANGANESE BALANCE, TISSUE MANGANESE CONCENTRATIONS AND GROWTH . . . . . . . . . . . . . . . . 206 x11 Figure 1.1 1.2 1.3 1.4 1.5 2.1 2.2 2.3 2I4 2.5 3.1 3.2 3.3 3.4 3.5 LIST OF FIGURES Influence of dietary copper levels on hematocrit (Expt 0 1) o o e ......... o o o o o o o o o 0 Influence of copper intake on hemoglobin (Expt. 1) Influence of dietary copper levels on mean corpuscular hemoglobin concentration (Expt. 1) . . . . . . . . . Influence of copper intake on ceruloplasmin activity Page 65 (Exp: I 1) I I I I I I I I I I I I I I I I I I I I I I I I 72 Influence of dietary copper levels on plasma copper concentrations (Expt. 1) . . . . . . . . . . . . . . . . . 74 Effect of copper intake on hematocrit (Expt. 2) . . . . . 87 Effect of dietary copper levels on hemoglobin (Expt. 2) . 91 Effect of copper intake on mean corpuscular hemoglobin concentration (Expt. 2) . . . . . . . . . . . . . . . . . 92 Effect of copper intake on ceruloplasmin activity (EmtI 2) I I I I I I I I I I I I I I I I I I I I I I I I 94 Effect of copper intake on plasma copper levels (Emt I 2) I I I I I I I I I I I I I I I I I I I I I I I I 95 Response of hematocrit to varying levels of dietary copper (Expt. 3) . . . . . . . . . . . . . . . . . . . 107 Response of hemoglobin to dietary copper levels (EmtI 3) I I I I I I I I I I I I I I I I I I I I I I I I 109 Response of mean corpuscular hemoglobin concentration to copper intake (Ewt I 3) I I I I I I I I I I I I I I I I I 111 Response of ceruloplasmin activity to dietary copper levels (Expt. 3) . . . . . . . . . . . . . . . . . . . . 119 Response of plasma copper levels to copper intake (ExptI 3) I I I I I I I I I I I I I I I I I I I I I I I I 120 Effect of dietary manganese concentration on hematocrit (EmtI 1) I I I I I I I I I I I I I I I I I I I I I I I I 143 xiii Figure Page 4.2 Influence of manganese intake on hemoglobin (Expt. 1) . . 145 4.3 Response of mean corpuscular hemoglobin concentration to dietary manganese levels (Expt. 1) . . . . . . . . . . 147 4.4 Effect of manganese intake on serum alkaline phosphatase act1v1ty (ExptI 1) I I I I I I I I I I I I I I I I I I I 150 4.5 Response of serum manganese levels to dietary manganese (ExptI 1) I I I I I I I I I I I I I I I I I I I I I I I I 152 xiv INTRODUCTION Although copper was known to occur in the tissues of plants and animals as early as the 1800's its biological importance was not recog- nized until the reports of McHargue (1925a, 1925b, 1926) and the conclusive evidence furnished by Hart and coworkers (1928) that copper was essential for mammalian hemoglobin biosynthesis. This breakthrough generated much research which resulted in the description of other meta- bolic functions of copper such as its involvement in cardiovascular structure, connective tissue and bone collagen synthesis, myelin forma- tion, enzyme systems and reproduction. The literature is filled with reports of copper interaction with other minerals such as molybdenum, calcium, iron and zinc, and other dietary factors such as sulfate, pro- teins and lipids. Another aspect of research on copper that sparked global interest was the European reports by Barber and coworkers (1955) followed by those of Braude (1965) that a high dietary level of copper up to 250 ppm caused a significant improvement in body weight gain and feed conversion effi- ciency of swine. Attempts to duplicate these trials resulted in conflict- ing reports from the United States and Canada. Effects of high level copper feeding ranged from depressed performance, slight improvement in body weight gain accompanied by decreased feed efficiency, to highly significant overall improvement in performance. These contradictory reports notwithstanding, it was clearly established that copper is ‘essential for optimal performance in livestock. However, the minimum 1 amount of copper required by growing pigs has not been precisely estab- lished. Because of the effects of interaction between copper and other minerals it is difficult to define precisely the minimum copper require— ment of baby pigs. It seems therefore that the most practical approach would be to maintain other dietary factors at an optimal level while varying the concentrations of copper in the diets. Semi-purified diets were used in the work reported here, since other dietary factors were easily controlled. The importance of defining the minimum requirement of copper for swine becomes clear when the side effects of high dietary dosages of copper on the environment are considered. Since copper is not biodegrad- able and tends to accumulate in the top 8 inches of the soil (Lagerwerff, 1967), it has therefore a very good chance of contaminating water sources and affecting aquatic life. Accumulation of copper in the environment arises from its widespread use as a fungicide in the form of Bordeaux mixture, its high concentration in swine waste from farms using high dietary copper levels, and other agricultural uses such as viniculture, control of potato blight, leaf spot of sugar beets and peach leaf-curl. Although the NCR942 Committee does not regard "lagoon and lake sterility" as a result of the antibiotic-like effects of copper as a very serious problem, the danger has been recognized. High concentrations of copper in the soil have been reported to interfere with the functions of the soil nitrifying flora (Gilbert, 1952). The use of swine waste contain— ing high levels of copper, if not monitored and modified, produces chronic toxicity in ruminants, particularly sheep. Although the threat of copper toxicity in humans arising from ingestion of meat products from animals fed high levels of copper is not very likely, a USDA regulation requires withdrawal of copper from finish- ing pigs 15 days prior to slaughter. Porcine muscle copper level is low and the effect of dietary copper is negligible; however, the liver concen- tration of copper is markedly increased by high copper levels in the diet, and the 15-day withdrawal provides sufficient time for depletion of excess copper in this organ. Based on the studies of Ullrey and associates (1960) the NRC (1973) suggested that 6 ppm copper is the requirement for baby pigs on natural corn-soy diets. Since the mechanism of action of copper at high dietary intakes is not clearly understood but is supposed to resemble the action of antibiotics, it is important that a distinction be made between minimum essential requirement of copper for optimal metabolic functions on the one hand and an antibiotic-like effect on intestinal microflora by high amounts of copper inducing increased performance on the other. The experiments reported in this dissertation were conducted to determine: 1. The minimum essential requirement of copper for baby pigs, using semi-purified diets and varying the levels of copper. 2. The effects of low levels of copper on body weight gain, feed consumption, feed conversion efficiency, tissue copper levels, some enzyme systems and copper balance in baby pigs. LITERATURE REVIEW A. Copper as an Essential Trace Element In the latter part of the twenties McHargue (1925a, 1925b) pro- vided evidence of the existence of copper in plant and animal tissues indicating that the livers of domestic animals, more than any other organ, contain copper in addition to manganese and zinc. He also showed that at birth bovine liver yields more copper than does the adult liver. At about the same period McHargue (1926) conducted studies using rats and presented data showing the importance of copper as a trace mineral supplement to animal diets. Two years later, Hart and coworkers in Wisconsin (1928) proved conclusively the essentiality of copper and demonstrated its vital role in mammalian hematopoiesis. These researchers induced nutritional anemia in rats by raising them on whole milk diets fortified with iron salts, and then reversed the anemia effectively by administration of iron and copper, but not by iron alone. This copper- iron therapy not only restored hemoglobin to normal levels but also caused increased vigor, increased appetite and smoother hair coat. Although they were not able to define the specific function of copper in hematopoiesis, it was hypothesized that copper, like iron in chlorophyll biosynthesis, might be acting as a catalyst somewhere in the biosynthetic pathway of hemoglobin; since the hemoglobin molecule does not incorporate copper. Subsequently, the unique position of copper in promoting 4 hemoglobin synthesis was confirmed by other workers (Elvehjem and Hart, 1929; Waddel et al., 1929; Elvehjem and Sherman, 1932) who showed that iron supplementation by itself failed to restore normal hemoglobin status in anemic rats, thereby establishing the obligatory need for copper. The uniqueness of copper in this role was further lent credence by Waddell et al. (1929) who conducted feeding trials using copper and twelve other transition elements. Further studies extended this phenom- enon to other species (Elvejhem and Hart, 1932; Schultze et al., 1936a, 1936b; Wilkerson, 1934). The possibility of other biological roles for copper motivated a great deal of research. Field studies with livestock were being con— ducted worldwide and simultaneously with laboratory investigations of copper at the cellular level in order to define its mechanism of action. Such laboratory probings uncovered the existence of many copper contain- ing proteins in mammalian tissues and cells. Some of these proteins, such as cerebrocuprein (Porter and Folch, 1957; Porter and Ainsworth, 1959); hemocuprein (Mann and Keilin, 1938); hepatocuprein (Shapiro et al., 1961); and neonatal hepatic mitochondrocuprein (Porter et al., 1962; Porter et al., 1964), were not thought to possess enzymatic activity. They are bluish-green soluble proteins containing two atoms of copper and zinc per molecule (Carrico and Deutsch, 1970). Erythrocuprein, cerebrocuprein and hepatocuprein were later shown by McCord and Fridovich (1969) to exhibit superoxide dismutase activity. Detailed studies of their physical, chemical and immunological properties led Carrico and Deutsch (1969) to report that erythrocuprein, hepatocuprein and cerebro- CEUDrein are identical and should be designated collectively as cytocu- IIreiJI. Although their specific physiologic functions are unknown they are thought to represent intermediate compounds in copper metabolism (Li and Vallee, 1973). Other copper proteins have been shown to exhibit oxidative and a variety of catalytic functions. Such copper-enzymes are monoamine oxidase, ceruloplasmin, tyrosinase, cytochrome oxidase, ascorbic acid oxidase and superoxide dismutase. Studies with copper deficient animals served to reveal the biochemical position of copper in these enzymes. Meanwhile field studies from different parts of the world impli— cated copper in some livestock diseases. Since these diseases responded to copper therapy they were regarded to be a consequence of copper deficiency. From Florida, Neal and associates (1931) described the "salt sick" of cattle and sheep, Sjollema (1933) reported the "lecksucht", a disease of sheep and cattle in Holland, "falling disease" of cattle was recorded in south-western Australia (Underwood, 1966), Bennetts and Chapman (1937) labelled a disease of lambs "enzootic neonatal ataxia", some areas of England and Scotland witnessed "swayback in sheep, and there were scattered reports of copper deficient livestock from Scandi- navia, tropical Africa, South America, and the Mediterranean regions (Underwood, 1966). Detailed histopathological examinations of these copper deficiency diseases indicated that, in addition to hemoglobin biosynthesis, copper was also involved in cardiovascular function, cere— brospinal myelination, connective tissue and bone formation, growth and reproduction, pigmentation, and adipose tissue metabolism. In its biological roles, copper was also found to interact with molybdenum and sulfate (Dick and Bull, 1945; Dick, 1952, 1954a, 1954b, 1956), zinc, iron, calcium (Hill and Matrone, 1962; Mills, 1968; Kline at aZ., 1972) and cadmium (Whanger and Weswig, 1970). B. Tissue Distribution of Copper According to the studies of Spray and Widdowson (1951) and Widdowson (1960) the young of most animal species possess a much higher level of copper per unit of body weight than the adults, and this level gradually declines as maturation progresses. Various species of animals have been surveyed and shown (Cunningham, 1931) to contain in their livers a greater fraction of total body copper than in any other organ or tissue at a given point during life. These liver levels can be as high as 79% of the total body supply in ruminants, especially in sheep (Dick, 1954). Tissue analyses by Cunningham (1931) and Smith (1967) of various species established that the liver, brain, kidneys, heart, and hair contain high concentrations of copper, while spleen, pancreas, muscles, skin, and bone are of intermediate copper levels, and prostrate, pituitary, thyroid and thymus glands show low levels. Analysis of samples from liver, spleen, and aorta show a fall in copper content as the adult stage is attained, whereas adult brain concentrates almost twice its fetal copper stores (Schroeder et al., 1966). Variations in dietary copper concentrations exert a direct effect on the levels of copper in blood, liver, kidney and spleen, but the bone shows a more significant reduction in forms of copper particularly the chicken bone as reported by Rucker et a1. (1969). Different species show a.wide variety of eye copper levels. Examination of fresh water fishes, frogs and mammals (Bowness et al., 1952b; Bowness and Mbrton, 1952a) :tndicated that the highest levels of eye copper are found in the pig- mented areas like choroid, retina and iris. These levels range from 1:3.5 ppm (on the dry basis) in sheep choroid to 105 ppm in the iris of freshwater trout. Hair copper concentrations have been reported to range from a mean value of 7.8 ppm in black hair of dairy cattle (Anke, 1967) to 10 to 47 ppm in black and white hair from ten different species (Goss and Green, 1955). Levels below 8 ppm copper in cattle hair were associated with copper deficiency by Van Koetsveld (1958), but this association did not hold true in studies with copper-deficient cattle (Cunningham and Hogan, 1958) and rats (Dreosti and Quicke, 1966). Depig— mentation of hair or achromotrichia is a condition exhibited by a wide range of species including rats, guinea pigs, rabbits, sheep, cattle and goats at dietary intakes low enough to induce copper deficiency; this phenomenon of loss of hair color has not been noticed in pigs (Underwood, 1971). A breakdown in synthesis of melanin from tyrosine, a reaction catalyzed by a copper enzyme, has been suggested as a probable cause of achromotrichia (Roper, 1928). Supplementation of the deficient diet with adequate copper quickly reverses this condition. In blood, a great proportion of copper (85-90%) is bound with an a-2 globulin as ceruloplasmin (Wintrobe et al., 1953), and about 10 to 152 is associated with red blood cells (Kimmel et al., 1959, Markowitz et al., 1959). About two-thirds of total erythrocyte copper is bound to a protein formerly known as erythrocuprein (superoxide dismutase) in humans (Shields et aZ., 1961), and hemocuprein (superoxide dismutase) in ‘bowine erythrocytes (Mann and Keilin, 1938). In addition to ceruloplas- tnin and erythrocuprein, the blood contains copper enzymes such as mono- amine oxidase and cytochrome oxidase. The normal blood levels of copper vary from 0.5 to 1.5 meg/ml in species like rats, dogs, pigs, sheep, cattle and humans (Beck, 1961). C. Metabolism of Copper 1. Absorption Various workers have shown that the region(s) 0f the gastro- intestinal tract from which copper is absorbed varies among species of animals. Bowland et al. (1961) reported that most of the copper ingested is absorbed from the small intestine and colon in pigs; in the rat, absorption is from the stomach and small intestine (Van Campen and Mitchell, 1965); from the jejunum in dogs (Sacks et aZ., 1943); and in chicks from the duodenum (Starcher, 1969). In man the net percentage of copper absorbed is slightly over 30% of the amount ingested (Cartwright and Wintrobe, 1964). Absorption of copper from the gastrointestinal tract has been shown to be affected by several factors. Starcher (1969), using radio- active copper, reported that, in the chick, binding of copper to a duo- denal protein constitutes an important phase in copper absorption. As the calcium content of the diet increases, a marked decrease in utiliza- tion of dietary copper results (Kirchgessner and Grassman, 1970). This is because high dietary intakes of calcium salts raise the pH of intes- tinal contents, thereby depressing copper absorption as shown in sheep (Dick, 1954). Ferrous sulfide also depresses copper absorption by inducing formation of insoluble copper sulfide. Studies on the availability of copper from various copper com- pounds indicate that for pigs (Bowland et al., 1961) and sheep (Lassiter and Bell, 1960) the water soluble forms of copper, particularly sulfate, nitrate, carbonate and chloride, are more effectively utilized than are copper sulfide, cuprous oxide and cupric oxide. 10 Copper absorption is also depressed by certain organic substances which form complexes with copper, rendering it unavailable for absorption. Phytate present in soybean proteins was shown by Davis and coworkers (1962) to reduce absorption of copper. In chicks, increased dietary ascorbic acid (Hill and Starcher, 1965) exacerbates copper deficiency by suppressing intestinal absorption of the element. This phenomenon also occurs in rabbits (Hunt and Carlton, 1965) and in rats (Van Campen and Gross, 1968). Interference with normal absorption of copper from the gut has been reported to involve other minerals such as mercury (Van Campen, 1966), silver (Hill et aZ., 1964), cadmium (Hill et al., 1963; Van Campen, 1966), zinc (Starcher, 1969), molybdenum (Matrone, 1970) and sulfate sulfur (Dick, 1954). The mode of action of some of these elements has not been elucidated (e.g., mercury and molybdenum), however zinc and cadmium have been reported by Starcher (1969) to depress copper absorp- tion by binding competitively to the duodenal mucosal protein, thereby displacing copper. Binding to mucosal protein is an essential step in copper absorption (Starcher, 1969). Molybdenum probably acts as a result of formation of a copper—molybdenum complex which is absorbed, trans- ported and excreted undissociated (Matrone, 1970); in this bound form, the copper of the copper—molybdenum unit becomes unavailable for utiliza- tion by the animal. Amino acids were shown by Kirchgessner and Grassman (1970) to affect the absorption of copper from rat gastrointestinal tract. Combi- nation of amino acids with copper results in the formation of copper- amino complexes which are then transported uncleaved to the point of absorption. These researchers further indicated that the ease of absorption decreased with increased complexity of the amino acid 11 polymers, monomeric copper—amino acids being more easily absorbed than dimeric and so on. Configuration of amino acids was also shown to affect copper absorption, the copper—L-amino acid complexes being better absorbed than D-forms. 2. Transport and Tissue Storage Upon absorption from the gut, copper becomes bound loosely to serum albumin (Bearn and Kunkel, 1954) forming the small direct—reacting fraction of serum copper. This copper-albumin forms a pool which assumes pivotal importance in that it receives copper from many sources; absorbed copper from the intestine enters into it before being distributed to the liver, bone marrow, kidneys, erythrocytes and other tissues (Cartwright and Wintrobe, 1964); the pool also receives copper from these tissues (Bush et al., 1956b). 0n reaching the liver parenchymal cells, copper is taken up by hepatic mitochondria, microsomes and soluble fractions for storage or for synthesis of erythrocuprein, ceruloplasmin, and other cellular copper-enzymes (Underwood, 1971). Ceruloplasmin synthesis goes on in the liver (Markowitz et al., 1955; Sternlieb et aZ., 1962) after which it is released into the blood- stream; while erythrocuprein, according to Bush and ;oworkers (1956), is ‘probably synthesized in the bone marrow normoblasts. Under normal physiological conditions, ceruloplasmin copper has been shown (Bush at aZ., 1956b) not to exchange with the copper in the direct-reacting fraction in the blood. 3. Excretion The biliary system is the major pathway for the excretion of COPPen'in pigs and dogs (Mahoney et al., 1955; Bowland et al., 1961). in mice: (Gitlin et al., 1960), in humans (Van Ravestyn, 1944) and in 12 chickens (Beck, 1961). Liver copper is secreted through bile, back into the intestine, and small quantities of copper are excreted directly into the gastrointestinal tract - from whence they are voided as fecal copper along with a high percentage of unabsorbed dietary copper (Cartwright and Wintrobe, 1964); a smaller amount is excreted through the urinary system. Biliary-obstruction studies performed on dogs (Mahoney et al., 1955) and man (Bush at aZ., 1955) show that of the excreted copper about 802 is biliary, 162 is from direct secretion into the gut, and about 4% is urinary. In man, negligible amounts of copper are lost through sweat (Hamilton and Mitchell, 1949). Estimated copper turnover in man is as follows (Cartwright and Wintrobe, 1964): of the 2.0 to 5.0 mg copper ingested daily, 0.6 to 1.6 mg (322) is absorbed; 0.5 to 1.2 mg is excreted in the bile, 0.1 to 0.3 mg passes directly into the intestines, and 0.01 to 0.06 mg appears in the urine. The daily turnover of copper through ceruloplasmin is of the order of 0.5 mg (Sternlieb et aZ., 1961). D. Metabolic Roles of Copper 1. Hematopoiesis Following the pioneer studies of Hart (1928) and others in the early thirties, in establishing the role of copper as promoter of hemo- globin synthesis, more detailed research has described other aspects of copper function in blood metabolism. These aspects were studied by means of experimental induction of copper deficiency in various animal species. 13 The serum copper level below which normal hematopoiesis is arrested has been determined as 0.10 to 0.12 mcg/ml in sheep (Marston at al., 1948) and 0.2 meg/m1 in pigs (Schultze, 1936b; Lahey et aZ., 1952). The mechanism by which copper deficiency caused subnormal hemoglobin levels eluded early workers, until Chase and coworkers (1952a) provided evidence that, in copper-deficient rats, absorption of iron from the gut is impaired. In a copper-deficient state, depressed hemoglobin levels can result from impairment of iron metabolism, protoporphyrin and heme synthesis or biosynthesis of globin. But since it has been shown that in copper deficiency there is normal protein synthesis and hence normal production of globin (Gallagher et al., 1956; Dreosti and Quicke, 1968), and heme (Lee et al., 1968), the cause of impaired hemoglobin synthesis must be associated with iron metabolism. This observation was already confirmed by Gubler et al., (1952) and Chase et al. (1952b) who induced copper deficiency in swine on milk diets and showed impaired iron absorp- tion from the gastrointestinal tract, incomplete mobilization of iron from the tissues and marked inability of the animals to utilize paren- terally administered iron for hemoglobin biosynthesis even when the iron was presented to bone marrow in normal quantities. Wintrobe and co- workers (1953) reported a major depletion of total body iron from the hemoglobin compartment in copper deficiency by reason of a depressed movement of iron from.tissues to plasma; administration of copper resulted in a sharp rise in plasma iron. The postulation by some researchers (Gubler et al., 1952; Chase et al., 1952a, 1952b; Wintrobe et al., 1953) that copper deficiency induced hypoferremia arose from impaired mobilization of iron from tissues to plasma was later confirmed by others, and more recently by l4 Osaki et a1. (1956) and Ragan et al. (1969) who obtained data to show that iron transfer from the tissues to plasma requires the enzymatic conversion of iron from the ferrous to the ferric state by ceruloplasmin. Ability of ceruloplasmin to convert iron into the ferric state is by virtue of its feroxidase activity (Evans and Abraham, 1973; Gray and Daniel, 1973). Oxidation of ferrous to ferric ions occurs before iron is incorporated into the carrier-protein apotransferrin for delivery to the site of hemoglobin synthesis. Failure of this oxidation step because of lack of ceruloplasmin inhibits iron transfer (Wintrobe et al., 1953; Lee et al., 1968), and it accumulates as non-hemoglobin iron in bone marrow, liver and the reticuloendothelial system, thereby becoming un- available for hemoglobin formation (Cartwright et al., 1956). To increase the circulating and available iron from its storage sites, a release must be effected by copper (Marston and Allen, 1967). A consequence of this deranged hemoglobin production is anemia. In swine, rabbits and rats, anemia is microcytic and hypochromic (Foster, 1931; Fitz-Hugh et al., 1933; Smith and Ellis, 1944; Smith et al., 1944a, 1944b; Lahey et aZ., 1952), accompanied by leukopenia, neutropenia and hypocupremia (Wintrobe et al., 1953). Copper deficiency anemia in swine is indistinguishable from anemia of iron deficiency (Lahey et al., 1952). In dogs and chicks the anemia is normocytic and normochromic (Van Wyk at al., 1953; Maas et al., 1944; Matrone, 1960); in ruminants grazing copper- deficient pastures, e.g., cattle and sheep, macrocytic and hypochromic (Cunningham, 1946; Bennetts et al., 1941; Marston et aZ., 1948); in lambs, microcytic and hypochromic (Beck and Bennetts, 1942). In addition to anemia, the bone marrow undergoes erythroid (normoblastic) hyperplasia with the exception that, in the pig (Baxter and Van Wyk, 1953), there is 15 depressed reticulocytosis (Lahey et aZ., 1953). Unless adequate copper levels are administered, copper deficiency anemia shows no reaction to iron therapy (Cartwright, 1956; Schultze et al., 1936a; Elvehjem, 1932). 2. Cardiovascular Integrity Severe copper deprivation in cattle was reported by Bennetts and associates (1942a, 1948) to cause myocardial fibrosis which leads to sudden death, known as "falling disease". Believed to be a consequence of cardiac failure precipitated by physical exertion, falling disease occurs almost exclusively in Western Australia. The myocardium becomes infiltrated by collagen, thus causing a loss of its architecture, and adversely affecting its function. Species specificity seems the case with this disease since it is not observed in sheep and horses exposed to the same conditions (Davis, 1950); however, in pigs cardiac hypertrophy has been observed as a result of copper deficiency, but it is not identical to nor a typical characteristic of falling disease (Gubler et al., 1957). Cardiac hypertrophy seems to be due to reduced cytochrome oxidase activity which causes myocardial enlargement and, according to Li and Vallee (1973), is a compensatory mechanism for the reduced respiratory activity. Studies done on other species have produced cardiovascular mal- function. In chicks raised on low copper diets (0.8 ppm Cu) there was widespread subcutaneous and internal hemorrhage (Carlton and Henderson, 1963; Savage et al., 1966), the aorta showed greatly thickened walls, a small lumen, fragmented elastic laminae and dissecting aneurysms; mortality rate was high (Simpson and Harm, 1964; O'Dell et al., 19613; Carlton and Henderson, 1963). There were also cases of spontaneous aortic rupture, 16 loss of elastic fibers, fibrosis of myocardium and vessels, and hemo- pericardium, due to unsupported vasa vasorum, by diapedesis or rhexis (Carlton and Henderson, 1963). In turkeys, dietary copper levels below 1.0 ppm resulted in frequent aortic rupture and internal hemorrhage from smaller vessels (McSherry et al., 1954). Pigs showed high incidence of aortic rupture (Weissman et al., 1961; Carnes et al., 1961; Weissman et aZ., 1963), aortic fragility and reduced aortic tensile strength (Coulson and Linker, 1968; Smith et al., 1968). Aneurysms and loss of aortic structure were demonstrated by Everson and coworkers (1967) in guinea pigs, and O'Dell and coworkers (1961b) produced failure of elastic tissue in newborn rats. Histopathological analyses of affected tissues revealed diminished elastin and increased lysine contents of elastin (Kimball et al., 1962; Starcher et al., 1964). Increased elastin lysine is due to failure of conversion of lysine to desmosine and isodesmosine, the cross linking residues of elastin (O'Dell et al., 1966) and a failure of the incor- poration mechanism to attach newly formed proelastin into aortic fibers (Partridge et aZ., 1964). These abnormalities result in accumulation of lysine, the precursor of proelastin. The lysine to desmosine conversion is catalysed by copper containing amine oxidase whose activity is depressed in aorta, serum and heart of copper-deficient animals (Bird et al., 1966; Blaschko et al., 1965; Mills et aZ., 1966; Hill et al., 1967). Amine oxidase acts by oxidatively deaminating the epsilon-amino group of lysine residues in elastin (Hill et al., 1968). Other workers (Partridge et al., 1964; Starcher et al., Weissman et al., 1963) reported increased swelling and solubility in hydrolytic agents of aortic and heart valvular elastin from copper-starved animals, and accumulation of non-elastin, l7 non-collagen protein and soluble collagen. Significantly lower concen- trations of desmosine and isodesmosine were found by 0'De11 and associates (1966) in copper-deficient animals. 3. Bone and Connective Tissue Metabolism Copper-deficient animals have been shown to display abnormal bone conditions. Teague and Carpenter (1951) described unusual leg formations, loss of rigidity in leg joints, excessively flexed hocks and crooked forelegs in young pigs as signs that were reversed by copper therapy. Similar bone defects resulting in fractures and animals with squatty and shorter than normal body structures were reported by other workers in swine (Lahey et al., 1951; Follis et al., 1955). In chicks there were unsteady gait, complete paralysis and sharp bending of the proximal end of the metatarsus (O'Dell et aZ., 1961a; Gallagher, 1957; Rucker et aZ., 1969a, 1969b). In rabbits, Hunt et al. (1966) reported medial curvature of radius and ulna, cortical thinning, increased width, distortion of epiphyseal cartilage and bone destruction; in turkeys, the bones were fragile and deformed (Rucker et aZ., 1969a). To a lesser extent there were various forms of defects observed in mice (Guggenheim et al., 1964), dogs (Baxter and Van Wyk, 1953) and foals (Bennetts, 1932). In ruminants grazing copper-deficient pastures, Cunningham (1950) and Davis (1950) observed brittle bones that fractured easily even though they appeared normal in form and shaft thickness. Upon histochemical examination, affected bones showed osteoporosis (Carlton and Henderson, 1964; Rucker et al., 1969a), failure of bone deposition in cartilage matrix, unusually thin cortices, wide epiphyses, deficient trabeculae, and spontaneous fractures due to inadequate stabilization of fibrous proteins (Baxter and Van Wyk, 1953; Carnes, 18 1971). Osteoporosis was a consequence of deranged osteoblastic activity accompanied by cessation of osteoclastic function (Carlton and Henderson, 1964). Gross bone lesions have been shown to be due to metabolic defects at the cellular level which are caused by copper deficiency. The cupro- enzymes that maintain the structural integrity of bone and connective tissue are known to be markedly reduced; depressed cytochrome and amine oxidase activities have been reported by several workers (Hunt at aZ., 1966; Rucker et aZ., 1969a; Waino et aZ., 1959). Chou and associates (1968) described decreased amine oxidase activity in tendon and cartilage of copper-deficient chicks. Subnormal activities of these enzymes result in a derangement in connective tissue metabolism. Rucker and coworkers (1969b) stated that, in copper-deficient chick bone, amine oxidase which functions in oxidative deamination of lysyl residues to u-amino adipic acid-A-semialdehyde residues in collagen synthesis is inhibited, much like in vascular tissues. This inhibition results in a concomitant decrease in intramolecular cross-linking of elastin (O'Dell et aZ., 1966; Kim and Hill, 1966; Hill et aZ., 1967). Consequently the copper-deficient bones yielded more collagen which contained more aldehyde and was more soluble than did control bones (Rucker et aZ., 1969a). Connective tissue abnormality was initially identified by O'Dell and associates (1961) in young chicks, and in pigs by Shields and co- workers (1962). That deranged biosynthesis of collagen and elastin cul- minates in connective tissue and bone lesions has been confirmed by several workers using swine (Weissman et aZ., 1963, 1965; Smith et aZ., 1968), chicks (O'Dell*et aZ., 1966; Miller et aZ., 1965; Chou et aZ., 1968; Rucker et aZ., 1969a, 1969b, 1969c) and turkeys (Savage et aZ., 19 1966). More recently Carnes (1971) summarized these observations: "...in copper deficiency the connective tissue showed diminished tensile strength of elastin and collagen due to impaired polypeptide chain cross- linkages, ... the proelastin lacked crosslinks and had increased lysine residues, diminished aldehyde precursors of cross links and an inhibition of a late step in biosynthesis of elastin and collagen as already reported by Hill (1969) and Carnes (1968)." 4. Enzyme Activity a. Ceruloplasmin. This enzyme, also known as polyphenol oxidase, was described by Holmberg and Laurell (1947) as a serum copper-containing alpha-globulin, blue in color, with a molecular weight of 151,000. It contains 8 atoms of copper and 4 units of the hemocuprein of Mann and Keilin (1938). Of the two types of copper present in serum, direct and indirect acting, ceruloplasmin contains indirect reacting copper, which is tightly bound and will not react with chelating agents unless the molecule is destroyed (Milne and Weswig, 1968; Cartwright and Wintrobe, 1964) to liberate the copper from the protein by acid hydrolysis (Wintrobe et aZ., 1953; Marceau and Aspin, 1972). It was shown by Wintrobe and associates (1953) that, irrespective of copper status in animals, the ratio of direct reacting copper and ceruloplasmin copper is the same. About 802 of copper in serum is in the form of ceruloplasmin in most animals such as rats, pigs, sheep, dogs and humans (Butler, 1963; Milne and weswig, 1968; Wintrobe and Cartwright, 1953; Cartwright, 1950). Thus, Todd (1970) concluded, the activity of ceruloplasmin gives a strong indication of serum and whole blood copper concentration. In chicks, 20 only a small portion of plasma copper is contained in ceruloplasmin (Starcher and Hill, 1965); and turkeys have no measurable ceruloplasmin oxidase activity (Wiederanders, 1968) and in fact possess the lowest reported serum copper levels (Evans and Wiederanders, 1967). Cerulo- plasmin functions in: 1. transfering iron from cells to plasma by promoting the rate of iron saturation of transferrin and stimulating utilization of iron (Osaki et aZ., 1966). This function has been confirmed by Ragan and coworkers (1969) and by Gray and Abraham (1973) who established that ceruloplasmin facilitates iron transfer as a result of its ferroxidase activity. 2. donating copper to extrahepatic tissues (Owen, 1965). 3. maintaining hepatic copper homeostasis as shown in cases of post-adrenalectomy and post-hypophysectomy states; in rats increases due to adrenalectomy were reported in hepatic copper concentration and serum ceruloplasmin (Evans et aZ., 1970; Gregoriadis and Sourkes, 1970). The hepatic copper level increases were a consequence of decreased biliary copper excretion (Evans et aZ., 1970). 4. oxidation of various substances such as polyphenols (Holmberg and Laurell, 1948), serotonin and epinephrine (Martin et aZ., 1964). In vitro reactions indicate that the best substrate with which to measure oxidase activity is p-phenylenediamine. In addition to these functions, ceruloplasmin has been shown by Hampton et a1. (1972) and by Gary and Daniel (1973) to possess histaminase activity. Evans and Wiederanders (1967) established a correlation between total plasma copper and ceruloplasmin oxidase activity; lower plasma copper concentration was associated with low oxidase activity of 21 ceruloplasmin. In rats kept on diets unsupplemented with copper, cerulo- plasmin activity was significantly depressed when compared to control values (Milne and Weswig, 1968; Starcher and Hill, 1965; Evans and Abraham, 1973). In a study with swine raised on a copper-deficient milk diet, Williams and associates (1975) reported that ceruloplasmin concen— tration at 8 weeks was 8% of control values, and decreased further to less than 1% of controls at 12 weeks. b. Superoxide Dismutase. McCord and Fridovich (1969) described an enzyme purified from bovine erythrocytes which catalyzes the dispro— portionation or dismutation of univalently reduced oxygen or superoxide radicals. Known as superoxide dismutase (SOD), it functions according to the following: I- I- + g 02 + 02 + 2H 02 + H202 The enzyme contains two equivalents of copper per mole (McCord and Frido- vich, 1969) and an equal molar level of zinc (Carrico and Deutsch, 1970). It has been shown to be identical to the previously isolated copper- containing human erythrocuprein (Markowitz et aZ., 1959, Kimmel et aZ., 1959), bovine hemocuprein (Mann and Keilin, 1939), and equine hepatocu- prein (Mohamed and Greenberg, 1953). Superoxide dismutase is widely distributed within mammals and among microbes (McCord et aZ., 1971; Keele et aZ., 1970); in fact all cytochrome containing aerobes and some aero-tolerant anaerobes (except Lactobacillus plantarum) exhibit superoxide dismutase activity (McCord et aZ., 1971). It has been shown by McCord and Fridovich (1969) that in cata- lyzing the oxidation and reduction of the superoxide radical to H202 and 22 02, superoxide dismutase enables the organism to survive in the presence of molecular oxygen. This is achieved by prevention of Oé--induced mem- brane damage. An organism that lacks superoxide dismutase could survive in a molecular oxygen environment provided the superoxide radical does not accumulate in lethal amounts (McCord et aZ., 1971). The dependence of enzymatic activity of superoxide dismutase upon adequate dietary copper was demonstrated by Williams and associates (1975). Using an evaporated milk diet, these researchers induced copper deficiency in swine and reported an 85% drop in hepatic and erythrocyte superoxide dismutase activity in the copper-deficient animals whose plasma copper was 5 to 10% of controls. c. Cytochrome Oxidase. As the terminal member of the cytochrome system, cytochrome oxidase is capable of reducing oxygen. Basing their conclusion on a strong positive correlation between copper content, heme content and enzyme activity, several researchers (Eichel et aZ., 1950; Wainio et aZ., 1959) reported that cytochrome oxidase contains copper as a functional component of its structure, participates in electron trans- fer in the terminal steps of the respiratory chain, and contains a lipid (Wainio et aZ., 1959), present as mitochondrial lipid (White et aZ., 1959). Cytochrome oxidase was described by White and associates (1959) as a polymer of subunits with a molecular weight of 72,000, with each subunit containing one heme and one copper atom, and its activity due to its polymeric form. Cohn and Elvehjem (1934) reported a marked reduction in cyto- chrome oxidase in heart and liver of milk-anemic rats, and a restoration of its activity to normal following copper administration. Similar reductions in cytochrome oxidase activity in liver, heart and bone 23 marrow of copper-deficient rats were shown by Schultze (1939, 1941) and Gallagher et al. (1956a, 1956b) who also reported that copper therapy caused restoration of enzymatic function. In copper-deficient swine, an eight-fold reduction in heart cytochrome oxidase and three-fold decrease in liver cytochrome oxidase was observed by Gubler and coworkers (1957). Other investigators reported depressed brain cytochrome oxidase in lambs suffering from copper deficiency-induced swayback (Howell and Davison, 1959; Mills and Williams, 1962); in copper-deficient cattle (Mills et aZ., 1963; Poole, 1970); and in copper-deficient chickens (Hill and Matrone, 1961). Loss of cytochrome oxidase function has been attributed by Gallagher and associates (1956a) to failure of synthesis of heme a prosthetic group due to copper deficiency. d. Other Enzymes. That copper deficiency affects the activity of other copper-containing enzymes has been reported by several workers. Schultze and Kuiken (1941) and Adams (1953) fed rats diets deficient in copper and reported that catalase activity of liver, kidney and blood showed a significant decrease when compared to controls, and supplementa- tion of diets with copper restored activity of catalase to normal. In one set of experiments with swine, liver catalase activity was not affected by subnormal copper levels (Lahey et aZ., 1952), whereas Gubler and associates (1957) reported decreased catalase activity in copper- deprived swine; however, in both experiments low copper levels did not influence activity of catalase in kidney and erythrocytes. Monoamine oxidase activity was reported to be undetectable in plasma of copper-starved pigs, and its activity was normalized by dietary copper supplementation (Blaschko et aZ., 1965). The function of amine 24 oxidase was described by Partridge and coworkers (1964) as catalysts of oxidation of the epsilon amino group of lysine to aldehyde prior to con- densation to desmosine and isodesmosine (the crosslinkage group in elastin synthesis); and it has been established that amine oxidase con- tains copper (Yamada and Yasunobu, 1962; Hill and Mann, 1962; Buffoni and Blaschko, 1964). Kim and Hill (1966) subsequently confirmed that the role of copper in elastin synthesis is related to its role in amine oxidase activity. They reported that in copper deficiency amine oxidase activity was reduced in the aorta and addition of amine oxidase to a copper-deficient system stimulated synthesis of desmosine from lysine. In ewes and lambs, Mills and associates (1966) observed that low plasma amine oxidase activity was not associated closely with copper deficiency. Several researchers reported that copper is an essential functional component of the enzyme tyrosinase (Kubowitz, 1937, 1938; Allen and Bodine, 1941; Sreerangacher, 1944) and that the enzyme exhibits oxidase activity (Brown and Ward, 1959). The removal of copper from tyrosinase by cyanide treatment followed by dialysis resulted in a loss of 85% of its activity, and addition of cupric ions restored activity of the enzyme to 90% of original values (Lerner et aZ., 1950). In copper deficiency in all animal species except swine, failure of pigmentation in hair or wool (achromotrichia) seems to be a consequence of deranged conversion of tyrosine to melanin mediated by copper-containing tyrosin- ase (Underwood, 1966). In humans, albinism is characterized by a lack 0f detectable tyrosinase activity (Harris, 1959). Butyryl coenzyme A dehydrogenase was described by Mahler (1954) 88 a cuproflavoprotein since it contains riboflavin and copper. However, it was reported to be unaffected by copper starvation (Gubler et aZ., 25 1957) since upon dialysis to remove copper the copper-free butyryl co- enzme A dehydrogenase was still actively reduced by butyryl coenzyme A (Mahler, 1954). Subsequently, studies by Scheinberg and Sternlieb (1960) showed that copper was not a functional component of butyryl coenzyme A dehydrogenase, but probably was a contaminant. Uricase activity was studied in swine liver and was thought to be related to the presence of copper in the enzyme. The enzyme catalyzes conversion of uric acid to allantoin. 5. Nervous System Myelination A disorder of the nervous system in young and newborn lambs known as enzootic neonatal ataxia was first described in Australia by Bennetts (1932), and was known to be associated with tissue hypocuprosis. This condition can be prevented by administration of copper to the pregnant ewe (Bull at aZ., 1938). Later a clinically similar disease called "swayback" and exhibiting identical morphological signs as ataxia was reported by Innes (1936). Pathological signs include diffuse symmetrical degeneration of cerebral white matter, cerebral cavitation, secondary motor tract degeneration into the spinal cord, and degenerative changes in the neurons of the brain stem nuclei and spinal cord. There were also chromatolysis, myelin degeneration and cell necrosis (Innes and Shearer, 1940; Barlow et aZ., 1960a). That swayback and enzootic ataxia are not caused by a failure in the intermediary metabolism of copper or by una- ‘vailability of copper in pastures as previously postulated (Eden st aZ., .1945) was conclusively demonstrated by workers (Bennetts and Beck, 1942b; IBarlow et aZ., 1960b) who reported the diseases in Australian and English Pastures with excessively low copper levels. Earlier views that swayback was essentially a demyelinating 26 encephalopathy (Innes, 1936; Innes and Shearer, 1940) were challenged by the findings of Behrens and von Schulz (1959) and von Schulz and Behrens (1960). These researchers suggested that swayback was caused by venous stasis, edema, and perivascular cuffing. However, later investigations confirmed the reports of earlier workers and stated that cerebral lesions were not essential features of swayback, but rather scattered lesions of hyaline neuronal necrosis and nerve fiber degeneration in the brain and spinal cord (Spais et aZ., 1961; Fell et aZ., 1961; Howell et aZ., 1964). In the brain, neural lesions were found to persist after birth (Barlow, 1963a); and lesions in the white matter of the spinal cord indicate aplasia of myelin rather than demyelination (Howell et aZ., 1964). Mills and Fell (1960) examined lambs from ewes on a high sulfate- molybdenum diet and reported degeneration of cells in motor neurons of the red nucleus and ventral horns of gray matter in the spinal cord; there was demyelination in the cerebral cortex and spinal cord. These researchers state that ataxia was likely to develop in lambs if liver copper levels of ewes fell below 20 mcg/g and especially below 10 mcg/g. Butler and Barlow (1963) were unable to produce swayback in lambs by administering high levels of molybdenum and sulfate to pregnant ewes. A nervous disease was described by Roberts and associates (1963a, 1963b) in lambs, characterized by nervous lesions, cerebral edema, tissue hypocuprosis, and minor degrees of cavitation in cerebral white matter. Histopathological findings confirmed acute cerebral edema with severe widespread neuronal involvement, perivascular cuffing, cortical necrosis and cerebellar compressive effects attributable to pressure and or anoxia. The hypocuprosis was reversed by copper therapy. Sheep are not unique in showing nervous disorders due to 27 hypocuprosis. In guinea pigs fed a copper-deficient diet, Everson and coworkers (1967, 1968) reported ataxia, missing or malformed cerebellar folia, cerebellar agenesis, widespread hypomyelinogenesis as indicated by phospholipid determinations, and soft translucent areas of the cerebral cortex. In rats and chickens, copper deficiency was shown to cause two major metabolic disturbances: loss of cytochrome oxidase activity and suppression of phospholipid synthesis by the liver (Gallagher et aZ., 1956a; 1956b; Gallagher, 1957). In pigs with ataxia, low liver copper levels were found (Wilkie, 1959) and although no pathological reports were made, there was demyelination of all areas of the spinal cord except dorsal areas. The author reported ataxia in 2— to 3-week- old pigs, incoordinated hind quarters, and liver and blood copper levels of 9.8 and 0.25 ppm, respectively; these conditions were reversed by ad- ministration of copper. McGavin and associates (1962) made field observations in Australia and reported that pigs with liver copper levels of 3, 9, 10 and 14 ppm showed marked spinal demyelination which affected the dorsal spinocerebellar tract, dorsal, lateral and ventral funiculi extending to the medulla oblongata, cerebellum and posterior cerebellar peduncle. In goats, signs of swayback have been described by Hedger et al. (1964) and by Owen et al. (1965). At necropsy, cells and tracts of the central nervous system showed typical swayback lesions, and cyto- chrome oxidase activity was low, accompanied by low blood and brain copper levels. Decreased cytochrome oxidase activity in brain and liver as a result of hypocuprosis was reported by Howell and Davison (1959) in swayback lambs, by Schultze (1939, 1941) in rats, and by Gubler et al. (1957) in pigs. The relationship between demyelination and cytochrome 28 oxidase was established by Gallagher (1957): low brain copper leads to decreased cytochrome oxidase activity (since the enzyme contains copper) in motor neurons which in turn leads to demyelination since phospholipid synthesis is depressed in copper deficiency; phospholipids are a component of myelin. 6. Fatty Acid Metabolism in Depot Fat Following reports by Barber et al. (1957) and Bellis (1961) that high dietary levels of copper adversely affected pork carcass grade, Taylor and Thomke (1964) fed 250 ppm copper to bacon pigs to determine the quality and properties of porcine depot fat. They observed a highly significant difference in iodine number of backfat between treated (250 ppm Cu) and control groups. Using vacuum techniques, the researchers showed that dietary copper caused softer backfat (i.e., with a lower melting point), and found this observation compatible with iodine values. The authors therefore speculated that high levels of dietary copper may affect absorption and transport of dietary fat in such a way as to cause changes in composition of depot fat, and that high liver copper levels may interfere with liver functions in fat metabolism. Experiments by Bowland and Castell (1964, 1965) yielded data to indicate that copper supplementation softens porcine backfat. However, their papers made no mention of specific biochemical processes involved, but alluded to a possibility of an interaction between copper and protein source to cause soft fat. Investigations by Braude (1965) failed to confirm these findings. However Elliot and Bowland (1968) reported that copper supplementation at 280 ppm caused a significant increase in proportion of unsaturated 29 fatty acids (UFA) in the outer and inner backfat, and perinephric fat at 26, 47 and 70 kg liveweight. This increase was accompanied by a corre- sponding decrease in proportion of saturated fatty acids (SFA), and there were no significant differences in fatty acid composition of these fats at 90 kg liveweight. The observed increase in proportion of UFA was probably due to increases in the 16:1 and 18:1 fatty acids with decreases in 16:0 and 18:0 acids. Softness of depot fat due to high levels of dietary copper was attributed by some workers (Moore et aZ., 1969; Christie and Moore, 1969) to changes in structure of triglycerides, and by others (Elliot and Bowland, 1968, 1969, 1970) to increases in major saturated fatty acids. Other investigations showed that in the depot fat of pigs on diets supplemented with 250 ppm copper there was a slightly higher concentration of oleic acid and lower concentration of stearic acid (Moore et aZ., 1968), a low proportion of stearic acid, more palmit- oleic acid and a higher oleic:stearic acid ratio (Castell et aZ., 1975). Several researchers (Amer and Elliot, l973a, l973b; Myres and Bowland, 1972, 1973, 1975; Ho et aZ., 1973, 1974, 1975; Castell et aZ., 1975) published confirmatory reports that porcine depot fat softness induced by diets containing high levels of copper was caused by increased proportions of unsaturated fatty acids and a concomitant decrease in proportions of saturated fatty acids. In addition to increased percent- ages of UFA in pig backfat resulting from feeding 6 levels of copper (125, 150, 175, 200, 250 ppm), soft fat has been linked to an increase in oxidative susceptibility and decreased keepability of fatty pork (Amer and Elliot, l973b), a condition which was countered by supplemental vitamin E (Amer and Elliot, 1973a). These depot fat changes were thought by Myres and Bowland (1973) 30 to be related to a disturbance in the balance between lipolysis and re- esterification of fatty acids. More specifically, Ho and Elliot (1973) presented data to suggest that increased 18:1 and decreased 18:0 fatty acids in depot fat of copper-supplemented pigs were related to an en- hancing effect of copper on specific activities of both hepatic and adipose stearoyl-CoA desaturase systems. Later, Ho and Elliot (1974) implicated copper involvement in the entire fatty acyl desaturase system, and more recently the authors suggested that UFA increases were due to the role of copper in desaturation reactions as a component of some cuproprotein enzyme system or metalloprotein (Ho and Elliot, 1975). It appears from the work done so far that copper is definitely involved in fatty acid metabolism in porcine depot fat, but the exact mechanism is yet to be clearly defined. 7. Reproduction Although the mechanism by which copper influences fertility and reproduction in animals is not well understood, various workers have shown that subnormal levels of dietary copper adversely affect repro- duction. Keil and Nelson (1931) placed rats on milk diets supplemented with iron in form of ferric chloride and observed that reproduction occured only when copper sulfate was added to the diet. Bennetts and associates (1942a, 1948) reported that low copper status of dairy cows resulted in a high incidence of infertility, decreased milk production and retarded growth and development of the young. Dutt and Mills (1960) raised female rats on copper-deficient diets and observed that the rats had normal estrous cycles; out of 18 pregnant rats, only 3 produced litters; necropsy showed unmistakable evidence of fetal resorption, and 31 uterine nodules representing previous sites of implantation of embryos were found in a majority of the rats. Studies by Hall and Howell (1969) and Howell and Hall (1969) showed that copper-deficient rats were successfully mated after exhibiting normal estrous periods but failed to produce litters. Pregnancy was not inhibited, but normal fetal de- velopment ceased on day 13 of pregnancy, fetal tissues disintegrated and the placenta underwent necrosis. These studies show conclusively that copper deficiency does not affect the estrous cycle, mating and conception in the rat, but for maintenance of pregnancy until production of normal litters, there must be adequate intakes of copper, otherwise the fetuses undergo resorption. In chickens, copper deficiency has been shown to affect repro- ductive perfomrance. Savage (1968) placed hens on low copper diets for 20 weeks and observed decreased egg production and copper content of eggs, plasma and liver; hatchability was decreased and approached zero at 14 weeks. There was early embryonic mortality following anemia and widespread hemorrhage, due probably to defective red blood cell and connective tissue formation (Simpson et aZ., 1967) during the early stages of embryonic life. E. Interaction of Copper with other Minerals 1. lggg_ The fact that copper deficiency produces anemia in animals provides evidence for an interaction between copper and iron. Effects exerted upon iron metabolism by copper have been extensively investigated by many researchers (Lahey et aZ., 1952; Cartwright et aZ., 1952, 1955, 1956; Gubler et aZ., 1952; Bush at aZ., 1955, 1956a; Chase at aZ., 19523, 32 1952b; Jensen et aZ., 1956). In rats, copper increased iron absorption (Chase at aZ., 1952a), and copper deficiency in pigs resulted in impaired iron absorption and reduced iron utilization in hemoglobin synthesis (Gubler et aZ., 1952) and caused iron deposits in the cells of the duodenal mucosa. These effects were reversed by administration of copper. Ceruloplasmin, a copper containing enzyme, is the agent through whose feroxidase activity copper affects iron metabolism. In swine suffering from copper-deficiency anemia, there were low serum iron, decreased iron uptake for hemoglobin synthesis, low liver iron (Gubler et aZ., 1952), and the erythrocyte life span was 20% that of controls (Jensen et aZ., 1956; Bush at aZ., 1956a). There was also an impaired ability to absorb iron from the gastrointestinal tract, and a slower rate of disappearance of radioactive iron from liver and bone marrow (Gubler st aZ., 1952). Since, in the rat, it is unlikely that copper exerts a direct effect on iron absorption (Cunningham, 1931; Houk et aZ., 1946; Chase at aZ., 1952a, 1952b), Matrone (1960) proposed the effect is indirect. A lack of copper induces a mucosal block which decreases iron absorption from the gastrointestinal tract during copper deficiency. This block is lifted during copper adequacy, thus allowing iron to enter hemoglobin synthesis; in this fashion absorption of iron is increased. In ruminants grazing copper deficient pastures, extensive iron deposits in the liver were reported by some workers (Marston et aZ., 1948; Underwood, 1956; Marston, 1952), and in tissues the iron was in the form of hemosiderin (Marston, 1952). Matrone and associates (1957), investigating copper deficiency in calves, reported that the level of 33 iron in serum and liver was not affected by dietary copper levels. These researchers did not find excessive iron deposits in the calves' livers. Available data seem to show that in ruminants dietary copper has slight influence on iron metabolism. 2. Molybdenum and Sulfate Ingestion of high levels of molybdenum by cattle grazing pastures normal in copper but high in molybdenum was established to be the cause of teartness in England (Ferguson et aZ., 1943); this condition was over- come by supplemental copper. It therefore became evident that an inter- action exists between copper and molybdenum since high molybdenum levels produced copper deficiency in situations where the copper level was adequate. Dick and Bull (1945) described hypercuprosis in sheep grazing low molybdenum pastures, and the condition was treated with supplemental molybdenum. Comar and coworkers (1949) investigating molybdenum-induced copper deficiency, administered radioactive copper and molybdenum to cattle and rats and observed an interference in bone metabolism caused by molybdenum; this action was thought to be due to lowered liver copper, molybdenum interference and inhibition of bone enzyme systems, and molyb- denum competition with phosphorus. The role of inorganic sulfate in ameliorating the toxic effects of molybdenum was first noted by Dick (1953) who fed lucerne bay to sheep and observed that decreased blood molybdenum levels were due to high levels of inorganic sulfate in the hay. He also reported that as the sulfate concentration increased, with dietary molybdenum held constant, blood molybdenum was lowered. Similar observations were discussed in rats by Miller and associates (1956); in sheep, Miller and Engel (1960) 34 observed increased blood copper concentrations, and copper losses in liJrer and blood were reported in cattle (Mylrea, 1958). High dietary Sulfate at constant dietary levels of molybdenum caused increases in blood copper of sheep, while in cattle molybdenum and sulfate favored copper losses (Miller and Engel, 1960). These authors reported that in rats increased dietary sulfate at constant molybdenum intake decreased blood copper; and in sheep induced molybdenum loss via urine, feces and tissues. Although dietary molybdenum and sulfate affected copper storage in ruminants, some workers reported that blood copper was not affected but there was tissue copper depletion in sheep (Suttle and Field, 1968; Marcilese et aZ., 1969, 1970). A copper-molybdenum-sulfate interaction has been shown to successfully prevent copper accumulation in the liver (Dick, 1953; wynne and McClymont, 1955; Cunningham et aZ., 1959). Such interaction if it exists should be able to eliminate excessive hepatic copper levels in swine. Experiments performed with pigs showed that such an interaction between copper, sulfate and molybdenum does exist, but differs from the one in ruminants since there was no reduction in tissue copper levels (Gipp et aZ., 1967; Hays and Kline, 1969; Kline et aZ., 1971, 1972, 1973; DeGoey et aZ., 1971). Depressing effects of high dietary copper (500 ppm) however, were partially overcome in pigs by addition of sulfide to the diet with no apparent response due to molybdenum. Levels of 450 to 700 ppm sulfide were added to diets containing 250 ppm copper and 1800 ppm sulfide added to 500 ppm copper resulted in liver copper concentra- tions similar to those of pigs fed low copper levels (15 ppm) (Kline et aZ., 1973). In studies with sheep, Dick (1954) showed that sulfide re- duced absorption and retention of ingested copper, and Mills (1960) 35 reported that rats developed anemia when fed sulfide and molybdenum. Anemia was a result of sulfide-induced hypocuprosis causing a decrease in available copper needed for hemoglobin synthesis. Research done with pigs on corn-soy diets containing 11 to 18 ppm copper showed that addition of 0.4% sulfate significantly increased plasma copper concentrations while 50 to 1500 ppm molybdenum significantly depressed liver copper up- take and a combination of sulfate and molybdenum increased liver storage of copper (Dale et aZ., 1973; Standish at aZ., 1975). Although the site of action and mechanism at which molybdenum and sulfate influence copper metabolism is not known (Dale et aZ., 1973) it has been shown that in the gastrointestinal tract molybdenum forms a complex with copper in the ratio of 3 to 4 (Dowdy and Matrone, 1968a, 1968b) and 1 to l in vitro (Neilands et aZ., 1948; Britton and German, 1931). Formation of this copper-molybdenum complex may render copper biologically unavailable and inactive in the sheep, chick, and pig. 3. Calcium and Zinc Any interaction between copper and calcium.must be indirect and has not been clearly defined. However Marston et al. (1948) and Macpherson and Hemingway (1968) reported that copper requirements of sheep grazing highly calcareous or limed soils were significantly in- creased. Copper absorption in sheep was greatly depressed by high intakes of dietary calcium carbonate (90g/day) which limited storage of copper in livers of adult sheep (Dick, 1954). It is thought that calcium carbonate depresses copper absorption by increasing the pH of the intes- tine (Underwood, 1971). In pigs, Hoefer and coworkers (1960) reported that at high calcium levels copper significantly increased growth rate, reduced incidence and severity of parakeratosis but was less effective 36 than zinc. Antagonism between copper and zinc with respect to liver storage was reported earlier by some researchers (Van Reen, 1957; Allen et aZ., 1958; Davis, 1958). Ritchie and associates (1963) showed that 100 ppm zinc added to a diet containing 250 ppm copper prevented parakeratosis, reduced liver copper concentrations and protected against copper toxico- sis resulting from 250 ppm copper. Toxicity signs included anemia, internal hemorrhage, jaundice, yellow cirrhotic livers, weakness, gastric ulceration and incoordination. Similar signs were reported by Gordon and Luke (1957), O'Hara et al. (1960), wallace et al. (1960), Buntain (1961), and Allen and Harding (1962). In pigs, 500 ppm zinc or 750 ppm iron in presence of 750 ppm copper was shown to eliminate jaundice, normalize serum copper and restore aspartate transaminase activity to normal; how- ever only 750 ppm iron afforded protection against anemia induced by high copper concentrations. In the absence of zinc and iron, 425 ppm copper caused severe toxicosis in swine, the effects of which were eliminated by addition of zinc and iron; in zinc deficiency, 250 ppm copper caused toxicosis and exaggerated parakeratosis which resulted from the zinc deficiency (Suttle and Mills, 1966b). The mode of action by which zinc antagonizes copper is not clearly understood, however Starcher (1969) investigated the mechanism of copper absorption from the gastrointestinal tract of chicks and proposed a scheme: it appears that copper is bound to a duodenal protein with an approximate molecular weight of 10,000; this constitutes an important step in the process of copper absorption. Zinc inhibits copper absorption by binding to, and competing for, the same protein-binding sites in the duodenum required for copper. 37 4. Other Minerals Cadmium at high dietary doses has been reported to adversely depress copper uptake from the gastrointestinal tract and influence tissue distribution of copper (Hill et aZ., 1963; Van Campen, 1966). In chicks, silver exacerbated the effects of copper deficiency (Hill et aZ., 1964), and in rats mercury slightly suppressed the uptake of copper 6l’Cu) from the gastrointestinal tract (Van Campen, 1966). Whanger and Weswig (1970) reported that in rat diets containing 6 ppm copper, addition of 100 ppm cadmium, 200 ppm silver, 500 ppm molybdenum or 10,000 ppm sulfate showed silver to be the strongest antagonist of copper, followed by cadmium, molybdenum, zinc and sulfate in that order. Other elements such as nickel, tungsten, vanadium, chromium, rhenium, uranium and tantalum did not exert any observable influence on copper retention (Cunningham, 1950; Dick, 1954). F. High Level Copper Feeding Initial studies were conducted by Braude (1945, 1948) to show the growth-promoting effects of copper when used as a dietary supplement for pigs. The pigs showed a craving for copper, and a preference when given a choice, for diets supplemented with copper; these observations were confirmed by Mitchell (1953). Following reports by Barber and associates (1955) that addition of high levels of copper (250) ppm in the form of copper sulfate to diets of growing pigs resulted in improved growth rate, numerous workers conducted studies using various levels of copper: 100, 125, 150, 200, 250 ppm, and higher. These studies reported improved growth rate and overall performance with levels up to 250 ppm (Bowler et aZ., 1955; Barber et aZ., 1960; Allen et aZ., 1961; Barber et 38 aZ., 1961; Lucas at aZ., 1962a; Lucas and Calder, 1957a), and thereby verified the findings of Barber (1955). Most of these investigations were done by European workers. Daily gains of growing pigs were significantly improved by addition of 250 ppm copper to the diets, and the increases were of the same magnitude as those obtained by Barber and coworkers (1955b) with antibiotics; further comparative effects of copper and antibiotics were confirmed subsequently by Bowler et al. (1955). Addition of 230 ppm copper by Schurch (1956) caused increased weight gains and improved feed utilization. Barber (1957) later confirmed previous findings in which copper exerted effects similar to those obtained from chlortetracycline and oxytetracycline. The results obtained by Lucas and Calder (1957a), Bellis (1971) and Bunch et al. (1963) with high level copper supplementation indicated that growth response reached a peak and plateaued when pigs attained 50 to 60 kg bodyweight, and that a decline in response arose as a consequence of copper accumulation in the liver. Workers in several countries have reported (Lucas at aZ., 1962a; Bunch et aZ., 1963; Wallace, 1967; Drouliscos et aZ., 1970) that copper supplementation at high dietary levels exerts relatively more pronounced effects on growing pigs by increasing digestive and/or metabolic efficiency. Others (Mitchell, 1953; Barber et aZ., 1962; Braude, 1965; Young et aZ., 1970) attribute the effect of high level copper to a stimulatory effect on feed consumption especially in young pigs. Variation in responses to increased copper levels was indicated in reviews made by Braude (1965), and wallace (1967). The extent of these responses seems to be influenced particularly by the level of protein in the diet (Wallace at aZ., 1960; 39 Lucas at aZ., 1962b; King, 1964; Combs et aZ., 1966); while the source of dietary protein has been implicated (Barber et aZ., 1962; Beames and Lloyd, 1965; Drouliscos st aZ., 1970). Levels of copper up to 250 ppm in the diet were shown to improve growth rate during the growing phase of pigs, but removal of copper after eight weeks was shown by Miller at al. (1969) and other workers (NOR-42 Committee on Swine Nutrition, 1974; Gipp et aZ., 1973) not to affect rate of gain or feed efficiency. Gipp and associates (1973) reported that the addition of 250 ppm copper to corn-soy diets did not consistently affect rate of body weight gain during starter, grower, finisher, or overall weaning—to-market periods and carcass measurements. While some researchers in Europe and the United States were observing improved performance as a result of high copper feeding, contradictory reports appeared in the literature. No beneficial growth response was observed in pigs fed 200 or 250 ppm copper (Lucas and Calder, 1957b; Teague and Grifo, 1966; Livingstone and Livingston, 1968; Parris and McDonald, 1969; Elliot and Bowland, 1970; Amer and Elliot, 1973). Ho and associates (1975) reported that the presence of high levels of dietary copper caused deterioration in both average daily gain and feed conversion. 'Canadian investigators failed to show consistent benefits similar to those reported by European workers. Mostly, high level copper feeding yielded no improvements (Castell and Bowland, 1968; Drouliscos, 1970; Young et aZ., 1970; Castell et aZ., 1973). Earlier studies by Bass et al. (1956) and Davis (1958) had shown irregular growth effects and possible toxicity when copper was fed to swine in high concentrations. Elliot and Amer (1973) obtained an improved growth rate but it was nonsignificant, and only up to 23, 46 or 69 kg live weight; 40 at 92 kg, growth was depressed significantly, and upon removal of supple- mental copper from the diet at 23, 46 or 69 kg live weight, growth was depressed. Withdrawal of copper from the diet between 46 and 57 kg live weight did not affect performance but allowed liver concentration of copper to return to normal by slaughter weight (Teague and Grifo, 1966). Some Canadian workers obtained beneficial results with 250 ppm copper in starter diets of pigs and only up to 50 kg live weight (Beames and Lloyd, 1965; Young and Jamieson, 1970). Recently Braude and Ryder (1973) re-confirmed the optimum level of copper supplementation to be 250 mg copper per kilogram of diet. However, the improved performance was slightly less than the 8.1% for average daily gain, and 5.4% for feed converstion efficiency obtained by Braude (1965) earlier. Despite some reports to the contrary (Livingstone and Livingston, 1968; Elliot and Bowland, 1970; Amer and Elliot, 1973), many workers have shown overwhelmingly that copper is essential for growth and improvement of performance in pigs especially at a dietary level of 250 ppm (Braude, 1945; Barber et aZ., 1955; Wallace, 1967; Braude and Ryder, 1973; Young and Jamieson, 1970; Ho and Elliot, 1973; Braude et aZ., 1970). The effect on growth and performance produced when swine diets are supple- mented by copper sulfate has been shown by researchers (Barber et aZ., 1957; Bowland et aZ., 1961; Hawbaker et aZ., 1959) to be due to the copper radical and not the sulfate. The mechanism by which copper exerts its beneficial action at high dietary levels is not clearly under- stood. Hawbaker and coworkers (1961) postulated that the beneficial action of copper is probably due to its antibiotic-like action on the intestinal microflora. WOrk done by other researchers opposes this theory by showing that, as a resulf of fecal flora studies (Fuller et aZ., 41 1960) and gastrointestinal tract flora count (Smith and Jones, 1963), copper causes a change in microflora from one type to another, but not a population decrease. G. Copper Requirements 1. Pigs Although numerous studies have been conducted using various levels of copper in pig diets, as yet the minimum copper requirements of pigs have not been precisely established. Ullrey and associates (1960) fed baby pigs diets containing 6, l6 and 106 ppm copper and observed no significant differences in treatments in growth rate, feed efficiency or hematology. Based upon this report the National Research Council (1973) recommended 6 mg of dietary copper per kg of diet, and stated that 0.1 to 0.15 mg per kg body weight is near the minimum require- ment. British Agricultural Research Council (1967) estimated that 4 ppm copper is adequate for growing pigs up to 90 kg liveweight. The fact that most natural swine rations analyze higher than 4 to 6 ppm copper seems to render copper supplementation of normal rations unnecessary (Underwood, 1971). As previously discussed, interaction of copper with other minerals (calcium, zinc, sulfate, molybdenum, cadmium and iron) and various dietary factors such as protein, affects its absorption and utilization, and therefore influences its requirement. According to Underwood (1966, 1971) the basic requirement for copper is one at which all the factors affecting its utilization are at optimal levels. Responses of pigs to high copper intakes (500 to 750 ppm) have been shown to be influenced by protein sources (Combs et aZ., 1966; Suttle 42 and Mills, 1966a; Young et aZ., 1970; Parris and McDonald, 1968), and dietary protein levels (Combs et aZ., 1966; Wallace et aZ., 1960). Suttle and Mills (1966a) conducted studies using soybean meal, dried skim milk and white-fish meal as protein sources and suggested that the influence of protein source is probably due to differences in calcium content of the various proteins. 2. Sheep_and Cattle Dick (1954) estimated that the copper requirement of sheep was 1.0 mg copper per day or less. As in pigs, the interaction between copper and other minerals influences the amount of copper required by ruminants. In South Australia, sheep grazing pastures containing 3 ppm copper and growing on calcareous soils were reported to develop signs of copper deficiency (Marston et aZ., 1948a, 1948b). Administration of 8 ppm copper prevented copper deficiency but was insufficient for adequate wool keratinization and maintenance of normal blood copper levels. Intakes of high concentrations of calcium carbonate and moderate molybdenum and sulfate by wool-sheep place their copper requirement at 10 ppm; the minimum for cattle and cross bred sheep is 4 ppm; and for Merino sheep 6 ppm (Underwood, 1971). High intakes of sulfate and molybdenum at 0.5 mg per day affect retention of copper in wool-sheep (Dick, 1954). Based on the effects of interaction among molybdenum, sulfate, calcium and copper, and the findings of several workers (Spais et aZ., 1968; Allcroft and Parker, 1949; Hunter et aZ., 1945; Innes and Shearer, 1940), it is difficult to specifically define the copper requirements of ruminants. In Australia, field studies showed that application of 5 to 7 kg of copper sulfate per hectare to pasture in- creases its copper content and alleviates copper deficiency (Underwood, 43 1971). 3. Other Species In rats fed milk diets fortified with iron and manganese, Schultze and coworkers (1934) found that supplements of 0.01 to 0.05 mg copper per day were optimal for promoting growth and synthesis of hemo— globin. Copper deficiency has been produced in rats by several researchers using diets which contained less than 1 ppm copper. Studies by Mills and Murray (1960) established the minimum copper requirements for 70 g rats as 1 ppm for hemoglobin formation, 3 ppm for growth, and 10 ppm for maintenance of normal melanin concentration in hair; reproduction and lactation would be maintained by 50 ppm. Everson and associates (1967) kept female guinea pigs on diets containing 0.5 to 0.7 ppm copper and reported normal reproduction when compared to control females on 6 ppm copper. However, anemia and hair depigmentation later developed and growth of young was adversely depressed. Poultry diets containing 4 to 5 ppm copper are generally sufficient, and all normal poultry rations contain more than 5 ppm copper (Underwood, 1971). EXPERIMENTAL PROCEDURES A. Introduction Three trials involving 52 baby pigs were conducted to determine the minimum copper requirement of baby pigs on semi-purified diets. Baby pigs were obtained from Yorkshire, Hampshire and Yorkshire-Hampshire crossbred herds from the Michigan State University Swine Research Farm, where the experiments were performed. High-protein casein was used as the protein source and mineral and vitamin premixes were made from re- agent grade compounds. Energy sources included glucose and fat. The room temperature was initially 30°C but was lowered to 25°C later in the course of the trials. All diets were stored at a temperature of 4°C. 8. Experiments 1. Experiment 1 Sixteen 7-day-old baby pigs were taken from sows on low-copper gestation and lactation diets (3.6 ppm copper, Table A-2) and raised individually in stainless steel cages. They were adjusted to the basal purified diet for 4 days during which period they were fed twice daily and given water ad Zibitum. At the end of the adjustment period the pigs were randomly alloted to four experimental diets as shown in Table 1. The diets were supplemented with anhydrous copper sulfate resulting in the following dietary copper levels: diet 1 (basal), 1.3 ppm; diet 2, 3.2 ppm; diet 3, 5.6 ppm; diet 4, 9.3 ppm. 44 TABLE 1. COMPOSITION OF DIETS USED IN EXPERIMENT 1 45 Ingredient l 2 3 4 Caseina 2100 2100 2100 2100 Cereloseb 3500 3360 3220 2940 a-Cellulosec 350 350 350 350 Mineral premixd 420 420 420 420 Larde 350 350 350 350 Fat soluble vitaminsf 70 70 70 70 Water soluble vitaminsf 140 140 140 140 Corn 0113 70 70 7o 70 Copper premixh _____Q _1_13_g __2_8_0_ _§_§_(_)_ 7000 7000 7000 7000 Copper COHC- (analyzed, ppm)1 1.3 3.2 5.6 9.3 aHigh protein casein, General Biochemicals, Chagrin Falls, Ohio. bDextrose 2001, CPC International, Englewood Cliffs, New Jersey. cSolka-Floc, Brown Co., Berlin, New Hampshire. dPremix made from reagent grade compounds (See Table A-4). eArmour Co., Detroit, Michigan. fSee Table A—4. gMazola, Best Foods, Englewood Cliffs, New Jersey. hCopper premix in cerelose containing 100 ppm Cu from anhydrous CuSO . iAs fed basis. 46 Initial weights of pigs were taken and blood parameters including plasma copper levels were determined at this time. Subsequently they were weighed and blood samples taken bi-weekly from the anterior vena cava for hemoglobin, hematocrit, ceruloplasmin and plasma copper deter- minations. All feed was weighed each time and individual feed consump- tion recorded. Some pigs suffered from diarrhea, but addition of neo- mycin to the drinking water brought it under control. Three pigs died of undetermined causes, one from each of treatments 2, 3 and 4. After 6 weeks a copper balance trial was conducted. Pigs were adjusted to being fed semi-liquid diets (feed was mixed with water to make a slurry) and to being reared in stainless steel metabolism cages and also to the frequent handling necessitated by transfer to and from the feeding cages for 3 days prior to a 5-day collection of feces and urine. During each feeding, pigs were removed from the metabolism (collection) cages and placed in the feeding cages. Their snouts were wiped clean after feeding to avoid contamination of feces and urine with feed. A fine wire screen was used to separate feces from urine, and the urine was collected in polyethylene buckets (acidified with 6N HCl) through a funnel stuffed with glass wool. Throughout the collection period, feed intake was kept constant and unconsumed feed was collected, air-dried and weighed. For each pig total feces output was oven-dried at 50°C for 48 hours, weighed, ground and stored in airtight polyethylene sacs, and total urine output was recorded, sampled and stored in acid washed polyethylene bottles at 4°C. At the conclusion of the trial, all pigs were killed by exsangu- ation after anesthesia using intravenous injection of sodium pentabar- bital. Gross and microscopic pathological observations were made. 47 Various tissues, organs and glands were removed and weighed. They were then placed in polyethylene sacs and frozen immediately over dry ice, and later stored at -2000 until used for analysis. Hair samples were removed from the loin of each pig and the 7th rib was dissected out at the costochondral junction, fixed in formalin and submitted for histo- pathological examination. 2. Experiment 2 Fifteen 3-day-old baby pigs from sows on low-copper (3.6 ppm copper) gestation and lactation diets were adjusted to a basal semi- purified diet for 9 days in stainless steel cages. The casein used in this basal diet was washed with Na EDTA to remove as much of the copper 2 as possible. After adjustment the pigs were allotted to 3 experimental diets (Table 2) with copper levels as follows: diet 1 (basal), 0.6 ppm; diet 2, 1.9 ppm; diet 3, 2.8 ppm. Six pigs died, two from each of the treatments. A balance trial was conducted after 28 days, and at the end of the trials the pigs were killed, postmortem examinations performed and tissues, organs and glands collected, weighed and stored at -20°C. The tarsal and metatarsal bones were submitted for histopathological examination. 3 . Experiment 3 The same procedure used in the previous trials was followed except: a. Twenty week-old baby pigs were assigned to 4 dietary treatments analyzing 0.9 ppm; 2.0 ppm; 4.0 ppm and 4.9 ppm copper (Table 3). b. Pigs were group fed and distilled deionized water was provided 48 TABLE 2. COMPOSITION OF DIETS USED IN EXPERIMENT 2 Ingredient 1 2 3 Caseina 2100a 2100 2100 Cerelose 3300 3265 3230 a-Cellulose 350 350 350 Cu-free mineral premixb 420 420 420 Lard 350 350 350 Fat soluble vitamin premixC 70 70 70 Water soluble vitamin premixd 140 140 140 Corn oil 70 70 70 Copper premixe o 35 7o Selenium premixf 100 100 100 Chromium premixg _199_ ‘_199 __100 7000 7000 7000 Copper conc. (analyzed, ppm) 0.6 1.9 2.8 aCasein treated with NaZEDTA. bSee Appendix, A, Table A-3. c’dSee Appendix A, Table A-4. eCopper premix in cerelose containing 100 ppm Cu from anhydrous CuSO4. fSelenium premix in cerelose containing 7 ppm Selenium from NZSeO3' gChromium premix in cerelose containing 70 ppm Chromium from CrC13°6H20. 49 TABLE 3. COMPOSITION OF DIETS USED IN EXPERIMENT 3 laggedient l 2 3 4 Casein 2100 2100 2100 2100 Cerelose 3300 3230 3090 2950 u-Cellulose 350 350 350 350 Cu-free mineral premix8 420 420 420 420 Lard 350 350 350 350 Fat soluble vitamin premixb 70 70 70 70 Water soluble vitamin premixc 140 140 140 140 Corn oil 70 70 70 70 Copper premixd 0 70 210 350 Selenium premixe - 100 100 100 100 Chromium premixf _199_ _199_ _199' ._199 7000 7000 7000 7000 Copper conc. (analyzed, ppm) 0.9 2.0 4.0 4.9 8See Appendix A, Table A-3. b’cSee Appendix A, Table A—4. dCopper premix in cerelose containing 100 ppm Cu from anhydrous CuSOA. eSelenium premix in cerelose containing 7 ppm selenium from NaZSeOB. fChromium premix in cerelose containing 70 ppm chromium from CrC13°6H20. 50 as drinking water. c. Casein was used untreated with NaZEDTA. d. A balance trial was conducted after 65 days for 5 days following 4 days of adjustment. C. Analytical Methods 1. Hematology a. Hemoglobin. Determination of hemoglobin was by the cyanmet- hemoglobin method of Crosby et al. (1954). Using a dry, clean and cali- brated Sahli hemoglobin pipette, 0.02 ml whole blood was measured into 5 ml Drabkin's solution.1 The blood and solution were mixed thoroughly on a Vortex-Genie2 device and allowed to stand for 10 minutes at room temperature to allow formation of cyanmethemoglobin. Optical density (GB) of the sample was determined on a Coleman Junior 11 spectrophoto- meter set at a wavelength of 540 nanometers. Hemoglobin concentration was calculated as follows: (OD540) x (Standard hemoglobin factor of Drabkin's solution) = hemoglobin concentration in g / 100 ml. b. Hematocrit. Hematocrit was determined using the method of McGovern et al. (1955). A capillary tube (75 mm long x 2 mm diameter) 1Drabkin's solution was made up by dissolving 1.0 g sodium bicarbonate (NaHCOB), 0.05 g potassium cyanide (KCN) and 0.2 g potassium ferricyanide (K3Fe [CN]6) in deionized distilled water and diluting it to 1 liter. 2Scientific Industries, Inc., Springfield, Mass. 51 containing dried heparin was filled by capillary attraction with blood to within 1 or 2 cm of the end. The unfilled end of the tube was heat- sealed over a micro-burner gas flame for 2 to 4 seconds. Constant rotation of the tube was maintained to ensure a flat inner base of the tube. After sealing, the tube was identified and placed with the sealed end in contact with the peripheral rim of the centrifuge headl. The flat removable centrifuge head cover was then screwed in place, the auto— matic timer set for 5 minutes and the motor turned on. At the end of 5 minutes of centrifugation at 10,000 rpm, the motor was automatically turned off and the hematocrit read promptly. Hematocrit was expressed as percentage of whole blood. c. Centrifugation. About 10 to 12 ml of blood were withdrawn from the pig's vena cava and immediately placed in a heparinized and acid-washed centrifuge tube. The tube was then shaken thoroughly to mix the blood with heparin and to prevent clotting. The blood sample was next centrifuged at 2000 x G for 12 minutes. Cell-free plasma was care— fully decanted, using disposable pipettes, into acid-washed vials. About 3 ml were kept on ice and used immediately for determination of ceruloplasmin, and the rest was stored at -20°C for subsequent mineral analysis. The red cells in experiment 3 were stored at 4°C for assay of superoxide dismutase activity. 2. Physical Determinations a. Treatment of casein. In order to produce very lowbcopper casein for use in the basal diets, as much copper as possible was 1International Hemacrit Centrifuge, International Equipment Company, Boston, Massachusetts. 52 removed from high-protein casein using a modified method of Shanklin at al. (1968). High-protein casein was treated by suspending it in deionized distilled water (120 g casein per liter of water). The slurry was mechanically stirred in a polyethylene container while in a water-bath at 50°C. The pH of the slurry was adjusted to 4.6, the isoelectric point of casein, with 0.1 N hydrochloric acid. Disodium ethylenediamine- tetraacetate (NaZEDTA) was added at a level of 0.5% of the protein (0.52 g per 120 g casein), and the mixture was stirred for 45 minutes. The casein-NaZEDTA mixture was then allowed to settle and the supernatant siphoned off. The casein was resuspended in deionized, distilled water and treated again with Na2EDTA as previously described. This procedure was repeated a total of 5 times for each batch of casein. Disodium EDTA was removed from the casein by repeated washing with deionized distilled water until the absence of EDTA in the supernatant was indicated by formation of calcium oxalate precipitate when a drop of a saturated solution of calcium chloride was added to 10 m1 of supernatant which had been combined previously with 5 ml of an ammonium oxalate solution and adjusted to pH 11.0 with sodium hydroxide solution. Finally as much water as possible was removed from the casein by filtering, after which the casein was dried in an oven at 50°C and finely ground before use in the purified diets. Analysis of treated and untreated casein and the diets used in Experiment 2 yielded the copper concentration values presented in Table 4. b. Bone mechanics. Left femurs were removed from pig carcasses and cleaned of all muscle, connective tissue and periosteum and stored in air-tight polyethylene sacs at -20°C. Before use, the bones were 53 TABLE 4. AVERAGE COPPER CONTENT OF TREATED AND UNTREATED CASEIN AND DIETS 1 Casein or diet Copper (ppm) Untreated high—protein casein 1.6 NazEDTA treated high—protein casein 0.3 Untreated high-protein casein diet 1.3 NazEDTA treated high-protein casein diet 0.6 thawed at room temperature. Strength characteristics--bending moment, moment of inertia, maximal load, maximal stress, and Young's modulus of e1asticity--were determined using an Instron Testing Instrument, Model TT CMLZ. The instrument was equipped with an FM-compression load cell having 250 kg full scale. Cross-head speed was 0.5 cm per minute and chart speed 1.0 cm per minute. A broad, flat porcelain plate was used as a base for the two fulcra supporting the femur. Deflection and maxi- mum load were recorded automatically on the chart. In calculating strength characteristics of the femurs the formulas of Miller et al. (1962) were used and are as follows: Maximal bending moment, M Wl/4 n/64 (303 - bd3) Moment of inertia, I Maximal stress, S MD/ZI Young's modulus of elasticity, E W13/481Y 1As fed basis. 2Instron Engineering Corporation, Canton, Massachusetts. 54 where 2 ll maximal load (kg) 1 = distance between fulcra (cm) B = outer horizontal diameter (cm) b = inner horizontal diameter (cm) D = outer vertical diameter (cm) d = inner vertical diameter (cm) Y = deflection at center of bone when load W is applied (cm) 3. Chemical Analyses a. Plasma copper. Plasma stored at -20°C was thawed at room temperature and diluted 1:6 with deionized distilled water. Using arti- ficially prepared serum as standardsl, copper concentration was deter- mined with the aid of an atomic absorption spectrophotometer at a wave- length of 324.7 nm. Plasma copper was expressed in micrograms per 100 ml. b. Tissue cqpper. Slices were made from frozen tissues and organs without thawing. Using a Polytronz, homogenates were prepared by blending slices 1:2 with deionized distilled water while placing ice 1Artificial serum standards were prepared to contain the following: Std. l: 2000 ppm Na, 100 ppm K, 1.0 ppm Cu, 0.5 ppm Zn, 50 ppm Ca, 20 ppm Mg, 25 ppm P, and 1.0 ppm Fe. Std. 2: 3000 ppm Na, 200 ppm K, 1.5 ppm Cu, 1.0 ppm Zn, 100 ppm Ca, 40 ppm Mg, 50 ppm P, and 2.0 ppm Fe. Std. 3: 4000 ppm Na, 300 ppm.K, 2.0 ppm Cu, 1.5 ppm Zn, 150 ppm Ca, 60 ppm Mg, 75 ppm P, and 3.0 ppm Fe. 2Brinkmann Instruments, Westbury, New York. 55 around the base of the blender beaker. The homogenates were weighed into 250 ml Phillips beakers in duplicate and wet ashed. Digestion con- sisted of the addition of 60 ml concentrated (12N) nitric acid to the homogenates followed by heating on a hot plate until almost dry. Upon cooling, 7 ml of 72% perchloric acid were added to each beaker which was then covered by a watch glass to prevent excessive evaporation while on the hot plate. Oxidation was continued until the reaction was completed. The watch glass was removed when dense white fumes appeared. Heating was continued until the volume was reduced to 2 to 3 ml. After cooling, each solution was made up to volume by addition of deionized distilled water. Standards were prepared using the same procedure. Copper content of samples was determined by atomic absorption spectrophotometry at a wavelength of 324.7 nm and expressed as parts per million. c. Feed and feces. Samples of feed and feces were finely ground in a Wiley milll, weighed into tared acid-washed 250 ml Phillips beakers in duplicate and digested using the wet ashing procedure already described for tissues. Analysis was done by atomic absorption spectro- photometry and copper concentration was expressed as parts per million (ppm). d. Urine. Urine samples previously stored at 4°C were poured into beakers and shaken thoroughly for homogeneity. Ten milliliters of each sample were pipetted into 250 m1 Phillips beakers in duplicate and digested according to the wet ashing procedure. Digested samples were 1Arthur H. Thomas Co., Philadelphia, Pennsylvania. 56 diluted to volume with deionized distilled water and copper content was determined by atomic absorption spectrophotometry and expressed as ppm. e. flair. Samples of hair were soaked in deionized distilled water in 200 m1 beakers for 45 minutes and drained on filter papers. They were then immersed in 95% ethanol for 45 minutes in order to remove adhering debris and foreign materials. Upon removal from ethanol the samples were air-dried at a temperature of 50°C and subjected to wet ashing. Copper content was determined by atomic absorption spectrophoto- metry and expressed as ppm. f. Bone ash. Broken left femur samples from 2b above were cut longitudinally and laterally into approximately 8 pieces with a band saw. The pieces were weighed on tared filter paper, identified with pencil on a tag, and bone and tag were wrapped in cheesecloth. The wrapped pieces of bone were placed in a Soxhlet extractor and extracted with absolute ethanol for 24 hours1 to remove water, and with anhydrous diethyl ether for 24 hours2 to remove fat. The wrapped bones were removed, placed on a covered tray and allowed to dry in a hood until odor free. They were then placed in porcelain crucibles, oven dried, and weighed before being ashed in a muffle furnace at 600°C for 18 hours. Upon cooling the ash was weighed and percent ash was calculated as follows: Weight of ash Weight of dry fat free bone z ash on fat free basis - 1Alcohol was poured over bones until extractor was filled 1% to 2 times; 20 amp rheostat was set at 65. 2Anhydrous diethyl ether was poured as for alcohol; 20 amp rheo- stat was set at 35. 57 3. Bone copper. Ash from f above was finely ground using mortar and pestle, and approximately 300 mg of the well mixed powdered ash was dissolved in 5 ml of 6N hydrochloric acid. Two milliliter aliquots of the resulting ash solution were diluted 1:3 with deionized distilled water and copper content was determined by atomic absorption spectropho- tometry at a wavelength of 324.7 nm and expressed as ppm on a dry fat free basis. 4. Enzymolggy a. Plasma ceruloplasmin. Plasma ceruloplasmin activity was assayed on freshly obtained plasma using a modified method of Smith and Wright (1968). Buffer solution was prepared by dissolving 32.816 g sodium acetate and 1.55 g disodium ethylenediaminetetraacetate in de- ionized distilled water and diluting to 500 ml. The substrate, p-phenyl- enediamine dihydrochloride (PPD) was used without further purification. The working solution had a pH of 5.4 and was adjusted to a final pH of the reaction mixture of 6.3. Buffer and plasma were maintained at a temperature of 37°C in a water-bath before using, but the substrate (PPD) was kept at room temperature to minimize autooxidation. To 2.4 ml buffer and 0.1 m1 substrate, 0.5 ml plasma was added and mixed on a Vortex-Genie device. The mixture was incubated at 37°C for 5 minutes. After incu- bation 1 m1 of 0.3 mM sodium hydroxide solution, freshly prepared and kept at 0°C, was added to stop the enzyme reaction. The final mixture was thoroughly shaken and transferred into a calorimeter cell (l-cm). Optical density of the sample was read at a wavelength of 540 nm, and ceruloplasmin activity was expressed in terms of optical density per minute. Blanks were also measured for optical density, i.e., autooxida- tion in the absence of plasma enzyme, and actual enzyme activity 58 determined as follows: Ceruloplasmin activity = C j _ E in ODSQO/min ODSQO Of sample ODSQO Of Blank b. Brain and erythrocyte superoxide dismutase. A modified method of McCord and Fridovich (1969) was used to assay for superoxide dismutase activity in brain tissue and red blood cells as follows: (1) Buffer. This consisted of 250 m1 of 1.5 M KHZPO4 and 973 ml of 1.5 M K HPO and 1.12 g of Na EDTA adjusted to a pH of 7.8. 2 4 2 (2) Xanthine. This solution was made by dissolving 130.5 mg sodium salt of xanthine and 16.7 mg NaCN in deionized distilled water and diluting to 500 ml. (3) ggytochrome c. In 10 ml deionized distilled water, 37.2 mg ferricytochrome c (Sigma type III or equivalent) were dissolved and the solution stored in the dark. (4) Xanthine oxidase. About 0.1 ml xanthine oxidase and 10 mg catalase were dissolved in 10 ml deionized distilled water and adjusted to give 0.250 A change per 10 minutes. (5) Sample preparation. (a) Red blood cells. About 0.1 ml of washed red blood cells was agitated in 1.0 m1 deionized distilled water. (b) Brain tissue. About 1.0 g tissue was homogenized in 10 ml deionized distilled water and the homogenate was spun at about 20,000 x G for 15 minutes. The supernatant was diluted 1:7 with deion- ized distilled water. 59 (6) Assay. A sample and control cuvette were analyzed simultaneously at 550 nm and 25°C. The control cuvette contained 2.6 ml deionized distilled water, 0.1 ml buffer solution, 0.1 m1 cytochrome c solution, 0.1 ml xanthine solution; the sample cuvette contained 2.5 m1 deionized distilled water, 0.1 ml buffer solution, 0.1 m1 cytochrome c 0.1 ml xanthine solution and 0.1 ml sample. Initial absorbances were set and 0.05 ml xanthine oxidase solution rapidly added to control and sample cuvettes, mixed thoroughly and the reaction followed for 5 to 10 minutes. The recorder was set at 0.5 A full scale and travelled 0.2 in/min. The initial slopes of the resulting curves were used to calcu- late the change in Absorbance/lO minutes. The quantity of superoxide dismutase which gives 50% inhibition of the blank rate is defined as one unit. Sample rate Control rate 100 Z Inhibition - 1 - Units of Superoxide dismutase activity = Z Inhibition/ 50‘ Inhibition/ Unit Superoxide dismutase activity was calculated per g protein for brain tissue and per ml red blood cells for blood. D. Statistical Analyses The data from Experiments 1, 2 and 3 were subjected to a one-way analysis of variance using the Unequal-l format on a CDC1 6500 computer at the Michigan State University Computer Laboratory. Simple correlations were also calculated and the levels of significance of differences be- tween means were determined using the Bonferroni t-statistics test with 1Control Data Corporation, Minneapolis, Minnesota. 60 the following formula: Y-Y tB - l 2 .1 MSE(-1—+l) r1 r2 where §i = mean for treatment 1 T' = mean for treatment 2 M5 = error mean square r = number of observations in treatment 1 r = number of observations in treatment 2 RESULTS AND DISCUSSION A. (Experiment 1: Copper requirement of baby pigs on semi-purified diets supplemented with varying levels of copper to yield upon analysis: 1.3 ppm, 3.2 ppm, 5.6 ppm and 9.3 ppm copper Data for growth and food utilization are presented in Table 5. Feed intake, average daily gain and feed per gain were not significantly affected by dietary copper levels. Weight gain of pigs was at a normal rate. Ullrey and associates (1960) conducted baby pig studies with 6, l6 and 106 ppm copper and Gipp et al. (1973) with 2, 10 and 250 ppm copper and reported that average daily gain and feed per unit of gain were not significantly affected by treatment. Three weeks into the trial, two of the pigs on the basal diet exhibited signs of nervousness and developed leg problems and the inability to walk forward. These signs, however, disappeared before the fifth week. Similar observations were made by Teague and Carpenter (1951) and by Lahey et a1. (1952) but these workers reported that the leg problems persisted through the trial. Hematocrit, hemoglobin and mean corpuscular hemoglobin concentration values are summarized in Table 6. Although hematocrit values increased in the second, fourth and eighth weeks, except for values in diet one, and dropped in the sixth week (Figure 1.1), there was no consistent effect due to treatment. Hemoglobin, like the hematocrit, did not show any trend caused by dietary copper levels (Figure 1.2); there was 61 62 TABLE 5. THE EFFECT OF DIETARY COPPER LEVELS ON GROWTH (EXPT. l) Diet no. 1 2 3 4 Cu conc., ppm1 1.3 3.2 5.6 9.3 No. of pigs 4 3 3 3 Avg. init. wt., kg 1.28:0.09 1.20:0.11 1.42:0.11 1.23:0.11 Avg. final wt., kg 12.89i0.89 12.8211.02 13.4211.02 11.69i1.02 Avg. daily gain, g 276:0.01 282:0.02 273:0.02 269:0.02 Feed/gain 1.28 1.30 1.25 1.31 1Expressed on as fed basis. 63 TABLE 6. THE EFFECT OF DIETARY COPPER LEVELS ON HEMATOCRIT, HEMOGLOBIN AND MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (EXPT. 1) Diet no. 1 2 3 4 Cu conc., ppm 1.3 3.2 5.6 9.3 No. of pigs 4 3 3 3 l Hct. , Z . Initial 24.9il.63 30.6:2.31 30.211.89 30.5il.89 2 weeks 41.0:2.68 39.7:3.09 38.9:3.09 34.9:3.09 4 weeks 37.0il.38 40.3il.59 40.5il.59 36.7il.59 6 weeks 34.6il.97 36.7:2.28 35.412.28 34.2:2.28 8 weeks 38.0tl.lS 42.4:1.15 40.5il.15 39.011.40 HbELg/loo ml Initial 6.9i0.53 8.5:0.6l 8.8:0.6l 8.5:0.6l 2 weeks 12.6:0.60 12.3:0.70 12.510.70b 10.8i0.70d 1. weeks 11.422032 13.110.373 13.8i0.37 'c 12.020.37 6 weeks ll.3i0.70 ll.7i0.81 l3.0i0.81 12.8i0.81 8 weeks ll.li0.60 13.610.60 12.6i0.60 12.9i0.73 3 MCHC 2 2 Initial 27.7i0.59 27.3:0.83 29.410.68 27.6:0.68 2 weeks 20.812.22 30.912.56 32.7:2.56 31.6:2.56 4 weeks 31.1il.63 32.7il.89 34.2il.89 32.811.89 6 weeks 41.612.22 32.0:2.57 36.9:2.57 38.0:2.57 8 weeks 29.4il.43 32.2il.43 31.1il.43 32.9il.75 aSignificantly greater than least value (P<0.05). b c,d l 2Hemoglobin. Hematocrit. Significantly different (P<0.05). 3Mean corpuscular hemoglobin concentration. Significantly greater than least value (P<0.01). 64 Figure 1.1. Influence of dietary copper levels on hematocrit (mmt.l). 65 .H.H ..amfim v.85 3.55233 :0 £60 on we mm 3 q d - So can mg v 55 30 ES mg m 35 ..... \ 30 ES Nam 65 ll :6 5% ma: _ 85 Ii. . is mm 0? n? 9;, ‘moogowaH 66 Figure 1.2. Influence of copper intake on hemoglobin (Expt. 1). 67 .~.H ..awaa £20 .0288th3 co goo mm ms mm v. 0 so 58 09320 s so 53 m9 905 ..-..- - . :6 ES momma ll \ .m 30 can 0: _ 85 ll . Iw oovfi ‘wqonbowaH 68 however, a slight rise in the second and third weeks. In the fourth week hemoglobin values of pigs on the second and third treatments were significantly (P<0.05 and P<0.01, respectively) greater than correspond- ing values under the first treatment and there was a significant (P<0.05) difference between treatments three and four. Mean corpuscular hemoglobin concentration rose steadily after the second week and then dropped in the eighth week except for the second treatment (Figure 1.3). These observations are in agreement with the results of Ullrey and co- workers (1960) who reported that baby pig hematology was not signifi- cantly affected by 6, l6 and 106 ppm dietary copper. Gipp and associ- ates (1973) observed no significant effects on hematocrit, hemoglobin and mean corpuscular hemoglobin concentration when young pigs were fed 2 ppm dietary copper. Ceruloplasmin activity and plasma copper concentrations are shown in Table 7. Ceruloplasmin activity of pigs on the basal diet was significantly (P<0.01) depressed, dropping from an initial value of 0.17 OD/min to 0.02 OD/min in the sixth week and rising to 0.05 by the end of the trial (Figure 1.4). There was, however, no significant reduction within the rest of the treatments. Pigs on the basal diet exhibited ceruloplasmin values which were significantly (P<0.01) lower than corresponding values in treatments 2, 3 and 4, but there was no signif- icant difference between treatments 2, 3 and 4. Identical results were reported by Gipp and coworkers (1973) who observed a significant (P<0.01) reduction in ceruloplasmin of pigs kept on diets containing 2 ppm copper. Williams and associates (1975) reported significantly (P<0.01) depressed ceruloplasmin activity down to 12 of control in pigs fed lowbcopper diets. Plasma copper concentrations were significantly 69 TABLE 7. THE EFFECT OF DIETARY COPPER LEVELS ON CERULOPLASMIN ACTIVITY AND PLASMA COPPER CONCENTRATION (EXPT. 1) Diet no. 1 2 3 4 Cu conc., ppm 1.3 3.2 5.6 9.3 No. of pigs 4 3 3 3 1 pp. , OD/min 2 weeks 0 17:0.04 0.22:0.05b 0.42:0.05: 0.23:0.05b 4 weeks 0 03s0.02 0.20:0.03b 0.26:0.03b 0.21:0.03b 6 weeks 0.02:0.02 0.21:0.03b 0.23:0.03b 0.24:0.03b 8 weeks 0 05:0.01 0.28:0.01 0.23:0.01 0.24:0.02 P1. Cu2, meg/100 ml Initial 19.914.83 27.1:5.58 25.515.583 34.215.58b 2 weeks 49.2:20.9 134.4:24.2b d 164.7:24.2b c 19:34:24.2b a weeks 55.4:16.3 202.6:18.8b’ 256.2:18.8b’ 198.6:18.8b’c 6 weeks 51.316.78 188.317.83b 228.5:7.83b 201.6:7.83b 8 weeks 41.4:15.4 189.0:15.4 191.3:15.4 199.3:18.8 aSignificantly greater than least value (P<0.05). b c,d g 1Ceruloplasmin activity. 2Plasma copper concentration. Significantly different (P<0.05). Significantly greater than least value (P<0.01). 70 Figure 1.3. Influence of dietary copper levels on mean corpuscular hemoglobin concentration (Expt. 1). 71 .m.H anomam 220 BEmEtmaxm :0 £69 mm Nw q d I I I ’I o I \ I ’ \ I I \\ -\- ‘ I \ \\ Li I \ \ \\\\\ ‘ mm v. 0 ‘\ ~\ \ :6 see 09 v65 :6 see 0.9 m 85 ..... Go 58 NE N 85 ll: :6 58 m: _ $5 l..l.. “3 CU Om U) N) «x, "ouoo ugqmbowaH JD|nOSDdJOO uoaw 0 <1' U7 Sf 72 .40- .35 r- .E E \ i- O .30 0 >2 1?: .25- .2 '23 < .5 .20- g 0\ —-— Diet I (l.3 ppm Cu) 2 '5 —— DietZ (3.2 ppm Cu) 8- ' h ‘ ----- Diet3 (5.6 ppm Cu) 'é \‘ Diet4 (9.3 ppm Cu) 8 IO- \ .05- ‘ '/o L ‘/ -‘._/ '000 I4 is 42 56 Days an Experimental Diets Figure 1.4. Influence of copper intake on ceruloplasmin (Expt. 1). 73 (P<0.01) increased by treatments 2 and 3 containing 3.2 and 5.6 ppm copper, respectively, when compared to treatment 1 up to the fourth week; there was a decline thereafter (Figure 1.5). Plasma copper of pigs on diet 4 reached a peak at the sixth week and subsequently declined slightly. There was a significant (P<0.01) difference between plasma copper levels of pigs on diet 1 and pigs on diets 2, 3 and 4, but no significant difference between plasma copper values of pigs on diets 2, 3 and 4. Depressed plasma copper concentrations in pigs have been reported by Gipp et al. (1973) and Williams et al. (1975) who showed that law dietary copper (2 ppm and less) significantly (P<0.01) reduced plasma copper. Dreosti and Quicke (1968) provided evidence to indicate that plasma copper was a sensitive index of copper status. Tissue copper concentrations and brain superoxide dismutase activity are presented in Table 8. Liver, kidney and heart copper values were not significantly influenced by dietary copper. This is at variance with previous results which established that dietary copper strongly influences the levels of copper in the liver (Schultze et aZ., 1936a; Lahey at aZ., 1952; Wintrobe et aZ., 1953; Dempsey et aZ., 1958; Ullrey et aZ., 1960). Spleen copper showed a significant (P<0.05) difference between treatment 1 and treatments 2 and 4. Brain copper concentration of pigs on the basal diet was significantly (P<0.05) lower than that of pigs on diet 4, but not on diets 2 and 3; brain copper of pigs on diets 3 and 4 were significantly (P<0.05) different. There was a significant (P<0.01) difference in hair copper between treatment 1 and treatments 2 and 3, and between treatment 1 and treat- ment 4 (P<0.05). Brain superoxide dismutase activity did not exhibit any significant treatment effects, an observation not in keeping with to 74 260- ’A“ 240 “x‘ ‘\ 220 \\ E 200 be\ C) l/’ “-..'____ ____ 9 I80 \ O ‘5’ ISO 33’ (1 I40 a . o —-— Diet | (L3 ppm Cu) 0 '20 —— Diet 2(32 ppm Cu) O —---- Diet 3(5.6 ppm Cu) g IOO Diet4(9.3 ppm Cu) 2 ‘1 so 60 . _ ~-~a\~\ 4O ". 204 I00 I4 28 42 56 Days an Experimental Diets Figure 1.5. Influence of dietary copper levels on plasma copper concentrations (Expt. 1). 75 TABLE 8. THE EFFECT OF DIETARY COPPER LEVELS ON TISSUE COPPER CONCENTRATIONS AND BRAIN SUPEROXIDE DISMUTASE ACTIVITY (EXPT. 1) Diet no. 1 2 3 4 Cu conc., ppm 1.3 3.2 5.6 9.3 No. of pigs 4 3 3 3 1 Tissue Cu, ppm Liver 69.1:17.04 73.2:19.67 52.6:19.67 104.9il9.67 Kidney 64.8:9.55 46.7ill.03d 64.1ill.03 57.2ill.03d Spleen 60.919.57 93.1ill.OS 76.2:ll.05 88.8ill.05 Heart 22.9:10.4é 24.3:12.06 36.5:12.03 20.3:12.0g Brain 6.1:2.86 8.83:3.30c 8.3::3.30c 22.3:3.30d Hair 10.2iO.65 14.5i0.76 15.0i0.76 13.8i0.76 2 Brain SOD , units/m8 protein 3.3:0.78 4.9:o.90 4.8:0.90 3.310.90 1Expressed on dry basis. a cSignificantly greater than least value (P<0.01). d ,b Significantly greater than least value (P<0.05). 2Superoxide dismutase. Significantly different (P<0.05). 76 the results of Williams and associates (1975) who reported an 85% drop in hepatic and erythrocyte superoxide dismutase activity in copper deficient swine. Results of the copper balance trial are given in Table 9. Copper intake, by virtue of increasing dietary copper concentration, was significantly (P<0.01) different between diet 1 and diets 3 and 4. Fecal copper of pigs on diet 4 was significantly (P<0.01) greater than that of pigs on diet 1. There was a significant (P<0.05) difference in urinary copper as percent of copper intake between diet 1 and diet 4; urinary copper (Z) of pigs on diets 2 and 3 was greater, though not significantly, than corresponding values in diet 4. Absolute copper retention was influenced by dietary copper -- pigs on diets 3 and 4 retained significantly (P<0.05 and P<0.01, respectively) more copper than did pigs on diet 1. Percent copper retention was not significantly affected by treatments. Strength characteristics of the left femur estimated by physical and chemical measurements are summarized in Table 10. Pigs on the basal diet had significantly (P<0.05) greater femur vertical and horizontal diameters1 (D and B, respectively) than those of pigs on diet 3. Elas- ticity of femurs an treatments 2 and 3 was significantly (P<0.05) greater than elasticity values for treatment 4. Other physical charac- teristics, such as femur weight, maximum load and maximum stress, showed no significant treatment effects. Percent femur ash was not signifi- cantly different between treatments, but femur copper concentration of 1Measured at mid-shaft of femur with the lateral and medial condyles facing downwards. 77 TABLE 9. THE EFFECT OF DIETARY COPPER LEVELS ON COPPER BALANCE (EXPT. 1) Diet no. 1 2 3 4 SEM1 Cu conc., ppm 1.3 3.2 5.6 9.3 No. of pigs 3 3 3 3 Cu intake, mg/day 0.98 1.82 3.428 5.84b 0.25 Cu excretion, mg/day b Fecal 0.42 0.91 1.21 2.88 0.24 Urinary 0.08 0.09 0.11 0.09 0.02 Cu excretion, Z of intake Fecal 42.9 50.0 35.4 49.3 Urinary 7.7c 5.1 3.3 1.6 Cu retention, mg/day 0.48 0.82 2.10c 2.87a 0.37 Cu retention, Z of intake 49.5 44.9 61.4 49.1 5.94 1Standard error of the mean (t). aSignificantly greater than least two values (P<0.01). bSignificantly greater than least three values (P<0.01). cSignificantly greater than least value (P<0.05). 78 .moumsc3oc wofiomm maaawooo Hmuauma was Hugues sues ummnmluaa um consume: .ma>ow asp ou aflhvaoo Hmavaaiwwa asu Baum aocuuman H .mammn aauwiuom .huv so mammaunxmo n .Amo.ovmv aoHa> unwed saga haumauw hauamawmaawwmu was.on~.e as.one.s He.ons.m ~m.one.e ans .su mH.Hem.oe mH.Hnm.He mH.Hnm.~e ~o.aeo.ae N .ssnssoo sn< omuaumwuauawumno Hmufiaanu HH.~se.m nHH.~sH.eH nHH.~ns.eH Nw.Hns.oa «as\wx coca .eswossnnan so scenes: Ho.st0sm Ho.Hssesm Ho.Hsnmem es.~mnsme Nau\mx .nnowun asasxnz Ho.onwa.o Ho.ones.o Ho.onss.o Ho.oso~.o as .nsswsss so Osman: He.~Hem.Nm He.NHse.e~H He.~flwm.sea ms.oans.o~H asiwx . asses museums annexe: ea.mw~.se es.mns.sm ea.mnm.es me.eno.sm as .eon annexe: e~.onoe.e sm.onms.m s~.ono~.m m~.osms.m as .AAV semen; mo.on~e.a mo.ossm.H mo.onoe.a n~o.onee.~ av anaeswu> ~o.os~e.a No.0ssm.H No.onoe.H n~o.onme.H Ame stnonwwom Eu .Huauaaaav Hoauauxm em.mwe.~m em.mnm.mm eN.mwm.Hs Hm.~n~.as w .snnwe .usmesz mafiumwuauaoumno Hmawmmnm m m m e nwse so .oz m.m s.m ~.m m.H ans ..ssou no a m N H .ao omen as .amwa m32mm HhMQ mmH mo moHHmHMMHUmA mmmmcu wm< Qz< ZHZm< 92¢ zHZmaomoqommo .Mmmmoo <=m oBu umooa coco Houoouw mauaooamwowamo .Amo.ovmv ooao> uoooa onsu Houoouw hauoooamfiowam A .Aao.ovmv ooao> uoooa coco nouoauw mauoooamwawamo amN.eNAN.N0N omN.eNam.e0N sNN.NNam.NN NN.0NNN.0 axons 0N 800.0NAN.NNN 000.0Na0.N0N n0e.NNa0.00 00.0Naa.m names 0 00.0N 0N.00N 0.n0.N0N _ 80.0NN 0.Ne names 0 Ne.NN 0N.00N nN.00N sN.00N N.00 names e 00.0N 8N.0NN na.0NN sm.eNN N.0NN asses N N0.NH 0.00N e.N0 N.00N 0.00 NnNaNsH Na 00N\mwa .Nao .Nm U000.330 0000.0AN0.0 000.0a00.0 N00.0a00.0 axons 0N U000.0300 se00.0a00.0 000.0AN0.0 000.0AN0.0 names 0 N00.0 800.0 88.0 sm0.0 No.0 names 0 000.0 n00.0 n00.0 a00.0 No.0 names a 000.0 00.0 00.0 m0.0 00.0 names N 000.0 00.0 00.0 00.0 00.0 NnNstN sNa\00 4N.ao m m m 0 mass 00 .oz 0.0 0.0 0.N 0.0 one ..aaoa so 2mm 0 m N N .os sass Am .aexmv onHHHU< ZHZmma mmmmoo MMfiuoo aaaooaooaouooa .A00.0ve0 asnwoooae aNunnaamaszma.e A.Q.Hzoov 0N mqm I \ I . I \ 30 can 0.: 320 I so can 0.: m .20 ..... So can 0N: N .20 ll :6 can 0.9 _ 8.0 l-l / i. (H. {I ll 5. I!) Q ugw/ao ‘Kungiov UleDldOIDJGO 120 250 8 6: o IOO Plasma Copper conc., mag/IOO ml 50 r —-- Diet l (0.9 ppm Cu) \ —— Diet2(2.0 ppm Cu) ' '''' Diet3(4.0 ppm Cu; \ - Diet4(4.9 ppm Cu ‘\ ” . . . \‘— - -- 3 0 I4 28 42 56 70 Days an Experimental Diets Figure 3.5. Response of plasma copper levels to copper intake (Expt. 3). 121 influence tissue copper concentrations, especially of liver (Dempsey at aZ., 1958; Ullrey et aZ., 1960; Sutter et aZ., 1958). Data from copper balance trials are given in Table 22. There was a significant (P<0.01) difference in copper intake due to treatment. Absolute copper excretion and copper excretion as percent of intake were not significantly different between diets. Absolute copper reten- tion showed a significant (P<0.01) difference between diet 4 and diets l and 2 while copper retention as percent of intake did not differ significantly between treatments. Physical and chemical measurements of the left femur are summar- ized in Table 23. None of the characteristics measured showed any significant effects due to treatment. Rucker et al. (1969a) reported that in copper deficient chicks bane copper content was SOZ of controls; data obtained in this trial indicate that dietary copper had no influ- ence on bone ash and copper content, or that the low copper basal diet was not fed long enough to deplete bone copper stores. Table 24 shows correlations between copper balance, plasma copper, ceruloplasmin and average daily gain. Plasma copper levels after the second week are strongly and significantly (P<0.05 or P<0.01) correlated with ceruloplasmin activity. Copper intake and absolute copper retention were significantly (P<0.05 or P<0.01) correlated with plasma copper concentration. Average daily gain was significantly (P<0.01) correlated with initial ceruloplasmin, fecal copper and urinary copper, and negatively but significantly (P<0.05) correlated with per- cent copper retention. The correlation between copper intake and ceru- loplasmin on weeks 6, 8 and 10 was highly significant (P<0.01); and ceruloplasmin activity on weeks 6, 8 and 10 was significantly correlated 122 .Aao.ovmv mooao> noun» umooa coco noumouw mauooowmfiamwmo .Aao.ovmv mo=Ho> oau uoooa sonu Houoouw aauoooamfiowwm o .AHo.ovmV ooam> omooa saga Houoouw haoaoowmacmamo Nw.mNnN.oN Nw.maao.om NN.MHHN.N¢ No.0NHo.qm oxauaa mo N .ooauoouou :0 omN.onma.N mN.onoH.N mN.owom.o om.owcq.o Noo\wa .ooNuaouou =0 me.ano.e ac.aew.N me.aum.c mm.Hnm.c anodes: mm.NHHm.mN mm.NHHo.o< mm.NNHe.Nm mw.mana.flq Hooom oxouoa mo N .oouuouoxo :0 mo.onHH.o mo.onoo.o mo.onoo.o .mo.onmo.o Nassau: mH.onan.o mH.onmo.H ma.onmm.o mN.onoN.o Nooom hao\we .oofiuouoxo =0 000.0N0N.N poo.osmN.N noo.onmN.N No.0amN.o mne\ws .oxnoca no N m m N mwwo «0 .oz o.q 0.0 o.N m.o Eon ..oaoo :0 q n N N .oa unfin Am .eaxmv mozma Mummou wM 00.0 00.0 00.0 00.0 00.0 000 0000000000 80 .uouoaowo Hoauouxm 00.N0 0.00 0.00 0.00 0.00 N .00000 .000003 oofiumwuouoouono Hoowmmnm 0 0 0 0 000a 00 .oz m.0 0.0 o.N m.o Boo ..oooo :0 :00 0 m N 0 .oo gown 00 .00000 szmh Emma mmH mo mOHHmHMMHONH mmmmou MMdHMHn mo Hommmm mmfi .MN MAN< 02¢ 2H2m<420422m0 .mmmmoo <2mwg Nm. 00. mm. 00. NN. no. 0n. me. 00. N0. mm. 0N. oNN. omn. awn. mm. no. «N. 0mm. 0mm 000” 0000. won” noun 000. new. 0N0. me. 00. 0m. 0000. new. 000 new. 00 00 oN0. 0mm. 000. mm. NN.: NN. 000. 000. 0000. owe. me. 00.1 000. 000. cm. N0. 00.1 0m oN0. 0H0 «mo 00 one mm 0x003 0x003 0x003 mxoos axooa HaNuHoH 0x003 03003 oxoos 03003 03003 HoNuNcH 00 m 0 0 N 00 m 0 0 N lNufi>Nuom owamoamoaouou uommoo afimoam 00 .amxmv NB0>000< 2020000000000 020 000000 020000 .02000000200200 000000 000000 2003000 020000000000 .oqm mqm<8 127 demyelination of nerve fibers in the white matter. This spongiosis appeared in the white matter in various areas of the brain but was most prominent in the white matter of the cerebellum. Again this observation cannot be explained since incoordination and other nervous disorders were not grossly evident. The metatarsal of one pig on the basal diet demonstrated a severe change histologically - cartilaginous cells persisted throughout the marrow cavity and there was excess osteoid in the region of spicules; one pig on diet 3 and two on diet 4 showed very slight to slight changes. CONCLUSIONS In the light of the results obtained from these three experiments and within the limits of experimental error and the limits imposed by degree of adequacy and sensitivity of the parameters studied, the follow- ing conclusions have been drawn: 1. The levels of dietary copper used in these studies (0.6 to 9.3 ppm) did not significantly influence average daily gain and feed conver- sion efficiency, and hence did not significantly affect growth. 2. Although tissue copper concentrations were slightly affected by dietary copper levels, the tissues were not markedly depleted of their stores as a result of low dietary copper, nor did they exhibit any gross or microscopic lesions attributable to low capper intakes. 3. The morphological and histochemical integrity of the skeletal system, as shown by histopathological examination of the rib, tarsus and metatarsus and by the strength characteristics of the left femur, was not significantly impaired by low-copper diets, thereby indicating that bone copper reserves were not significantly affected. 4. Brain and erythrocyte superoxide dismutase activity did not respond significantly to dietary copper levels; it is likely that, despite the low copper intakes, there was an adequate amount of this element in the brain and red blood cells to sustain activity of this enzyme; or superoxide dismutase activity in brain and erythrocyte might not be as sensitive a measure of copper deficiency as was suggested by 128 129 others. 5. Liver copper was not severely depleted by low dietary copper since analysis showed that liver copper concentrations were within the range of normal adult levels, although young pigs may normally have some- what higher liver copper concentrations than adults have. 6. Grossly, anemia was not evident, but there was a consistent lowering of hemoglobin levels by diets low in copper (0.6, 0.9, 1.3 ppm copper). Although these differences were judged as statistically non— significant, the effect of low copper was considerable since hemoglobin levels should be more or less constant if the supply of copper is adequate. Such low hemoglobin values can be construed as signs of sub- clinical anemia. In addition, plasma copper levels of pigs on low copper diets were significantly and consistently depressed and in one instance fell (5.2 mcg/lOO ml) much below the minimum hemopoietic level of 20 meg/100 ml. Based on these criteria -- hemoglobin and plasma copper -- anemia and copper deficiency were biochemically present. Such a sub- clinical condition was also indicated by a significant depression of ceruloplasmin activity, an observation which suggests that on low dietary copper, ceruloplasmin activity cannot be sustained. 7. In spite of the low-copper diets (0.6, 0.9, 1.3 ppm copper), pigs demonstrated positive absolute copper retention, showing that copper loss via urine and feces did not exceed copper absorption. 8. It is conceivable that the inconsistent responses of the para- meters chosen were because some were not sensitive indicators of copper status at such low dietary levels, e.g., average daily gain, soft tissue and skeletal copper concentration, and erythrocyte and brain superoxide dismutase activity, while hemoglobin, ceruloplasmin, plasma copper and 130 copper balance were sensitive measures. 9. Based on these parameters, the minimum copper requirement for the baby pig is very low and is probably between 3.0 and 4.0 ppm on an as-fed basis, or between 3.4 and 4.6 ppm on a dry basis. MANGANESE INTRODUCTION Manganese requirements of pigs for growth, skeletal development and fertility have been placed at conflicting levels and have not been defined precisely. Levels of 11 to 14 ppm manganese in corn-soybean diets promoted growth but caused leg stiffness and lameness while 50 ppm manganese (Keith et aZ., 1942) and 60 pm (Miller et aZ., 1940) prevented skeletal malformations without curing them. Experiments by Johnson (1940, 1944) indicated that 0.3 ppm manganese resulted in satisfactory growth but caused poor reproduction and depressed tissue manganese while 6 ppm promoted successful reproduction. He also reported that diets containing 7 to 10 ppm manganese maintained adequate performance of pigs from weaning to market. Diets containing 12 ppm manganese were reported to be inadequate for reproduction but maintained bone and somatic growth (Grummer et aZ., 1950), and when the same diets were supplemented with 40, 80 and 160 ppm manganese, optimum performance was recorded with the 40 ppm level, indicating that higher levels did not improve gains. Different performance results were reported for growing pigs placed on dietary manganese levels ranging from 0.5 to 34 ppm (Plumlee et aZ., 1956), and for pigs taken from sows on 70 to 90 ppm manganese (Speer et aZ., 1952). For baby pigs, the manganese requirement for optimal growth was placed at 0.4 ppm by Leibholz et al. (1962). Normal reproduction and farrowing were reported for sows on 80 to 117 ppm manganese 131 132 (Leibholz at aZ., 1962) and for sows on 6 to 100 ppm manganese (Newland et aZ., 1961). Underwood (1971) has concluded that growth of pigs is maintained on very low dietary intakes of manganese at the expense of tissue manganese, but such low levels for prolonged periods cannot promote fertility and reproduction. A level of 20 ppm manganese has been recommended for normal growth of baby pigs by the NRC (1973). The trial reported here was conducted to determine the manganese requirement of baby pigs as a follow-up to the work of Kayongo-Male (1974) who reported that this requirement is between 3 and 6 ppm. Semi- purified diets were used along with various manganese levels. Manganese balance, serum and tissue manganese concentrations, serum alkaline phosphatase and bone strength characteristics were used as indicators of manganese status. EXPERIMENTAL PROCEDURES A. Introduction An experiment was conducted as a followbup to "Experiment 4" previously conducted by Kayongo-Male (1974) to determine the manganese requirement of baby pigs from sows fed a lowhmanganese diet (13.9 ppm Mn, Table B-2). The Michigan State University swine farm furnished the pigs and facilities used in this trial. Diets were semi-purified, the protein being supplied by high-protein casein. B. Experiment 1 (Mn) Sixteen 8-day-old baby pigs were taken from sows on a low manga- nese diet, housed and fed as previously described for Experiment 1 in the copper trials. They were adapted to the basal diet shown in Table 25 for 5 days. After adaptation, the pigs were allotted at random to the four diets shown in Table 25 which contained the following manganese levels: diet 1 (basal), 0.9 ppm; diet 2, 2.2 ppm; diet 3, 3.8 ppm; diet 4, 7.4 ppm. The pigs were kept on these diets for a 42-day growth trial. Blood samples were taken initially and on days 7, 14, 28 and 42 of the trial for determination of hemoglobin, hematocrit, alkaline phos- phatase activity and serum manganese. At the end of the growth trial, the pigs were placed in stain- less steel metabolism cages for a 2-day adjustment period followed by a 3-day collection of feces and urine. The pigs were then killed, weights 133 134 TABLE 25. COMPOSITION OF DIETS USED IN EXPERIMENT 1 (Mn) Ingredient Basal B + 1.5 ppm B + 3 ppm B + 6 ppm Casein 2100 2100 2100 2100 Cerelose 3500 3360 3220 2940 a-Cellulose 350 350 350 350 Low Mn-Mineral premixa 420 420 420 420 Lard 350 350 350 350 Fat sol. vit. premixb 70 70 70 70 water sol. vit. premixc 140 140 140 140 Corn 011 70 70 70 70 Mn premixd _____Q _lfl)_ ___2__8_Q __5fl)_ 7000 7000 7000 7000 Mn Conc. (analyzed, ppm)1 0.9 2.2 3.8 7.4 8See Appendix B, Table B-1. b ’cSee Appendix A, Table A-4. dManganese premix in cerelose containing 100 ppm.Mn from MnSOA'HZO, J. T. Baker reagent grade. 1Expressed on as-fed basis. 135 of various organs, tissues and glands were obtained, and liver, kidney and muscle were saved for mineral analysis. The left femur was taken for measurement of bone strength characteristics, and rib 7, metacarpal and radiocarpal joints were fixed in formalin solution and submitted for histopathological examination. C. Analytical Methods Hemoglobin, hematocrit, bone strength characteristics, bone ash, tissue, feed, feces and urine manganese were determined according to procedures previously described for the copper trials. 1. Bone minerals a. Manganese. Ashed bone was finely ground and approximately 300 mg of powdered ash were dissolved in 5 ml of 6N HCl. Two milliliter aliquots of the acid-ash solution were diluted 1:2 with strontium mixture A;, and the manganese concentration was determined by atomic absorption spectrophotometry at 279.4 nm, and expressed as ppm on a dry, fat-free basis. b. Calcium and magnesium. Aliquots of the acis-ash solution were diluted 1:20 with deionized, distilled water. The resulting solutions were further diluted 1:100 with strontium.mixture B2 and calcium and magnesium levels determined by atomic absorption spectro- 1Dissolve 60.86 g SrC12°6H20 and 10.0 g NaCl in 1 liter of deionized distilled water to yield 20,000 ppm Sr. 2Dissolve 30.5 g SrClz'GHZO and 5.0 3 NaCl in 1 liter of deion- ized distilled water to yield 10,000 ppm Sr. 136 photometry at 422.7 and 285.2 nm, respectively. Calcium and magnesium concentrations were expressed as a percentage of dry, fat—free bone. c. Phosphorus. Aliquots of the acid-ash solution were diluted 1:200 with deionized, distilled water, and phosphorus was determined by the colorimetric method of Gomorri (1942). To 0.5 ml of the 1:200 diluted solution, 2.5 ml of MS solution1 were added, followed by 0.25 ml of Elon solutionz. A.water blank was treated in the same manner as was the sample and both were incubated for 45 minutes and analyzed for phosphorus using a Coleman Junior II spectrophotometer. Optical density was recorded at 700 nm and phosphorus concentration was expressed as percentage of dry, fat-free bone. 2. Serum manganese Determination of serum manganese was performed by flameless atomic absorption spectrophotometry on the Instrumentation Laboratories, Inc., Medel 455 flameless atomizer connected to an IL Model 453 atomic absorption unit. Duplicates of 10 ul aliquots of undiluted serum were dried at 100 to 6000 over a period of 3 minutes, pyrolized at 12000 for 20 seconds and analyzed at 24000 for 15 seconds. A clean cycle was auto- matically completed at the end of each analytical period. To each serum aliquot was added 10 ul deionized distilled water or 10 ul standard manganese solutiona. Since the volume of serum used was the same as the 1Dissolve 5.0 g (NaZMo04°H20) in 500 m1 d.d. water, add 14 ml of lZN H2804 and make up to 1 liter. 2Dissolve 1.0 g Elon (p-methyl-amino-phenosulfate) in 100 ml of 32 NaHSO3 and filter. Keep refrigerated. 3Standards of 5, 10, 20 and 30 ppm Mn were made from a stock solution of 1000 ppm. 137 addition volume and the slope was 1.0, serum manganese concentration in parts per billion, was read directly from the signal print—out recorded on a Jahrmann SC 1200—R strip chart. The flameless atomizer was purged with argon (at 20 psi) at the end of each cycle. 3. Serum a1kaline_phosphatase The activity of serum alkaline phosphatase was determined accord- ing to the procedure outlined in Sigma Technical Bulletin No. 194 (1963). Assays were done using freshly obtained serum. Solutions used were ob- tained from Sigma1 or were freshly prepared as follows: a. Sodium hydroxide. In 1 liter of deionized distilled water 0.8 g anhydrous sodium hydroxide was dissolved to give approximately a 0.02N solution. b.. Standard solution ofgp-nitrophenol. This was obtained as Sigma Stock No. 104-1 (10 mM/liter) and was kept in the dark at 4°C. c. Working standard solution. Into a 100 ml volumetric flask 0.5 ml p-nitrophenol standard solution was pipetted followed by 0.02N sodium hydroxide solution to 100 ml; the flask was thoroughly shaken to mix the solutions. d. Sigma 104 phosphatase substrate. The substrate, p-nitrophenyl phosphate, was obtained from Sigma and stored at 4°C. e. Stock substrate solution. In 10 ml deionized distilled water, 0.04 g substrate was dissolved; this was enough for 20 determina- tions. 1Sigma Chemical Company, St. Louis, Missouri. 138 f. Alkaline buffer solution. About 0.75 g glycine from Sigma was dissolved in approximately 25 ml deionized distilled water. Three to 5 ml 1N NaOH were added, followed by a solution of 0.0203 g MgC12'6H20 in 5 ml deionized distilled water. The solution was made up to 90 ml with water and the pH adjusted to 10.5. Water was added to make a final volume of 100 ml, and the solution was stored at 4°C. g. Alkaline buffered substrate. The alkaline buffer solution was mixed 1:1 with the stock substrate solution. h. .Asgay. Into a test tube, 0.05 ml serum and 1 ml buffer sub- strate were added, shaken on a vortex and immediately placed in a water bath at 38°C. Reagent blanks were similarly treated using deionized distilled water in place of serum. Exactly 30 minutes later, 10 ml 0.02N NaOH solution were added and mixed on a vortex. This stopped the enzyme activity and allowed the color produced to be measured. The color was formed by liberation of p-nitrophenol by the enzyme alkaline phosphatase. Optical density of the samples was determined on the Beckman DU Spectro- photometer1 at 410 nm using reagent blanks as reference. Units of alkaline phosphatase (AP) were determined by using the calculated stan- dard factor. Addition of 0.1 m1 HCl to each sample resulted in a color- less solution, and a second optical density and AP units were determined. 1Beckman Instruments, Inc., Fullerton, California. 139 The true corrected AP activity was calculated as follows: Alkaline Phosphatase activity (Sigma Units/ml) 0 2 x AP units of h _ AP units of colored sample colorless sample D. Statistical Analyses The data were subjected to a onedway analysis of variance using the Unequal-l format on a CDC 6500 computer at the Michigan State Univer- sity Computer Laboratory. Simple correlations were also calculated and the levels of significance of differences between means were determined using the Bonferroni t-statistics test. RESULTS AND DISCUSSION A. Experiment 1: Manganese requirement of baby pigs on semi-purified diets supplemented with manganese sulfate to yield upon analysis: 0.9 ppm, 2.2ppm, 3.8 ppm and 7.4 ppm manganese The effects of dietary manganese levels on growth are shown in Table 26. Average daily gain of pigs on the diet containing 3.8 ppm manganese was the highest (362 g) followed by the diet containing 7.4 ppm manganese (295 g). Pigs on the basal diet (0.9 ppm Mn) gained weight at the same rate as did those on diet 2 (2.2 ppm Mn). However, there were no significant differences in average daily gain and feed efficiency due to dietary manganese levels. Hematocrit, hemoglobin and mean corpuscular hemoglobin concen- tration values are presented in Table 27. Hematocrit values of pigs on diets 1 and 3 rose steadily, the first peaking on the fourth week, the third on the sixth week; for diets 2 and 4, hematocrit fluctuated (Figure 4.1). These values do not represent any significant treatment effects. Hemoglobin was not significantly affected by dietary manganese levels up to week 4. In week 6, hemoglobin was significantly (P<0.05) higher on diet 4 than on diets l and 3 (Figure 4.2). Mean corpuscular hemoglobin concentration rose until the fourth week in pigs on diets l and 2, while the rise continued into week 6 for pigs on diet 4; for diet 3 the values fluctuated (Figure 4.3). MCHC value for pigs on diet 4 was significantly (P<0.05) higher than the corresponding values for pigs on diets l and 2, while MCHC value for diet 3 was significantly (P<0.01) 140 141 TABLE 26. THE EFFECT OF DIETARY MANGANESE LEVELS ON GROWTH (EXPT. 1) Diet no. 1 l 2 3 4 Mn conc., ppm 0.9 2.2 3.8 7.4 No. of pigs 4 4 3 4 Avg. init. wt., kg 2.71:0.14 2.43:0.14 2.69:0.16 2.4510.14 Avg. final wt., kg l3.28i0.72 l3.23i0.72 15.90i0.83 13.85i0.72 Avg. daily gain, g 288 285 362 295 Feed/gain 1.35 1.42 1.30 1.32 1Expressed on as-fed basis. 142 TABLE 27. THE EFFECT OF DIETARY MANGANESE LEVELS ON HEMATOCRIT, HEMOGLOBIN AND MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (EXPT. 1) Diet no. 1 2 3 4 Mn conc., ppm 0.9 2.2 3.8 7.4 No. of pigs 4 4 3 4 l Hct. , Z Initial 36.0il.28 37.3il.28 37.111.48 37.9il.28 7 days 37.3il.l8 38.3il.18 37.8il.36 39.1:1.18 14 days 39.4il.54 36.6il.54 38.1il.78 37 3il.54 28 days 44.4il.47 41.8tl.47 40.8il.69 40.5il.47 42 days 39.9i0.72 40.4i0.72 41.5:0.83 39.1iO.72 1113.2, g/100 ml Initial 10.0i0.26 9.4:0.26 10.5i0.30 9.5:0.26 7 days ll.0i0.29 11.1:0.29 12.0i0.34 ll.4i0.29 14 days ll.9i0.62 10.8i0.72 ll.5i0.72 ll.8i0.62 28 days l4.5i0.58 l4.2i0.58 13.8:0.67 l3.5i0.58 42 days 12.0:0.45 12.6i0.45 12.0t0.52 l4.li0.45 3 MCHC , Z Initial 27.8il.04 25.2il.04 28.3il.20 25.231.04 7 days 29.7tl.40 29.0il.40 31.8il.62 29.2il.40 14 days 30.110.73 29.6:0.84 30.110.84 31.5:0.73 28 days 32.710.75 33.9:0.75c 33.8:0.87e 33.2:0.75b d 42 days 30.1il.l7 31.1tl.l7 28.9il.35 36.1il.17 ’ aSignificantly greater than least two values (P<0.05). b d ’cSignificantly different (P<0.05). ’eSignificantly different (P<0.01). 1Hematocrit. 2Hemoglobin. 3Mean corpuscular hemoglobin concentration. 143 45 1 --— Diet l (0.9 ppm Mn) — — Diet2(2.2 ppm Mn) ------ Diet3(3.8 ppm Mn) Diet4(7.4 ppm Mn) * / f‘ )0‘ :5. '- l /"..’ \\‘ L. I g 40 / V- 3 E Q) I Tr"",¢r"”' \\\‘// 35 1 1 1 1 o 7 :4 28 42 Days on Experimental Diets Figure 4.1. Effect of dietary manganese concentrations on hematocrit (Expt. 1). 144 Figure 4.2. Influence of manganese intake on hemoglobin (Expt. 1). 145 .0.0 000000 0005 BEmEtmaxm :0 0000 00 00 v. 0 o 1 - Es. 58 3:120 \ as. 5% 0.000 E0 ..... - v. /0 as. 5% 0.000 020 II as. 58 0.0: 020 l.| L m. Iwoovb ‘quIbowaH 146 Figure 4.3. Response of mean corpuscular hemoglobin concentration to dietary manganese levels (Expt. 1). 147 .m.q munwfim 0.05 .20250090 :0 0000 00 00 S 0. d o 8 Om U) N) % "ouoo ugqolbowaH JDIDOSDdJOQ uoaw 22 500 05320 E). 500 0.00 m .20 ..---- 22 500 0.000 .20 II as. 500 0.00 _ .05 l-l l O V 148 lower than the value for diet 4. The effects of dietary manganese levels on serum alkaline phos— phatase activity and serum manganese are summarized in Table 28. Initial alkaline phosphatase activity was high -- 40.3, 59.7, 44.0 and 52.1 Sigma units for treatments 1, 2, 3 and 4, respectively -— but decreased dramatically until it reached 4.9, 6.0, 5.6 and 6.5 Sigma units, respectively (Figure 4.4)- Lassiter et al. (1970) reported a rise in serum alkaline phosphatase activity in response to dietary manganese. There were, however, no significant differences between treatments. Serum manganese showed no consistent effects due to dietary levels of manganese. After a drop in the first week, all the treatments produced an increase in the second week which persisted in treatments 2 and 4. Treatments 1 and 3 caused fluctuations in serum manganese levels (Figure 4-5)- However, on the sixth week serum manganese levels were lower on diet 1 than on all other diets -- 8.0, 10.0, 12.0 and 12.3 ppb for diets l, 2, 3 and 4, respectively -- but this difference was not significant. Significant responses of serum manganese to dietary manganese were reported by Hawkins et al. (1955), Plumlee et al. (1956), Newland and Davis (1961) and Rojas et al. (1965) in swine and ruminants. Table 29 shows the result of the manganese balance trial, and values of selected fecal and urinary minerals. Dietary manganese levels significantly (P<0.01) influenced manganese intake, fecal and urinary manganese excretion and manganese retention. Manganese intake was sig- nificantly (P<0.05 or P<0.01) different between the four diets. Abso- lute fecal manganese excretion was significantly (P<0.05) different between diets 1 and 2, and significantly (P<0.01) different between diets l, 3 and 4. Absolute urinary manganese excretion was higher on 149 TABLE 28. THE EFFECT OF DIETARY MANGANESE LEVELS ON SERUM ALKALINE PHOSPHATASE ACTIVITY AND SERUM MANGANESE CONCENTRATION (EXPT. 1) Diet no. 1 2 3 4 Mn conc., ppm 0.9 2.2 3.8 7.4 No. of pigs 4 4 3 4 1 SAP ,fiSigga units Initial 40.319.03 59.719.03 44.0110.42 52.119.03 7 days 30.7:2.80 38.5i2.80 32.4:3.23 37.9:2.80 14 days l8.2il.32 19.411.32 l8.0il.53 17.6il.32 28 days 9.110.67 9.810.67 8.9:0.77 9.6:0.67 42 days 4.9il.l4 6.011.14 5.6il.32 6.5il.14 Serum MnZLppb Initial 10.8il.23 10.3il.42 7.311.42 11.711.42 7 days 7.0il.09 7.511.09 7.7il.26 6.5tl.O9 14 days 8.5il.04 9.8il.20 9.3il.04 8.3tl.04 28 days 7.812.18 10.8:2.18 8.5:3.08 11.012.18 42 days 8.0il.38 10.011.19 12 0:1.38 12.3il.19 1Serum alkaline phosphatase. 2 Serum manganese concentration. 150 0 so ..- "E —-— Diet l (0.9 ppm Mn) 3 \ —— Duet 2 (2.2 ppm Mn) 0 \ - ----- Diet 3 (3.8 ppm Mn) g 50 -—- Diet 4(74 ppm Mn) '0 \\ 3:; \\ 0 4o '25 \‘\;:“ u: " s ‘s 5} \\ \ 0% 30 - \1“\ . - \\ .s \ s \ 2 ~ ‘\\ g '0 " “‘s 35 ‘ ""§:::; (I) _ . o 7 :4 is 42 Days an Experimental Diets Figure 4.4. Effect of manganese intake on serum alkaline phosphatase activity (Expt. 1). 151 Figure 4.5. Response of serum manganese levels to dietary manganese (Expt. 1). 152 .0.0 «0:000 0005 BEoEtoaxm 00 0000 as. ea 0.00 N 020 Ill 32 Ea 0.00 _ 020 |-I 00 00 v. 0 00 0 \» 0. . . ..--.. III‘I‘.’ \‘I. ....I ll! \\ \ m ...... \\ \\ II I \\\\ m s In. \\ \ / C] \s \ 0 O— s / \ __ .. 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