DIETARY 66min. us RELAszst-iw m. zmc Am PAMKEMTQM m THE PEG The“: for Hm Doqm «3‘ pk. D. MICHIGAN STATE URIE 5331?”? Bastian D. Ritchie 1964 THES‘S IIHIITWWI'II‘IHWWWIIFHIITWRI 3 1293 00858 0882 A C '_Is: This is to certify that the *I'-~ thesis entitled ’WTARY COPPER: I'I'S RELATIONSHIP TO . 1 “'“sz'C"'AND PARAKERATOSIS IN THE PIG ‘45.“... 3—.- n -o -- ‘ " ‘0‘- -o a _ .9 . . presented by Harlan D. Ritchie ’ has been accepted towards fulfillment of the requirements for Ph D degree “Animal Husbandry / Major fiofessor Date February 20, 1961; 0-169 LIBRARY Michigan State University L In.”»-—_._ .hno—d.‘ .WH‘-.—r‘—-‘-'-" _.- _‘4 — . fl‘". . _-_... —-'-.._.~_ PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE fl ‘I‘ Q I“! P \J flier“ %% = —l MSU Is An Affirmetive Action/Equal Opportunity Institution cMchH.‘ ABSTRACT DIETARY COPPER: ITS RELATIONSHIP TO ZINC AND RARAKERATOSIS IN THE PIG by Harlan D. Ritchie Five experiments, involving a total of nine trials and #38 weanling pigs, were conducted to investigate relationships between porcine parakeratosis and various dietary trace elements, particularly copper and zinc. Interrelationships between these two elements and iron were also studied. Copper was added at two levels (125 and 250 ppm) and in two forms (sulfate and oxide) to diets which varied in calcium and zinc content. Copper was also compared with a broad-spectrum antibiotic as a dietary growth stimulant. In the first experiment, parakeratosis occurred on low-zinc basal diets (28-39 ppm Zn) at three levels of calcium intake (0.55, 1.05, and 1.31% Ga). -Compared to healthy pigs, parakeratotic animals were characterized in most instances by depressed growth rate, higher serum gamma globulin fraction, lower serum albumin fraction, and lower serum alkaline phosphatase activity, in addition to skin lesions typical of this syndrome. Lower hemoglobin and hematocrit, and higher total serum protein were also noted in several parakeratotic lots. Zinc supple- mentation (50 or 75 ppm) was completely effective in preventing para- keratosis; copper (125 ppm) was completely effective at 0.55 and 1.05% calcium levels and partially effective at 1.31% level; iron (100 ppm) was partially effective at lowest level but was ineffective at a higher calcium level. Copper-fed pigs exhibited significantly higher liver copper co: with coppe basal lev: I: t D9; (54- I‘ e.-. .9 J. Harlan D. Ritchie copper concentration than control pigs. When zinc was fed in combination with copper, storage of copper in the liver was reduced to that of the basal level. In the second experiment, two trials were conducted to study effect of adding two levels of copper sulfate (125 and 250 ppm Cu), with or without supplemental zinc (100 ppm), to high-calcium, low-zinc basal diets. In each trial, the parakeratotic basal lot was divided after several weeks to study effects of copper therapy. In one trial, the higher copper level was almost completely effective in preventing parakeratosis, while in the other it was only partially effective; the lower level was partially effective in both trials. Copper toxicity occurred at the 250 ppm level, but there was little or no evidence of it at the lower copper level. Supplemental zinc profoundly reduced liver copper levels and appeared to furnish complete protection against copper toxicity. Supplemental copper significantly lowered liver iron storage, but this effect was overcome when zinc was added to the ration. Copper had little influence on liver zinc stores. Either 125 or 250 ppm of dietary copper therapy in the basal lots brought about a marked recovery from parakeratosis. Copper sulfate (125 ppm Cu) and chlortetracycline (10 mg./lb.) were added to a normal-calcium (0.63%) basal diet in the third experi- ment. Neither supplement alone accomplished a significant increase in growth rate. However, joint supplementation resulted in a signi- ficant response (P<:;05) in daily gain. There were no significant differences in hematology. In the fourth experiment, high-level (250 ppm Cu) copper sulfate or capper supplemen‘ . :cmanc Harlan D. Ritchie or copper oxide was added to a normal-calcium diet, with and without supplemental zinc. There were no significant differences in per- formance or hematology. No clinical symptoms of copper toxicity were noted, but there was a high incidence (90%) of cirrhotic livers in pigs fed copper sulfate with no added zinc; a much lower incidence (30%) was observed in those fed copper oxide alone. Copper oxide-fed pigs stored significantly less liver copper than those fed copper sulfate. Both forms significantly reduced liver iron storage, copper sulfate having a greater effect than copper oxide. Neither form significantly increased loin copper levels. Two trials were conducted in the fifth experiment to test the hypothesis that parakeratosis-producing diets inhibit activity of phytase or phosphatase in the intestine of the pig. Results were highly variable; in only one instance was there a significant difference favoring acceptance of the hypothesis. DIETARY COPPER: ITS RELATIONSHIP TO ZINC AND PARAKERATOSIS IN THE PIG By Harlan Di Ritchie A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 196a I‘ a . A“ .".‘ an ..-\... .- Tne I . E a . c .-J a c . .. l C. he 4% s at 2. x l» .c .3 a a Q. .7 ‘ IJX. ‘ ‘c G d. F} | ,-~ ‘- La in $3,354 ACKNOWLEDGMENTS The author is deeply grateful to Drs. J. A. Hoefer, R. W. Luecke, E. R. Miller, and E. P. Reineke for their counsel throughout his graduate study, and for their attentive reading of this manuscript. The advice of Dr. R. U. Byerrum, who also served on the guidance committee, is greatly appreciated. The writer is indebted to Drs. R. W. Luecke, D. E. Ullrey, and E. R. Miller, whose laboratories furnished the facilities and materials which made this research possible. Sincere gratitude is expressed to Dr. R. H. Nelson and the Animal Husbandry Department of Michigan State University for the use of facilities and animals and for financial support through an assistant instructorship. Special thanks are due Miss Betty Baltzer for her patient guidance of this neophyte investigator through the rigors of various chemical analyses. The writer is indebted to Drs. D. A. Schmidt, H. Rothenbacher, and C. E. Whiteman for their pathological work in this study. Thanks are also due Prof. L. J. Bratzler for the use of the meat laboratory and its personnel in the slaughter of the experimental animals. The author wishes to thank Mrs. D. R. Green, Mrs. A. M. Whitmore, Mr. C. L. Zutaut, Mrs. 0. A. Thompson,and Mrs. I. Ackerman for their assistance in certain of the biochemical determinations. He is also grateful to Mr. G. B. Stafford and others at the swine farm for their “work in the rearing and management of the pigs. Merck and Company, Rahway, N. J., Chas. Pfizer and Company, Terre ii Haute, Ind., and American Cyanamid Company, Pearl River, N. Y., are acknowledged for their generous supply of certain ration components. The writer is grateful to Miss Marie Stehlik, who skillfully and efficiently typed this manuscript. The author wishes to acknowledge the encouragement of his parents, who early in life impressed upon him the importance of a sound education. This writer is especially indebted to his wife, Lou, who has spent all six of her married years as a bread-winning student wife. Her knowledge of the English language has been of invaluable assistance throughout his graduate studies. Furthermore, her boundless energy and enthusiasm have always been an inspiration to him. iii \r- L Datum! Urn-r- . 1 ' -~..|u 7.. 2.1:??? “ “‘t I Harlan D. Ritchie candidate for the degree of Doctor of Philosophy DISSERTATION: Dietary Copper: Its Relationship to Zinc and Parakeratosis in the Pig OUTLINE OF STUDIES: Main Area: Animal Husbandry (Animal Nutrition) Minor Areas: Biochemistry, Physiology BIOGRAPHICAL ITEMS: Born: August 3, 1935; Albert City, Iowa Undergraduate studies: Iowa State University, 1953-1957 Graduate studies: Michigan State University, 1957-196h EXPERIENCE: Assistant Instructor, Michigan State University, l957-l96h MEMBER: American Society of Animal Science Society of Sigma Xi Alpha Zeta iv In: ,. H LIL B} 9-7, sci . A, I Q M Q» A ‘ C.\ C C x C CL\ C C \ .Nc h \ v a we .5. pm. an E Do as D . E Do U o WI. no. I. II. III. TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . . . . . Biochemical Role and Metabolism of Copper . . . . . . Biochemical Role and Metabolism of Zinc . . . . . . . High-Level Dietary Copper . . . . . . . . . . . . . Zinc and Porcine Parakeratosis . . . . . . . . . Copper and Zinc Interrelationships . . . . . . . . . . MERMTAL PROCEDURE 0 O O O O O O O C O O O O O O Gener 8.1. O O o O o s o o e 0 e o O 0 o 0 O o s s e 0 Biochemical Determinations . . . . . . . . . . . . Experiment I. Supplementation of Low- and High- Calcium Diets With Copper, Zinc, and Iron . . . . Experiment II. Addition of Copper Sulfate and Zinc Oxide to a High-Calcium Diet . . . . . . . . . . Experiment III. Addition of Copper Sulfate and Chlortetracycline to a Normal-Calcium Diet . . Experiment IV. Addition of Copper Sulfate, Copper Oxide, and Zinc Oxide to a Normal-Calcium Diet . . Experiment V. Intestinal Phytase and Phosphatase Activity of Pigs Fed Various Levels of Calcium, With and Without Supplemental Zinc and Copper . . RESUDTS AND DISCUSSION . . . . . . . . . . . . . . . . Experiment I . . . . . . . . . . . . . . . . . . . . Experiment II . . . . . . . . . . . . . . . . Experiment III . . . . . . . . . . . . . . . . . . . . Experiment IV . . . . . . . . . . . . . . . . . . . . 12 19 30 to ALL Ah 1+5 53 55 57 58 60 62 62 71+ ' 95 97 SUI COT BIZ TABLE OF CONTENTS (continued) Experiment V . . . . . . . . . . . . . . . . . . . . . . . . 102 V. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 VI. CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . 112 VII. BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . 11A VIII} APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . 128 vi n 4| E Tu PN/ ‘ A us‘v‘. “ \‘. a) «(J ‘ls 10. ll. 12. 13. 1h. LIST OF TABLES Exp. 1. -- Composition of basal diets . . . . . . . . . . Exp. 1, Trial A. -- Response of pigs on low-calcium diet to supplemental zinc, iron, and COpper (13 weeks) . . . . . . . . . . . . . . . . . . . . . . . Exp. 1, Trial B. -- Response of pigs on high-calcium diet to supplemental zinc, iron, and copper (11" weekS) O O 0 O O O 0 O O O C O O O O O I O O O O O 0 Exp. 1, Trial B. -- Effect of supplemental zinc, iron, and copper on serum protein values . . . . . . . . . . . Exp. 1, Trial B. -- Effect of supplemental zinc, iron, and copper on trace element content of pigs' livers . . Exp. 1, Trial C. -- Effect of supplemental copper and zinc at varying levels of dietary calcium (12 weeks) . . Exp. II, Trial A. -- Response of pigs fed a high-calcium diet to supplemental copper and zinc, 0-10 weeks . . . Exp. II, Trial A. -- Effect of supplemental copper and zinc on pig performance for last 5 weeks (10th-15th week) and for entire trial (0-15th week) . . . . . . . . . . . Exp. II, Trial A. -- Hematology of pigs fed high levels Of Copper and. Zinc O O O O I O O O O O O I I O O O O 0 Exp. II, Trial.A. -- Trace element content of livers from pigs fed high-calcium diet supplemented with copper and Zinc 0 O O I O O O O O O O O O O 0 O 0 l O 0 O 0 O 0 Exp. II, Trial B. -- Effect of supplemental copper and ' zinc on pig growth and hematology, 0-8 weeks . . . . . . Exp. II, Trial B. -- Effect of supplemental copper and zinc during last 6 weeks (8th-Iuth week) and during entire trial (0-1’+th week) . . . . . . . . . . . . . . . Exp. II, Trial B. -- Trace element content of livers from pigs fed supplemental copper and zinc . . . . . . . Exp. III. -- Response of pigs to copper sulfate and chlortetracycline, alone and in combination (1h weeks) . vii 65 67 .69 .72 75 77 82 83 87 89 92 96 Table 15. 16. 17. 18. LIST OF TABLES (Continued) Page Exp. IV. -- Comparison of two forms of copper added to a normal-calcium diet with and without supplemental zinc (13 weeks) . . . . . . . . . . . . . . . . . . . . . 98 Exp. IV. -— Trace element analyses of liver and loin from pigs fed copper sulfate and copper oxide with and without Zinc O I O O O O O 0 O O O O O O O O I O 0 O O O O 99 Exp. V., Trial A. -- Effect of supplemental copper and zinc with varying calcium levels on growth, hematology, and intestinal phytase and phosphatase (8 weeks) . . . . 103 Exp. V., Trial B. -- Effect of normal and parakeratotic diets on intestinal phosphatase activity (7 weeks) . . . 105 viii LIST OF FIGURES Figure 1. Exp. II, Trial A. -- Typical effect of copper therapy on parakeratosis . . . . . . . . . . . . 2. Exp. II, Trial B. -- Growth curves of pigs fed high- or normal-calcium diets supplemented with either c0pper or zinc . . . . . . . . . . . . . . . . . ix L)\ . Exp . Exp . Exp . Exp. Exp. Exp. E1133 , LIST OF APPENDIX TABLES Exp. II. a- Composition of basal diets . . . . . . . . . Exp. III. -- Composition of basal diet . . . . . . . . . Exp. IV. -- Composition of basal diet . . . . . . . . . Exp. V. -- Composition of basal diets . . . . . . . . . Exp. II, Trial A. —- Copper, zinc, and iron content of liver S O O O O O O O O O O O O Q 0 O O O O 0 O O O 0 Exp. II, Trial B. -- Copper, zinc, and iron content of livers O O I 0 O O O O O O O O O O O O O O O O O 0 O 0 Exp. II, Trial B. -- Histopathological examination of livers from pigs fed two levels of copper sulfate with and without supplemental zinc . . . . . . . . . . Exp. IV. -- Trace element analyses of livers and loins . 132 133 13A .135 Recent scents i.. of the tract P. v~ Fl c: the eife I. INTRODUCTION Recent years have seen an expanding interest in the role of trace elements in animal nutrition, both in this country and abroad. Most of the trace element research has involved two approaches: (1) studies of the effects on animals of basal diets in which the element under in- vestigation is absent or present in abnormal or (2) applied studies of naturally occurring animal diseases that were found to be caused in part or in tgtg'by a deficiency or an excess of one or more trace elements. Much of the attention in monogastric species, particularly rats and swine, has been focused on three elements--iron, coppergand zinc. This thesis comprises a study of these elements, especially the latter two, in the nutrition of the growing pig. The Wisconsin research group was the first to demonstrate that copper and zinc are dietary essentials for the rat (Hart gt 21., 1928; Todd 23 31., 193A). These findings were subsequently extended to other species. Teague and Carpenter (1951) demonstrated the need for a small amount of copper in the diet of the pig and described symptoms of copper deficiency. Tucker and Salmon (1955) were the first to show that porcine parakeratosis could be prevented or cured by adding zinc to the ration. It was also reported by these and many other investigators that high levels of dietary calcium aggravate the parakeratotic condition; however, the mechanism involved has not been satisfactorily elucidated. -1- As a r lgSSaibycy feeiing of throughout of copper-- aiied to p: -2- .AS a result of the work of Barber and his colleagues (Barber gt 21., l955a,b,c, 1957, 1960; Bowler gt‘gl., 1955) and Lucas and Calder (l957a,b), feeding of copper salts to growing swine has become rather widespread throughout Great Britain. These investigators found that a high level of copper-~equivalent to many times the published dietary requirement-- added to pig rations, improved performance to much the same extent as an antibiotic. Conversely, in this country Wallace gt El' (1960) report- ed that levels of copper sulfate similar to those used in the British studies depressed growth rate and produced copper toxicity. It was discovered in one trial, however, that supplemental copper was effective in preventing parakeratosis. This effect of copper on parakeratosis has also been reported by Hoefer 23 El' (1960). Other workers have found copper to have little or no effect in preventing this disease (Hennig, 1960; O'Hara at 21., 1960; Priebe etngl., 1961; Smith gt 21., 1962), although Bunch gt El° (1963) have reported that zinc may be necessary for maximum response to high levels of copper. .In constrast to the negative results of the Florida station, Iowa investigators have found 250 ppm copper as copper sulfate or copper oxide to stimulate rate of gain and feed conversion during the growing period, with no gross symptoms of toxicity (Hawbaker gt 21., 1961; Bunch gt 21., 1961, 1963). However, copper sulfate gave no response after 125 1b. live weight, and, compared to copper oxide, resulted in lower hemoglobin values and a much higher sterage of liver copper. Variable results were obtained with copper carbonate (Bunch et_gl., 1962). the Brit: :crcer tc ‘aii- 1' -lv-’ -- cf the 5‘. f w- ’ i an . y- ‘vvme- .' J. sac &: .' D HSV- «orb- \. Fh‘ ’- .wfiI‘S a: . "Y: r... -w ‘v-.o§-\,‘d*' A $2 ‘ "" awn --:. . ‘ ‘ V. ‘ iv 'Z‘cfs- ‘. ”“‘fi‘) r A. ”anal .. t.-. ea; ‘ ‘f‘hq‘fi I»~.v.;~ V. a ,1 ‘ s.. "Q ~ "W :8 V I 13" I Mfia m1 .9 . _ ‘ H‘ " ‘rr '1 .Q‘Av y‘. 3“ g. f‘r‘; a“ ‘ an; ' “‘\ ‘l ‘. #q de A‘ P: Q». Coincident with the increased feeding of copper sulfate to pigs in the British Commonwealth, there have been several published reports of copper toxicity (Gordon and Luke, 1957; O'Hara gt 21., 1960; Buntain, 1961; Allcroft _e_t_ _a_1., 1961 ; Allen and Harding, 1962). The severity of the symptoms tends to be rather closely related to the level of copper in the tissues, especially the liver. In this respect, it is interesting to note that the liver copper values reported in these papers are considerably higher than those in the Iowa work, where no toxicity was found. A number of experiments in this country have recently brought to light another possible causative factor in the parakeratosis syndrome, namely, phytic acid (Plumlee at 21., 1960; Green.gt_§l,, 1961; Oberleas 33 9.1.; 1961, l962a,b; Smith _a_p _a_1., 1962). It has been demonstrated that the phytic acid in soybean meal diets renders the zinc in these diets less available. Using the results of an in_vit£g study as evidence, Missouri researchers (Oberleas gt 21., 1962b) have postulated that the action of phytate on zinc availability takes place in the gastrointestinal tract, but this has not been confirmed else- where. These workers have also suggested that the deleterious effect of high dietary calcium on zinc utilization is mediated through the phytic acid content of the diet. The work presented in this dissertation was initiated to: (1) determine the effects of supplemental copper fed at various levels and in different forms to growing swine and its interaction with other elements, namelm.zinc, calcium, and iron; (2) elucidate the role of calcium a: L order 1 5:11 patho] growth and Ritchie et -h- calcium and its relationship to zinc and copper in porcine parakeratosis. In order to attain these objectives, various physiological, biochemical, and pathological observations were made, in addition to collecting growth and dietary intake data. The reports of Hoefer gt 21. (1960) and Ritchie 93 9.1- (1961, 1962, 1963) comprise portion of this thesis. fit The :11 ‘5 m‘fient :01 1 \ 1 “928) W 3" 0 ‘1‘. q 3:. suppiene: ' .' W °‘” ‘ .: Species was elsewhere (I pat Cf man: 0 1 0‘ Copper a "+. a -dglorl 0. SE II. REVIEW OF LITERATURE Biochemical Role and Metabolism of Copper The first conclusive evidence that copper is an essential dietary nutrient for animals emerged from the classical studies of Hart gt El° (1928). This group of investigators demonstrated that it was necessary to supplement highly purified iron salts with copper in order to restore normal levels of hemoglobin in the blood of anemic rats. The fact that copper is in some way essential for normal hematopoiesis in a number of species was subsequently verified by further work at Wisconsin and elsewhere (Elvehjem, 1935). This discovery stimulated research on the part of many workers who have attempted to define the biological role of copper at the cellular level. These studies have led to the iso- lation of several copper protein compounds some of which have been Shown to be enzymes with oxidative activity. Holmberg and Laurell (19h8) isolated, crystallized, and character- ized a plasma copper protein named ceruloplasmin which normally con- stitutes a high proportion of the plasma copper of Swine, rats, dogs, and humans (Cartwright _ep 21.” 1950; Wintrobe gt 51,, 1953). This compound is an eta-globulin; it contains about 0.3% copper which corresponds to eight atoms of copper per molecule. Ceruloplasmin is a true oxidase but its physiological role as such has not been elucidated. The copper of ceruloplasmin is tightly bound to the globulin moiety. Scheinberg and Sternlieb (1960), in their compre- -5- tensive re ‘ .ransccr - (1:99. .11- - .155 O» hensive review of copper metabolism, point out that most investigators agree that there is a small proportion of plasma copper which is not a part of ceruloplasmin. This is present as free cupric ion which is in equilibrium with copper loosely bound to albumin. -When copper first enters the plasma from the gastrointestinal tract it is probably in this form. Thereafter, it is transferred progressively, presumably in the liver (Lang and Renschler, 1958), to ceruloplasmin (Bearn and Kunkel, 195A). The loosely bound copper is probably that portion in transport (Gubler, 1956), for, unlike ceruloplasmin copper, it can diffuse freely across semipermeable membranes. Copper in red blood cells is found in at least two forms. A minute portion is in diffusion-equilibrium with plasma (Bush st 21., 1956a). The major portion is not diffusible and is tightly bound to a copper protein of red cells, erythrocuprein (Markowitz gt 21., 1959) or hemocuprein (Mann and Keilin, 1938). Red cell copper is less labile than plasma copper, the latter being a more reliable indicator of changes in the copper status of an animal than whole blood copper (Underwood, 1962). In the copper-deficient pig, the fall in plasma copper is greater than the fall in red cell copper (Wintrobe £3 31., 1953). The physiological function of red cell copper remains unknown. Unlike ceruloplasmin it has no oxidase activity. Cytochrome oxidase is a well-known component of the electron transfer system, which is concerned with cellular oxidation (Keilin and Hartree, 1939). 'Wainio _ep 21- (1959) have r‘ather’definitely demonstrated that this protein is.a copper-containing enzyme., Moreover,there is con- siderable evidence showing an early and marked loss of cytochrome oxidase act chick (Schc 19:6 ). I. lcss of cy‘. a . If F-V‘. On A a» y 1 4. VAL)» I . ‘ .2..I'*‘f‘" \ T : Vcbu‘ . ‘P. 'v- ‘L"e; We". \— ._ . 3 T“r~'a.‘, '4‘. ‘VJ if. *7. . u..e CC) 1.1‘)‘ A‘ s v nUL, l «fie Vh‘A‘ . J ‘J 9e if .' A c.- ‘ 0 ‘v Uri: \ ~ vs s'Lr‘e ] oxidase activity in the tissues of the copper-deficient rat, pig, and chick (Schultze, 1939, l9hl; Gubler gt 21., 1957; Gallagher 23.21., 1956a). The latter group has reported evidence which indicates that the loss of cytochrome oxidase activity results from a failure of synthesis of its prosthetic group, hemecX, rather than of its protein component. Underwood (1962) believes that the synthesis of the heme group of cytochrome oxidase must be regarded as one of the basic functions of copper, unrelated to a general suppression of iron metabolism. There is no evidence that the role of copper in erythrogenesis is related to this particular function. Tyrosinase is a mammalian copper-containing metalloenzyme which exhibits oxidase activity (Brown and Ward, 1959). It is thought to be involved in the conversion of tyrosine to melanin, a pigment of the integument. Albino humans, for example, possess no detectable tyrosinase activity (Harris, 1959). Furthermore, achromatrichia has been observed in the copper-deficient rat, rabbit, cat, dog, goat, sheep, and cattle, but not in the pig (Underwood, 1962). Porter and Folch (1957) have isolated a copper protein named cerebrocuprein from bovine and human brains. Its physiologic function, if any, is unknown. The functions of hepatocuprein (Mann and Keilin, 1938) and a copper protein isolated from horse liver (Mohamed and Greenberg, 195%) are un- lknown. It is highly possible that these are the same protein. Uricase catalyzes the oxidation of uric acid to allantoin. A porcine hepatic uricase was isolated by Mahler gt El' (1955), who re- ported that its activity appears to be related to the presence of copper in the en: clproenzyl Buty: Trirase h; seimnt w: :96 ). L d“ I ' JESS: 5~PPe is ya “on fret: LANA) . w . as not 1 3‘ a I h.‘ \. euge Wick n -8- in the enzyme. Nevertheless some doubt persists that uricase is a cuproenzyme. Butyryl coenzyme.A dehydrogenase and 5-aminolevulinic acid de- hydrase had been previously reported as being copper proteins but sub- sequent work refuted the earlier observations (Scheinberg and Sternlieb, 1960). In this case, copper was apparently a contaminant. The activity of catalase, the hemin-protein which catalyzes the decomposition of hydrogen peroxide, has been reported by Gubler gt El° (1957) to be lowered in the liver of copper-deficient pigs; but other researchers have not found a consistent reduction in catalase activity. The specific role of copper in hematopoiesis and erythrogenesis has not been satisfactorily resolved. Elvehjem (1935) concluded that copper is not concerned with assimilation of iron but with transformation of ingested iron into hemoglobin. Schultze (l9h0) also indicated that copper is not necessary for absorption and storage but is needed for utilization of iron by the blood-forming organs and for mobilization of iron from the tissues. However, in a more recent study (Bush gt 21., 1956b), it was observed that the transport and mobilization of iron was not impaired in copper-deficient pigs but that there was a reduced ability to absorb iron from the gastrointestinal tract. In his review, Matrone (1960) maintains that the effect of copper on iron absorption, in the rat at least, is largely indirect. He suggests that hemoglobin synthesis is blocked in copper deficiency so that absorbed iron can only enter the tissue iron pool, which eventually sets up a "mucosal block," decreasing iron absorption. The exact point at which copper exerts an influence on hemoglobin formation that the a the actiVi the biosfl containiné subsequem Copper is the free 6 Kalli! inCI there is E the blood the incorl ficiency :' of hemin-I (Subler g as a resui Hit. the : formation has been extremely elusive. Iodice gp‘gl. (1958) suggested that the anemia of copper deficiency may be related to a reduction in the activity of O-aminolevulinic acid dehydrase, which is involved in the biosynthesis of hemoglobin and which they showed to be a copper- containing enzyme. However, as mentioned previously, copper content was subsequently shown to be unrelated to the activity of this enzyme. Copper is apparently not necessary for protoporphyrin synthesis, Since the free erythrocyte protoporphyrin of copper-deficient sheep is act- ually increased (Allen, 1956). .Anderson and Tove (1958) reported that there is a lowered incorporation 33:11339 of radioglycine into heme in the blood of copper-deficient chicks; the addition of c0pper restores the incorporation of glycine toward normal. ~Conversely, cOpper de- ficiency in swine is associated with a decrease in the concentration of hemin-proteins only when an increased requirement is evident '(Gublerlgtwgl., 1957). For example, hemoglObin levels may be reduced as a result of the inability of the hematopoietic tissues to keep up with the increase demanded by a shortened red cell survival time (Bush 23 21., 1956b). The latter workers suggest that copper is an essential component of adult red cells and that a certain minimum of copper must be available both for their production and for the maintenance of their integrity in the circulation. underwood (1962) states that "the evidence so far available is therefore inadequate to implicate copper unequivocally with any stage in the biosynthesis of hemoglobin and that the anemia of copper de- ficiency must be explained, for the time being, on the basis of im- paired erythrocyte maturation and a reduced survival time of the nature er Cope :echaeism :; a. (1 rather se flexed he acted her. :28 bones fi'iie epip‘ 5! 31332281280 ‘fim . y-»irr;nz ""3"“ a de: PTCbably ( 3‘4- . an}! acic -10- mature erythrocytes produced." Copper apparently plays a role in normal bone development but the mechanism is not understood. Teague and Carpenter (1951) and Follis EEHEI° (1955) have reported that young pigs deprived of copper exhibit rather severe deformities of the limbs, characterized by excessively flexed hocks and crooked forelegs. Baxter and Van Wyk (1953) have noted bone deformities and fractures in young copper-deficient dogs. The bones showabnormally thin cortices, deficient trabeculae, and wide epiphyses, while serum calcium and phosphorus levels are normal. Spontaneous fractures and osteoporosis have been observed in copper- deficient cattle and sheep (Cunningham, 1950). It has been suggested, without proof, that the bone abnormalities in copper deficiency result from excessive resorption of bone or decreased deposition of bone matrix, or both (Adelstein and Vallee, 1962). In studies designed to measure defects in synthetic processes occurring in copper deficiency, Gallagher §t_al. (1956b) demonstrated that a deficiency in phosphatidic acids in copper-deficient rats was probably due to a lowered capacity for coupling coenzyme A-activated fatty acids to glycerophosphate. They suggested that this discovery may be important in understanding the pathogenesis of the demyelina- ting disease, "swayback" (neonatal enzootic ataxia), which is seen in newborn lambs dropped by ewes which are cepper-deficient (Cunningham, 1950). va this defect in phospholipid synthesis had occurred in utggg at a time when myelin, which is rich in phospholipid, was being formed, demyelination might have resulted. In addition, deficiency in cytochrome oxidase activity may be involved in producing demyelination, which can 1mm t° j ataxia has In 51'- 1efect in thought tc which nor: Balan absorpti" e regarding :agzitude Comer _e_ a iose of Cu 3?. intravel reivew of 4 normal conc although In via the bi: Bell (1960‘ and only 0. ”ii.“ um swine -11- which can be caused by agents, such as potassium cyanide, that are known to inhibit cytochrome oxidase. .A disease similar to enzootic ataxia has been observed in pigs and calves (Cunningham, 1950). In Sheep suffering from naturally occurring copper deficiency, a defect in wool-keratin has been noted (Underwood, 1962). This has been thought to be related to an abnormality in the cross-linkages of keratin which normally occur through disulfide bridges. Balance studies utilizing radiocopper have been valuable in tracing absorptive and metabolic movements of copper; but little is known regarding the biochemical mechanisms involved in these movements. The magnitude of copper absorption in the ruminant has been estimated by Comar gt El' (l9h8). Within 5 days after administration, 75% of an oral 6h'was found in the feces and 5% in the urine, while 3% of dose of Cu an intravenous dose was found in the feces and 3% in the urine. In a reivew of all species by Underwood (1962), it was reported that under normal conditions 90% or more of ingested copper appears in the feces; although most of this consists of unabsorbed copper, active excretion via the bile occurs in all Species. Likewise, with sheep, Lassiter and Bell (1960) found about 90% of the Cu6h eliminated through the feces and only 0.5-1.0% in the urine 96 hours following oral dosing. Studies with swine have been somewhat variable. In young growing pigs, Buescher 23 El. (1961) reported that within 96 hours after an oral dose of Cu6h labelled cupric sulfate, 6h% of the dose was excreted via the feces, l.h% by way of the urine, and 3h% was retained. However, Bowland at El. (1961) found that only 5% of an oral dose of labelled cupric sulfate was absorbed in young pigs. It was calculated by these workers that the bile coul: of the to“ liver was dose of 1: ing to Saj binding 5: accmdlat; affected 1 Zinc Crgacisms of stars reported . 38:1: Of 21' 1932.). 2,, illg I‘Elat for the n. L11955,I the Slmdr. diSCOVery go‘fl‘th a“ I ‘ix Keil. 33931331 r0. Co. . ls’cltueI A’e’Si‘ole 1 bile could account for up to h0$ of the excretion; only a small fraction of the total excretion occurred in the urine. They observed that the sliver was the major site of storage, containing hh-Sl% of the total dose of labelled copper 2h hours after intravenous injection. .Accord- ing to Saltman‘gpigl. (1959), it appears that there are specific copper- binding sites on or in the parenchymal cells of the liver, and the accumulation of copper is not directly coupled to metabolic energy or affected by sulfhydryl activators or inhibitors. Biochemical Role and Metabolism of Zinc Zinc was first demonstrated to be an essential nutrient for living organisms when Raulin (1869) showed that it is necessary for the growth of.A§pergillus niger. It was not until 1934 that convincing evidence was reported concerning the need for zinc for the normal growth and develop- ment of mice (Bertrand and Bhattacherjee, 193k) and rats (Todd gt al., 1931+). With the improvement of the purified diet technique for study- ing relatively uncomplicated zinc deficiency, the importance of zinc for the normal development of these species became even more apparent. In 1955, Tucker and Salmon showed that zinc deficiency is the basis of the syndrome in swine known as parakeratosis. Following this significant discovery, O’Dell 23 al. (1958) reported that zinc is essential for growth and normal develoPment of birds. Keilin and Mann (l9h0) presented the first evidence of a funda- mental role for zinc in metabolisms They showed that this element is a constituent of the enzyme carbonic anhydrase, which catalyzes the re- versible reaction between carbon dioxide and water. Zinc was shown to site up its act‘- other hi Val enzyme p: Ale: equine l; -13- make up 0.33% of the enzyme molecule and was found to be essential for its activity. Since that time, zinc has been found to exist in several other highly purified enzyme systems. Vallee and Neurath (l95h, 1955) have demonstrated that zinc is an integral, structural part of the pancreatic carboxypeptidase molecule; it appears that one atom of zinc is firmly bound to one molecule of enzyme protein. Alcohol dehydrogenase of yeast (Vallee and Koch, l955a,b) and of equine liver (Theorell 2’9. _a_l_., 1955; Vallee and Koch, 1956) was found to contain four and two moles of zinc per mole of protein, respectively. Furthermore, it is thought that each zinc atom serves as a locus of reversible attachment for a molecule of the coenzyme, DPN (Hayes and Velick, 1951i). Glutamic dehydrogenase, which catalyzes the reversible oxidative deamination of glutamate, was reported by Adelstein (1957) to be a zinc metalloenzyme. The average zinc content was 3.h-: 1.0 atoms per mole- cule. Like alcohol dehydrogenase, it appears to bind one DPN molecule per atom of zinc. Further studies by Vallee gpugl. (1956) indicated that other pyridine nucleotide dehydrogenases may prove to be zinc metalloenzymes. Mathies (1958) has established that alkaline phosphatase from swine kidneys is a zinc enzyme. The best preparations contained 0.15% of firmly bound zinc. It was surmised that one molecule of the enzyme contains several atoms of zinc. The isolation of a zinc-dependent hexokinase from.Neurospora crassa led to the suggestion that this is a zinc metalloenzyme (Medina and Nichol not ident: described In at whose acti Mcst of ti: aetivated are: m‘gin Yeast aldo PhOSp'natas In ‘51 and looseL 3:131 the la- lc‘c‘sellv' box Port. Neii Detezm hm" Eryn a1318$ , In 3‘ a Feral] CEHtage of (v ‘Vallee 6t \ . nucleotide. 1&th with ‘ Hui-78:1 ] mush]; how -1h- and Nicholas, 1957). However, the criteria for this conclusion were not identical with those employed to evaluate the zinc metalloenzymes described above. In addition to the metalloenzymes, there are several other enzymes whose activity is increased by the addition of zinc ions (Vallee, 1959). Most of them.lack specificity as evidenced by the fact that they are activated by other ions also. Some of these zinc enzyme complexes are: arginase, carnosinase, histidine deaminase, lecithinase, enolase, yeast aldolase, oxalacetic decarboxylase, serum and intestinal alkaline phosphatase, and several peptidases. In blood serum, zinc exists in at least two fractions--firmly and loosely bound zinc. The former is reported to amount to about 3h$ and the latter to 66% of the total zinc content (Vikbladh, 1951). The loosely bound complex appears to be concerned primarily with zinc trans- port. Neither substance has been shown to exhibit enzymatic properties. Determinations of zinc content and carbonic anhydrase activity of human erythrocytes have implied that they are mutually dependent vari- ables. .In a number of pathological conditions the two parameters vary in a parallel fashion, allowing the interpretation that a large per- centage of erythrocyte zinc is an integral part of carbonic anhydrase (Vallee g: 51., l9h9). Since erythrocytes are rich sources of pyridine nucleotide-dependent dehydrogenases, it is possible that the fraction of zinc.that does not form.part of the carbonic anhydrase may be assoc- iated with some of these enzymes (Vallee 93H21., 1956). Human leucocytes have been reported to contain a zinc metallo- protein; however, no enzymatic activity in this complex nor any corre- * tion betw zinc-contai: Severe cantly incrl (Underwood, z'mc-defici. 0n the e‘fita‘cly s‘m reduction :1 erythrocyte -15- lation between the zinc content of leucocytes and the activity of several zinc-containing enzymes has been demonstrated (Vallee, 1959). Several studies have shown that large oral doses of zinc signifi- cantly increase whole blood and plasma zinc in rats, rabbits, and cats (Underwood, 1962). JMiller and Miller (1962) recently reported that zinc-deficient calves exhibit a reduced blood zinc content. 0n the other hand, zinc-deficient rats and chicks have not in- evitably shown a reduction in plasma zinc concentration, nor has a reduction in blood carbonic anhydrase activity, and therefore in erythrocyte zinc, been detected in this condition (Hove gppgl., l9h0b; O'Dell gt 31., 1958). In pigs, Hoekstra 3t _a_1_. (1956) showed that adding 50 ppm of zinc to a zinc-deficient diet increased the zinc concentration of blood plasma but had no effect on the zinc content of erythrocytes. Since the report by Bertrand and Vladesco (1921), several groups of workers have confirmed the finding of high concentrations of zinc in the male sex organs and fluids of various species. The biochemical forms and functions of zinc in these glands and their secretions have not been determined. Zinc deficiency in rats (Millar 23.21., 1958, 1960) results in degeneration of the testes, hypoplasia of the coagulat- ing glands, the seminal vesicles and_pro$tate, and relative or complete decrease in the numbers of sperm in the epididymes. All changes pro- duced by zinc deficiency, except the testicular atrophy, were reversed When supplemental zinc was added to the diet. It appears that the ' impaired development of the male accessory sex organs in rats may be a result of severe inanition which in turn causes reduced gonadotropin 8ecretion from the pituitary and a consequent fall in androgen production. It is not the femal suggests iue direc (1963) pr have been several y The ' hie’h conc: EV‘31“, the (Underwoog Since “0 contain 4 9. {element -16- It is not clear why the inanition of zinc deficiency does not affect the female reproductive system in a similar manner. The evidence suggests that the testicular atrophy associated with zinc deficiency is due directly to the lack of this element. Recently,-Prasad g£_gl. (1963) presented some interesting evidence that zinc deficiency may have been responsible for the hypogonadism and dwarfism observed in several young male patients in Egypt. The tissues of the eye, especially the choroid, are known to contain high concentrations of zinc in a wide range of animal species. How- ever, the exact function of zinc in eye tissues remains unexplained (Underwood, 1962). Since the early work of Lutz (1926), skin and hair have been known to contain high zinc levels compared to most soft tissues of the body. Alopecia and parakeratotic skin lesions are characteristic of dietary zinc deficiency in all species, but consistent and conclusive evidence is lacking that these deficiency symptoms are associated with a signifi- cant reduction in the levels of zinc in the integument (Underwood, 1962). In addition to showing symptoms manifested in other species, zinc- deficient chicks exhibit an abnormal respiration and a shortening and thickening of the long bones (O'Dell gt 31,, 1958). The abnormal bone condition appears to arise from a failure of cartilage cell development in the epiphyseal plate region of the long bones and decreased osteo- blastic activity in the thin bony collar. Keinholz 33 gl. (1961) altered the development of chicken embryos by withholding zinc from the maternal diet; the major defect was grossly impaired skeletal development. These authors noted that excess dietary calcium aggravated the zinc deficiency, which is in agreement with some other studies of calcium and as reviewed Enzyme 1:1" fruitful of zinc. E remains wit Similar f in chicks (0'1: 8 reduced c Subnornal 1 Ed in zinc. PhCSPha‘case calcium.and zinc interactions in the nutrition of swine and poultry, as reviewed by Forbes (1960). Enzyme studies with zinc-deficient animals have not been particular- ly fruitful in correlating deficiency symptoms with biochemical functions of zinc. Hove‘gp'gl. (19h0b) reported that carbonic anhydrase activity remains within normal limits in the blood of zinc-deficient rats. Similar findings have been reported in the blood of zinc-deficient chicks (O'Dell'gp‘gl., 1958), but Miller and Miller (1962) demonstrated a reduced carbonic anhydrase activity in calves fed a low-zinc diet. Subnormal levels of intestinal and kidney phosphatase have been report- ed in zinc-deficient rats (Hove £3 21., l9h0a) and of serum alkaline phosphatase in parakeratotic pigs (Luecke g§‘§1., 1957; Newland gpflgl., 1958). Other workers have not been able to correlate levels of the latter enzyme with the incidence of parakeratosis in pigs (Stevenson and Earle, 1956) or with symptoms of zinc deficiency in chicks (Morrison and Sarett, 1958). More recently, however, Earle gt El' (1961) have reported decreased alkaline phosphatase activity in several different tissues of pigs suffering from parakeratosis. Need for further studies on the activity of zinc enzymes in tissues of zinc-deficient animals would seem to be indicated. Balance studies using radioactive Zn65 have been valuable in tracing the absorption, excretion, and metabolic pathways of zinc, although the results have been variable and frequently difficult to interpret. In steers, an average of 70% of an oral dose was recovered in the feces and only 0.3% in the urine, while 20% of an intravenous dose was found in the feces and 0.2% in the urine during a 6-day balance trial (Feaster 5'2 31:, 1951+: and high“Zin< intestine is mounts Via 1 In swine with the zinc that in pics atscrbed; ”hi 58% of the 21 were obtained Degree c or combinatic carbonate, ox Whereas the Peterson and not been con have all bee Parakeratosi -18- . gpugl., 195k). This pattern of excretion was followed on both normal- and high-zinc diets. Zinc that is absorbed and reexcreted into the intestine is done so chiefly via the pancreatic juice and only in minute amounts via the bile (Sheline _e_t g_l_., 1943). In swine and rats, apparent absorption of zinc varies inversely with the zinc content of the diet. Whiting and Bezeau (1958) reported that in pigs receiving 3h ppm of zinc, 21% of the zinc was apparently absorbed, while Beardsley (1958) found that in pigs fed only 9 ppm, 68% of the zinc intake was absorbed. Qualitatively similar results were obtained by Forbes and Yohe (1960) with rats. Degree of zinc absorption appears to vary with the chemical form or combination in which it is ingested. Zinc in the form of the sulfate, carbonate, oxide, or metal seems to be equally available to the chick, whereas the zinc in certain ores is poorly utilized (Edwards, 1959; Roberson and Schaible, 1960). Although similar comparative studies have not been conducted with swine, the carbonate, oxide, and sulfate forms have all been used successfully in the prevention and treatment of parakeratosis. Different protein sources also vary in their capacity to supply zinc for the needs of chicks, rats, and pigs. Soybean protein, which contains substantial amounts of phytic acid, is a less effective source of zinc for these species than casein or egg white, which contain none of this compound (O'Dell and Savage, 1960 ; Forbes and Yohe, 1960; Oberleas'gt‘gl., 1962a). The soft tissues of the body constitute a large pool of exhange- able zinc which is in equilibrium with the zinc of plasma. Feaster et_al. (195h) found that the most rapid accumulation and turnover rate of retained Zii adrenals in st kidney cortex, :rations of zi was supplement geater increa En'ard _e_': :55.“ :se from salts to treat no time dren: 3" .0: enteric d; Us ing var of retained zinc occurs in the pancreas, liver, kidney, pituitary, and adrenals in steers. In swine, Hoekstra 23.21. (1956) showed that liver, kidney cortex, spleen, and small intestine all contained similar concen- trations of zinc in pigs fed a low-zinc basal ration. When this diet was supplemented with zinc, the liver and kidney cortex exhibited a much greater increase in zinc concentration than the other organs studied. High-Level Dietaryggpperl Evvard‘§§‘_l. (1928) were perhaps the first to report a beneficial response from copper supplementation of swine rations. The use of copper salts to treat various disorders of swine is probably quite old and at one time drenching with a solution of copper sulfate was popular therapy for enteric disturbances (Luecke _e_t_ 21., 1963). Using various plates and salt bricks, Braude (19h8) demonstrated that young pigs fed a normal diet exhibited a craving for metallic copper. Mitchell (1953) reported that nursing pigs given their choice of either a basal creep meal or one supplemented with 25 gm. copper sulfate per 100 lb. of ration (150 ppm copper) overwhelmingly preferred the one containing added copper. However, in a subsequent trial (Barber $3.21., 1955b), copper supplementation failed to have any effect on either growth rate or creep feed consumption during the suckling period. ' lThe compounds»-copper sulfate, copper sulfide, and copper carbonate-- whenever used in this thesis, refer to the cupric form of copper (oxida- tion state of +2). However, the commercial copper oxide used in this thesis is a mixture of the cupric (+2) and cuprous (+1) forms. Widespl by the 8.1mm ing pigs we: copper sulfa weeks of an eight feedir growth pr one 1.! 2‘ "Vs-er et al as effective rate of pigs 5331936? Tit S In Gem 0? pigs and 1 ‘ltilization. “iainst an if i: that count 1°?me aiding a mining of J£0 gnu/tom) m'iotic. $de lamented “Cat 0f the ‘18:?! \“fl feej -20- Widespread interest in copper supplementation was precipitated by the announcement of Barber'gtwgl. (1955c) that daily gains of grow- ing pigs were improved by feeding 250 ppm of supplementary copper as copper sulfate; this improvement was established during the first 8 weeks of an 18aweek trial. In a more extensive experiment involving eight feeding centers in England, Bowler 239 _a_l. (1955) confirmed the growth promoting properties of 250 ppm of copper. Another report by Barber gt_al, (1955a) indicated that this level of copper was equally as effective as chlortetracycline in significantly increasing the growth rate of pigs, but they found no additive response from feeding the two supplements in combination. In Germany, Schurch (1956) added 230 ppm of copper to the ration of pigs and noted an increased weight gain and an improved feed utilization. He suggested that the copper may have protected the pigs against an infection which affected the controls. In more recent work in that country, Hennig (1960) did not obtain a significant response from adding 265 ppm of copper as copper sulfate to a growing pig diet consisting of potato silage plus a grain-soybean meal supplement. Barber 5.3; 9.1; (1957) confirmed their previous work by demonstrat- ing that the effect of 0.1% copper sulfate (250 ppm copper) was comparable to that Obtained from oxytetracycline (10 gm./ton) or chlortetracycline (20 gm./ton) and that there was no additive effect from copper plus an antibiotic. rLiver copper concentration of the pigs receiving copper- supplemented rations averaged 109 ppm, wet basis, which was eight times that of the controls. Smaller increases in other tissues from the supplemented pigs were observed. They also reported that the 3g; libitum feeding of rations containing 0.5% or 1.0% copper sulfate for a period of and body wei sated a sati reductiOn or In the scat allowed creaseCi rate 551 :0 resull no effect or cent j: whi: weight, Luce respcfises tc and P631311: but chlortei 333 ppm of c controls. F firing the E irg phase (J cop-per conce to have 25 1 ‘. I be“: Us. e fed cc o. “flee EfOI‘e it -21- a period of from 3 to 5 weeks caused a rapid reduction in feed intake and body weight gains. However, no mortality occurred and all pigs re- sumed a satisfactory growth rate and feed intake immediately following reduction or omission of supplemental copper from the diet. vIn the studies cited up to this point, the pigs were fed in a way that allowed some expression of appetite. In several instances the in- creased rate of gain from the use of supplemental copper sulfate appear- ed to result from an increase in feed consumption, there being little or no effect on the efficiency of feed utilization. However, in an experi- ment in which pigs were fed according to a fixed scale based on live weight, Lucas and Calder (1957a) were still able to obtain growth responses to copper supplementation. They found that copper sulfate and penicillin together were no better than either one fed separately, but chlortetracycline added to control diets already supplemented with 250 ppm of copper significantly improved performance over that of the controls. Most of the response to copper supplementation was observed during the growing period (to to 100 lb.) rather than during the finish- ing phase (100 to 200 1b.). These investigators also determined liver copper concentrations. On a dry matter basis, they found the controls to have 25 ppm; the pigs fed copper from #0 to 100 1b., 82 ppm; and those fed copper from ho to 200 1b., 506 ppm of copper in the liver. Therefore it was possible to markedly reduce accumulation of liver copper by removing the copper supplement after 100 lb. live weight. In a subsequent study,-Lucas and Calder (1957b) fed five levels of copper sulfate, ranging from 0.012% (31 ppm copper) to 0.2% (500 ppm copper), to pigs from weaning to market weight. Before 100 1b., the highest levels of copper caused the greatest improvements in rate of gain a copper le feed effi the entir bignest a approach : copper), on a dry I have 155:3 I its the 0. increases an al‘itl'me Lucas observed t -22- of gain and feed efficiency. Between 100 and 200 lbs, none.of.the- copper levels improved performance,and reductions in rate of gain and feed efficiency with the 0.2% level suggested marginal toxicity. Over 'the entire experimental period, rate of gain and feed efficiency were highest at the 0.05% level, but overall treatment differences did not approach statistical significance. Up to the 0.025% level (62 ppm copper), there was no increase in liver copper over that of the controls. On a dry matter basis, pigs receiving 0.05% copper sulfate were found to have 163 ppm of liver copper; those fed 0.1%, 575 ppm; and those receiv- ing the 0.2% level, 3,085 ppm. Thus, above a certain level, further increases in dietary c0pper appear to result in a geometric, rather than an arithmetic, increase in concentration of copper in the liver. Lucas gpugl. (1961), in a continuation of their earlier work, observed that growing pigs fed rations supplemented with 250 ppm of copper gained more rapidly than those supplemented with 16, 62, or 125 ppm. .As before, copper additions had no influence on performance from 100 to 200 1b. live weight. Comparing this trial with the pre- vious study (Lucas and Calder, 1957b), it was concluded that in pigs fed according to a fixed scale the greatest consistent increases in growth rate and feed efficiency occurred at the 250 ppm level of copper supplementation. In the Netherlands, Darmners go 31, (1959) have observed an im- proved rate of gain and feed conversion from high levels of copper supplementation. They found the response was greater with 250 than with 125 ppm of copper as copper sulfate, but there was little dif- ference between 250 and-187 ppm. A Tasmanian study conducted by Fagan.§p El. (1961) demonstrated crease ‘ SIZHCTS t u (D O t 9 1‘ larger 0 a . pen era: .7“ ,~ 4. :‘Cl.url rat ‘ r -23- that 250 ppm of copper in the sulfate form increased the growth rate of pigs in nine out of ten experiments but the magnitude of this in- crease was statistically significant in only one experiment. The authors indicated that under Tasmanian conditions of pig feeding the use of high-level copper supplementation is associated with some danger of copper toxicity. In oneof their trials a pig died from perforation of the digestive tract, which they believed was due to the ingestion of a particle of copper sulfate as a result of inadequate mixing of the ration. Barber 2: El° (1960) reexamined the effects of copper sulfate (250 ppm copper) and antibiotics fed separately or in combination. -Pooled results from three experiments Showed that copper sulfate alone significantly increased growth rate, feed consumption, and feed efficiency. Oxytetracycline or chlortetracycline, added to a diet already supplemented with copper sulfate, significantly increased growth rate and feed efficiency but not feed consumption. More recently, however, a coordinated trial carried out at 21 centers revealed that copper sulfate and oxytetracycline stimulated perfor- mance when fed alone but that there was no further improvement when both supplements were fed together (Braude $3.21,,l962). It is in- teresting to note that in the latter trial copper sulfate stimulated growth rate and feed efficiency significantly greater than did the antibiotic. Bellis (1961) concluded from his study that the mode of action of copper and antibiotics is similar, and that in general farm practice in England there is unlikely to be any economic advantage in supple- menting growing pig diets containing 250 ppm copper with chlortetra- cycline. T1 gay sometime joint apple In this have been sc ed by invest reported by alone and i: 1 . v‘: +18 n U‘ ~01"?- r511 -2h- cycline. The author suggested, however, that the bacterial environment may sometimes be such that additional benefit could be obtained by joint supplementation. In this country, effects of feeding high levels of c0pper to swine have been somewhat more variable and less conclusive than those report- ed by investigators in Great Britain. .A study at the Arkansas station reported by Scott (1958), involving levels of 50 and 250 ppm of copper alone and in combination with chlortetracycline and/or brewers dried yeast, showed that only the antibiotic increased gains significantly. Investigators at the Florida station (Wallace gp‘gl., 1960) observed that copper levels of 250 ppm or higher proved toxic and that consistent improvement in performance was not obtained at any level of copper supplementation. It was shown that elevating the protein content of the corn-soybean meal basal diet from 15 to 20 or 25% significantly reduced the toxic effects of 750 ppm of supplemental copper; this agrees with the observations oancCall and Davis (1961) in the rat. Hawbaker 33 El. (1961), on the other hand, reported a significant improvement in growth rate of pigs fed supplemental copper sulfate, and no toxicity was observed. They found that 250 ppm copper was the Optimum level for maximal response in growth rate and feed conversion. A combination of copper sulfate and antibiotic produced an additive effect with respect to rate of gain, but no such response was obtained when copper sulfate and an arsenical were fed jointly. Liver copper values for the copper-fed pigs were 10 times higher (293 ppm) than those of the untreated controls but there was no significant difference in loin copper values. In comparing the seemingly divergent results of the Iowa and Florida studies, it may be of some significance that in all but the than tho: associate had a sig They sugg supplenen animals. In f: the effecil baby pig I COpper) tc COPPGI‘ ox: 1n improx'rj' but the instance the Iowa trials were of shorter duration (h2-56 days) than those of the Florida workers (70-82 days). Hawbaker and his associates also made fecal flora counts and reported that copper sulfate had a significant effect on the numbers of certain types of microorganisms. They suggested that the alteratiOns in the fecal microflora of cOpper- supplemented pigs might account for the improved performance of these animals. In further work at the Iowa station, Bunch gp‘gl. (1961) compared the effects of copper sulfate, copper oxide, and chlortetracycline on baby pig performance. They again found 0.1% copper sulfate (250 ppm copper) to be the optimum supplemental level for this form of copper. Copper oxide compared favorably with similar levels of copper sulfate in improving performance. Moreover, copper oxide had the advantage of depositing much less copper in the tissues (liver and loin) than the sulfate form. Copper sulfate exhibited the further disadvantage of depressing hemoglobin concentrations when added to the diet at the rate of 250 ppm copper or higher. These results confirmed those of Hawbaker 23H2i° (1959), who showed that the copper radical, and not the sulfate radical of copper sulfate, was producing the growth res- ponse. Allen gp_§l. (1961) studied various aspects of high-level copper supplementation and found that copper carbonate was equally as effective as copper.su1fate in improving performance of growing pigs. Buescher .23.2le (1961) conducted a radiocopper balance experiment in which they found that the availability of copper whether as sulfate, carbonate, or oxide was similar for swine. Barber 23 El. (1961) compared copper sulfate with copper sulfide (013), a powing I sulfate i stores of ra#: L‘Cn €er as -26- (CuS), a very insoluble compound, and reported that the response of growing pigs to sulfide was much less than that to the sulfate. The sulfate form, but not sulfide, significantly increased liver and kidney stores of copper. It was suggested that the effectiveness of copper in promoting growth is related to the amount of soluble copper in the gut, but whether the site of action is systemic, enteric, or both, remains unknown. Using labelled copper compounds in a balance study, Bowland gingl. (1961) proved conclusively that a higher percentage of copper sulfate is absorbed from the gastrointestinal tract than copper sulfide. In an attempt to resolve the discrepancies reported in the response of pigs to copper supplementation in the United Kingdom and the United States, Lucas 33 El. (1962a) compared the effect of adding copper sulfate to a barley-fish meal diet and a corn-soybean meal diet. The former ingredients are commonly used in the U. K. and other European countries, while the latter are basic components of U. S. swine rations. Both diets were hand-fed according to a fixed scale based on live weight. Copper sulfate fed at a rate of 250 ppm of supplemental copper similarly stimulated growth rate of pigs on either ration from no to 100 lb..1ive weight. From 100 1b. to market weight, copper sulfate added to either ration exerted no influence on rate of gain. The copper supplement slightly improved the efficiency of utilization of the barley-fish meal ration but had no effect on the corn-soy diet. Barber §p_gg, (1962) designed a similar experiment in which they compared copper supplementation of barley-fish meal and barley-soybean meal rations. In contrast to the results described above, these workers Observed a significantly greater response when copper was added IE on 4" iv Us; & to ‘ £119 More an» C v 59",“- vbu‘w a . I’D“ to»... ‘f r U.‘ .. to the diet containing fish meal than when added to the all-vegetable diet. In this trial, however, the pigs were not restricted but fed to appetite. Copper-supplemented pigs fed the fish meal diet consumed more feed than those on the soybean meal ration but this alone did not account for the difference in growth rate. This same research group had previously shown that the response to copper is slightly greater when pigs are fed ad libitum than when they are hand-fed (Allen gt 21., 1961). King (1960) studied the effect of environmental temperature on the response to copper sulfate and antibiotics. He found the response to these growth stimulants was greater when pigs were maintained at 5&0 F. than when they were kept at 650 F. In a more recent experiment, King (1963) compared the response to copper sulfate of pigs fed two dif- ferent levels of water mixed with their feed. He concluded that the level of added water had no significant influence on the response to supplemental copper. The effect of 0.1% copper sulfate in the diet of earlyaweaned pigs was investigated by Lucas 23.21. (1962b). They found that the perfor- mance of pigs weaned at 10 days of age and fed to MS lb. live weight was improved by the feeding of copper sulfate. When chlortetracycline was added to the copper-supplemented ration, there was a slight additive response. In this country, Meade 23 El° (1961) also reported a response from adding 50 to 200 ppm of copper as copper sulfate to the diet of earlyaweaned pigs. They found that pigs weaned at 23 days of age and fed a copper-supplemented ration for #6 days gained lO-l5% faster than the controls; there was a further small improvement from including oxytetracycline in the diet. Bunch strating t1 tility to From 125 t while coppe [K :zese inve: -r3.:’ 00‘ ‘W -.-.‘V - ‘ a “ "I :U. \A‘ x fiA :Vsbdh‘nc "“b -28- Bunch 23 El. (1963) recently confirmed their earlier work by demon- strating that cOpper oxide and c0pper sulfate are similar in their ability to stimulate weight gains of pigs from 1h to 125 lb. live weight. From 125 to 200 1b., however, c0pper sulfate slightly depressed gains while copper oxide increased daily gain compared to the control pigs. These investigators also confirmed their previous finding that pigs fed copper sulfate deposit significantly higher concentrations of copper in the liver than do those fed the oxide form; on the other hand, they found no difference in the COpper content of the loin muscle. In other work in the U..S., Miller and Barnhart:(l961).reported that 0.1% copper sulfate resulted in a considerable improvement in daily gain and that it was comparable to various antibiotics in this respect. A search of the literature indicates that Ogilvie (l9h2) was probably the first to describe what was suspected of being copper poisoning in pigs. Nothing further was reported for several years until Gordon and Luke (1957) found evidence of an outbreak in which it was known that the feed had been supplemented with COpper sulfate, although it was not possible to ascertain the amount of copper that had been fed. Since then, several reports of copper toxicity have paralleled the increased use of copper sulfate as a growth stimulant in the United Kingdom and elsewhere. Indications of marginal toxicity and liver copper levels up to 3,000 ppm were reported by Dammers E: El“ (1959) in pigs fed wet meal containing0.3% copper sulfate. Two of these livers had a grayish- brown appearance and showed some connective tissue proliferation. The pigs on this ration grew slowly. .At levels of supplementation of -29- 0.2% and below, no toxicity was apparent. O'Hara 23 El. (1960) in Australia reported the occurrence of copper poisoning in young pigs receiving a diet supplemented with copper sulfate at the rate of 250 ppm c0pper for a period of 16 weeks. Buntain (1961) gave a detailed account of the death of 23 fattening pigs and condemnation of several carcasses in a herd of 200 pigs fed a diet containing approximately 130 ppm of copper. Livers and kidneys had up to 2,200 and 950 ppm copper, respectively, compared with normal values of about 50 and 25 ppm. It was suggested that the dangerously high copper levels found in these organs from all pigs examined, to- gether with some other unidentified toxic factor in the diet, may have caused the losses. Allcroft 22H2l° (1961) compared diets containing various levels of added copper sulfate fed ad libitum.to pigs from weaning to market weight. No significant increase in growth rate was obtained on diets containing 0.06 to 0.16% supplemental copper sulfate; additions of 0.2 and 0.h% reduced weight gains significantly, caused jaundice and the death of three out of seven pigs. In spite of careful mixing, these workers found wide variations in the copper content of the feed at each level of supplementation. Liver copper accumulation increased sharply on diets containing more then 0.06% added copper sulfate but no toxic symptoms were produced up to the 0.16% level. The authors con- cluded from their results that while high liver copper levels pg; g3 do not necessarily produce toxic symptoms it appears that above a certain liver concentration, other factors may precipitate a syndrome attributable to copper toxicity. Allen and Harding (1962) experimentally poisoned pigs by feeding v diets contai oxide, or cc signs were , . " ‘:R‘ a Ar)“ 1‘3 “i "‘"J v- ’0 3'- ’7‘ ‘V‘Tr bcCuueu U‘bb H LEQa ‘ in ..- .. ”‘- fla’fln 6 -uk ub“1‘;e 32.1 :u\ -30- diets containing up to 1,000 ppm of copper as copper sulfate, copper oxide, or commercial mineral mixture. The first indication of toxicity was a rise in blood copper concentration. .The chief clinical signs were jaundice, dullness, weakness, anemia, trembling, and respir- atory distress. The principal macroscopic lesions were jaundice, poorly clotted blood, yellOw to orange liver, pulmonary edema, ulceration of the esophageal zone of the stomach, and blood-stained intestinal contents. Distinctive histological changes were found in the liver and kidneys. Similar findings have been reported in one or more of the studies re- ferred to previously. .Allen and Harding believed the cause of death in the poisoned pigs to have been the anemia brought about by intravascular hemolysis and loss of blood into the alimentary tract. Hemingway (1962) and others have criticized Allen and Harding's paper because the work was based on only one pig per treatment. In the first of two trials, Bunch gt El° (1962) reported that copper carbonate added to the diet at levels of 250 and 500 ppm copper depressed gains and hemoglobin concentrations. In the second trial, pigs fed the 250 ppm copper gained faster and exhibited higher hemoglobin levels than either the controls or those receiving the 500 ppm of copper. Pigs fed the highest level of copper carbonate showed lower weight gains and depressed levels of hemoglobin, ceruloplasmin,and liver iron compared to the other two groups; they also had significantly higher concentrations of liver cOpper. Zinc and Porcine Parakeratosis According to Kernkamp and Ferrin (1953), the syndrome which they called parakeratosis was widespread among swine herds in this country. Tcey descr and sugges characteri these incr :cs': sever Depenil-.g ‘ . Q :Larrnea. I ‘. ‘0 V ...e liseas 2.2K: 31113 ie“? ~ ‘01“: -31- They described the gross symptoms and histopathology of this disease and suggested that it was of nutritional origin. The condition is characterized by a dermatosis in which the skin becomes thickened and heavily incrusted, with deep fissures breaking the incrusted areas; these incrustations begin on the ventral abdominal wall and become most severe in the regions of the legs, thighs, ears, and head. Depending upon the severity of the disease, it is often accompanied by diarrhea, anorexia, and retardation of growth. .In extremely severe cases the disease occasionally terminates in death, but in mild or moderately affected pigs spontaneous recovery may eventually take place. Parakera- tosis has been observed primarily in swine receiving rations composed of corn and a vegetable protein supplement. Such rations have often, but not always, been fortified with a mineral mixture to the extent that the total calcium content of the diet was in excess of the pub- lished requirement. This disease is most apt to occur in young rapidly growing pigs after they have been weaned. Raper and Curtin (1953) reported that when supplemental cObalt and zinc were added to a corn—cottonseed meal ration no symptom of the disease was observed. Then, Tucker and Salmon (1955) made the important discovery that symptoms of parakeratosis could be prevented or cured by zinc supplementation. In addition, these investigators showed that high levels of calcium and/or phosphorus aggravated the condition. Their work prompted studies elsewhere concerning the influence of various nutritional factors, particularly zinc, on parakeratosis in swine. Luecke SEMEl° (1956) were among the first to confirm the fact that zinc supplementation (20 ppm as zinc carbonate) prevents parakeratosis and that raising the calcium level of the diet increases the incidence of the di added 50 from 0.8 prevent t but a Sieve cases of 1 a I"? V? & ' 4.1. l" (,0 f \ ‘A: :U‘A.“ 0‘1.‘ ‘4“, I! “A «tI‘al " -32- of the disease. Lewis g _a_il_. (1956) reported similar results when they added 50 ppm of zinc in sulfate form to corn-soy rations that ranged from 0.8 to 1.h% calcium. .This level of zinc supplementation did not prevent the appearance of parakeratosis in all of the experimental pigs, but a level of 100 ppm was completely effective. The latter workers also demonstrated the therapeutic effect of zinc treatment on established cases of parakeratosis; pigs injected with zinc sulfate responded simi- larly to those fed this compound, but the injections caused severe local irritation. Phosphorus supplementation had no significant effect on weight gains but significantly decreased skin lesions when added to the 0.8% calcium.basal ration. Bellis and Philp (1957) have reported similar findings. This would appear to vindicate phosphorus as a causative-agent in the parakeratotic syndrome. Hoekstra‘gp El. (1956) observed that 50 ppm of supplemental zinc as zinc sulfate had no effect on the zinc content of erythrocytes, spleen, intestine, or pancreas of pigs fed either a normal (0.8%) or high (l.h%) calcium ration. But zinc additions did increase the con- centration of zinc in blood plasma, liver, and kidney. As the authors point out, the latter results could represent either a return to "normal" concentration or simply an increase due to excess dietary zinc. They declined to speculate as to which of these alternatives may be the more plausible one.) However, their plasma zinc values for-10 "normal" pigs would suggest that the parakeratotic pigs were subnormal in this respects. The.high-calcium ration had.little effect on tissue zinc elevels when no additional zinc was fed, but when supplemental zinc was added the zinc concentations of liver and kidney were decreased compared with those from pigs fed the normal-calciumeplus-zinc diet. Excess calc i1 -‘png-e‘; web‘- rn is 9:- ...an a r afi‘:qr~°-‘ ”9-8.1.,1 V". L and did I calcium.did not appear to influence the pH of the intestinal tract and thereby reduce the absorption of zinc. Stevenson and Earle (1956) concluded from their studies on paraker- atosis that in diets for growing pigs which contain up to 1.0% calcium, the minimum zinc content for prevention of this disease is between hh and 80 ppm. -They found depressed levels of hemoglobin, serum inorganic phosphorus and blood sugar, and a shift in serum.albumin-globulin ratio in parakeratotic pigs. The depression of various blood components may be a reflection of the inanition which accompanies parakeratosis rather than a result of zinc deficiency pg; g3. Serum alkaline phosphatase activity was more variable among parakeratotic than among normal pigs and did not appear to be related to gross symptoms of the disease. 0n the other.hand,‘:luecke.‘_e_t_.'g;.y(1957)'fand Newland. 33:31, (1958) found that the activity of this enzyme was lowered in pigs afflicted with para- keratosis and elevated in zinc-supplemented pigs. Luecke g} 3g,.(l957) were surprised to find that pigs receiving a ration which contained only 0.5% calcium, a level that is below.N.R.C. (1959) recommendations for growing pigs, exhibited a h0% incidence of parakeratosis. In a previous trial, pigs fed a diet containing 0.65% calcium had shown an incidence of only 10%. The explanation for these somewhat incompatible observations may at least partially rest on the fact that the low—calcium ration contained less zinc than the normal- calcium diet (32 vs. h5 ppm). ,It was evident from this experiment that low calcium levels are no insurance against parakeratosis and that supplementation of the ration with zinc is a more effective means of prevention. Similar results were obtained by Lewis 23 El° (1957b). They concluded that zinc supplementation appears to be a much more -3h- effective method of controlling parakeratosis than limiting calcium in- take below National Research Council recommendations. Their investi- gations also revealed that when the amount of calcium in rations is increased from 0.5% to 0.8% and then to 1.2%, the zinc concentrations in bone, hair, kidney, and liver declined significantly, but the zinc of plasma, pancreas, skin, and intestine did not show a similar change. They surmised that excessive amounts of calcium probably reduced the zinc content of tissues by hindering zinc absorption from the gastro- intestinal tract. No significant increase in the zinc concentration of the skin, where parakeratotic lesions occur, was noted when the zinc content of the ration was increased to 128 or to 1,028 ppm. How- ever, these amounts of supplemental zinc produced varying degrees of increase in the zinc concentration of pancreas, liver, hair, bone, blood plasma, kidney, and intestine. It was suggested that none of the body components analyzed represent the site at which zinc exerts its beneficial effect on parakeratosis. In a subsequent trial, Lewis gt 3;. (1957a) demonstrated that zinc was readily removed from an ip_zip£g solution during the precipitation of calcium phosphates brought about by increaSing from 3 to 6 the pH Of solutions of calcium, phosphate, and zinc. These conditions were said to simulate somewhat the change in pH of ingesta during its passage from the pig's stomach to his small intestine. Increasing the Ca:P ratio of these solutions markedly increased the amount of zinc removed from solution. The authors suggested that this type of phenomenon may explain the detrimental effect of a high-calcium diet on parakeratosis. In this same experiment, they also observed that method of feeding influenced the severity of parakeratosis induced by feeding a high- -F J“ (“3... ..v-‘ n. - us a C ‘Q ‘7‘ s a n. a n1 ‘ ~ ‘ as... ‘ . Vs'fi -35.... calcium ration. §g_libitum feeding of the dry diet was more detrimental than hand-feeding the same ration wet; however, the latter method by no means prevented parakeratosis. .In more recent work, the Wisconsin group (Smith 23 21., 1960a) autoclaved a high-calcium diet in order to rule out the possibility that any infectious agent in the feed might influence the development of parakeratosis. Cu:>Fe. At the final bleeding, the treatment differences were not as great which may have been a reflection of the general improvement in the condition of the parakeratotic pigs. In general, the Skin lesions were most severe at about 8 weeks, followed by some recovery even though no therapeutic measures were taken. -67- Total serum.protein and the albumin and gamma globulin fractions 'were influenced in a significant manner by all trace element additions TABLE # Exp. I, Trial B. -- Effect of supplemental zinc, iron, and copper on serum protein values Lot no. and treatmenta Std. 39 #0 #1 #2 #37 ## error Basal of Item (1.05% Ca) +Zn +Fe +Zn+Fe +Cu +Zn+Cu meansb 53-day values Total serum protein, gm./100 ml.c 7.#2 6.99 6.88 6.62 6.79 6.71 0.19# Albumin, %(1 32.0 #0.9 39.1 #0.# #3.2 #2.0 2.28 cxglobulin, % 2#.5 23.9 23.6 25.5 22.6 22.# 1.22 Bglobulin, % 12.1 10.6 11.3 12.5 12.1 10.9 0.69 Yglobulin, %e 31.# 2#.6 25.9 22.8 22.1 2#.7 1.7# Final (98-day) values Total serum protein, gm./100 ml. 7.#6 7.00 7.11 7.05 7.09 7.03 0.1#2 Albumin, %8 27.0 #8.0 37.7 #6.8 ##.2 #8.2 2.2# O‘globulin, % 21.3 17.3 21.5 19.# 19.7 18.3 1.30 .Bglobulin, % 13.0 12.9 12.3 12.# 12.0 12.6 0.71 Yglobulin, %h 38.6 21.8 28.5 21.# 2#.0 21.0 2.00 aZinc added at rate of 50 ppm; iron, 100 ppm; and copper, 125 ppm. Based on 38 degrees of freedom. 0Lot 39 significantly greater than #2, #3, and ## (Pr<.05). ( C1I..;t 39 significantly less than #3, ## (P<.01), and #0, #1, #2 P«<.05 . eLot 39 Si ificantly greater than all other lots: #2, #3 (P (.01), and #p, #1, ## flaw). Lot 39 significantly greater than #0 (P (.05). 8Lot 39 significantly less than all other lots (P<.Ol). Lot #1 significantly less than #0, #2, ## (P<.01), and #3 (P<.05). Lot 39 significantly greater than all other lots (P<.Ol). Lot #1 Significantly greater than #0, #2, and ## (P<:.05). as indicated in table #. At 53 days all supplemented lots exhibited a lower percentage of gamma globulins and a higher percentage of albumin -68- than the basal pigs. The net result was a Significantly higher total serum protein in the basal lot. Final values revealed a further increase in the gamma globulins and a decrease in the albumin fraction in the basal and iron-fed (lot #1) pigs, whereas the reverse was true in the remainder of the lots. Stevenson and Earle (1956) and Smith 33 El- (1960b) likewise have noted a reduction in the albumin:globulin ratio of parakeratotic pigs as compared to animals fed an adequate level of zinc. Except for the iron lot (#1), all of the supplemented lots in the present trial exhibited serum protein values that compare favorably with those reported by Miller £3 21. (1961) for healthy pigs of comparable ages. None of the experimental treatments had an appreciable influence on either the beta or alpha globulin fractions. This too is in agreement with Smith EEwEl° (1960b). Table 5 Shows the results of the trace element analyses of the livers. Also included are values obtained from the livers of 10 healthy, presumably "normal," pigs fed low-calcium diets (mean, O.##% Ca) with no trace element supplementation. The pigs fed 125 ppm of c0pper alone (lot #3) exhibited a significantly higher (P<:.Ol) concentration of copper in the liver than those in the remaining lots. There was apparently an interaction between copper and zinc, for the mean liver copper concen- tration of lot ## was not significantly higher than that of lots re- ceiving no additional copper. It was not surprising to find that 50 ppm of additional zinc Significantly increased (P<:.01) the liver zinc con- centrations in all lots that received the zinc supplement. However, neither copper nor iron supplementation had a significant effect on liver zinc levels. Thus it appears that the role of copper in pre- venting parakeratosis may not be explained on the basis of an ability -69- .300 5 or can 30V .3 d- ssfi aossoam Essa-oneness .3 p3 .30.va me one 29v .3 13 sons. moo-H afiessoaeasman ms boa .AHo.v_mv ms paooxo msoa aosso Has sons nnoa sasssosaAcmsn mm eon-r .AHo.V.mv nsoa cones sonso sons soomoam aasssoaoasman as one .ms .0: macaw .AHo.V.mV nsoa Assoc Has ones soosoam sasssoseasman ms soar .pan03 hoop Mom oopooaaoo can: moocoaoMMflo pomoflmflawflm Ozm .Edwoamo &::.o dowwao>o noes: mpmflo come .Eoomonm mo mooamoo mm so domwmo .Emm mmH .aommoo was mean OOH aooafl mama Om mo open pm wooed oowmn .mfimon pomflozihad «Sam ca commoamxmd Pauper m.mm one was New mmm mmm mam seam .soAH Haunts m.aa mam mos 0mm was emm moa mama .osam Humm m.sa mm ems ma NH ea ma page .aoaaoo -- m.mm . Hema mesa omma mafia Hmma ram o.em -.ea ao>sq oa .. m m e a e a mafia .oz mmwm omomma do+oN+ 50+ om+oN+ om+ oN+ lNoo fimo.av EopH saswhoos mo Homom Ito songs as me we Hen. 0:1» mm mm + sooz .opm ppmofipmwae one .os poq omam>wa .mwfim mo poopooo pooaoao momap so ammnoo poo «ooafl .oofiu Hopooaoammdm mo pummem I: .m awake «H .mxm m mumda to influence zinc storage, at least within the liver. However, in a later trial (Exp. II, trial B) it will be seen that in one instance, copper supplementation resulted in a significant increase in liver zinc (see table 13). In light of reports published in recent years, the liver iron values in this trial are rather difficult to interpret. As mentioned in the REVIEW OF LITERATURE section, studies in this country have in- dicated there is a loss of liver iron when additional zinc is fed; however, it should be noted that the levels of supplemental zinc were much higher than in the present investigation. In Great Britain, Cassidy and Eva (1958b) have observed a reduction in liver iron as dietary copper level is increased. Neither of these phenomena occurred in this trial. There was in fact a significant increase (P<{.01) in liver iron concentration over that of the basal pigs in all supplemented lots with the exception of the pigs fed only additional copper (lot #3). The increase was especially marked in the lots fed zinc in conjunction with copper or iron. One may surmise that this was merely a reflection of a decreased ability on the part of the debilitated basal pigs to assimilate dietary iron. This conjecture is supported by the fact that the mean liver iron level of the basal lot was less than half that of the "normal" pigs. On the other hand, it may be argued that when iron was added to the basal diet in lot #1, the "subnormal" parakera- totic pigs in that lot were able to absorb and store the supplemental iron to a considerable extent. Trial C Results for trial C are reported in table 6. At the low-calcium level there was a nonsignificant improvement in rate of gain when either -71- zinc (lot #6) or copper (lot #7) was added to the ration. Only one mild case of parakeratosis developed in the basal lot (#5) and none in the zinc- or copper-supplemented groups. This is in contrast to the high incidence of severe parakeratosis that developed in lot 31 (trial A) on a very Similar basal ration. »Extreme variation in the incidence of parakeratosis and performance of pigs fed seemingly identical rations has been reported by Hoefer and Pond (1961). Pigs receiving the high-calcium basal ration (lot #8) made very poor growth and all but one pig in this lot were afflicted with severe parakeratosis. Supplementation of this diet with 75 ppm of zinc (lot #9) resulted in improved growth rate (P<(.01) and complete suppression of the dermatosis. When 125 ppm of copper were added to the high-calcium basal ration (lot 50), the effect on growth rate was less than that of supplemental zinc but still highly significant (Poaoa wfim mooo .Eoomoam mo mooawoo N: no oomomo .mH poo ma mpoa ovofi mace: OH Hopmm ooofi>flo mm: AHmmmpv H pogo .899 CH mam ocHN one hommoo mo mCOfipfloom HweooEoHQQSm damn -- om.m aH.m mm.m Hm.m Hm.m :. -- sssm\eooa ero.o mm.a mm.a mm.a Hm.a oa.a -- mm.o n.oH .sasm aassm .mxz mano .oooofisomaom -- o o O NH mm mm we cross ma -- o o o o o OOH me memos oa & .mflmopoaoxohom -- om.m aw.m em.s mm.s om.m se.m ma.m casw\eooa sma.o ms.a pm.a ss.a mm.a Hm.a rm.a se.o m.oa .sanw eases -- s.epa e.opa H.wma m.mma m.osa o.moa m.pw .oH ..e: roostma -- m.maa H.mHH s.eoa m.oaa s.mm w.em m.ow e.oa -.ss roos.oa n: m m m we we a : mwflm .oz cosmos 0mm 50+ mma 56+ ooa 0mm mma 0mm Ase em.av sosH mo ooa eN+ ooa oN+ oN + so + so + do + Hmmmm .HOHHm m m J m N am: Di .Upm mpcoEpmoap com .0: 90A . - Axooa opmHuov Hmflpp mafipoo pom pom Axum: QPmHISPOHV . mxooz m pmma Moe moomanmaom Mag :0 ooww pow Hommoo HwPooEoHamom mo poowmm I: .< Hofihe .HH .mxm m mumdfi -ya- skin lesions and they were very mild. However, several pigs in this lot exhibited symptoms of copper toxicity--severe anemia, internal hemorrhages, jaundice, yellow cirrhotic livers, gastric ulceration, loss of weight, weakness, and incoordination. These symptoms resemble those reported by other workers (see REVIEW OF LITERATURE section). Two pigs in lot 3 died suddenly during the 13th week and another died after the 15th week. Although accurate diagnoses were difficult because of extensive post-mortem changes, it appeared likely that these three pigs died as a result of c0pper toxicosis. Two other pigs in this lot were close to death when they were submitted for autopsy after the 15th week. There was little doubt that these two pigs were suffering from the toxic effects of copper. Dyspnea was observed in one of these pigs after being driven a Short distance. A similar observation was made by O'Hara §p_al. (1960) who reported respiratory distress in pigs suffering from copper toxicity as a result of feeding 250 ppm of copper. In the latter study, the authors noted a sudden liberation of copper into the blood and proposed that this was associated with the development of a severe anemia which in turn resulted in anoxia, circulatory failure, and death. One of the three remaining pigs in lot 3 had an enlarged cirrhotic liver, and the carcass was condemmed by the inspecting veterinarian due to a pronounced, generalized icterus. The other two pigs appeared normal in every respect except for the "muddy" color of their livers. Studies at the Florida station have demonstrated that copper toxicity in rats (McCall and Davis, 1961) and swine (Wallace gp‘al., 1960) may be alleviated by increasing the protein content of the ration to relatively high levels. Perhaps some degree of protection against copper toxicity was removed from lot 3 when their dietary protein was -79... reduced to 12.5% after the 12th week. It was apparent in this trial that zinc offered considerable protection against copper toxicity Since none of the pigs in lot 6 showed any signs of toxic effects. Furthermore, there was no evidence of toxicity in pigs receiving 125 ppm of copper, with or without zinc (lots 2 and 5). Had the trial continued for a longer period of time, it would have been enlightening to observe if and when the pigs in lot 1B (250 ppm Cu) might have Shown symptoms of copper poisoning. Results of this trial would seem to confirm the data of Wallace §p_al. (1960) which suggest that the margin between safe and toxic copper levels is relatively narrow. It is also apparent that effects of feeding high—copper levels should not be evaluated on the basis of short-term trials.- For example, copper toxicity in lot 3 and the onset of parakeratosis in lot 2 would not have been observed had the present trial been terminated after the first period (10 weeks). A summary of the entire trial in table 8 shows there was a trend for improved growth rate as the dietary copper level was increased and as zinc was added to the ration. The animals in lot 6 gained signifi- cantly faster (P<.05) than those in lot 2, and the difference in gain between lots 2 and 5 approached Significance. Feed conversion ratios for the supplemented lots were virtually identical. The effect of copper therapy on parakeratosis is illustrated in figure 1. These two pigs were typical of their respective lots, both at 10 and at 15 weeks. Hematological data of lots LA through 6 may be compared in table 9. Hemogldbin and hematocrit values of pigs in lot 3 were significantly lower than those of the other treatments, which was probably a reflection .QH mm no.3 comp panoz 2H mooohommflo ".3603 m .Hom hog-moo HmpooEonm-sm mo SHE Omw pom we: pomHH so mHm oHHg-s. «confirm. Hammp HEM op op ooosHp-Loo pmoH co mHm .mxomz OH now P036 Ham-mp ooHNIEOH .eBHononewH: mnH>Hoooh .833 TH OB mean 93 woowHoS menH apom .mHmopmHodem-H so hmmnoonv hoamoo mo Poommo HmOH-EHB .i :4. HmHMB «HH dam .H MmbOHh -81- of the copper toxicity prevalent in that lot. Although the differences between the other lots were not statistically Significant, there was a tendency for hemoglobin and hematocrit values to decline as dietary copper was increased, and to rise when the ration was supplemented with zinc. Bunch pp 21. (1961, 1963) and Wallace E£.§l' (1960) have also shown that hemoglobin concentrations are depressed by highecopper diets. However, neither group of investigators found zinc to be of benefit in altering the subnormal hemoglobin levels found in pigs fed 200 or 250 ppm of supplemental COpper even though weight gains were stimulated slightly. Simek 23 El: (1961) have reported that pigs fed high levels of copper sulfate exhibited lower hemoglobin concentrations but higher hemato- crits. This is contradictory to the present data except for lots 1A and 1B, where such a relationship was found. Table 10 summarizes the results of the trace element analyses of the livers. Individual values are given in appendix table 5. Table 10 Shows that by adding 125 or 250 ppm of copper to the basal ration, liver copper levels were increased 11- and 60-fold, respectively. Concentration of liver copper in lot 3 was comparable to levels (68# to 1,800 ppm) observed by O'Hara 23 a1. (1960) in the livers of pigs suffering from chronic copper poisoning. Buntain (1961) and Gordon and Luke (1957) have found liver copper levels of 710 to 2,200 ppm and 2,160 to 2,500 ppm, respectively, in pigs that apparently died as a result of copper toxicity. Allcroft 22.2l' (1961) reported a mean liver copper level of 1,725 ppm in pigs fed a diet containing 0.1% copper sulfate for approximately 5 months. On the other hand, Iowa investigators (Hawbaker :3 al., 1961; Bunch gp_g;., 1961, 1963) have ~82- .SOVO m J .m ens .AmOVaV O .3 :3 ”mPOH Hospo HHm some Hoon thCOOHwHome m pogo .AHO.u.mV O .m .s .m one AmO.uva ea ooH cone soSoH easesosescwsm m sons .Eonooaw wo mooawmp Om so ommmmo .mH Ooh HO mp3 AHmmmn .Hpoqn .Emm sH mam ocHN Ono smmmoo mo mcoHpHOOm HmpcoEon om HHOH sz3 com mmHm Mo hWOHOmeom I: .< HmHaB .HH..mxm . m mqmde -83- .Amo.uvmv m eoH omen soesosm aHossOHeHsmHm O eon .AHO.V av m poo mH coop Hopooam thomOHmHomHm m Ono-¢H mPoH .AHO.uvmV O Oct Am .mH coop Hopmoaw thomOHmHome m 90H .AHO.u.mv m use am am AmH aaH coop aopmosw aHpooOHmHomHm : 90H: .AHO.uvmv mpoH Honpo soap smpnosm aHOSOOHmHome O can .m A: macaw .AHO.Uvmv m use «O «OH crop Hopmoaw hHPomOHmHome O ass .m .mH noon .AHO.uvmv neoH noeeo HHm sons soomoam aHsssOHaHsmHn m eoHa .PQwHoS muon How Ompomaaoo one: moooosommHO HomOHmHome ozo .Eowmoaa mo mooawov O: so Oommmo .mH one dH mpoH opoH mace: OH Hopmm OoOH>HO mm: AHmmmov H pogo .Emm EH mam ooHN poo Hmmmoo mo mooHpHpom HmpooEmHmmom HHdn .mHmOQ pzmHmeihHo «Sam oH commoamxmm H.Hs mmm Om: . emm om mmm mmH mam seam .soaH O.sm mam mam eOm OmH mOH mm mm mama .osHN O.»O OH: O» OH wHHH rem ems mm page .soaaoo H.He NOHH OmmH mamH mHMH emmH mmHH OOOH o.sm ..s> aosHH nu m m m m w z z mem .02 enacts Omm sO+ mmH sO+ OOH Omm mmH Omm Ase em.Hv sosH no OOH sN+ OOH sN+ as + so + so + so + Honom scene .m «m s m m .mH .oaH .Opm ppooEpmme pom .oo 90H wooHN poo Hommoo 39H: OopooEonmom poHO ESHOHmousto pom mem Eonm mao>HH mo poopooo pooEoHo moose nu .< HoHHB aHH .mxm OH mqmde _gh- found no evidence of toxicity in pigs fed 0.1% copper sulfate. The liver copper values reported in their work have accordingly been some- what lower than those in table 10 and in the Australian and British studies cited above. It appears, therefore, that copper concentration of the liver may be a reasonably good diagnostic measure of copper toxicity. At any rate, the data of Allcroft gp_p;. (1961) and Dammers EE.§l' (1959) indicate that it is a more accurate measure of the copper status of the animal than either kidney or blood copper values. Allcroft 33 El. (1961) found that blood copper levels did not Show a significant rise until the supplemental intake of copper sulfate reached 0.2% of the diet. Their studies also demonstrated that kidney copper concentrations did not exhibit a significant increase until the diet was supplemented with 0.12% copper sulfate. Liver copper levels were dramatically reduced when zinc was added to the copper-supplemented rations. This is compatible with the observation that pigs receiving 250 ppm of copper with no added zinc developed symptoms of copper toxicity, whereas those receiving the same level of c0pper with added zinc showed no evidence of toxicity. The fact that the basal diets fed in the Iowa experiments, mentioned above, were well fortified with supplemental zinc (81.6 or 163.2 ppm) may be the explanation behind their reports of low liver copper levels and no toxicity in pigs fed 0.1% copper sulfate. Molybdenum content of the diet has also been found to influence tissue copper levels (Dick, 1956). Perhaps the Iowa rations were higher than normal in molybdenum content, which may have caused a reduction in liver c0pper storage. However, this is purely conjecture, as there were no molybdenum analyses reported for the rations fed in any of the studies -85- reviewed in this thesis. Davis (1958) has reported that a high level of copper in the liver, such as may occur with copper toxiCity, will result in an almost complete elimination of zinc from liver tissue. However, in this trial, increased levels of copper resulted in no depression of liver zinc; in fact there was a slight but not Significant tendency for the storage of liver zinc to increase as copper increased. As in Experiment I (trial B), the lower level of copper sulfate (125 ppm Cu) did not reduce the concentration of liver iron below that of the controls. However, the higher level (250 ppm Cu), both with and without supplemental zinc, did bring about a significant reduction (P<.Ol) in liver iron. Trial B It was decided that the results obtained in trial A merited further investigation. Trial B was essentially a repeat of the preceding one, with four exceptions: (1) ingredients of animal origin were deleted from the basal diet; (2) when the basal lot (30) was divided after 8 weeks, lots 30A and 30B were Switched from the high-calcium basal diet to one which was lower (1.16% vs. 0.65) in calcium content, but the diets of the remaining lots were not altered; (3) the level of supplemental copper sulfate (125 ppm Cu) used as therapy in lot 30B was half the level fed to lot 1B in trial A; and (#) in addition to the values obtained at the time of slaughter, hematology was also studied midway through the experimental period. Table 11 shows that the basal pigs (lot 30) performed very poorly during the first 8 weeks. These pigs began showing symptoms of parakeratosis at the third week,and by the sixth week’six out of the -86- nine cases were quite severe. In contrast to trial.A, parakeratotic lesions appeared in the copper-supplemented lots (31 and 32) at the end of the fourth week. By the eighth week, two pigs in lot 31 and three in lot 32 were considered to have severe cases of parakeratosis. Their reduced rates of growth in comparison to lots 33 through 35 (P.(.01) were a reflection of their condition. It is apparent from the data that supplemental copper was at least partially effective in preventing parakeratosis. However, its influence was not as profound as in trial A, where the 250 ppm level furnished almost complete protection and 125 ppm delayed the disease for 1# weeks. Perhaps the slightly higher level of zinc in the basal diet in trial A (see appendix table 1) furnished the copper— supplemented pigs in that trial with some additional protection against parakeratosis. At 8 weeks the hemoglobin values of the basal pigs were significant- ly lower than those of the other groups, with the exception of the pigs receiving 250 ppm of copper alone (lot 32). The hemoglobin level of the latter group was significantly lower than that of the zinc-fed lot (33). The hematocrit results were similar to those for hemoglobin. Qualitatively, these data resemble the 15-week values of the previous trial, in that there was a Slight but not significant tendency for hematological values to regress with increasing copper levels and to improve with zinc supplementation. -87- .AmO.V.aV mm sons nnoH aHessoHOHsmHn mm ens Om msoHo .32va mm ones nnoH .HHsssoHeHsmHm mm soH .RQVO mm :m .Hm eon AHOY: mm sees. snoH aHsssOHoHcm-Hn Om eoHo .22va mm ens .sm .mm oat anoH sHessOHoHsmHn mm ens Hm nooH .AHO.uvmv nsoH assoc HHs ones nnOH aHessOHoHsmHn Om soHe .Poofipmosp op OmpmHmpod we: SOHSE mmmoHpOHanpo: mo endpoop oo>oaoa me ooOo .aooooam mo homewoo mm so Oommmp .599 CH mam ooHN pom Hmmmoo mo mooHPHooo HepooEOHmmdm HHoH eoHono .mOm pow ¢Om mpOH OpoH mxooz m Hopmm OoOH>HO mm: AHmmmpv Om poqp .899 op who oops pom Ho99oo mo mGOproom HmpooEoH99dm HH¢O -- mm.m mm.m mm.m Hm.m mm.m .. -- sHsm\eooa OmH.O ms.H mH.H ms.H pm.O om.O .. Om.O m.OH .oHsm HHHsO .mp3 :HnO emoomEhomao9 -- O O O s m H O nao>HH OHoossaHo .oz OH.H O.HH m.Hs O.mH m.Om H.Os m.Os O.mm e .sHAOopssom Hm.O m.mH m.mH H.HH m.mH m.mH O.mH s.mH o.Hs OOH\.em .sHoonosom -- O O O Om OO O: OO neoo3.HH .. O O O Om Om OOH om armor m g .mpmoponoxwaom -- mm.m mo.m “ OO.H mO.H mm.H mm.m mm.H sHsm\eooa psH.O mm.H mm.H Oo.H H0.0 mm.O mm.H Om.O m.OH .anm aHHsn .. m.OpH m.mpH s.msH s.mHH m.HHH H.HHH m.mp .oH -.pa HomersH -- H.HOH m.mOH m.mOH m.ps m.ps O.HH O.ss m.OH ..ps roosvm .. OH OH m OH OH m m ana .oz cosmos Omm so+ mmH so+ OOH Omm mmH mmH .Hso am0.00 sooH mo OOH om+ OOH cm+ on + so + so + so + Honsm noses mm Hm mm mm Hm omOm oaOm .Opm opoospmmpp one .0: poH kl: Axons oszuOV.HmHHp ohppoo mopaso one Axons anHuopwv mxoms O pmmH moppso ooHN pow H099oo HmpooEOH99om mo poommm in .m HmHHB .HH .9xm NH mnde -90- indicate any anemic condition. Furthermore, the liver copper concentra- tion found in this animal--696 ppm-ewes below the average of the lot (see table 13). Nevertheless, post-mortem findings were highly suggestive of copper toxicosis. In the other pigs, there were no indications of copper toxicity until they were slaughtered. At that time, four of the pigs fed 250 ppm of copper alone (lot 32) showed cirrhotic livers, as indicated in table 12. One of these carcasses was condemmed due to a generalized icterus. One liver in lot 30B and two livers in lot 31 exhibited cirrhosis. None of the remaining lots showed any gross ab- normalities of the liver. Growth curves of several representative treatments for the entire trial are portrayed in figure 2. The marked effect of copper therapy in stimulating the growth rate of parakeratotic pigs is evident in this illustration. It may be seen in table 13 that 250 ppm of copper, with or with- out zinc supplementation, significantly increased (P<:.Ol) liver copper stores over those of all other lots. Contrary to previous results, addition of zinc oxide to the copper-supplemented rations did not cause a significant reduction in liver copper concentration owing to a great amount of variation within lots, but the difference between lots 32 and 35 approached statistical Significance. Nine of the ten pigs in lot 32 had a liver copper concentration of 500 ppm or higher, whereas only three of the animals in lot 35 exhibited values of over 500 ppm. However, the highest liver copper level in the entire trial (1,819 ppm) was found in one of the pigs in lot 35. Individual values for liver copper, zinc, and iron are given in appendix table 6. The lots receiving additional dietary zinc showed a significantly -91- .ooHN so po99oo pooppo opp: OopooEOH99dm mpopo ESHOHwonHOEpoo Ho romp: pom mwp9 90 mo>poO zpsopw u: .m HwHHB .HH .9xm .m mmDOHm pooEpmopp no name: is H OH O O s m o _ H _ _ _ oopopppop m9nHo-EV\‘ \.. \o\o\\ \0\\\ \o\0 \ 0% \q\e\o \ o \ \\\\ I] O \o\c \ \ \x\ W O\. \ O \\\ \\1\ \\ ..\\\\ xx. \.\.\ \ o\ ..\\ \\ .\ m \\ o\ \\ L m .A \\ O\ \\\\ O M. \ .\ .. \.\ n \. IONH \\ \\\ \\ \x as sea OOH + Hones cosmos .mm and :21--- \ \ .\ so see mmH + Hones cosmos .Hm soH . 1 \xxx Haunt-pp .90 E99. mm.H + medn OOIHOEHOZ «mom pQH I II II OmH \.\\ Human Afimm.ov mOqushoz «dom poH .Ilildllln . Hones $3.3 cosmos .Om Hos l omH -92- aHoss so HosoH ssHOHso HHpssOHoHsmHn mom soH mm eoH .Amo. V9v mm asap .Hopwopm .AmO.va «Om ens .HHO. v.3 mm .mm .Hm sons sossoam .HHsesoEHsmHn .HmO.uvmv mom son .AHO.V.aV mm .mm .Hm .aOm sass sopsosm aHpssOHmHsmHn Hm ooHH .HHO.UvmV npoH moans sospo cone sossosm HHpssoHoHsmHm mm ens .Hm .mm .mOm upoHs .AHO.V.mv mpOH aospo HHs sass sarcasm sHpssOHeHsmHn mm one mm npon .powpos hoop pom oopooppoo ompa mmooopommpo pCOOHMHomHm oz.p .HopomomHm onHoO meH Hm pOH 8099 mao>HH oseo .Eoomopp mo moopwoo Om so Oommmo .COHpmH Hemmp AfimH.HV ESHOHoouompo oo ooooHpooo mpOH Hmopo m*mO.O op decodes coop no: popo .mOm pom ¢Om mpOH opoH mama: w Hopwo omOH>Ho mm: AHmmMQV Om poHo .E99 up can oopm pow H099oo mo mooproom HmpooEoH99dm HH4Q .mpwmp pompmzimpo .899 up commop9xmm O.mH eHm mHm OOH mOm mHm Hom Hmm Haas .eosH O.Hm Omm oom Omm mOH mOH mmm HOH seas .osHN H.mOH Hpm Hm Hm mom mm HO Om mesa .aoaaoo m.mm mOmH ommH mpmH mmmH mmOH mmHH opm e.sm ..ps sosHH .. OH om O OH OH m m mmHH .oz encore Omm so+ mmH so+ OOH Omm mmH mmH Hso emo.Ov soOH so OOH sN+ OOH sN+ on + so + so + so + Hsnsm noses mm Hm mm mm Hm omOm oaOm .Opm ppooapoopp one .oo pop mooHN pom Ho99oo HmpeoaoH99om O09 me9 Soam mHo>HH 90 poopooo pooSOHo momma I: .m HOHHE «HH .9xm MH mnmde -93 .. higher (Pi(.01) liver zinc storage, which is consistent with previous results. However, lot 30B (125 ppm Cu therapy) also exhibited a Signifi- cantly higher (P <.01) concentration of liver zinc than the other lots receiving no added zinc. In fact, the mean value for lot 30B was nearly as high as for the lots fed supplemental zinc. Because of this unexpected finding, the livers from this lot were assayed again for zinc; the repeat values were virtually the same as the originals. The higher feed consumption of the pigs in lot 30B may have partially, but not entirely, accounted for the difference. If indeed the therapeutic 00pper fed to this lot was responsible for an increased utilization of dietary zinc, then this fact could at least in part account for the marked recovery from parakeratosis noted in these pigs. The small number of pigs involved and the great amount of variation in the liver zinc values in this lot preclude such a conclusion. In general, the liver iron data resemble those of the previous trial. Except for lot 3#, all of the copper-supplemented lots were lower in liver iron than the pigs supplemented only with zinc (lot 33); some of these differences were Significant, while others were not. Tissues from 57 pigs were examined histopathologically in this trial. In general, there were few, if any, changes in the spleen, heart, lung, or kidney that could be attributed to the experimental treatments. In the liver, however, some histological changes were noted in those pigs fed supplemental copper sulfate. These observa- tions are summarized in the following paragraphs, and the individual pig data are compiled in appendix table 7. Lot 30A (Basal). -- The livers of five animals were examined microscopically. .All were essentially normal except for the occurrence -9h- of small foci of dark-staining lymphocyte-like cells. These were interpreted to be hemopoietic centers. Lots 30B and 31 (l25ppm Cu)x-- Fifteen livers from pigs fed 125 ppm of supplemental copper were examined. Nine of these were essentially normal. In five of the other six, there was a questionable but slight in- crease in connective tissue in the periphery of the lobules (inter- lobular septa). In the remaining liver, it was judged that there was a definite slight increase in the amount of interlobular connective tissue. Iot_32 (250 ppm Cu). -- Seven livers from this lot underwent examination. One liver was essentially normal, but in the other Six there was an increase in connective tissue peripherolobularly. Further- more, there were focal areas of fibrosis in two of these livers. Eosinophils were present in these fibrotic areas. Lot 33 (100 ppm Zn). -- Nine livers were examined and all were essentially normal except for the presence of hemopoietic centers in several of them. Lot 3# (lOOppm Zn + 125 ppm Cu). -- Seven liver Specimens were examined and six were essentially normal. There was an increased amount of connective tissue in the liver of one animal. Lot 35 (100 ppm Zn + 250 ppm Cu). -- Eight livers were examined and six were essentially normal. In one liver, a slight increase in the amount of connective tissue in the interlobular septa was noted. In another, a marked degree of fibrosis was observed; the connective tissue was not only increased in the interlobular septa but also tended to replace entire lobules. General. -- All seven of the livers that were grossly cirrhotic (see table 12) were likewise among those that showed an increase in -95... fibrous connective tissue when examined microsc0pically. Moreover, it was apparent upon histological examination that the lots having the highest incidence of fibrotic livers were those which had the highest accumulation of liver copper (see table 13). Experiment III The results of Experiment III are summarized in table l#. One pig in lot 3 became unthrifty and died 22 days after the start of the trial. This pig was submitted for autopsy, and liver changes suggestive of an acute toxicity snydrome were observed. It is possible that the copper-supplemented diet the pig had been consuming could have contri- buted to his death; however, it seems improbable that a toxic condition would develop so early in the experiment. On the other hand, Fagan _e_‘l_:_ El. (1961) attributed the death of a single pig in a lot of 20 pigs receiving 0.1% copper sulfate to the ingestion of a particle of copper sulfate in feed which apparently had not been thoroughly mixed. They cautioned against the dangers of improper mixing of feeds supplemented with copper sulfate. In the present experiment, perhaps the pig in question consumed a portion of feed which contained an unusually high concentration of copper sulfate. Unfortunately, the liver from this animal was not obtained for copper assay. Values for average daily gain indicate there was a small additive response to the combination of copper sulfate and chlortetracycline. All three treated lots out-gained the basal pigs, but only the dif- ference between lots 2 and 5 was statistically significant (P<<.05). Iowa workers have observed an additive response with respect to growth rate from a c0pper sulfate-antibiotic combination (see REVIEW OF -96- LITERATURE section). British reports have varied on this point, but fairly recent work by Bellis (1961) and by Braude gp 21. (1962) in- dicates there is little or no advantage in supplementing British pig diets, containing 250 ppm of copper, with a broad-Spectrum antibiotic. There were no appreciable differences in feed efficiency except for lot #, which required approximately 7% more feed per pound of gain than the other groups. None of the differences in hemoglobin or hematocrit were statisti- cally significant. TABLE 1# Exp. III.-- Response of pigs to copper sulfate and chlortetracycline, alone and in combination (1# weeks) Lot no. and treatment Std. 2 3 h 5 error Basal + CTCa + Cu + CTC of Item (0.63% Ca) 10 mg./lb. 125 ppm + Cu meansb No. pigs 8 8 7c 8 -- Initial wt., lb. 36.5 36.0 38.0 36.0 _— Final wt., lb. 18#.# 196.9 195.0 200.0 -- Daily gain, lb.d 1.51 1.6# 1.60 1.67 0.052 Feed/gain 3.0# 3.06 3.26 3.03 -- Hemoglobin, mg./100 ml.e 13.0 13.# 13.7 12.9 0.#8 Hematocrit, %e 39.1 39.6 #0.5 38.6 l.#8 aChlortetracycline. bBased on 27 degrees of freedom. COne pig died suddenly after 22 days. Upon autopsy, it was concluded: "the liver changes suggest an acute toxicity syndrome." dLot 5 significantly greater than lot 2 (P<<.05). 8No significant differences between lots. -97- Experiment IV Experiment IV was designed to compare two common inorganic forms of copper, namely, the sulfate and oxide salts. It is evident in table 15 that the treatment differences in daily gain were small and nonsignificant. Pigs in the basal lot grew normally and displayed no symptoms of parakeratosis in spite of the fact that their diet contained only 30 ppm of zinc. It is noteworthy that in Experiment I (trial A), a high incidence of parakeratosis occurred in pigs fed a basal ration similar in calcium and zinc content to the present one. This again suggests that there are other factors involved in the etiology of the parakeratotic syndrome. Neither copper sulfate nor copper oxide caused a significant reduction in hemoglobin or hematocrit. This is consistent with the fact that there were no clinical symptoms of copper toxicity at any time during the experiment. When the pigs were slaughtered, however, gross examination of the livers revealed that there were varying degrees of hepatic damage resulting from the copper-supplemented diets. Lot ll (CuSOu alone) displayed by far the highest incidence of cirrhotic livers--90%. The pigs fed copper oxide alone (lot 12) showed con- siderably less cirrhosis (30%), followed by those lots which were fed zinc in combination with copper (lots lh and 15). The liver copper values (table 16 and appendix table 8) appeared to be directly related to the incidence of cirrhosis. Lot 11 exhibited a significantly higher (P<:.Ol) concentration of liver copper than all the other groups. Lots 12 and 1h were also significantly greater (P<’.Ol) than the lots ranking below them. The addition of 100 ppm of zinc to -98- .mpOH cmmzpmn moccmHMMMHO pQOOHchme ozHU .choEdocm OHCOHQO 09 odd OH 90H Scam do>oamh me mQOo .EOOomhw go momawmc mm so dmmmmn .Emm OOH mo opus 039 pm odHN mama Omm mo opwh map as deuce mm: aommoow -- H H O m O O nabeHH eroanaHe mo .02 mm.O s.sm m.sm O.sm m.Om H.sm m.Om em .pHneoeeabm mm.O :.mH H.mH H.mH O.mH O.HH O.mH O.Ha OOH\.ms .aHBOHmosem -- me.m OO.m mw.m Op.m ma.m Om.m eHaw\ebbm HmO.O PO.H mm.H mm.H HO.H mm.H mm.H O.mH .eHem sHHnO -- H.HOH H.OeH H.OsH m.meH H.OOH O.asH .BH ..93 HeeHm -- m.Om m.Om O.Hm m.Om 0.0m m.Hm .eH ..p> HerHeH -- OH OH OH OH OH em nmaa .02 became oaO + 4488 + an + Odo + Omao + HaO *O0.0V smeH M0 2N + mm + mewm hoahm mH :H MH NH HH OH .Opw dpcoEpwohp and .0c 90H Amxomz MHV ocHN HapcoamHmmdm psoans and ngB pme EdHononHmEHoqnw op deuce Hommoo mo mahom 03¢ mo QOmHmeEOO u: .>H .mxm mH mqmdfi -99.. .AmO.O u AV mGOHpmecoocoo Hommoo Ho>HH Ono cHoH soospop coppmHopHoo on ma: opone .mpOH coozpop moocopomch padoHpHmmHm ozCH .HHO.V& HH one .mH .HH aefi nopaonm aHpeooHeHomHn mH poH .AHO.uvmv npoH nonpo HHo soap aopooam aHpoooHeHaan MH one OH mpon .AHO.v.mv opoH been» nospo coup nopoonm aHpeoonHamHn mH oeo.:H .mH opoHo .AHO.V.mV mH ego .mH .OH gasp nopeonm aHpeoonHemHn :H one mH npoH .AHO.V.mv npoH aoapo HHo soap aopnoam aHpeoonHemHn HH poHo .HHO.V.av mH .mH one .HHO.V.aV HH .HH .OH "nooH nonpo HHo soap nnoH sHpeooHeHemHn mH poHo .hHo>HpoommoH «mode> :HOH was po>HH Mom Eocoopp p0 mmopwoo :H was Mm so Oomwmo .899 OOH mo opwa 03p pm ocHN mama Omm mo mpwp ogp pa downs was HoQQOOp .mHmwn ppmHoznhpO «Egg up commopmxmw mmm.O mm.m pH.m mm.m m4.m mm.m OH.m saga .noaaoo .: m m m a H m mmHa .oz mothmcd CHoH m.mm Hm: HOH me pmH OHH mmm wean MeonH O.mH HHm mmm mmm HHH OOH mmH sag ooHN a.mOH mam mmm Om HOO mpmH Om be a .noaaoo m.OH HHmH mmmH mmHH HONH wOmH OHMH e.am ..p3 ao>HHy -- OH OH OH OH OH O mmHa .oz wommeod Ho>HH omcmoE 050 + somdo+ QN + 050 + zomdo + Ame ROO.OV BopH mo QN + mm + Hammm aoaao mH eH mH mH HH OH .Opm ppcoapmopp Odo .oc poH wosHN pdonpps Ono app: mOon HoQQOO Ono opmedm Hommoo cop mmpm Soap CHOH Ono Ho>HH mo mothwsw pcoEoHo mowHB In .>H .mxm OH mqmde -100- the diets of pigs fed either form of copper resulted in a significant reduction in the accumulation of liver copper. It is worthy of note that the oxide-fed pigs stored 70% and h5% less liver copper than those fed sulfate, with and without zinc, respectively. This is in agreement with the work of Bunch 33 El‘ (1961, 1963), who showed that pigs fed supplemental copper sulfate accumulated more than twice as much copper in their livers as those fed copper oxide. These workers also reported that supplemental zinc was necessary for maximum growth response to copper supplementation. This was not observed in the present experiment but was evident in previous trials. It is somewhat surprising that no clinical symptoms of copper toxi- city were observed in lot 11 when it is considered that their mean liver copper concentration was even slightly higher than that of pigs fed the same level of copper sulfate in Experiment II, trial A (1,572 vs. 1,hh8 ppm); the latter pigs showed definite clinical symptoms of a toxicosis. However, there is undoubtedly a considerable amount of variation among animals in their ability to tolerate high levels of dietary copper. Wallace gt El' (1960) have reached this conclusion from their studies on high-level copper feeding. As was anticipated, the zinc-supplemented lots--l3, la, and l5--had significantly higher liver storage levels of this element than did the unsupplemented lots-—lO, 11, 12. Neither copper oxide nor copper sulfate had a significant influence on liver zinc values. Both forms of copper significantly depressed the storage of liver iron. Copper sulfate had a greater effect in this respect than COpper oxide, especially when supplemental zinc was fed (lot 1h vs. lot 15). When zinc was introduced into the diet, the depressant effect of dietary -lOl- copper on liver iron storage was partially overcome, which agrees with Experiment II. From the data compiled to date, it appears that within the liver of the pig there is competition between these three elements for storage sites. When a sufficient amount of copper is absorbed, iron storage appears to be inhibited. When zinc is added to a copper- supplemented diet, uptake of copper by the liver is depressed. This in turn could be the explanation for the higher iron levels observed in such livers, compared to livers from pigs fed copper alone. The data indicate that the interaction between copper and zinc is not reciprocal; that is, the copper levels employed in these experiments have not re- sulted in a lowered uptake of zinc. It cannot be said whether the inter- action between copper and iron is reciprocal because high levels of these elements were not fed jointly; however, there was no depression of liver c0pper when 100 ppm of iron was added to the basal ration in Experiment I (table 5). There is no evidence in the literature that high levels of dietary iron interfere with liver copper storage. These competitive interactions may be considerably more complex than this writer has just indicated. Furthermore, any conclusions drawn from the results of this work cannot be justifiably applied to situations where the supplemental trace element levels differ from those employed here. It was decided to assay loin samples from 20 of the pigs in this experiment in order to learn whether high levels of dietary copper re- sult in an increased deposition of copper in skeletal muscle tissue. Each lot was randomly sampled. It is obvious from the data in table 16 that neither form of supplemental copper significantly increased the concentration of copper in the longissimus dorsi. Compared to the liver, -102- the loin values are extremely low, the highest individual value being 3.69 ppm (see appendix table 8). Bunch gt El. (1963) have reported that levels of loin copper were increased significantly in pigs fed 250 ppm of supplemental copper from either the sulfate or oxide salts. Their mean values were somewhat higher than those reported here, ranging from 5.7 ppm for their basal lot to 10.3 ppm for pigs receiving copper oxide; however, the latter concentration could hardly be considered dangerous for human consumption. Barber gt 21. (1957) have pointed out that calf liver, containing 150 ppm of copper, is not considered unfit for human consumption. In the present trial, there was no correlation between loin and liver copper concentrations (r = 0.02). Experiment V Trial A The results of trial A are summarized in table 17. The high-calcium basal pigs (lot 27) grew significantly slower than the other lots and all but one pig in this lot exhibited symptoms of parakeratosis. Skin lesions began to appear after 3 weeks and became very severe after 8 weeks, when the trial was terminated and the pigs slaughtered. The normal-calcium basal pigs (lot 26) performed as well as those fed the same diet supplemented with zinc (lot 31), even though two pigs in the former lot developed a mild dermatosis. Supplementing the high~ calcium ration with 125 ppm of copper enabled the pigs in lot 29 to grow almost as rapidly as those in the other supplemented groups. By the time thevaere slaughtered, four of these pigs exhibited mild skin lesions which were perceptible only upon close examination. Thus, the .AHO.dvmv mm pOH supp umoH mecuoHpchHm Fm poqn .cHopopm .wE pom odponmoosm ochwMocH .moE mu commohmxo th>Hpo¢w .AHO.Uvav Om use AmO.uvav om soap nnoH sHpoooHoHsan pm poHo .mpOH sookpop woocopopMHO pcuopppcmpm ozo .AHO.hvmv npoH aoepo HHo soap nnoH aHpsooHoHean pm poHO .pcoEHHoQXo ogp op OopuHohc: on 0p anuommu noH£3 mmochpHanps: op odd Om Ocu «mm «Om mpOH mo nouo EOHp Oo>oEoH mpg oCOo .MH co omupunmmonm ocu flmm so omupmnm «Souoohp mo moopwod m: co Oomup mMOHOpqun ucu Epsoawp .Smm OOH mo opus oap pu ocHN “Ema mmH p0 opup onp pu Oouvu mu: hogmoou mm.m .. -- -- .. m.Hm s.mo .monopoemnoaa HosHpnopsH Om.e p.mm H.mm O.Hm p.em m.:m p.mm u.oonopasa HooHpnoch em.o O.mm m.os m.Om p.mm m.Om m.om ea .ensooossom mm.O N.HH m.mH O.HH O.HH :.HH O.mH o.HE OOH\.Ew «sHQOHonom Ly n: O O :: O mm mm R amHmopupoxupum no .u -- Hm.m mp.m mu.m Hu.m om.e Hm.m oHou\ooom mw0.0 Hm.H :m.H OH.H mm.H w:.O Hm.H O.pH acHuw kHHHuQ I: m.OOH :.wOH m.Om O.MOH 0.00 m.HOH .pH ..p3 Hucpm -- p.mm O.mm O.mm H.mm H.mm m.mm .oH ..ps HoHpHsH a: m w m w m w omem .oz O.mcuoE :N so + cN 50 ON A&©m.Hv ARHm.OV EopH MO + $0 + no + $0 + 60 do do Hoppo Hufihoz nmpm :mHm QmHm anm Huahoz .opm Hm Om mm mm em mm uWMoEpuoap ccu .oc poq Amxooa mv omupusmwonm ucu omuphnm Huchmoch cau NhMOHopqun «npzoaw co mHo>oH EdHoHuo wcpzpu> nppz ocHN dcu pommoo HupcoEoHQQSm mo pooppm I: .< HuHHB s> .mxm NH mum¢a -th- ability of supplemental copper to partially alleviate the symptoms of parakeratosis was reconfirmed (see tables 2, 3, 6, 7, 8, 11, and 12). It is interesting to note that the high-calcium-plus-zinc lot (28) gained just as rapidly as its normal-calcium counterpart (lot 31). There was a slight additive response in growth rate when copper and zinc were combined in lot 30, but it was not statistically significant. None of the differences in hemoglobin were significant. The mean hematocrit value of the high-calcium basal lot was significantly lower than that of lot 26 (P< .05) and lot 30 (P< .01). The blood picture of the copper-supplemented pigs was not depressed, as it was in Experiment II. Thirteen of the intestinal tissue extracts were not assayed immediately and became contaminated with microbial growth which meant that it was necessary to discard them. Fortunately, each lot was about equally represented in this loss. There were no significant differences between lots with respect to intestinal phytase activity. The results of the assay were highly variable, which is probably indicative of in- accuracies inherent in the method. In this trial, intestinal phosphatase assays were performed only on the normal- and high-calcium basal lots (26 and 27). The high-calcium pigs exhibited a significantly lower intestinal phosphatase activity than those fed the normal-calcium diet. Trial B This trial was conducted to further test the hypothesis that high- calcium, parakeratotic diets inhibit the activity of phytase or phospha- tase in the intestine of the pig. The data are summarized in table 18. -105- As mentioned in the EXPERIMENTAL PROCEDURE section, it was necessary to add zinc to the low-calcium diet after 3 weeks to prevent TABLE 18 Exp. V, Trial B. -- Effect of normal and parakeratotic diets on intestinal phosphatase activity (7 weeks) Lot no. and treatment 26a 21 Std. Low High error Ca Ca of Item (0.53% Ca) (l.h2% Ca)_p meansb No. pigs 7 7 -- Initial wt., lb. 23.0 23.0 -- Final wt., lb. 79.0 h7.7 _- Daily gain, 1b.c 1.1M 0.50 0.058 Feed/gain 2.uu 3.u3 -— Parakeratosis, % O 100 -- Intestinal phosphatased 53.2 61.0 3.36 aIt was necessary to add 100 ppm of zinc to the lot 20 ration after 21 days in order to prevent parakeratosis completely. Based on 12 degrees of freedom. CLot 20 significantly greater than lot 21 (P‘<.Ol). Activity expressed as mcg. inorganic phosphorus per mg. protein. the development of parakeratosis in lot 20. After 7 weeks, the weight gains of the high-calcium pigs (lot 21) had come to a virtual stand- still and all of them showed symptoms of parakeratosis. At this time, both lots were slaughtered, and assays for phytase and phosphatase were performed on a section of small intestine from each pig. Unfortunately, none of the intestinal samples exhibited phytase activity. The sodium phytate substrate was apparently unstable during -106- storage, for its inorganic phosphorus content was found to be three times higher than it had been originally (0.93% vs. 0.31%). Perhaps all of the hydrolyzable phosphate groups on the sodium phytate underwent a non- enzymatic hydrolysis during storage; or rather, something may have occurred during the preparation of the tissue extracts to destroy their phytase activity. Conversely, sodium-beta-glycerophosphate, used as substrate in the phosphatase assay, is apparently a very stable compound; no trace of inorganic phosphorus was detected in the substrate blanks during the assay of this enzyme. Contrary to trial A, the high-calcium pigs in this trial showed a higher intestinal phosphatase activity than the low-calcium pigs. How- ever, the difference was not statistically significant. -107- V. SUMMARY Experiment I Three trials, involving a total of 158 weanling pigs, were con— ducted to study the effect of adding copper (125 ppm), zinc (50 or 75 ppm), or iron (100 ppm) to basal diets varying in calcium content (0.55, 1.05, and 1.31%). Zinc-copper and zinc—iron combinations were employed in two of these trials. Parakeratosis occurred in the basal pigs at all three levels of calcium intake. Compared to healthy pigs, the parakeratotic animals were characterized in most instances by depressed growth rate, higher serum gamma globulin fraction, lower serum albumin fraction, and lower serum alkaline phosphatase activity, in addition to the skin lesions typical of this syndrome. Lower hemoglobin and hematocrit, and higher total serum protein values were also noted in several lots in which parakeratosis was present. Zinc supplementation was completely effective in preventing parakeratosis. Supplemental copper was completely effective at the 0.55 and 1.05% calcium levels and partially effective at the 1.31% level. Iron partially prevented the disease at the lowest level of calcium intake but had no effect when it was added to a ration having a higher calcium content. Besides eliciting an improvement in rate of gain and skin condition, these trace elements produced highly significant responses in certain of the biochemical observations. The albumin and gamma globulin fractions and alkaline phOSphatase activities of the serum were particularly sensitive to zinc and to copper supplementation. A combination of zinc and iron was not as beneficial in improving -108- growth rate as zinc alone, which suggests an interaction between these two elements. A zinc-copper combination proved no more effective in stimulating body weight gains than zinc alone. Pigs fed 125 ppm of supplemental copper for 1h weeks exhibited a significantly higher CP