The Woman's College of The University of North Carolina LIBRARY CQ M no.S2A COLLEGE COLLECTION Gift of "lta Kennedy otewart THE EFFECT OF ZINC TOXICITY ON THE CALCIUM, PHOSPHORUS, AND MAGNESIUM CONTENT OF BONE AND BLOOD IN YOUNG RATS by Alta Kennedy Stewart L>> -.., A Thesis Submitted to the Faculty of the Graduate School at The Woman's College of the University of North Carolina in Partial Fulfillment of the Requirements for the Degree Master of Science Greensboro June, 1962 Approved by 4^<.ew«~ Director Y 4 APPROVAL SHEET This thesis has been approved by the following committee of the Faculty of the Graduate School at the Woman's College of the University of North Carolina, Greensboro, North Carolina. Oral Examination Committee Members t^fel^M Oale of Examination ?^ri8 JOSEPH RUZICKA BALTIMORE, MD. - GREEN^BORO.^C, -%** Title THE WVX <* ZDIC PHP" °S Vol. & Date Call No. Color 2573 Special Instructions: NT Woodell 3391 /; a to my advisor, Dr. Aden Magee, tion of this study and to the lomi Albanese, Miss Marguerite suggestions and helpful A.K.S. iii ACKNOWLEDGEMENT I wish to express my appreciation to my advisor, Dr. Aden Magee, for his guidance and help in the completion of this study and to the members of my advisory committee, Dr. Naomi Albanese, Miss Marguerite Felton and Miss Sandra Spahr, for their suggestions and helpful criticisms. A.K.S. iii TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION 1 II. REVIEW OF LITERATURE 3 III. EXPERIMENTAL PROCEDURES 7 Growth, Bone Formation and Blood Composition of Rats Fed High Levels of Zinc 7 Net Retention of Calcium, Phosphorus and Magnesium. . . 11 IV. RESULTS AND DISCUSSION 13 Experiment 1 13 Experiment II " Experiment III 18 Experiment IV Experiment V z' V. GENERAL DISCUSSION 32 Effect of Zinc on Growth 32 Influence of Zinc on Bone Mineralization 35 Effect of Zinc on Magnesium Metabolism and Utilization 38 VI. SUMMARY AND RECOMMENDATIONS 39 39 Summary Recommendations BIBLIOGRAPHY 43 iv CHAPTER APPENDIX A. Detailed Data Showing Effects of Treatments Tested in the Various Experiments. . . . APPENDIX B. Metabolism Data APPENDIX C. Analyses of Variance Data PAGE 48 63 75 LIST OF TABLES TABLE PAGE I. Composition of the Basal Diet 9 II. Effect of Zinc on Growth, Serum Composition and Bone Mineralization (Experiment I) M III. Progressive Stages in the Effect of Zinc on Growth, Serum Composition and Bone Mineralization (Experiment II) 17 IV. Effect of Various Supplements on Rats Fed High Levels of Zinc (Experiment III) 20 V. Effect of Calcium and Phosphorus Supplements on Rats Fed High Levels of Zinc (Experiment IV) 24 VI. Calcium, Phosphorus, and Magnesium Balance Summary (Experiment V) vi LIST OF FIGURES FIGURE PAGE 1. Arrangement of Apparatus Used in Metabolism Experiment 2. Effect of Zinc on Net Retention of Calcium, Phosphorus, and Magnesium 12 29 I vii CHAPTER I INTRODUCTION Aristotle believed the food of plants and animals to be com- posed of four "essential elements"--earth, air, fire and water. While our present day concepts of nutrition differ radically from such early beliefs, information in this branch of science is by no means complete. One area where information is lacking is that of the trace elements. Over the last hundred years, and especially in the last thirty years, a steadily increasing number of minerals, occurring in minute amounts in the animal body, have had definite physiological roles assigned to them. Two different kinds of investigations have advanced out under- standing of the role of these minerals: (1) the investigation of various naturally occurring diseases of man and animal shown to be due to a dietary deficiency, or excess, of a particular trace element and (2) the investigation of the effects on animals of specially constituted diets, deliberately designed to have an abnormally low, or high, content of the trace mineral under study. Of the twenty-two trace elements known at the present time, six have been shown to be essential for the maintenance of the higher forms of animal life. Zinc is one of these six. It was early discovered in plant and animal tissue. Its universal occurrence in nature, frequently 2 in concentrations approaching that of iron, has established its funda- mental importance to all living cells and has stimulated research into the specific physiological functions with which it is concerned. Its influence on growth, its presence in various enzyme systems and the discovery of biochemical lesions resulting from its deficiency or excess, have all provided basic data which will demand verification and expan- sion. The extent of the changes which occur in zinc-fed animals is only now being recognized. Although gross symptoms have been described, the sites and the modes of interference of zinc in processes such as bone development and mineralization have not been clarified; nor has informa- tion been provided on possible ways and means of alleviating these harmful effects. This study was undertaken with the purpose of extending present information in this area of investigation and of providing additional knowledge on the physiological reaction of the animal body to stress. It will attempt to clarify, if possible, the mechanism(s) of the inter- ference of zinc with calcium and phosphorus in bone mineralization and to determine the effect of zinc toxicity on the metabolism of magnesium in young rats. CHAPTER II REVIEW OF LITERATURE The work of Todd, Elvehjetn and Hart, in 1934, gave the first indication that zinc was essential for the normal growth of animals. In 1937, Sutton and Nelson reported that the limits of tolerence of 2 young rats to zinc was between 0.57. and 1.0% of the diet. They observed that levels of dietary zinc below 0.57. resulted in no adverse effects on the animals, while animals receiving 0.57. had reduced growth rates, poor reproduction and lowered hemoglobin levels. When rats were fed 1.0% zinc, death occurred in ten to twelve weeks. More recently, Van Reen has reported growth depression in young rats on an 3 intake of 0.37. of zinc. High levels of dietary zinc have been shown to interfere with copper metabolism, to inhibit certain enzyme systems, and to interfere with iron metabolism in young rats. Smith and Larson observed that the depression in hemoglobin associated with zinc toxicity could be allevi- ated by supplements of dietary copper.4 Van Reen noted a beneficial XW.R. Todd, C.A. Elvehjem and E.B. Hart, Am. J. Physiol., 107. 146 (1934). 2W.R. Sutton and V.E. Nelson, Proc. Soc. Exptl. Biol. Med., 36, 211 (1937). 3R. Van Reen, Arch. Biochem. Biophys., 46, 337 (1953). 4S.E. Smith and E.J. Larson, J. Biol. Chem. , 163.29 (1946). 4 effect of copper supplimentation on the depressed activity of liver cytochrome oxidase and catalase. Duncan e£ a_l. reported that copper overcame the decreased heart cytochrome oxidase activity associated with zinc toxicity. Grant-Frost and Underwood observed that lowered copper levels of the kidney and the liver of zinc-fed rats could be overcome by additional dietary copper. An early and marked loss of o liver iron has responded favorably to iron supplementation. Magee and Matrone, studying effects of zinc toxicity on hemoglobin formation, liver copper and iron content, and heart cytochrome oxidase activity, concluded that zinc interfered with copper metabolism by decreasing the utilization and increasing the excretion of copper. Interference with iron utilization appeared to be a direct effect rather than one mediated 9 through the copper metabolism. In 1951, Sadasivan reported that high levels of dietary zinc were associated with decreases in the calcium, phosphorus and total ash con- tent of the femur of rats. He also noted a significant increase in Van Reen, op_. cit. 6G.D. Duncan, L.F. Gray and L.J. Daniel, Proc. Soc. Exptl. Biol. Med., 83, 625 (1953). 7D.R. Grant-Frost and E.J. Underwood, Australian J. Exptl. Biol. Med. Scl.,' 36, 339 (1958). 8D.H. Cox and D.L. Harris, J. Nutrition, 70, 514 (1960). 9A.C. Magee and G. Matrone, J. Nutrition, 72, 233 (1960). 10V. Sadasivan, Biochem. J., 48, 527 (1951). phosphorus excretion and a decrease in intestinal phosphatase activity of rats fed 0.57. and 1.0% zinc.11' U Similar toxic effects have been observed in other animals. Gross bone changes and an arthritic condition characterized by swollen joints have been reported in the weanling pig. ' The results of Thompson and co-workers indicated a lowered net retention of both calcium and phosphorus in lambs fed supplements of zinc sulfate. Although these latter studies indicate that high levels of dietary zinc prevent the normal development and the mineralization of the bone in various species, the exact mechanism(s) of this zinc inter- ference remains an enigma. The apparant antagonistic relationship between zinc and calcium is controversial. As Forbes has pointed out, this relationship may not exist under all conditions. Although an interrelationship between zinc and phosphorus is indicated, information concerning this aspect of the problem is lacking. There is also the possibility that interrelationships exist between zinc and other minerals necessary for bone development. Since magnesium has been shown UV. Sadasivan, Biochem. J., 49, 186 (1951). 12V. Sadasivan, Biochem. J., 52, 452 (1952). UR.E.R. Grimmett, I.G. Mclntosh, E.M. Wall and C.S.M. Hopkirk, New Zealand J. Agr., 54, 216 (1937).. 14M.F. Brink, D.E. Decker, S.W. Terrill and A.H. Jensen, J. Animal Sci., 18, 836 (1959). 15. 5A.H. Thompson, S.L. Hansard and M.C. Bell, J. Animal Sci., 18, 187 (1959). 16 R.M. Forbes, Federation Proceedings, 19, 643 (1960). to be an essential constituent in bone formation and is closely associated with calcium and phosphorus metabolism, knowledge of the effect of excess zinc on magnesium would be desirable. It is evident that much more information must be made available before a final, definite statement can be made on the interference of zinc in bone mineralization. CHAPTER III EXPERIMENTAL PROCEDURES The primary objectives of this study were to investigate (a) the nature of the interference of zinc in bone mineralization, (b) the possibility of changes in blood calcium and phosphorus associated with zinc toxicity, and (c) the possibility of zinc inter- ference with magnesium metabolism. The series of four experiments, which constitute Part I of the study, were designed to show the effect of zinc toxicity on levels of calcium, phosphorus and magnesium in bone and blood in the presence and the absence of other dietary supplements, and to determine the relative time when changes in bone calcium, phosphorus and magnesium begin to occur. Changes in bone zinc were also studied. In Part II of the study, metabolism units were utilized to determine the effect of zinc toxicity on the net retention of calcium, phosphorus and magnesium. I. GROWTH, BONE FORMATION AND BLOOD COMPOSITION OF RATS FED HIGH LEVELS OF ZINC Since this phase of the study consisted of four experiments, procedures pertaining to a specific experiment will be discussed separately. Several procedures were common to all of these experiments, and these procedures are discussed in the following paragraphs. Weanling male albino rats, descendants of the Sprague-Dawley strain, were used in all phases of the study. Animals of a particular experiment were housed in individual wire-bottom cages in a controlled enivronment of 74 - 78 and 407. to 507. humidity. Food and water were offered ad_ libitum. Animals used in each experiment were randomized into replications according to initial body weights. Animals and test treatments within a replication were assigned at random to individual cages. The length of each experiment was four weeks. A weekly weight record was kept for all animals. The basal diet, shown in Table I, also served as the control treatment. This diet, by analysis, contained 0.527. calcium, 0.557. phosphorus, 0.04% magnesium and 10 ppm. of zinc. In all experiments, supplements were added to the basal diet at the expense of starch. Zinc was fed as the carbonate; supplements of calcium, phosphorus and magnesium were in the carbonate, phosphate and sulfate forms, respec- tively. At the end of each experiment, blood samples were obtained from the animals by means of a heart puncture. All blood samples from rats receiving the same treatment in a particular experiment were pooled. Serum was obtained from each pooled sample of blood by centrifuging. A serum filtrate was prepared using specified amounts of blood serum, distilled water and 20% trichloracetic acid. Measured amounts of the filtrate were taken for mineral determinations. Purchased from Holtzman Company, Madison, Wisconsin. TABLE I COMPOSITION OF THE BASAL DIET Constituents Per cent Casein Cornstarch c Vegetable fat d Mineral mix Vitamin mix Cellulose Oleum percomorphum 19 63 10 4 2 2 8 Vitamin Test," Nutritional Biochemicals Corporation, Cleveland, Ohio. bGlobe Easy-flow Conrstarch 3366, Greensboro, North Carolina. c"Crisco," Proctor and Gamble Company, Cincinnati, Ohio. Salt Mixture W, Nutritional Biochemicals Corporation, Cleveland, Ohio. eEach 100 gm. of vitamin mix contained the following: 1 mg. biotin, 5 mg. folic acid, 0.1 mg. of 0.17. vitamin B12 (with mannitol), 25 mg. thiamine, 25 mg. pyridoxine, 50 mg. 2-methyl napthoquinone, 50 mg. riboflavin, 50 mg. nicotinic acid, 150 mg. Ca-panthothenate, 500 mg. p-amino benzoic acid, 5 gm. inositol, 7.5 gm. choline, 30gn. DL-methionine, and 56.6 gm. corn starch. f"Alphacel," Nutritional Biochemicals Corporation, Cleveland, Ohio. 8Each 1000 gm. of diet contained 24 drops of oleum percomorphum, Mead Johnson and Company, Evansville, Indiana. 10 Rats from randomly selected replications were sacrificed, and the femurs of each animal were removed. The bones were cleaned of muscle and fat by rubbing with a rough cloth, dried at 35 C, and weighed on an analytical balance. The bones were prepared for mineral analysis by wet ashing with nitric and perchloric acids on a hot plate. The ash of each pair of bones was dissolved in 0.6 N HCl and brought to a volume of 100 ml. with distilled water. Five ml. of this solution were redi- luted to 100 ml., and appropriate aliquots of the second dilution were taken for the calcium and the phosphorus determinations. Appropriate aliquots of the first dilution of each bone sample were taken for the magnesium and the zinc determinations. 2 Calcium was determined by the method of Weybrew et al., phos- phorus by the method of Simonson et al•, and magnesium by a method outlined by Simonson et al.4 The method of McCall, Davis and Stearns was used to determine zinc. Due to the high alkalinity of the bone samples, a slight modi- fication in the calcium procedure was necessary. In order to maintain the required PH of 5 and to prevent co-precipitation of magnesium with calcium, three drops of 6N HCl were added to the test sample immediately after the addition of the brom cresol green indicator. 2J.A. Weybrew, G. Matrone and H.M. Baxley, Anal. Chem., 20, 759 (1948). 3D.G. Simonson, M. Wertman, L.M. Westover and J.W. Mehl, j. Biol- Chem., 166, 747 (1946). 4D.G. Simonson, L.M. Westover and M. Wertman, J. Biol. Chem., 169, 39 (1947). 5J.T. McCall, G.K. Davis and T.W. Stearns, Anal. Chem., 30, 1345 (1958). LI II. NET RETENTION OF CALCIUM, PHOSPHORUS AND MAGNESIUM In the metabolism experiment, four animals received the basal diet and four animals a diet containing 0.75% of zinc. To insure uniform adaptation, all animals were maintained on the basal diet for a preliminary period of five days. Each animal used in this experiment was placed in an individual metabolism cage. The cages were fixed as shown in Figure I, so that the urine and feces could be collected separately. During the experimental period of four weeks duration, fresh amounts of feed were given daily, and the food consumption of each animal was measured. Water was available at all times. To reduce food spillage, the feed intake was limited to fifteen grams during the first and second weeks and to twenty grams during the third and fourth weeks. The experimental period was divided into four consecutive seven-day collection periods. All urine and feces samples from an individual animal were pooled for each seven-day period. Measured amounts of the pooled urine and feces were ashed, dissolved in 6N HC1, and made up to volume with distilled water. Calcium, phosphorus, magnesium and zinc determinations were made on appropriate aliquots of these samples by the methods previously described. 12 FIGURE 1 ARRANGEMENT OF APPARATUS USED IN METABOLISM EXPERIMENT CHAPTER IV RESULTS AND DISCUSSION Experiment I The experiment was designed to test the effects of three levels of dietary zinc on (1) growth rate, (2) calcium, phosphorus, magnesium and zinc content of bone, and (3) calcium, phosphorus and magnesium levels in the blood. Although a minimum level of 0.57. of dietary zinc is usually necessary to cause a significant reduction in the growth of rats, there is the possibility that a lower level of zinc will signifi- cantly change the mineral composition of the bone and the blood. Thus, 0.25%, 0.50% and 0.75% levels of dietary zinc were used as the test treatments in this experiment. Detailed data from the experiment are given in Appendix A, Tables 1-5 while the means of growth rate, bone calcium, phosphorus, magnesium and zinc, and serum calcium, phosphorus and magnesium are shown in Table II. Analyses of the data (Appendix C, Table 1) revealed that the addition of 0.75% of zinc to the diet resulted in a highly significant decrease (p L 0.01) in weight gain and a significant decrease (p £ 0.05) in bone calcium and bone phosphorus. The data also indicate that a level of 0.25% of dietary zinc is associated with a highly significant increase (p £ 0.01) in the deposition of zinc in the bone, although no significant changes occurred in the content of bone calcium and I'll— -I III— TABLE II EFFECT OF ZINC ON GROWTH, SERUM COMPOSITION, AND BONE MINERALIZATION (EXPERIMENT I) Level o dietary f Weight gain3 at 4 weeks Bone constit „ a jents Serum constituents zinc Ca P Mg Zn Ca P Mg I gms mg./gra. dry weight rag./100 ml. None 188 146.29 66.04 3.45 0.09 13.73 11.92 3.27 0.25 197 143.82 54.01 3.37 0.58 13.74 12.72 3.07 0.50 161b 131.37 40.78 3.44 1.34 10.31 13.40 3.39 0.75 113b 107.41 39.57 3.44 2.34 12.15 11.92 2.87 a Each figure is the mean of 6 animals unless otherwise indicated. b Mean of 7 anirif Is "Each figure represents the determination for a pooled sample of serum from 6 animals. 15 phosphorus at this level of zinc intake. The dividing line for the toxic effect of zinc on these two minerals would appear to fall between the 0.257. and the 0.507. levels. Zinc had no significant effect on the magnesium content of the bones. The effect of increased dietary zinc on serum calcium, phosphorus and magnesium concentrations was inconsistent. Although serum calcium values were depressed at the two highest levels of zinc intake, serum phosphorus levels were either increased or remained at control levels. The changes which occurred in serum magnesium showed no distinctive pattern of increase or decrease. Failure to note any consistent effects, in calcium at least, may be due to the fact that serum levels of this mineral are primarily controlled by the parathyroid hormone and are not 1 readily influenced by dietary intake. Experiment II Although Cox and Harris have reported that the effect of zinc toxicity on certain body constituents was evident within the first week, no experiments have been conducted to determine how soon signif- icant changes begin to occur in the metabolism of calcium, phosphorus and magnesium in animals maintained on high zinc diets. In order to obtain this information, sixty-four animals were divided equally into four groups. One group was maintained as controls; the other three groups were fed diets containing 0.257., 0.50% and 0.757. of zinc, HM. Crampton and L.E. Lloyd, "Fundamentals of Nutrition," W.H. Freeman and Company, San Francisco, 1959, p. 221. Cox and Harris, loc. cit. 16 respectively. At weekly intervals, four animals from each group were sacrificed and levels of bone calcium, phosphorus, magnesium and zinc and serum calcium, phosphorus and magnesium were determined. The data (Table III and Appendix A, Tables 6-9) showed a signifi- cantly reduced weight gain (p £ 0.01. in animals receiving zinc supple- mented diets. This harmful effect upon weight gain, noticed in the second week, resulted in a final mean gain of 172 grams for the control animals but only 155 grams, 125 grams and 112 grams for animals receiv- ing diets containing 0.257., 0.507. and 0.757. of zinc, respectively. Of the minerals studied, the clearest and most definite patterns were found in the calcium and zinc values in the bone. As the zinc content of the diet increased, progressively larger amounts of zinc were deposited in the bone accompanied by a progressive lowering of calcium levels. Since the changes in the concentrations of these two minerals were judged highly significant (p £0.01) during the first week of the experimental period, it could be assumed that the toxic effect of zinc on calcium was an immediate one. As before, 0.757. zinc produced the greatest lowering in the calcium content of the bone. At this intake, calcium values were only slightly higher during the fourth week of the experiment than during the first week. This would indicate one of two possibilities: either a progressive slowing in the rate at which calcium was deposited in the bone, or an increased rate of with- drawal of calcium from the bone. Phosphorus presented a more variable pattern and one difficult to interpret. A puzzling drop in control values occurred during the latter part of the experiment. From the results obtained in TABLE III PROGRESSIVE STAGES IN THE EFFECT OF ZINC ON GROWTH, SERUM COMPOSITION, AND BONE MINERALIZATION (EXPERIMENT II) Treatment Weight8 gain Bone constituents c Serum constituents Ca P Mg Zn Ca P Mg gm. mg ./gm/ dry weight mg/100 ml. Control First week 39 140.85 26.19 2.57 .13 9.58 12.95 2.40 Second week 87 150.57 34.41 2.59 .08 12.79 12.75 2.66 Third week 134 156.01 36.56 2.81 .13 11.05 12.76 2.76 Fourth week 172 167.72 17.75 2.56 .15 11.39 12.15 1.57 0.257. Zinc First week 33 131.79 21.31 2.72 .40 10.02 13.16 2.56 Second week 76 143.39 42.32 2.54 .54 12.70 12.42 2.67 Third week 121 144.58 20.10 2.75 .77 13.00 13.93 3.05 Fourth week 155 154.23 19.90 2.60 .78 11.17 11.60 2.50 0.507. Zinc First week 31 113.19 36.56 2.56 .61 9.63 11.91 2.27 Second week 67 122.50 23.94 3.21 .90 10.35 12.96 2.87 Third week 107 142.32 25.38 2.71 1.77 11.10 12.99 3.14 Fourth week 125 143.62 39.86 2.96 1.68 10.01 11.95 1.73 0.75% Zinc First week 27 116.06 16.88 2.50 1.01 9.75 11.66 2.39 Second week 61 114.19 43.18 2.54 1.46 11.83 11.58 2.60 Third week 88 125.56 29.87 2.83 2.39 11.01 11.99 2.47 Fourth week 112 121.66 19.81 2.55 2.39 10.93 11.18 3.03 Each figure represents the mean of 16, 12, 8 and 4 animals for weeks 1,2,3 and 4, respectively. Each figure is the mean of 4 animals. Each figure represents the determination for a pooled sample of serum from 4 animals. 18 Experiment I, a continued rise in these values would have been expected. Although the pattern of change at each individual level of zinc was not a distinct one, the overall picture was a final bone content of phos- phorus equal to, or only slightly higher than, the levels of phosphorus observed during the first week. At the lowest and highest levels of zinc intake, the effect of zinc was noticeable during the third week; at the intermediate level, during the second week. However, the changes which did occur were not significantly different from each other in any one week of the experiment. The considerable variation observed in animals on the same treatment could have been a major reason for the failure to detect significant changes in bone phosphorus. Bone mag- nesium values showed variation from week to week and from diet to diet, but the observed differences were found to be not statistically signif- icant. Zinc, regardless of the concentration used, appeared to have no effect on the levels of magnesium in the bone. No distinct pattern could be defined for the serum constituents under investigation. Only during the fourth week was there any apparent trend. At this time, the serum calcium and phosphorus levels of rats fed the high zinc diets were slightly and consistantly below those of the control group. With magnesium, the trend was one of increased serum levels associated with the feeding of zinc. Experiment III The results of Experiment I and Experiment II showed a definite effect on bone mineralization at the higher levels of zinc intake. The major change noted was a decreased calcium and phosphorus content of the bone. Since Toepher and Sherman obtained improved levels of bone 19 calcium and phosphorus using calcium supplements under conditions of normal nutrition, there was the possibility that increasing the dietary level of calcium and phosphorus would counteract the toxic zinc effects 3 observed in bone. In order to test this possibility, Experiment III was designed to show the effects of adding calcium and phosphorus supplements to high zinc diets. In this experiment, supplemental levels of 0.4% each of calcium and phosphorus were added to diets con- taining 0.75% zinc. Supplements of 0.08% of magnesium were also utilized. The results of Experiment III are presented in Table IV and Appendix A, Tables 10-14. Supporting the previous findings, statistical analysis of the data revealed that increased levels of zinc are associ- ated with a highly significant decrease (p L 0.01) in growth rate. The growth data indicated that the control animals gained approximately twice as much as animals receiving 0.75% zinc in the diet. When sup- plements of 0.4% calcium, 0.4% phosphorus and 0.08% magnesium were added to the high zinc diet, alone or in combination, an improved weight gain occurred. Greatest improvement was noted in those animals receiving a diet supplemented with both calcium and phosphorus followed by the com- bination aupplement of calcium, phosphorus and magnesium. The addition of either calcium, calcium plus magnesium, or phosphorus plus magnesium resulted in intermediate increases in weight gain of approximately the same magnitude. No significant improvement in weight gain was observed when supplements of either phosphorus or magnesium were added to the 3E. Toepher and E. Sherman, J. Biol. Chem., 115, 684 (1936). 3 « E H t^ CO ON o -o o oo £ PH 00 CO •^ OS 4) b -a 'X5 co 00 I to CO CM 3 c •rl Ul W eg -M ao 4) s 4J 01 O J-1 M 00 • 3 w d i to 0J u H CM =0 0 m o os >0 en OS cs in CM 0> o> | N s-« sO CO CM CO U-l VO CM ^ in in OS CM OS OS ON 10 CM Os CO CO CM 10 U H weight Week 1 1 135 .08 130 .49 146 .48 148 .13 560 .18 140 .05 2 127 .19 135 .38 128 .27 136 .32 527 .16 131 .79 3 107 .76 117 .01 106 .70 121 .27 452 .74 113 .19 4 114 .26 126 .02 119 .47 104 .48 464 .23 116 .06 Total 484 .29 508 .90 500 .92 510 .20 2004 .31 Week 2 1 149 .19 147 .51 151 .87 153 .69 602 .26 150 57 2 152 .89 128 .65 146 .86 145 .16 573 .56 143 39 3 97 94 126 .94 133 .22 131 88 489 .98 122 50 4 114 70 106 .55 112 .32 123 20 456 .77 114 19 Total 514 72 509 65 544 27 553 93 2122 57 Week 3 1 148 46 162 64 144 61 168 32 624 03 156 01 2 133 07 156 05 153 20 136 00 578 32 144 58 3 142 28 121 93 155 47 149 58 569 26 142 32 4 112 65 139 59 135 42 114 49 502 15 125 56 Total 536 46 580 21 588 70 568 39 2273 76 Week 4 1 160 87 179 75 179 42 150 83 670 87 167. 72 2 159. 99 150 58 151 21 155. 12 616 90 154. 23 3 156. 05 137 37 136 65 144. 42 574 49 143. 62 4 126. 39 114 98 117 22 128. 04 486 63 121. 66 Total 603. 30 582 68 584 50 578. 41 2348 89 55 TABLE 7. EFFECT OF TREATMENTS TESTED IN EXPERIMENT II ON BONE PHOSPHORUS LEVELS Treatments 1 - Control 2 - 0.25% Zinc 3 - 0.57. Zinc 4 - 0.75% Zinc Replications Treatment Total Mean mg./gm. dry weight Week 1 1 24 .36 26 .99 32 .95 20 .44 104 .74 26 .19 2 13 .17 15 .02 16 .02 41 .01 85 .22 21 .31 3 25 .14 28 .38 35 .36 42 .47 131 .35 32 .84 4 11 .78 18 .06 17 .22 20 .44 57 .50 16 .88 Total 74 .45 88 .45 101 .55 124 .36 388 .81 Week 2 1 17 .94 41 .68 18 .06 56 .96 134 .64 34 .41 2 79 .73 46 .46 17 .40 25 .68 169 .27 42 .32 3 14 .43 19 .23 15 .06 47 .04 95 .76 23 .94 4 50 .09 34 .05 59 .20 29 .37 172 .71 43 18 Total 162 19 141 .42 109 .72 159 05 572 38 Week 3 1 11 40 35 86 25 .93 73 05 146 24 36 56 2 15 59 21 29 18 00 25 53 80 41 20 10 3 37 62 21 88 15 04 26 99 101 53 25 38 4 34 94 39 78 15 21 29 54 119 47 29 87 Total 99 55 118 81 74 18 155 11 447 65 Week 4 1 22. 99 18. 35 14 73 14. 94 71 01 17. 75 2 17. 79 15. 34 25 22 21. 26 79. 61 19. 90 3 43. 50 37. 09 45. 90 32. 94 159. 43 39. 86 4 16. 03 25. 10 20. 58 17. 54 79. 25 19. 81 Total 100. 31 95. 88 106. 43 86. 68 389. 30 56 TABLE 8. EFFECT OF TREATMENTS TESTED IN EXPERIMENT II ON BONE MAGNESIUM LEVELS Treatments 1 - Control 2 - 0.25% Zinc 3 - 0.57. Zinc 4 - 0.75% Zinc Treatment Replications 12 3 Total Mean Week 1 Week 2 Week 3 Week 4 1 2 3 4 Total 1 2 3 4 Total 1 2 3 4 Total 1 2 3 4 Total mg./gm. dry weight 2.61 2.21 2.81 2.67 10.29 2.57 2.54 2.78 2.82 2.74 10.89 2.72 2.28 2.82 2.44 2.70 10.25 2.56 2.30 2.43 2.67 2.61 10.01 2.50 9.73 10.23 10.73 10.72 41.41 2.11 2.69 2.67 2.87 10.35 2.59 2.13 2.42 2.67 2.94 10.16 2.54 3.90 3.73 2.76 2.46 12.86 3.21 2.43 2.55 2.42 2.76 10.16 2.54 9.57 11.39 10.53 11.03 43.52 2.39 2.88 2.65 3.33 11.25 2.81 2.88 2.64 2.71 2.78 11.01 2.75 2.91 2.75 2.56 2.64 10.86 2.71 2.78 3.06 2.79 2.70 11.33 2.83 0.96 11.33 10.71 11.45 44.45 2.44 2.45 2.38 2.97 10.24 2.56 2.60 2.19 2.81 2.80 10.40 2.60 2.93 3.10 2.86 2.93 11.82 2.96 2.54 2.86 2.56 2.25 10.21 2.55 10.51 10.60 10.61 10.95 42.67 TABLE 9. EFFECT OF TREATMENTS TESTED IN EXPERIMENT II ON BONE ZINC LEVELS 57 Treatments 1 - Control 2 - 0.25% Zinc 3 - 0.57. Zinc 4 - 0.757. Zinc Treatment Replications 1 2 Total Mean Week 1 Week 2 Week 3 Week 4 1 2 3 4 Total 1 2 3 4 Total 1 2 3 4 Total 1 2 3 4 Total mg./gm. dry weight 0.13 0.41 0.43 1.44 2.40 0.08 0.44 0.98 1.65 3.15 0.13 0.99 1.84 2.72 5.68 0.15 1.10 1.54 2.52 5.31 0.18 0.46 0.86 1.03 2.53 0.09 0.60 0.64 1.16 2.49 0.15 0.56 1.81 2.19 4.71 0.12 0.55 1.76 2.21 4.64 0.08 0.29 0.55 0.73 1.64 0.08 0.64 1.09 1.67 3.48 0.13 0.77 1.84 2.41 5.15 0.16 0.78 1.82 2.51 5.27 0.13 0.44 0.60 0.85 2.01 0.07 0.47 0.87 1.38 0.11 0.77 1.60 2.23 0.16 0.69 1.62 2.31 0.52 1.59 2.44 4.04 8.59 0.32 2.15 3.59 5.85 2.79 11.91 0.52 3.09 7.09 9.55 4.71 20.25 0.59 3.12 6.74 9.55 0.13 0.40 0.61 1.01 0.08 0.54 0.90 1.46 0.13 0.77 1.77 2.39 0.15 0.78 1.68 2.39 4.78 20.00 58 TABLE 10. EFFECT OF TREATMENTS TESTED IN EXPERIMENT III ON THE GROWTH OF RATS 1 - Control 2 - 0.757. Zn 3 - 0.75% Zn + 0.47. Ca 4 - 0.75% Zn + 0.4% P 5 - 0.757. Zn + 0.087. Mg Treatments 6 - 0.757. Zn + 0.4% Ca + 0.4% P 7 - 0.757. Zn + 0.4% Ca + 0.08% Mg 8 - 0.757. Zn + 0.47. P + 0.08% Mg 9 - 0.757. Zn + 0.47. Ca + 0.4% P + 0.087. Mg Replicat ions Treatment 1 2 3 4 5 6 Total Mean 4 week weight gain (g"J.) 1 198 204 (184)a 160 174 174 1094 182 2 86 76 105 101 85 132 585 97 3 103 121 127 165 134 106 756 126 4 104 104 103 71 136 118 636 106 5 117 105 142 120 108 87 679 113 6 157 163 133 142 166 164 925 154 7 126 125 107 136 157 128 779 129 8 150 114 151 98 91 164 768 128 9 134 129 170 97 139 170 839 139 Total 1175 1141 1222 1090 1190 1243 7061 ( )* indicates calculated missing plot value. 59 TABLE 11. EFFECT OF TREATMENTS TESTED IN EXPERIMENT III ON BONE CALCIUM LEVELS 1 - Control 2 - 0.75% Zn 3 - 0.757. Zn + 0.4% Ca 4 - 0.757. Zn + 0.4% P 5 - 0.757. Zn + 0.087. Mg Treatments 6 - 0.757. Zn + 0.4% Ca + 0.47. P 7 - 0.75% Zn + 0.47. Ca + 0.087. Mg 8 - 0.75% Zn + 0.47. P + 0.08% Mg 9 - 0.757. Zn + 0.47. Ca + 0.47. P + 0.087. Mg Replications Treatment 1 2 3 4 Total Mean mg./gm. dry weight 1 174.07 168.93 171.47 136.96 651.43 162.86 2 114.03 124.21 102.29 103.24 443.77 110.94 3 114.09 131.85 154.93 134.88 535.75 133.94 4 104.82 132.80 107.43 92.69 437.74 109.44 5 117.04 99.40 88.17 95.70 400.31 100.18 6 151.23 153.40 182.33 145.89 632.85 185.21 7 124.41 145.05 187.32 124.55 581.33 145.33 8 139.28 103.76 107.20 118.72 468.96 117.24 9 132.16 114.83 121.04 135.72 503.75 125.94 Total 1171.13 1174.23 1222.18 1088.35 4655.89 60 TABLE 12. EFFECT OF TREATMENTS TESTED IN EXPERIMENT III ON BONE PHOSPHORUS LEVELS 1 - Control 2 - 0.75% Zn 3 - 0.75% Zn + 0.4% Ca 4 - 0.757. Zn + 0.47. P 5 - 0.757. Zn + 0.08% Mg Treatments 6 - 0.75% Zn + 0.4% Ca + 0.4% P 7 - 0.75% Zn + 0.4% Ca + 0.08% Mg 8 - 0.757. Zn + 0.4% P + 0.08% Mg 9 - 0.75% Zn + 0.47. Ca + 0.4% P + 0.08% Mg Treatment Replications 2 3 4 Total Mean mg./gm. dry weight 1 49.54 44.94 38.40 40.64 173.52 43.38 2 31.52 20.44 31.81 17.79 101.56 25.39 3 45.58 20.64 24.42 17.32 107.96 26.99 4 36.05 37.26 25.02 29.77 128.10 32.03 5 52.52 29.41 38.29 20.22 140.44 35.11 6 50.46 38.62 29.03 32.74 150.85 37.71 7 41.39 26.54 34.95 47.80 150.68 37.67 8 40.59 45.42 51.29 24.62 161.92 40.48 9 35.04 30.29 35.78 33.95 135.06 33.77 Total 382.69 293.56 308.99 264.85 1250.09 61 TABLE 13. EFFECT OF TREATMENTS TESTED IN EXPERIMENT III ON BONE MAGNESIUM LEVELS 1 - Control 2 - 0.75% Zn 3 - 0.75% Zn + 0.4% Ca 4 - 0.75% Zn + 0.4% P 5 - 0.75% Zn + 0.08% Mg Treatments 6 - 0.75% Zn + 0.4% Ca + 0.4% P 7 - 0.75% Zn + 0.4% Ca + 0.08% Mg 8 - 0.75% Zn + 0.4% P + 0.08% Mg 9 - 0.75% Zn + 0.4% Ca + 0.4% P + 0.08% Mg Replications Treatment 1 2 3 4 Total Mean mg./gm. dry weight 1 3.21 2.70 3.20 2.80 11.91 2.98 2 2.93 3.15 3.14 2.74 11.96 2.99 3 3.08 2.71 2.77 3.10 11.67 2.92 4 2.93 2.79 2.73 2.63 11.07 2.77 5 3.70 2.86 3.24 3.00 12.80 3.20 6 3.17 2.70 3.33 2.81 12.01 3.00 7 3.44 3.43 3.62 3.44 13.93 3.48 8 3.25 2.69 3.69 2.84 12.46 3.11 9 3.54 2.81 3.00 3.37 12.73 3.18 Total 29.25 25.85 28.73 26.72 110.55 62 TABLE 14. EFFECT OF TREATMENTS TESTED IN EXPERIMENT III ON BONE ZINC LEVELS 1 - Control 2 - 0.75% Zn 3 - 0.75% Zn + 0.47. Ca 4 - 0.75% Zn + 0.4% P 5 - 0.75% Zn + 0.08% Mg Treatments 6 - 0.75% Zn + 0.4% Ca + 0.4% P 7 - 0.75% Zn + 0.4% Ca + 0.08% Mg 8 - 0.75% Zn + 0.4% P + 0.08% Mg 9 - 0.75% Zn + 0.4% Ca + 0.4% P + 0.08% Mg Replications Treatment 1 2 3 4 Total Mean mg./gm. dry weight 1 0.08 0.14 0.09 0.11 0.42 0.11 2 3.38 2.70 2.49 2.17 10.75 2.69 3 0.87 2.16 2.04 1.81 6.87 1.72 4 1.62 2.32 2.39 1.47 7.79 1.95 5 2.68 1.82 1.85 2.83 9.17 2.29 6 1.17 0.99 1.03 1.04 4.24 1.06 7 1.68 1.30 1.55 1.18 5.71 1.43 8 1.44 1.87 1.73 1.14 6.18 1.54 9 0.66 0.99 1.02 0.68 3.35 0.84 Total 13.57 14.30 14.20 12.41 54.49 63 TABLE 15. EFFECT OF TREATMENTS TESTED IN EXPERIMENT IV ON THE GROWTH OF RATS Treatments 1 - Control 2 - 0.75% Zn 3 - 0.757. Zn + 0.4% Ca 4 - 0.75% Zn + 0.47. P 5 - 0.75% Zn + 0.87. Ca 6 - 0.75% Zn + 0.8% P 7 - 0.75% Zn + 1.2% Ca 8 - 0.757. Zn + 1.2% P 9 - 0.757. Zn + 0.4% Ca + 0.4% P 10 - 0.75% Zn + 0.8% Ca + 0.8% P 11 - 0.75% Zn + 1.2% Ca + 1.2% P Replications Treatment 1 2 3 4 5 6 Total Mean 4 weeks weight gain (g«n.) 1 144 139 132 160 140 165 889 148 2 62 127 117 100 106 112 624 104 3 113 126 125 114 138 129 745 124 4 119 106 118 106 121 108 678 113 5 139 132 126 128 125 136 786 131 6 117 128 110 128 118 94 695 116 7 121 132 130 112 109 115 719 120 8 126 123 116 126 109 112 713 119 9 134 131 133 119 143 128 788 131 10 125 123 115 154 131 145 793 132 11 111 101 117 128 123 110 690 115 Total 1311 1368 1339 1375 1363 1354 8110 TABLE 16. EFFECT CF TREATMENTS TESTED IN EXPERIMENT IV ON BONE CALCIUM LEVELS Treatments 1 - Control 7 ■ • 0.75% Zn + 1.2% Ca 2 - 0.75% Zn 8 • • 0.75% Zn + 1.2% P 3 - 0.75* Zn + 0.4% Ca 9 • 0.75% Zn + 0.4% Ca + 0.4% P 4 - 0.751 Zn + 0.4% P 10 ■ 0.75% Zn + 0.8% Ca + 0.8% P 5 - 0.751 Zn + 0.8% Ca 11 ■ - 0.75% Zn + 1.2% Ca + 1.2% P 6 - 0.75% Zn + 0.8% P Replications Treatment 1 2 3 4 5 6 Total Mean mg ./gm. dry weight 1 176.70 174.12 151.60 150.61 156.78 170.36 980.17 163.36 2 108.21 136.20 107.47 98.27 118.28 130.02 698.45 116.41 3 126.85 140.91 119.30 116.27 147.28 143.10 793.71 132.29 4 112.11 103.99 118.09 113.80 132.58 129.14 709.71 118.29 5 147.43 138.28 160.64 138.05 152.65 157.65 894.70 149.12 6 128.60 130.88 129.63 125.33 123.72 109.28 747.44 124.57 7 147.69 146.97 152.94 147.81 132.53 152.26 880.20 146.70 8 127.33 144.53 124.67 115.36 129.41 114.76 756.06 126.01 9 139.18 158.75 163.18 118.10 146.16 150.50 875.87 145.98 10 156.02 145.53 115.26 153.31 148.38 160.88 879.38 146.56 11 151.07 149.44 170.09 159.37 146.16 163.84 939.97 156.66 Total 1521.19 1569.60 1512.87 1436.28 1533.93 1581.79 9155.66 ? 65 TABLE 17. EFFECT OF TREATMENTS TESTED IN EXPERIMENT IV ON BONE PHOSPHORUS LEVELS Treatments 1 - Control 2 - 0.75% Zn 3 - 0.75% Zn + 0. 4 - 0.75% Zn + 0. 5 - 0.75% Zn + 0. 6 - 0.75% Zn + 0. V7. Ca 4% P 8% Ca B% P 7 8 9 10 11 - 0.75% Zn + 1. - 0.75% Zn + 1. - 0.75% Zn + 0. - 0.75% Zn + 0. - 0.75% Zn + 1. 2% Ca 2% P 4% Ca + 0.4% P 8% Ca + 0.8% P 2% Ca + 1.2% P Replications Treatments 1 2 3 4 5 6 Total Mean mg ./gm. dry weight 1 42.64 43.07 41.78 52.45 35.74 28.74 244.42 40.74 2 30.83 27.02 39.73 38.82 25.60 30.76 192.76 32.13 3 44.46 38.86 27.72 27.72 42.70 29.82 211.28 35.21 4 44.35 40.40 29.82 24.94 25.13 27.75 192.39 32.06 5 35.34 40.52 23.89 18.73 37.52 26.86 182.86 30.48 6 23.90 34.10 28.55 39.53 31.42 15.82 173.32 28.89 7 36.80 39.15 41.08 25.90 25.17 32.59 200.69 33.45 8 46.02 31.86 22.86 19.59 37.75 30.96 189.04 31.51 9 31.67 26.64 30.77 32.16 26.06 22.57 169.87 28.31 10 40.66 46.32 23.23 29.58 51.66 36.24 227.72 37.95 11 32.88 43.80 38.57 21.68 24.57 30.61 192.11 32.02 Total 409.55 411.77 348.00 331.10 363.32 312.72 2176.46 66 TABLE 18. EFFECT OF TREATMENTS TESTED IN EXPERIMENT IV ON BONE MAGNESIUM LEVELS Treatments 1 - Control 2 - 0.75% Zn 3 - 0.75% Zn + 0.4% Ca 4 - 0.75% Zn + 0.4% P 5 - 0.75% Zn + 0.8% Ca 6 - 0.75% Zn + 0.8% P 7 - 0.75% Zn + 1.2% Ca 8 - 0.75% Zn + 1.2% P 9 - 0.75% Zn + 0.4% Ca + 0.4% P 10 - 0.75% Zn + 0.8% Ca + 0.8% P 11 - 0.75% Zn + 1.2% Ca + 1.2% P Replications Treatment 1 2 3 4 5 6 Total Mean mg./gra. dry we ight 1 1.35 2.18 2.25 2.99 2.89 2.80 14.46 2.41 2 1.40 2.40 2.29 2.31 2.69 2.82 13.91 2.32 3 2.80 2.12 1.86 1.75 2.82 1.75 13.10 2.18 4 1.96 2.48 2.49 2.05 2.35 2.38 13.71 2.28 5 1.70 2.45 1.63 2.25 2.82 2.22 13.07 2.18 6 2.52 2.19 1.84 2.29 2.54 2.21 13.59 2.27 7 1.39 2.32 2.12 2.67 2.49 2.61 13.60 2.27 8 1.56 2.31 1.67 2.12 1.95 1.85 11.46 1.91 9 3.04 1.83 2.04 2.50 2.65 2.43 14.49 2.42 10 1.41 1.47 1.86 1.61 2.68 1.68 10.71 1.79 11 1.01 0.84 1.39 1.54 2.23 (1.42)a 8.43 1.41 Total 20.14 22.59 21.44 24.08 28.11 24.17 140.53 '( ) indicates calculated missing plot value. b7 TABLE 19. EFFECT OF TREATMENTS TESTED IN EXPERIMENT IV ON BONE ZINC LEVELS Treatments 1 - Control 2 - 0.75% Zn 3 - 0.75% Zn + 0.4% Ca 4 - 0.75% Zn + 0.4% P 5 - 0.75% Zn + 0.8% Ca 6 - 0.75% Zn + 0.8% P 7 - 8 - 9 - 10 - 11 - 0.75% Zn + 1.2% Ca 0.75% Zn + 1.2% P 0.75% Zn + 0.4% Ca + 0.4% P 0.75% Zn + 0.8% Ca + 0.8% P 0.75% Zn + 1.2% Ca + 1.2% P Replications Treatment 1 2 3 4 5 6 Total Mean nig ./gra. dry weight 1 0.11 0.09 0.06 0.09 0.14 0.11 0.60 0.10 2 0.65 1.22 1.18 1.76 1.66 1.17 7.63 1.29 3 1.45 0.88 1.41 0.84 1.41 0.20 6.19 1.03 4 0.73 1.03 0.48 1.03 1.11 1.56 5.95 0.99 5 1.10 1.08 0.76 0.69 1.08 1.26 5.96 0.99 6 0.63 0.30 0.57 0.83 1.03 0.74 4.09 0.68 7 0.65 0.78 0.91 1.26 1.78 1.45 6.81 1.13 8 0.24 0.34 0.41 0.49 0.61 0.31 2.39 0.40 9 0.59 0.59 0.62 1.42 0.89 0.44 4.55 0.76 10 0.23 0.41 0.16 (0.49)a 0.36 0.49 2.16 0.28 11 0.15 0.08 0.27 0.30 0.48 0.30 1.57 0.26 Total 6.52 6.80 6.83 9.21 10.54 8.00 47.89 ( )a indicates calculated missing plot value. APPENDIX B METABOLISM DATA TABLE 1. EFFECT OF ZINC ON THE NET RETENTION OF CALCIUM (EXPERIMENT V) Mg. per total feed intake 363.94 Mg. excreted in the urine 0.49 Mg. excreted in the fecea 119.05 Net retention 244.40 Collection period 1 Control Replications 332.49 345.97 314.52 0.52 1.17 0.21 108.99 145.65 88.91 222.98 199.15 225.40 0.75% Zinc Replies itions 1 2 3 4 320.97 311.80 325.56 320.97 1.93 1.46 2.27 2.23 173.34 215.64 248.62 233.61 145.70 94.70 74.67 85.13 Mg. per total feed intake 399.89 Mg. excreted in the urine 0.58 Mg. excreted in the feces 196.38 Net retention 202.93 Collection period 2 Control Replications 404.38 0.54 131.81 272.03 386.41 0.38 110.61 275.42 4 384.16 0.47 151.22 232.47 0.75% Zinc Replications 1 2 3 4 348.48 1.44 295.39 51.65 371.41 1.32 311.66 58.43 353.07 3.41 342.65 8.01 362.24 1.24 310.15 50.85 3 Mg. per total feed intake 395.39 Mg. excreted tn the urine 1.29 Mg. excreted in the feces 233.90 Net retention 160.20 TABLE 1. (continued) Collection period 3 Control Replications 433.58 408.87 399.89 0.71 1.31 1.23 184.23 187.69 162.18 248.64 219.87 236.48 0.75Z Zinc Replications 1 2 3 378.29 378.29 389.75 3.14 2.87 1.38 260.69 245.79 327.68 114.46 129.63 60.69 371.41 1.11 294.45 75.85 Mg. per total feed intake 491.99 Mg. excreted in the urine 2.50 Mg. excreted in the feces 282.61 Net retention 206.88 Collection period 4 Control Replications 2 3 467.28 456.05 2.29 1.58 236.49 215.88 228.50 238.59 0.75% Zinc Replications 4 1 2 3 4 471.78 398.92 426.43 434.14 371.41 0.97 1.00 4.07 1.53 1.36 220.70 324.67 308.04 397.35 345.78 250.11 73.25 114.32 43.26 24.27 TABLE 2. EFFECT OF ZINC ON THE NET RETENTION OF PHOSPHORUS (EXPERIMENT V) Mg. per total feed intake 477.14 Mg. excreted in the urine 106.50 Mg. excreted in the feces 58.78 Net retention 311.86 Collection period 1 Control Replications 435.90 96.75 53.52 285.63 453.58 113.00 61.04 279.54 412.34 98.25 51.09 263.00 0.75X Zinc Replications 1 2 3 407.40 395.76 413.22 52.00 51.50 57.75 (150.02) 138.54 148.80 205.38 205.72 206.67 407.40 48.25 152.03 207.12 Mg. per total feed intake Mg. excreted in the urine Mg. excreted in the feces Net retention 1 524.26 151.75 (53.12)* 319.39 Collection period 2 Control Replications 530.15 149.75 56.73 323.67 506.59 133.00 62.36 311.23 503.65 136.75 68.62 298.28 0.75% Zinc Replications 1 2 3 442.32 471.42 448.14 77.58 87.50 139.50 193.40 237.00 182.31 171.13 146.92 126.33 459.78 110.50 189.28 159.80 TABLE 2. (continued) Collection period 3 Mg. per total feed intake Mg. excreted in the urine Mg. excreted in the feces Net retention Control 0 75X Zinc Replications Replies tionc 1 2 3 4 1 2 3 4 518 37 568 .44 536.04 524.26 480 .15 480 .15 494 70 471 42 191 00 170 .28 164.86 133.64 103 46 67 44 115 54 108 43 9/ /4 80 .91 75.48 73.92 216 .81 109 35 158 63 120 21 229 .63 317 .25 295.70 316.70 159 .88 303 36 220. 53 242 78 Mg. per total feed intake 645.02 Mg. excreted in the urine 205.40 Mg. excreted in the feces 151.73 Net retention 287.89 Collection period 4 Control Replications 2 3 4 1 612.62 597.90 618.51 506.34 193.62 179.49 169.37 92.01 95.32 75.70 94.59 192.93 323.68 342.71 354.55 221.40 ( ) indicates calculated missing plot value. 0.75Z Zinc Replications 2 3 541.26 75.07 133.47 332.72 538.35 119.36 222.55 196.44 471.42 116.66 188.41 166.35 TABLE 3. EFFECT OF ZINC ON THE NET RETENTION OF MAGNESIUM (EXPERIMENT V) Collection period 1 Mg. per total feed intake Mg. excreted in the urine Mg. excreted in the feces Net retention Control 0.75% Zinc Replications Replications 1 2 3 4 1 2 3 4 25.13 0.12 22.96 1.56 23.89 0.40 21.72 0.89 18.98 0.98 18.44 0.79 19.26 0.64 18.98 0.94 6.14 18.98 6.54 14.86 7.17 16.32 7.43 13.40 9.35 8.65 8.42 9.23 8.37 10.25 6.40 11.64 Mg. per total feed intake 27.62 Mg. excreted in the urine 0.19 Mg. excreted in the feces 8.36 Net retention 19.07 Collection period 2 Control Replications 27.93 26.69 26.53 20.61 0.11 (0.15)a 0.14 1.98 7.11 8.75 8.07 9.38 20.71 18.79 18.32 9.25 0.75X Zinc Replications 2 3 21.97 1.97 8.64 11.36 20.88 1.38 12.15 7.35 21.42 , (1.77)1 10.32 9.33 TABLE 3. (continued) Collection period 3 - Mg. per total feed intake Mg. excreted in the urine Mg. excreted in the feces Net retention Control 0.75% Zinc Replications Replications 1 2 3 4 1 2 3 i i 27 .31 29.94 28. ,24 27 .62 22.37 22.37 23.05 21 .97 1 .44 0.89 1. ,12 0 .92 1.64 1.52 0.34 0 .29 10 ./9 8.78 6. ,68 8 .48 8.45 12.18 11.37 11 .37 13 .08 20.27 20, .44 18 .22 12.28 8.67 11.34 10 .33 Collection period 4 Mg. per total feed intake Mg. excreted in the urine Mg. excreted in the feces Net retention Control 0.75% Zinc Replications Replications 1 2 3 4 1 2 3 t i 33. .98 32.27 31.50 32 .58 23.59 25.22 25.09 21 .97 1. .26 0.60 0.28 0 .49 1.28 1.01 0.97 1 .11 U .59 12.73 7.11 10 .56 6.87 13.49 11.11 9 .64 19 .11 18.94 24.11 21 .53 15.44 10.72 13.01 11 .22 ( ) indicates calculated missing plot value. APPENDIX C ANALYSES OF VARIANCE DATA 76 TABLE 1. ANALYSES OF VARIANCE OF DATA COLLECTED IN EXPERIMENT I Source Degrees of of Sum of squares Mean square Variation freedom Growth Total 23 32,769.8334 Replications 5 1,904.8334 380.9666 Treatments 3 25,593.8334 8,531.2777** 1,2 vs. 3,4 1 18,928.1666 18,928.1666** 1 vs. 2 1 225.3333 225.3333 3 vs. 4 1 6,440.3333 6,440.3333** Error 15 5,271.1666 351.4111 Bone calcium Total 23 14,339.6837 Replications 5 1,579.0199 315.8039 Treatments 3 6,027.9035 2,009.3011* 1,2 vs. 3,4 1 4,287.2247 4,287.2247** 1 vs. 2 1 0.6580 0.6580 3 vs. 4 1 1,740.0208 1,740.0208 Error 15 6,732.7603 448.8506 Bone phosphorus Total 23 7,136.3346 Replications 5 874.3784 174.8756 Treatments 3 2,795.6213 921.8737* 1,2 vs. 3,4 1 2,324.2080 2,324.2080** 1 vs. 2 1 434.2830 434.2830 3 vs. 4 1 7.1302 7.1302 Error 15 3,466.3349 234.8889 Bone magnesium Total 23 0.5834 Replications 5 0.1586 0.0317 Treatments 3 0.0280 0.0093 Error 15 0.3968 0.0264 77 TABLE 1. (continued) Source Degr ees of of Sum of squares Mean square Variation Freedom Bone zinc Total 23 18.5343 Replications 5 0.5957 0.1191 Treatments 3 17.2600 5.7533** 1,2 vs. 3,4 1 13.5751 13.5751** 1 vs. 2 1 0.7252 0.7252** 3 vs. 4 1 2.9601 2.9601** Error 15 0.6786 0.0452 ** Significant (p £. 0.05) Highly significant ( ^ 0.01) 78 TABLE 2. ANALYSES OF VARIANCE OF DATA COLLECTED IN EXPERIMENT II Source of variation Week 2 Total Replications Treatments Error Degrees of freedom Sum of squares Growth 47 11 3 33 7,530 887 4,735 1.908 Mean square 80.64 1,578.00 57.82 ** Week 1 Total Replications Treatments Error Bone calcium 15 3 3 9 2,656.2485 106.5641 251,078.6610 582.7708 35.5200 655.6378 64.7500 ft* Week 4 Total Replications Treatments Error Bone phosphorus 15 3,328.1154 3 872.5431 290.8477 3 583.9242 194.6414 9 1,871.6481 207.9609 Week 4 Total Replications Treatments Error Week 1 Total Replications Treatments Error Bone magnesium 15 1.1448 3 0.0281 3 0.4480 9 0.6687 Bone zinc 15 3 3 9 Significant (p ^ 0.05) ** Highly significant (p ^ 0.01) 2.0704 0.1215 1.6602 0.2887 0.0093 0.1493 0.6743 0.0405 0.5534** 0.6320 79 TABLE 3. ANALYSES OF VARIANCE OF DATA COLLECTED IN EXPERIMENT III Source of variation Degrees of freedom Sum of squares Mean square Growth Total 53 53,625.8149 Replications 5 1,765.7037 353.1407 Treatments 8 32,619.1482 4,077.3936** 2,4,5 vs. 3 ,7,8 1 4,511.3611 4,511.3611** 6,9 vs. 7 8 1 1,962.0416 1,962.0416* 6 vs. 9 1 616.3333 616.3333 Error 40 19,240.9630 481.0241 Bone calcium Total 35 23,789.7741 Replications 3 1,029.2579 343.0859 Treatments 8 15,931.5928 1,991.4491** 2,4,5 vs. 3, 6,7 1 9,130.2905 9,130.2905** 1 vs. 3 1 1,115.1552 1,115.1552 2 vs. 9 1 299.8000 299.8000 Error 24 6,828.9234 284.5384 Bone phosphorus Total 35 3,430.5605 Replications 3 43,409.0280 303.2289 Treatments 8 1,123.8380 104.4797* 2,3 vs. 5,9 1 272.0850 272.0850* 2,3 vs. 6,7 1 529.1150 529.1150** 1 vs. 6,7 1 86.2983 86.2983 Error 24 1,397.0358 58.2098 Bone magnes ium Total 35 3.5115 Replications 3 0.9478 0.3159 Treatments 8 1.2894 0.1611* 1,2,3,4 vs. 5,6,8,9 1 0.3591 0.3591* Error 24 1.2743 0.0530 TABI£ 3. (continued) 80 Source of variation Degrees of freedom Sum of s

The effect of zinc on rock bass feeding.

Abstract

Extracted data

The effect of zinc on rock bass feeding.

Abstract

Extracted data

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