S‘JME FACTORS AFFECTING MiNERAL UT‘ELEZATIOQN B‘i’ 'E‘HE BABY PR3 Thesis éer the Degree of Ph. E). MECHEE‘HW ST‘ATE UNWERSETY DELEEY G. HENBRICKS 1967 [“63" This is to certify that the thesis entitled SOME FACTORS AFFECTING MINERAL UTILIZATION BY THE BABY PIG presented by Deloy G. Hendricks has been accepted towards fulfillment of the requirements for M degree in Mfluséan 04y flfWéu Major professor Date SQFéMAzr‘ /2J /7é 7 0-169 SOME FACTORS AFFECTING MINERAL UTILIZATION BY THE BABY PIG Thesis for the Degree of Ph.D. MICHIGAN STATE UNIVERSITY Deloy G. Hendricks 1967 ABSTRACT SOME FACTORS AFFECTING MINERAL UTILIZATION BY THE BABY PIG by Deloy G. Hendricks Four experiments, involving a total of 52 baby pigs, were conducted to investigate the effects of source and level of protein, and level of ergocalciferol on mineral utilization by the baby pig. Some physiologi- cal effects of these various regimens were also studied. Baby pigs were taken from their dams at 3 days of age and, after a 4 day adaption to the dry diet and environmental conditions, were assigned to the various dietary treatments. Pigs were housed in wire bottomed metal cages in a room where no ultraviolet rays could enter. Serum was obtained from blood drawn from the anterior vena cava initially, at 3 weeks and again at 5 weeks on trial. A 3 day mineral balance study was conducted after which pigs were autopsied and weight of various organs and glands taken. Humeri, femurs and 2 ribs were taken for mineral and strength determinations. Performance, as measured by growth rate and feed efficiency, was no different when casein or soy was fed at levels providing 32% or 16% crude protein in the diet. Increasing ergocalciferol from 6.25 to 12.50 jug/kg diet did not improve rate of gain or feed efficiency. Food intake was decreased at the higher levels of protein intake irrespective of protein source. Serum inorganic phosphorus was depressed by high levels of isolated soy protein intake. Increasing the dietary intake of ergocalciferol did not overcome this depression nor the accompanying increase in serum alkaline phosphatase. By contrast, high levels of casein protein in the diet did not produce these changes in these two serum components. Three mineral balance studies, conducted after 5 weeks on a given regimen, showed that increasing isolated soy protein in the diet from 20% or 30% up to 40% had no effect on the retention or excretion of Ca, P or magnesium. Increasing ergocalciferol in the diet from 6.25 to 12.50/ug/kg diet did not improve the utilization of these minerals. A balance trial comparing casein with isolated soy protein at levels equal to 16% or 32% crude protein showed that casein enhanced retention of Ca, P and Mg when it comprised 40% of the diet. Isolated soy protein, how- ever, significantly depressed the retention of these minerals when fed at the high level. No consistent dietary treatment effects could be shown on the retention or excretion of Na, K, Fe, Zn, Mn, Cu, Co or chromium. Bone analyses showed decreasing specific gravity, ash, Ca, P, Mg and breaking strength as dietary protein increased when the protein source was isolated soy. Level of protein supplied by casein had no effect on these bone measurements. Ergocalciferol fed at 12.50/4g/kg diet was no more effective in preventing this depression in bone miner- alization than was 6.25/fig ergocalciferol/kg diet. Physiological responses observed, that were due to dietary treat- ment, included a mild hypertrophy of kidneys, liver and pancreas as pro- tein level in the diet increased. This was observed whether the protein was provided by casein or isolated soy. Ergocalciferol level in the diet had no effect on the physiological measurements taken. Influx and efflux of nutrients from various sections of the gastro- intestinal tract were studied by the chromic oxide indicator method. Influx of all nutrients studied was increased in the cranial small in- testine when casein was the source of dietary protein as compared to isolated soy. A compensatory efflux of these nutrients was observed to take place from the caudal half of the small intestine. Dietary protein level had no measurable effect on nutrient movement into or out of the gastrointestinal tract. SOME FACTORS AFFECTING MINERAL UTILIZATION BY THE BABY PIG by Deloy G. Hendricks A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 1967 449%é+ 3-3-99 ACKNOWLEDGEMENT The author expresses his sincere appreciation to Dr. E. R. Miller and Dr. D. E. Ullrey for their guidance and assistance throughout this ‘work, and for their critical reading of this manuscript. The writer ‘wishes to express his thanks to the members of his guidance committee, Drs. E. R. Miller, J. A. Hoefer, D. E. Ullrey, R. W. Luecke and E. P. Reineke for their suggestions and encouragement during the completion of this study. The author is deeply grateful to the Animal Husbandry Department of Michigan State University for the use of facilities and animals and for financial support through an assistantship. A sincere note of appreciation also goes to fellow graduate students, laboratory assis- tants and department secretaries, who have offered a great deal of assistance and encouragement during the course of this study. Special thanks are due Mrs. Kathryn Ide, who very skillfully and efficiently typed this manuscript. The author is very grateful to his parents for their fostering with- in him a love for animals and an interest in good nutritional practices.' Their continual encouragement toward the pursuit of experience and knowledge has been a motivating factor for this writer throughout his life. Above all, the author is indebted to his wife, Cora, whose patience, sacrifices and encouragement made the completion of this study possible. 11 Deloy G. Hendricks candidate for the degree of Doctor of Philosophy Dissertation: Some Factors Affecting Mineral Utilization by the Baby Pig Outline of Studies: Main area of study: .Animal Husbandry (Animal Nutrition) Supporting areas of study: Biochemistry, Physiology Biographical Items: Born: December 18, 1938, Pocatello, Idaho Undergraduate studies: University of Idaho, 1957—1961 Graduate studies: ZMichigan State University, 1963-1967 Experience: Lieutenant, United States Army, 1961-1963 Graduate.Assistant, Michigan State University, 1963 to present Member: American Society of Animal Sciences Alpha Zeta iii II. III. VII. VIII. TABLE OF CONTENTS INTRODUCTION . . . REVIEW OF LITERATURE . . . Protein Level . . . . . . . . . Protein Source . . Vitamin D Level . . . Nutrient Absorption and Secretion . . . . EXPERIMENTAL PROCEDURE . Introduction . General Conduct of Experiments . Chemical Analyses . . . . . . . Statistical Analyses . RESULTS AND DISCUSSION Experiment I. Effect of protein level on mineral utiliza- tion by the baby pig . . . Experiment II. Effect of level of protein and vitamin D on mineral utilization by the baby pig . Experiment III. Effect of level of protein and vitamin D on mineral utilization by the baby pig . Experiment IV. Effect of source and level of protein on mineral utilization by the baby pig . SUMMARY . . . . . . . . . . . . CONCLUSIONS . . . . BIBLIOGRAPHY . APPENDIX . . . . . iv 12 17 32 32 32 38 43 44 44 49 60 89 92 93 109 Table 10. ll. 12. 13. 14. LIST OF TABLES Composition of purified diets. Growth and serum analyses of baby pigs fed different levels of protein . . . . . . . . . . . . . . Daily calcium, phosphorus, magnesium and nitrogen ex- cretion and retention as affected by level of dietary protein . . . . Weight, density, composition and strength of bones from baby pigs fed different levels of protein . Organ weights of baby pigs fed different levels of protein. Growth and serum analyses of baby pigs fed two different levels of protein and ergocalciferol . Daily calcium, phosphorus, magnesium and nitrogen ex- cretion and retention as affected by level of dietary protein and ergocalciferol . Weight, density, composition and strength of bones from baby pigs fed different levels of protein and ergocalciferol . . . . . . . . . . . . Organ weights of baby pigs fed different levels of pro- tein and ergocalciferol . . . . . . . . . . . Growth and serum analyses of baby pigs fed different levels of protein and ergocalciferol . Daily calcium, phosphorus, magnesium, and nitrogen ex- cretion and retention as affected by level of dietary protein and ergocalciferol . Weight, density, composition and strength of bones from baby pigs fed different levels of protein and ergocalciferol . . . . . . . . . . . . . . . Organ weights of baby pigs fed different levels of pro- tein and ergocalciferol . . . . . . . . . . . . . Growth and serum analyses of baby pigs fed casein or soy protein at two different levels Page 33 45 46 47 49 51 52 53 55 56 57 58 62 63 LIST OF TABLES (Continued) Table 15 (A). Daily calcium, phosphorus, sodium, and potassium ex- cretion and retention as affected by source and level of dietary protein. . . . . 15 (B). Daily magnesium, iron, zinc, manganese, and copper excretion and retention as affected by source and level of dietary protein . . . . . . . 15 (C). Daily cobalt, nitrogen, energy, and chromium excretion and retention as affected by source and level of dietary protein . . . . . . . . . . . . 16. Weight, density, composition and strength of bones from baby pigs fed casein or soy protein at two different levels . . . . . . . . . . . . . . . . l7. Organ weights of baby pigs fed casein or soy protein at two different levels . vi Figgre 10. 11. LIST OF FIGURES Exp. I. -- Load-deflection curves for femurs from pigs fed different levels of soy protein (16%, 24%, or 32%) with 6.25/ug ergocalciferol/kg diet . Exp. II. -- Loadedeflection curves for femurs from pigs fed two different levels of soy protein (24% or 32%) and ergocalciferol (6.25 or 12.50/ug/kg diet). Exp. III. -- Load-deflection curves for femurs from pigs fed two different levels of soy protein (16% or 32%) and ergocalciferol (6.25 or 12.50/ug/kg diet). Exp. III. -- Photographs (4x) of cross-sections of femurs from pigs fed 16% soy protein diet with 6.25Axg ergo- calciferol/kg, 16% soy protein diet with 12.50/ug ergocalciferol/kg, 32% soy protein diet with 6. 25/ug ergocalciferol/kg” or 32% soy protein diet with 12.50 [big ergocalciferol/kg - . - - - Exp. IV. -- Load—deflection curves for femurs from pigs fed casein (C) or soy (S) at two different levels (16% or 32%) with 6.25/mg ergocalciferol/kg diet.. . Exp. IV. -- Photographs (5x) of cross- -sections of femurs from.pigs fed 16% casein, 32% casein, 16% soy,or 32% soy, with 6. 25/ug ergocalciferol/kg diet . . Exp. IV. -- Influx and efflux of dry matter along the GI tract of pigs fed casein or soy protein at two different levels of dietary protein . . . . . Exp. IV. -- Influx and efflux of calcium along the GI tract of pigs fed casein or soy protein at two .different levels of dietary protein . Exp. IV. -- Influx and efflux of phosphorus along the GI tract of pigs fed casein or soy protein at two different levels of dietary protein . Exp. IV. -- Influx and efflux of sodium along the GI tract of pigs fed casein or soy protein at two different levels of dietary protein . Exp. IV. -- Influx and efflux of potassium along the GI tract of pigs fed casein or soy protein at two different levels of dietary protein . vii Page 48 54 59 61 71 72 74 75 76 77 78 Figgre 12. 13. 14. 15. 16. 17. 18. LIST OF FIGURE (Continued) Exp. IV. -- Influx and efflux of magnesium along the GI tract of pigs fed casein or soy protein at two different levels of dietary protein . Exp. IV. -- Influx and efflux of iron along the GI tract of pigs fed casein or soy protein at two different levels of dietary protein . . Exp. IV. -- Influx and efflux of zinc along the GI tract of pigs fed casein or soy protein at two different levels of dietary protein . . . . . . . . Exp. IV. -- Influx and efflux of manganese along the GI tract of pigs fed casein or soy protein at two different levels of dietary protein . . . . . Exp. IV. -- Influx and efflux of copper along the GI tract of pigs fed casein or soy protein at two different levels of dietary protein . . . . . Exp. IV. -- Influx and efflux of cobalt along the GI tract of pigs fed casein or soy protein at two different levels of dietary protein . Exp. IV. -- Summary of net nutrient influx or efflux from sections of the gastrointestinal tract of baby pigs as determined by the Bergeim (1926) method. viii 79 80 81 82 83 84 87 Table 1. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. LIST OF APPENDIX TABLES Dilutions used for analyses . Intake and excreta of pigs during balance trial, Exp. IV. Slaughter sequence of pigs from Exp. IV . pH within the GI tract of baby pigs, Exp. IV Dry matter concentration within the GI tract of baby pigs, Exp 0 IV 0 O I O O I O O O O O O C I O O O O O I O o I Calcium concentration within the GI tract of baby pigs, Exp. IV . . . . . . . . . . . . . . . . . Phosphorus concentration within the GI tract of baby pig‘s, Expo IV 0 o o o o o o o o o I a o o 0 Sodium concentration within the GI tract of baby pigs, EXP I IV 0 O O O O O O O O 0 O O O O I O O O O I Potassium concentration within the GI tract of baby pigs, Exp. IV 0 O O O O O O O O O O O O I 0 0 Magnesium concentration within the GI tract of baby pigs, Exp 0 IV 0 O O I O O O O O O O O O O O O O D 0 Iron concentration within the GI tract of baby pigs, Exp 0 IV C O O C O O O C O O O C O O O O O I O Zinc concentration within the GI tract of baby pigs, Exp. IV . . . . . . . . . . . . . . . . . . . ' Manganese concentration within the GI tract of baby pigs, Exp 0 Iv I O O O O O O C O O O O O O O O O 0 Copper concentration within the GI tract of baby pigs, Exp. IV 0 O O O O O O O O O O O O O O I O O O Cobalt concentration within the GI tract of baby pigs, Exp 0 IV 0 O O O O O O O O O O O O O 0 C O O O o o Chromium concentration within the GI tract of baby pigs, Exp. IV . . . . . . . . . . . . . . . . . . . . . . Comparison of indicator and balance trial methods for de- termining apparent digestibility of nutrients, Exp. IV. Mineral mixtures used in purified diets . Vitamin mixtures used in purified diets . ix 111 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 I. INTRODUCTION This laboratory and others have been using purified diets to study various nutrient interactions. Reports by Carlson t al. (1964a, b) that bone ash in turkey poults was depressed by high levels of isolated soy protein in the diet were of interest to us. When soybean oil meal was substituted for the isolated soy preparation, it was discovered that lower levels of vitamin D3 were adequate for maximal growth. Miller al. (1965b) showed that purified diets containing casein required gg lower levels of vitamin D supplementation than similar diets composed of isolated soy protein. They carried out their studies on baby pigs and used serum Ca, P, alkaline phosphatase, and mineral deposition in bones as their criteria of vitamin D adequacy. This study was undertaken to determine the effects of source and level of protein and level of ergocalciferol on mineral utilization by the baby pig. Attempts were made to determine the digestive, absorp- tive, assimilative and excretive consequences of these factors. II. REVIEW OF LITERATURE A. Protein Level Economics would generally dictate that diets be utilized which are as low in protein as will keep the organism growing and healthy. A great deal of research has been carried out in all species to determine the minimal levels of protein that will still allow the subject to sat- isfactorily accomplish its purpose. A review of the literature shows very little work directly involving high levels of dietary protein and its possible metabolic and physiological effects. Manners and McCrea (1962), feeding casein diets to baby pigs, showed that as protein level ‘was increased from 20% to 45% of the diet, average daily gain and feed efficiency were maximum at 25% to 35% crude protein. Serum protein values were shown to respond directly to dietary protein level in the range of 20% to 35% crude protein in the diet. Becker g£‘§1.(1954b), 'working with pigs 1 to 4 weeks of age, showed that performance increased with increasing protein levels up to 22.4% of the diet. Pigs from 5 to 9 weeks of age gave adequate performance when fed a 12% milk protein diet. Peo gg‘gl. (1954), feeding a protein mixture of 50% soybean meal and 50% dried skim milk to pigs 1 to 8 weeks of age, showed that better gains were obtained with a high level of fat (10%) and high level of protein (30%). Sewell $5.51. (1953) reported satisfactory growth from suckling pigs fed simulated milk diets containing 24% and 28% protein. Sewell gtflgl. (1961) reported that the most rapid and efficient gains ‘were obtained with a combination of 20% protein and 8% added fat. There was a progressive decrease in the quantity of feed required to produce a unit of gain as protein level increased. In this latter trial, soy pro- tein was fed to pigs that were 3 to 7 weeks old. IMeade, t l. (1965) 2 found that corn-soybean meal diets containing 18% protein supported as rapid and efficient gains over a 6 week period in pigs weighing 13.4 kg initially as did mixtures of similar composition providing greater amounts of protein. Pigs fed diets containing less than 16% crude pro- tein during one or more of the successive 2 week periods of study were significantly less efficient in feed conversion. Jensen._£._l. (1957) fed casein-corn protein to pigs from 2 to 8 weeks of age and concluded that 16% to 16.6% protein would produce a rate of gain about equal to ~that obtained on higher protein levels. Feed required per pound of gain decreased markedly as protein level increased up to 16% to 16.6%. Above these levels, there appeared to be a trend toward further increase in feed efficiency. Reber 33.31. (1953) studied the influence of level of protein in rations fed to baby pigs from 2 to 7 weeks of age. They con- cluded that level of protein needed for maximum feed utilization and growth by baby pigs decreases as their body weight increases. A ration containing 41% protein produced maximum weight gains and feed efficiency for the young pigs. As pigs approached 8 weeks of age, a level of 20% protein appeared to be used as efficiently as higher levels. Noland and Scott (1960) showed that protein level in the diet linearly affected rate of gain of pigs from weaning to 75 pounds, but had little effect from 75 pounds to market weight. Levels tested were 12, 16 and 20% crude protein. Becker 35 £1. (1954a) showed that as crude protein in the diet increased, from 10% to 16%, average daily gain increased as did feed efficiency. However, when the pigs were over 100 pounds, maximum performance could be obtained when lower levels of protein were fed. Pond _£‘§1. (1960) showed that high protein rations (20% to 18%),when fed to growing and finishing swine, promoted signifi- cantly greater average daily gains than low protein rations (12% to 10%). The addition of 10% stabilized beef tallow increased rate of gain signi- ficantly with the higher protein rations but not with the low protein ration. Serum cholesterol was reduced and serum albumin and total serum protein were increased by feeding a high protein ration to pigs pre- viously consuming the low protein ration. Kropf g5 El. (1959) and Hale and Southwell (1967) reported that market pigs fed protein levels of 12% to 16% were more efficient in utilizing feed and had a higher yield of lean cuts than pigs fed lower levels of protein. Greeley _£‘§1. (1964) noted linear decreases in daily dry matter consumption and in dry matter consumed per unit of gain with increasing protein content from 13% to 19% in the diets of market hogs. As dietary protein level increased, there was a highly significant linear trend toward reduced efficiency of utilization of digestible protein. Wagner _E‘_l. (1963) observed in trials with growing swine that, as dietary protein level increased from 13% to 25%, average daily gain and feed required per pound of gain decreased. a1. (1937), by means of balance trials, studied the Woodman'gg effects of adding 12% of soybean meal at the expense of barley meal to a normal bacon pig diet. Nitrogen retention from the high-protein diet was no higher than from the normal-protein diet. No evidence was ob- tained to indicate that mineral metabolism (Ca, P, Cl) was affected by the higher level of protein. Armstrong and Mitchell (1955), after a series of nitrogen balance studies with growing swine concluded that fecal nitrogen output is linearly related to the protein level of the diet fed. Crampton and Rutherford (1954) had earlier shown this to be true for rats at dietary protein levels from 5% to 50% of the diet. Lloyd and Crampton (1955) and Whiting and Bezeau (1957) observed that the level of crude fiber in the diet as well as the level of crude pro- tein would affect the apparent digestibility of protein. Rippel _£,_1. (1965) found that in gravid gilts percent nitrogen retained plateaued at approximately 12.5% of dietary protein. They also found that serum protein was closely related to the level of dietary protein. 0f the individual serum components, albumin concentration closely resembled the changes in total protein. Forbes _£‘§1. (1958) found that the biological value of a protein depended on the protein concentration in the diet of the growing rat. As the dietary protein level increased, the biological value dropped. Sibbald gt 21. (1957) and.Meyer (1956), using the rat as the experimental animal, made observations similar to those made with swine in that the level of crude fiber in the diet limited the reten- tion of the nitrogenous portion of the diet. Sibbald ggugl. have shown that, at least in the rat, this is due to nitrogen intake being limited by energy concentration in the diet. Nitrogen content of the ration appeared to exert a negligible influence upon food consumption. Garcia and Roderuck (1964) showed that during ad libitum feeding, rats that consumed 464 mg N/5 days had a significantly higher intake of food than rats fed 1159 mg N/S days. Nitrogen retention.was greater at the higher level of N intake. There were no significant differences observed in liver weights. a1. (1962) found that approximately 15% to 18% protein Klavinsigg ‘was necessary to meet the normal needs of the rat as far as iron absorp- tion was concerned. When smaller amounts of protein were fed, the ab- t al. (1965) reported that sorption of iron was impaired. Abernathy 7 to 9 year old girls absorbed 2.5% to 25% of their dietary iron. Con- trary to the report of Klavins g3 g1., these studies failed to demon- strate any consistent effect of level of protein in the diet on iron abSorption. Protein intakes varied from 30% to 150% of the recommended allowances. Hegsted £5 31. (1948) reported that low protein diets (8% casein) with 2.2% ferric citrate added were toxic to rats. However, the addition of protein at higher levels prevented all evidences of iron toxicity. .McCall and Davis (1961) presented data which indicates that the dietary level of protein has a significant effect on the accumulation of copper in the livers of rats. An adequate or more than adequate level of dietary protein inhibited the accumulation of toxic levels of copper in the liver when large amounts of copper were ingested. No indication of copper deficiency was noted, however, when normal levels of copper 'were fed with high levels of protein. McCance _§ _1. (1942) in metabolic studies on adults, have shown that increasing the protein intake increased the amount of Ca and Mg absorbed from the gut and subsequently excreted in the urine. Colby and Frye (1951) increased the severity of magnesium deficiency syndrome in a1. (1963) rats by feeding high levels of protein (50% casein). Bunce 23 observed similar results with chicks as well as rats. A balance study in the rat failed to produce evidence of an effect of dietary protein on apparent absorption although urinary excretion of magnesium was in- creased with the higher protein diet (36% casein). a1. (1943) and Leathem (1951) reported that high Tepperman 25 dietary protein levels increased rat adrenal weights. Ingle t al. (1943), however, did not observe significantly higher adrenal gland weights in rats fed high amounts of protein, and in one experiment by Benua and Howard (1945) mice fed high levels of protein did not have hypertrOphied adrenals. Stoewsand and Scott (1964a) fed high protein diets to chicks and produced a stress as evidenced by hypertrophy, hyperactivity and depletion of phospholipid content of the adrenal cor- tex. No adrenal hypertrophy nor hyperactivity occurred when corti- costerone was injected daily in chicks fed high protein diets. Addis ggugl. (1951) showed that high levels of dietary protein (60%) caused renal hypertrophy. Differences were observed between sources of protein in the magnitude of the effect on the kidney. From calculated urea work load of the kidney, these workers concluded that the differ- ential effect of dietary protein upon kidney size is not due to any differences in the inherent nutritive value for the kidney, but is secondary to the work load imposed upon the kidney by urea excretion. Imondi and Bird (1967) observed that the size of the pancreas of chicks, when expressed as a percentage of the body weight, generally in- creased with increasing level of protein from 15% to 70% of the diet. When comparing casein and isolated soybean protein diets they concluded that the rapid growth of pancreatic tissue during protein repletion was not caused by any unique property of the soy protein. Tumbleson and Meade (1966) have shown that liver weight and percent nitrogen in the liver of young pigs increased significantly with in- creasing levels of dietary protein (8% to 20%). Tuba 3; a1. (1952), in a series of studies with the rat, found that serum alkaline phosphatase was not affected by protein level (0, 5, 10, 30, or 91% casein) nor protein source (casein, dried brewer's yeast or wheat gluten) in the diet. There was, however, a significant correlation between serum alkaline phosphatase activity and daily consumption of fat. Thomas and Combs (1967), in studies with young chicks, showed that, when the dietary protein level was reduced without changing the energy level, both total serum protein and albumin levels were reduced. How- ever, when the daily energy allowance was reduced without changing the protein intake, there was a rise in both serum protein and albumin levels. Stoewsand and Scott (1961) showed that the tibia bone ash of chicks fed high protein diets (71.5%) decreased as compared to that of chicks receiving a moderate level of protein (21.9%) in the diet. El-Maraghi a1. (1965) found that when the protein level in the diet was high, a 2; low calcium diet led to severe mineral-osteoporosis in the bones of young rats, but had only slight effect in the bones of older rats. Young rats given diets of high protein value had larger bones containing more mineral than litter-mates maintained on a low-protein regimen al- though both groups received the same amount of food and calcium. Im- proving the protein value of diets fed to adult rats whose bones had become rarefied led to a remineralization of their bones. Even in aged rats, matrix-osteoporosis brought about by diets of low protein value could be corrected by increasing the intake of protein. Silberberg and Silberberg (1952) observed that skeletal development was accelerated in growing mice fed diets containing 53% casein or 46% fish-protein as compared with the conditions seen in mice fed a 26% protein stock diet. Articular aging was retarded and the onset of degenerative joint disease ‘was delayed in the mice fed the protein-enriched diets. B. Protein Source Different sources of dietary protein vary quantitatively and qualitatively in their composition. Varying composition of amino acids, carbohydrates, fats, minerals and other constituents in different pro- tein sources can affect their usefulness in the diet. Jones and Pond (1964) found that, during the first 42 days after weaning, pigs fed diets containing dried whole milk gained significantly faster than pigs fed soybean meal rations. Pigs fed dried skim milk rations also gained faster than the pigs fed soybean meal rations, but the difference was not significant. Witczak 2;,gl. (1963) showed that pigs fed milk powder rations gained more rapidly than pigs fed either fishmeal or soya meal rations. Lewis _£,gl. (1955) observed that skim milk and casein diets produced significantly (P<0.05) heavier pigs at 5 weeks of age on significantly (PH<:0.05) less feed than Drackett soy diets. In an effort to improve the utilization of the soy diets, they added digestive enzymes to the basal soy diet. The average effect of all enzyme additions (1% pancreatin, 1% pepsin, .5% Star-zyme P, 1% papain, or 1% Mycozyme) was to significantly improve feed efficiency of the soy diets. The results of Maner g£.gl.(l96l), Cunningham and Brisson (1957), Alsmeyer 35 a1. (1957), Hudman and Peo (1957) and Calder t l. (1959), however, did not show that the addition of proteolytic enzymes to either liquid or solid diets containing soybean protein has a measur- t al. (1966) able effect on growth or protein digestibility. Barnes found that, on the basis of rate of return of serum protein values to normal and the rate of weight gain, casein was definitely superior to a fishmeal, a heat treated soybean meal and three different cottonseed meal preparations for the malnourished baby pig. Geurin _£_gl. (1951) reported that, for growing swine, dried whole egg and dried skim milk rations supported faster growth than rations with corn oil meal, soybean meal, tankage, corn gluten meal, or corn solubles plus soybean meal as 10 the primary protein sources. The quality of protein as measured by pro- tein efficiency was highest for skim milk, dried whole egg and corn oil a1. (1959) and Combs 25,31. (1963) found that young pigs meal. Hays g; 'were better able to utilize soybean protein at 5 to 8 weeks of age than at 2 to 3 weeks of age. These findings are in agreement with those of a1. (1957) when rations containing a mixture of milk, soybean Lloyd 23 and fish protein were studied with pigs at 3 and at 7 weeks of age. In contrast, there was very little improvement in the animals' ability to utilize the milk protein with increasing age. Sewell and West (1965), using pigs weaned at 21 days of age, saw no significant improvement in protein digestibility with age. These authors concluded that at least a part of the difference in response to protein from a skim milk source as compared to a soybean protein was due to the lactose content of the milk protein diets. Henry and Kon (1957) compared the casein and soy protein utilization by the rat at 2 different levels. They observed that the true digestibility of soy protein was about 10% less than that of casein, and protein level had no effect on the digestibility. Higher levels of protein resulted in lower biological values. Miller _£‘gl. (1965b), in work with the baby pig fed purified diets, did not note any differences in rate of gain or feed efficiency that ‘was due to protein source. They did show that diets containing casein required less vitamin D2 to support maximal bone mineralization in the baby pig than diets containing soy protein. iMineral balance studies carried out in this series of experiments showed that a greater percent of Ca and Mg were retained when casein was the protein source than when soy was fed. Morris, ggigl. (1967) reported Cu deficiency in rats fed Cu deficient 11 diets containing egg albumin or dried skim milk as the source of protein. Animals fed a similar soybean protein diet did not become deficient in copper. t al. (1957), in a series of nutrient utilization Lengemann studies showed that 45Ca'was absorbed one and one-half times as readily from milk diets as from CaCl2 solutions or CaCl2 plus grain by rats. Rabbits, however, did not show this response to the milk diet. Calcium retention was improved in an ll-year-old cow by the addition of dry skim milk to the ration. Forbes and Yohe (1960) found that the requirement of Zn by the rat depended on the protein source used in the diet. When 7 ppm Zn was provided by soy protein, the rat required 18 ppm in the total diet. ‘When casein provided 7 ppm Zn to the diet the total Zn requirement was only 12 ppm. If egg white provided 2 to 4 ppm of Zn, then the total Zn requirement was still 12 ppm. Apparent absorption values were 44%, 84%, .and 51% for Zn from isolated soy, casein and ZnC03, respectively. Smith a1. (1962) observed Zn deficiency symptoms in pigs receiving soy pro- 3.; tein rations (16 to 22 ppm Zn) but not in pigs fed milk protein rations (6 to 18 ppm Zn). Forbes (1964) found that substitution of soy protein for whole egg white protein in the diets of rats increased weight gain of animals fed Zn deficient diets. It decreased concentration of femur ash, Zn, Ca and P absorption and balance, and Mg, Zn and Fe absorption. Fitch 2£.§l- (1964) reported that, compared to casein, soybean protein in the diet caused a significant reduction in gastrointestinal absorp- tion of 59Fe. . Abernathy _5 El. (1965) reported that Fe absorption was reduced when diets containing no animal products were fed to preadolescent 12 children. Martin (1965), however, did not find any effect on Fe absorp- tion when the amount of plant protein in the diet was increased. She also reported no differences in Zn absorption due to the relative pro- portions of plant and animal protein in the diet. Jensen and Mraz (1966b) reported that tibia ash of chicks fed levels of 2.68% Ca and 1.45% P was significantly less than that of chicks fed a diet with casein-gelatin substituted for isolated soybean protein. Tuba and Dickie (1955) showed that casein and vitellin elevated the levels of intestinal alkaline phosphatase significantly above fast- ing levels in rats. Neither lactalbumin, egg albumin, zein, gelatin nor wheat gluten resulted in any intestinal enzyme response when fed six hours before sacrifice of the animal. 'Chachutowa 25,11. (1963) reported that pH values taken from caecal fistulae of pigs fed different sources of protein showed no differences due to protein source. Caecal ammonia values were lowest when skim milk was fed followed by soybean meal and fishmeal. Wilbur t El. (1960) reported the following pH values for pigs fed different diets. Starch diet Lactose diet Stomach 4.1 3.7 Duodenum 5.4 5.1 Ileum 6.3 6.8 Cecum 6.1 5.6 Rectum 7.0 6.8 C. Vitamin D Level Vitamin D level in the diet can affect rate of growth, serum Ca, P and alkaline phosphatase values, bone deposition and the absorption 13 and excretion of minerals as well as the levels of enzymes within the body. Researchers working with vitamin D have used one or more of these criteria as measures of vitamin D adequacy in their diets. t al. (1964a, 1965a, b), in studies aimed at studying the Miller vitamin D requirement of the baby pig and the effects of dietary vitamin D on the utilization of nutrients by the pig, used many of these parameters. They found that 100 IU of vitamin D2/kg of a diet, which 'was adequate in Ca and P, would support normal growth, feed efficiency, serum Ca, P, Mg and alkaline phosphatase, and adequate skeletal develop- ment with the absence of rachitic pathology. However, when the dietary protein was changed from casein to isolated soy protein it was found that 100 IU vitamin D2/kg of diet were not adequate to keep these cri- teria in the normal ranges. Wahlstrom and Stolte (1958) found that the addition of 90 USP units of vitamin D/lb to a mixed complete ration resulted in no differences in rate of gain, serum Ca and P, nor Ca, P or ash in femurs of pigs. They pointed out that all pigs had prior access to sunlight and that natural diets were fed; therefore, the controls which received no supplementary vitamin D could have stored adequate amounts of vitamin D. Johnson and Palmer (1939) showed that white pigs stored more vitamin D than colored pigs. Pigs fed low levels of vitamin D showed a reduced level of Ca in the serum. Baintner 35.51. (1963) showed that growth rate affects the vitamin D requirement of chicks. A.deficiency of vitamin D3 caused poor feed conversion and low values for Ca, Mg and P in bone and body tissues in chicks used in.these studies. Migicovsky (1957) observed that serum alkaline phosphatase values in vitamin D supplemented chicks were less than one-half that of non-treated chicks. ‘Vitamin D supplementation 14 (5000 IU) resulted in double the Ca in the bones as compared to the same chicks raised on a rachitogenic diet but not given the vitamin D supplement. Howland and Kramer (1921) observed that in children during the period of active rickets the serum Ca concentration may be normal or slightly reduced. The serum inorganic phosphorus, however, is regularly reduced sometimes to an extreme degree in active rickets. Pincus 25.211 (1954) found that vitamin D, when administered during the first weeks of life to infants fed a diet containing 3 to 4 times the amount of P found in human milk, tended to lower serum Ca levels. A trend toward lower serum Ca and higher serum P levels was noted in breast-fed infants receiving breast milk only. The effects of vitamin D on mineral absorption and utilization have been studied both in vivo and in vitro. Nicolaysen (1951), Schachter and Rosen (1959), and Hurwitz _£Hg1. (1967) have all shown that vitamin D treatment enhanced the transport of Ca in vitro from the mucosal to serosal surface of intestinal segments. Harrison and Harrison (1961) have shown this to be true with P as well. Nicolaysen reported that the upper part of the small intestine absorbed Ca at a higher rate than did the lower part. Schachter and Rosen concluded that the active transport mechanism is rather specific for Ca‘l'+ and 1Mg++. 'Harrison and Harrison found that the concentrative transport of P requires the presence of calcium. The complete removal of Ca from their everted loop system inhibited transfer of P against a concentra- tion difference and also eliminated the vitamin D effect. Hurwitz g£‘_l. found that, in the in vitro preparations, Ca was not transferred across the wall of the duodenum. In situ, however, the most rapid efflux of Ca occurred in the duodenal loop. Vitamin D treatment greatly enhanced 15 a1. (1966) and Taylor and Wasserman the efflux of calcium. Wasserman g; (1966) have reported a supernatant factor in homogenates of intestinal mucosa of the rachitic chick after vitamin D administration that has Ca binding activity in vitro. They propose that this factor may be an intra- cellular Ca carrier, the synthesis of which is promoted by vitamin D. Armstrong and'Varnum (1942) were unable to show any response of vitamin D treatment on P absorption. They concluded, however, that vitamin D may facilitate Ca absorption when a low Ca, high P diet was fed. Wasserman (1962) showed that vitamin D dosing of rachitic chicks did not alter the absorption of Na, K, Cu or Zn. It did, however in- crease the intestinal absorption of cesium and cobalt. Phosphate and phytate depressed 47Ca absorption in chicks with or without vitamin D supplementation. .Arensmeyer and Stroder (1963) showed that feeding irradiated milk to infants increased Ca retention by 33% and P retention by 48%. Nicolaysen (1937) stated that the action of vitamin D in the gut is confined to a direct action on the absorption of calcium. The 'well known reduced absorption of P in vitamin D deficiency is due to a precipitation by the increased amount of Ca in the bowel. Cohn and Greenberg (1939) found that the addition of vitamin D to the diet of rachitic rats resulted in an increase of only 10% to 15% in the net ab- sorption of phosphorus. Whiting and Bezeau (1958)'were not able to show a response of Ca absorption and retention to vitamin D treatment. The apparent absorption of P was decreased by the addition of both Ca or vitamin D to the diets. Zinc absorption was decreased by vitamin D but unaffected by dietary Ca level. Worker and Migicovsky (1961a, b) re— ported that vitamin D may exert an influence on the metabolism of Zn and cadmium. They showed that there was an increase in bone deposition 16 of Ca, Be, Mg, Sr and Ba from an oral dose of vitamin D treated chicks. Becker and Hoekstra (1966) concluded that the increased absorption of dietary Zn attributed to vitamin D probably results not from a direct effect of the vitamin, but from a homeostatic response to the increased need for Zn which accompanies enhanced skeletal growth and calcification. ‘Masuhara and Migicovsky (1963) increased Fe and Co absorption in chicks by feeding vitamin D when there were low levels of Ca in the diet but 'were not able to show any response to vitamin D treatment when Ca levels ‘were high. Krieger and Steenbock (1940) and Krieger g£_§l. (1940) showed that vitamin D will enhance the utilization of phytic acid phosphorus. The former report indicates that Ca-P ratio, however, was a more prevalent factor in determining the utilization of the organic phosphorus. t al. (1948) observed that adding vitamin D to a ration con- Spitzer taining nearly optimum inorganic phosphorus had little or no effect on P utilization as indicated by bone ash. However, the addition of vita- min D to a ration containing calcium phytate greatly enhanced P utiliza- tion. There were no differences in intestinal phytase activity attri- butable to vitamin D level, in rats receiving calcium phytate and varying amounts of vitamin D in their diet. Jensen and.Mraz (1966a) concluded that isolated soy protein interferes with the absorption of Ca or P or both of these elements in the chick. Their studies, however, showed that the interference was probably not in the absorption or metabolism of vitamin D because chicks previously fed an isolated soy protein diet could absorb radioactive Ca as readily as similar chicks previously fed casein-gelatin diets. Cheesman t 31. (1964, 1966) observed that phosphatase activity 17 'was decreased in the jejunum and kidneys of rats deprived of vitamin D. D. Nutrient Absorption and Secretion Many researchers have endeavored to explain the interactions of nutrients and how, where and under what conditions elements are absorbed from or secreted into the gastrointestinal tract. At a symposium on the interaction of mineral elements in nutrition and metabolism (1960) sponsored by the American Institute of Nutrition, some of the leading scientists gave reviews of work in their respective fields. Wasserman concluded from the numerous studies which he cited that there is no apparent simple uncomplicated relationship between the metabolism of Ca and phosphorus. Forbes attempted to bring together the research relat- ing to Zn and Ca interactions. He concluded that interference in Zn function by Ca occurs at the cellular level. The exact mechanisms causing the deficiency lesion(s) could not be described. O'Dell pointed out that when the diet is low in P,excess dietary Mg causes loss of Ca from the body. Also, consumption of excess Ca accentuates the symptoms of Mg deficiency in an animal consuming a deficient diet. Cotzias summarized the research dealing with Mn by pointing out that interactions do exist between.Mn and other important mineral nutrients, however, these interactions probably occur outside the body proper. Matrone con- cluded from his survey of research involving Fe and Cu that these ele- ments interact only at the level of hemoglobin formation. He pointed out that any interaction of these elements is probably at the cellular level. Miller and Engel pointed out that there is evidence that Zn, Mn, and Ca as well as Mo and 304 may be concerned with Cu metabolism. Manners and McCrea (1964) very thoroughly reviewed studies involved 18 in establishing the mineral requirements of early weaned pigs. They calculated from data on body composition and mineral retention by sowe reared piglets the following dietary mineral requirements of pigs 2 to 18 days old: Good utilization1 Poor utilization2 Ca 1.24% 1.73% P 0.66% 0.92% K 0.28% 0.39% Na 0.14% 0.19% Mg 375 ppm 525 ppm Fe 152 ppm 304 ppm Zn 102 ppm 204 ppm Cu 16.5 ppm 33.0 ppm Mn 1.76 ppm 3.51 ppm 1Assuming 70% utilization of major minerals and 20% utilization of trace minerals. 2Assuming 50% utilization of major minerals and 10% utilization of trace minerals. These figures are generally higher than requirement figures set forth by the National Research Council (1964). Moore and Tyler (1955a) critically reviewed methods of measuring Ca and P absorption by means of reference substances. They concluded that Bergeim's method (1926a) of studying the absorption and excretion of Ca and P is seriously limiting because (1) an entirely reliable reference substance is not known and (2) there is no satisfactory method known to distinguish between endogenous and exogenous Ca and P. Moore and Tyler (1955b), using 450a and 32P to mark Ca and P absorption or 19 secretion, concluded that Ca was absorbed most actively from the proxi- mal fourth of the small intestine and P from the proximal half. Both Ca and P were secreted into the lumen of the upper small intestine but ‘were reabsorbed from the lower segments of the small intestine. Neither Ca nor P appeared to be secreted through the wall of the large intestine. Yang and Thomas (1965), using both lignin and chromic oxide as reference substances reported that dry matter and organic matter were secreted into the omasum, abomasum and upper section of the small intestine of calves. Absorption of these materials occurred in the remainder of the tract. Efflux of P, Ca and ash exceeded influx in the rumen and lower GI tract but was variable in omasum and abomasum and influx ex- ceeded efflux in the upper section of the small intestine. Sodium net absorption occurred in the rumen, lower section of the small intestine and rectum, whereas net secretion occurred in the abomasum, upper sec- tion of the small intestine, large intestine and cecum. Ali (1967), using chromic oxide as a reference material in Ca studies with the rat, found that absorption occurred in all sections of the tract except the duodenum and stomach. Kvasnitskij (1951), using reentrant preparations on pigs in a very thorough series of studies, was able to determine di- gestion and absorption in various segments of the GI tract of the pig. He observed that 35% to 54% of the minerals taken in were absorbed in the stomach and small intestine and 15% to 23% of the mineral intake was excreted back into the large intestine. Kramer and Ingelfinger (1961) compared the characteristics of ileal effluence of ileostomized human subjects with those of normal feces. They concluded that the colon appears to absorb Na and water and excrete potassium. Herndon and Hove (1955) found that surgical removal of the 20 cecum of rabbits resulted in lowered Na and K utilization. Protein and fat digestibility were somewhat lower while ash and Ca were unchanged. Lloyd 33 a1. (1958) concluded that caecectomy had no deleterious effect on the growth rate of pigs fed either experimental or practical type rations. Differences in the ability of normal and caecectomized animals to digest various ration constituents were of small magnitude. Karel (1948) reviewed the research that had been done in the area of gastric absorption. He concluded that the stomach is definitely not an absorp- tive organ in the same sense as the intestine and cannot be of especial importance in supplying the nutritional needs of the normal organism. Its absorptive ability, however, has been grossly underestimated, particularly as regards substances physiologically active in minute amounts. Calcium Balance trials have been used a great deal in research to evaluate the utilization of various nutrients. De and Basu (1949) showed in human balance trials that the administration of a large dose of Ca, Mg, P, Fe, Cu or Mn with a basal diet increased the elimination of all other minerals to a considerable extent. The increased excretion of minerals was found to occur mainly through the feces. Widdowson _§._l. (1963) noted that P supplementation of breast-fed babies promoted, rather than hindered, the absorption of Ca and Mg. The urinary excre- tion of Ca, Mg and Sr was reduced when supplementary P was given. Miller E; 31. (1962) established the minimal requirement of Ca to be between 0.8 and 1.0% for the baby pig. They showed that, when growth and bone deve10pment were optimum, 77% to 82% of the dietary Ca was retained. Hansard 3; a1. (1961) showed that the amount of Ca normally 21 excreted in the urine is small with a fecal-urinary excretion ratio be- tween 22:1 and 50:1. As the pigs used in these studies became older, less and less of the Ca intake was absorbed. As age increased, the amount of endogenous Ca in the feces increased. Bergeim (1926b) studied rachitic and normal rats in an effort to determine at what level within the gastrointestinal tract Ca and P ab- sorption was impaired. Iron oxide was used as a reference material to determine absorption or secretion within the tract. Both normal and rachitic animals showed a considerable excretion of P into the intestines. Calcium absorption appeared to take place most rapidly where the excre- tion of P was most marked. In the lower bowel there was an approximate balance between excretion and absorption of Ca. Phosphorus was reab- sorbed at this level. Harrison and Harrison (1951) found that the most rapid rate of Ca absorption occurred within the first two to four hours of its administration. This absorption was from the proximal portion of the small intestine, and the amount of 45Ca absorbed during this inter- val‘was not influenced by vitamin D. Absorption of Ca from the distal portion of the intestine was found in rats receiving vitamin D, but not in untreated rachitic rats, except in animals in which the intestinal tract had previously been emptied of Ca by feeding a Ca-free diet. iMarcus and Lengemann (1962) showed that Ca absorption was greatly affected by the physical state of the dose. Calcium from a liquid dose was 45% absorbed and only 19% from a solid dose. The total relative percent of 858r and 450a absorbed in each of the gut components was estimated after absorption had ceased. The values obtained were as follows: 22 Solid dose Liquid dose Stomach 0% 0% Duodenum 8% 15% Jejunum 4% 23% Ileum 88% 62% Hurwitz and Bar (1965) reported exPerimental results which appear to indicate that the major portion of Ca and P absorption occurred in the anterior parts of the intestine. From P/91Y ratios in the duodenum, it was apparent that large quantities of endogenous P were emptied into this segment. The pattern of dry matter/le indicated absorption of dry matter along the entire intestine with a reduced rate at the posterios segments. Wallace SE 51. (1951) used radioactive Ca to study Ca excre- tion into the GI tract. They conclude that all segments of the tract participate in the excretion of calcium. The small intestine plays a major role in the excretion of 45Ca after an intramuscular injection. After a two-week period, 15%.and 41% of the injected dose had been re- covered in the feces of young and mature rats, respectively. Cramer (1964), however, after a series of studies using healed gut loops in dogs concluded that under physiological conditions, net Ca transfer is in one direction -- from gut lumen to blood. McCance and Widdowson (1939) had earlier concluded this in work with humans. Bronner and Harris (1956) suggested that bone salt formation and resorption are rate-limiting processes in a scheme of Ca metabolism that involves absorption, transport, bone salt formation, bone salt resorption and excretion. Wasserman t al. (1957) showed that singly administered L-lysine 23 and vitamin D promoted 450a absorption in the vitamin D-deficient rat. The combined effect of L-lysine and vitamin D was about the sum of the effects of the individual components. Wasserman and Taylor (1962) showed that the percent absorbed 47Ca deposited in the tibia varied with intraduodenal pH and vitamin D status of the chick. At low pH values (1.9, 2.0) there were no differences in the percent of duodenally ab- sorbed 47Ca accumulated by tibia in rachitic or vitamin D-treated chicks. However, at high pH values, proportionally less of the absorbed 47Ca ‘was deposited in rachitic tibia; pH waS‘without effect on uptake of 47Ca by tibia in the vitamin D-treated birds. Phosphorus Ammerman g£,§l. (1963) studied the absorption and retention of various phosphates by the use of radio active phosphorus. From an oral dose of P they found that 27% to 41% was absorbed by swine and 8% to 1F% was excreted in the urine. Net retention of P ranged from 20% to 30%. Cramer (1961) found that all parts of the intestinal tract of adult rats were able to absorb 32P. The rate of absorption was greatest in the duodenum, followed by the jejunum, ileum, colon and stomach in decreasing order. However, since 32F passed rapidly through the duo- denum and jejunum, less material was available to be absorbed, with the result that absorption was less effective in these segments than it was in the ileum. When the progress and rate of absorption was combined quantitatively, the greatest effective absorption was found to occur in the ileum (which absorbed 38% of the total), followed by the duodenum (29%), jejunum (25%) and colon (8%). McHardy and Parsons (1956) had earlier reported that P was absorbed more rapidly from the jejunum than 24 from the ileum. They also reported that the net absorption rate of in- organic phosphates increased with decreasing H+ concentration. Sodium and Potassium t 31. (1950) established the Na and K requirements of swine Meyer by using a combination of plasma studies and mineral balance trials. Growing pigs retained 80% to 90% of their dietary Na intake when Na levels in the diet did not exceed 0.09%. Potassium retention ranged from 40% to 50% of the dietary intake. Hamilton (1938) detected radioactive Na, Cl, Br and I in the hand 3 to 6 minutes after ingestion. Potassium was absorbed more slowly, requiring 6 to 15 minutes after ingestion to appear in the hand. The author concluded that these data indicated that these elements were ab- sorbed from the stomach or certainly in the upper small intestine. Reitemeier._£.§1. (1957a) and Code _£.§l. (1955) failed to observe any absorption of radioactive Na from the stomach and concluded that there ‘was a barrier to Na absorption in normal gastric mucosa. Reitemeier ,gg‘gl, found that when the radioactive Na did pass into the small in- testine it was readily absorbed. ‘McHardy and Parsons (1957) showed that alkaline conditions in the jejunum and ileum of the rat favored more rapid absorption of water and Na. Reitemeier g£_§l, (1957b) observed that labeled water (D20) uniformly passed more rapidly than 22Na from the contents of the small intestine into the blood stream. The mean initial rate of absorption of D 0 was about 20%/minute of that adminis- 2 tered and the mean rate of absorption of radio sodium was about 10%/min- ute. Visscher g£_§1. (1944) showed that Na+ moves in both directions across the intestinal epithelium. These authors reported that the rate of Na ion movement both out of and into the gut are positively correlated 25 with Na ion concentration in the small gut. The jejunum and ileum show greater variability in measured rates of Na movement than does the colon. Levitan _£ _1. (1962), using a constant perfusion fluid of 0.85% NaCl found that the entire colon absorbs water and Na and at the same time secretes potassium. Magnesium .A review by Hathaway (1962) summarizes the research carried out prior to that time involving Mg in human nutrition. Magnesium require- ments appear to be related to protein intake; i,e., higher Mg intakes are required for equivalent retentions when protein intakes are high. Its relation to Ca and P intakes and metabolism seems to be more compli- cated. O'Dell (1959) reviewed research involving the effect of other nutrients on the dietary requirement of Mg by non-ruminants. He re- ported that high levels of Ca, P, K and protein will all hasten the onset and severity of Mg deficiency. Dempsey g; 21. (1958) reported that fecal Mg is unrelated to Mg intake but was highly correlated with fecal nitrogen. Bartley 35.21. (1961) showed that retention of Mg, over a 7 day balance trial, increased linearly with increasing Mg intake. These researchers recommended 40 mg of Mg/100 g of ration as adequate to maintain health of pigs 3 to 6 weeks of age. Chutkow (1964), using 28Mg found that Mg is absorbed throughout the intestinal tract of rats and that probably more is absorbed in the ileum than in the jejunum. Over 70% of the total absorption of Mg occurs in the colon. The pattern of the intestinal excretion of endogenous Mg appeared to be the reverse of absorption, with most of the loss occurr- ing in the proximal gut. .Aikawa (1959) suggested that poor gastrointes- 26 tinal absorption of Mg accounts for its low renal excretion. He also commented that absorption does not appear to occur from the large intestine. Aikawa carried out his studies with rabbits. ‘Iggg Gubler (1956) reviewed the absorption and metabolism of iron. He emphasized that: (1) ferrous iron is generally absorbed to a greater extent than ferric iron, (2) there is usually an inverse correlation between the size of dose and the percentage of the dose that is absorbed, (3) in an acid medium (pH below 5), the Fe in foods and in ferric hydroxide is converted to the soluble ionic form, and the formation of insoluble and undissociated complexes is inhibited, (4) since ferric iron readily forms insoluble and undissociable complexes with phosphate ions, the presence of much phosphate in the diet can materially reduce the absorption of Fe, (5) phytic acid, by virtue of its ability to form insoluble Fe complexes, also inhibits Fe absorption. Barer and Fowler (1937) found that the amount of Fe excreted by men and women in the urine was fairly constant in the same individual regardless of the Fe intake. Schulz and Smith (1958), in balance studies ‘with children, determined that normal children absorbed about 10% of the Fe from.milk, eggs, chicken liver and Fe supplements added to commer- cially prepared infant's cereal. Iron deficient children absorbed 2 to 3 times as much food Fe as normal children. Normal children absorbed a1. (1962) ob- more Fe from milk than normal male adults. 'Bannerman‘gg served that rats receiving low levels of Fe (2 mg Fe/kg diet) absorbed 83% of a SO/ug ferrous iron dose. Rats supplemented with 240 mg Fe/kg diet absorbed only 8% of the test dose. 27 Copp and Greenberg (1946) used radioactive Fe to study the absorp- tion and excretion of iron. They concluded that Fe absorption took place in both the small and large intestine. Austoni and Greenberg (1940) had earlier shown that Fe moved through the GI tract of anemic rats more slowly than through normal rats. They suggested that the de- lay in the passage rate may be a factor in the increased absorption. .McCance (1937) and Widdowson and McCance (1937) concluded that the in- testine does not excrete iron. Hahn _£'g1. (1939) concurred that the excretion of Fe was negligible. Hahn £5,31. (1943) showed that Fe ab- sorption takes place largely in the stomach and duodenum. MaCallum (1894) had earlier observed that, when the dose was small, absorption occurred only in the part of the intestine adjacent to the pylorus. Arrowsmith and Minnich (1941), using an inflated balloon and a Miller- Abbott double lumen tube, were able to study Fe absorption at various levels within the human GI tract. Their data indicated that absorption occurred most readily in the stomach and duodenum, to a smaller degree in the jejunum and to an even lesser extent in the ileum. No evidence of absorption was noted when Fe salts were given rectally. Pearson 35 31. (1967) observed that the bulk of the Fe absorbed from a stock diet was absorbed by the first 30 cm of the intestine, with little absorption in the more distal portion of the gut. Whitehead and Bannerman (1964) concluded that the anemia of totally gastrectomized rats was caused by a quantitative defect in absorption while excretion of Fe continued at a normal or possibly increased rate. Koepke and Stewart (1964) pre- sented evidence for the existence of a substance in gastric juice from anemic dogs which facilitated Fe absorption from the GI tract of normal dogs. Duthie (1964) observed no significant decrease in Fe absorption 28 in dogs after removal of the duodenum. He was able to show absorptive superiority of the duodenum in rats only with large doses of Fe. Ohkawara g; 91. (1963), studying Fe absorption from the human large in- testine, presented evidence that the human subject can absorb small quantities of soluble ferrous Fe from the large intestine. VanCampen and Mitchell (1965) used ligated sections of the rat GI tract to study the absorption of Fe and other trace minerals. They re- 65 ported that Zn and 59Fe‘were taken up most rapidly from the duodenum, somewhat more slowly from the ileum and the mid-section and the least absorption occurred from the stomach. Capper64 absorption was greatest from the stomach and declined as the isotope was placed further away from the pylorus. £135 McCance and Widdowson (1942) reported that urinary excretion of Zn by adults did not vary with dietary intake nor was it raised by intra- venous injections. They also reported that some adults excreted addi- tional Zn in the feces when injected daily with 6 mg of Zinc. Sheline a1. (1943) obtained similar results with mice in that 50% of 652m gt; administered IV was recovered in the feces. Two percent of the adminis- tered dose was recovered in the urine of the mouse. Tribble and Scoular (1954) reported that human subjects excreted 8% of their dietary Zn in- take in the urine and 42% in the feces. Feaster g; _l. (1955) reported that only 5% of an oral tracer dose of 65anas retained by the adult rat. t al. (1958) and Beth and Hoekstra (1963, 1965) have Newland, studied the antagonistic effect of Ca on Zn absorption and utilization. 29 All authors concluded that their results indicate a decreased absorption of Zn with increased dietary calcium. a1. (1966), using intact strips of rat intestine, Sahagian 25 studied the absorption and interactions of Zn, Mn, Cd and Hg. The regional uptakes of Zn, Cd and Hg were least by the jejunum. Jejunum and ileum preparations took up Zn<fiHg<fiCd<€Mn. Zinc and Mn uptakes were diphasic, with initial rapid uptakes followed by slower continued up- takes for one hour. IManganese had no effect on Zn or Cd uptake. a1. (1956) observed that zinc is excreted in greater Birnstingle‘gg concentration and in greater quantity in pancreatic juice than in bile or duodenal sections. Ligation of the pancreatic ducts materially de- creased the 652n activity of otherwise intact duodenal aspirate. Man anese Vorab'eva (1965) confirmed the earlier observations of Everson and Daniels (1934) that (1) Mn retention varies inversely with age of children, (2) total urinary excretion is virtually constant irrespective of age, and (3) fecal Mn excretion varies directly with age, and there- fore with total dietary intake. North 25 l. (1960) in a study of Mn metabolism in women found that over 8 periods of 5 days each 9 college women retained 41% of their dietary Mn intake. Fecal Mn accounted for 91% of the total Mn excretion. Lang 2; 31. (1965) did not show any effect on.Mn retention when skim milk was added to the diet of young men fed all vegetable diets. Greenberg and Campbell (1940) and Green- berg gg‘gl. (1943) had earlier reported that very little of the Mn ab- sorbed by rats was excreted in the urine. Liebholz £5.31. (1962) showed that Mn intake was reflected by the 30 Mn content of bone, liver and hair of the baby pig. Tal and Guggenheim (1965) reared mice on a basal diet of meat which is poor in both Mn and Ca. They found that the addition of small amounts (2.5 to 5.0 mg/kg of meat) of Mn improved weight gain and calcification of bone and decreased incorporation of injected radio Ca into bone. Copper a1. (1961) showed that 60% of an oral dose of 64Cu was Buescher 33 recovered in the feces of swine. Only 1% to 3% was found to be excreted in the urine. Mahoney 25.31. (1955) had earlier found similar results in dogs. They concluded from their work that the major pathway of ex- cretion of Cu is through the biliary system. Bowland 3;.gl. (1961) con- cluded that Cu transfer across the gut wall apparently occurred mainly in the small intestine and the colon. They, too, found the feces to be the major route of Cu excretion with the bile accounting for up to 40% of the total excretion. VanRavesteyn (1944) stated that, in his studies ‘with man, the excretion of Cu via the bile and the feces does not appear to run parallel. He drew the conclusion that the Cu found in the stools, at least in part, is excreted through the intestinal wall. VanCampen (1966) and VanCampen and Scaife (1967) observed that Zn affected Cu uptake from the stomach and from the duodenum in the same manner and to about the same extent. In both cases high levels of Zn depressed 646u uptake, but did not produce any change in the tissue distribution pattern. Their data indicated that the depression of Cu absorption by high levels of Zn is mediated either in or on the intestine. Cobalt Kent and McCance (1941) suggested that the gut is the main channel 31 of excretion of Co in foods, probably because relatively little is ab- sorbed. Once the Co had reached the tissues, however, the process of elimination was mainly via urine. Greenberg g£_§l, (1943) concluded that the urine is the chief pathway of excretion of absorbed Co. They pointed out, however, that orally administered Co is only partially ab- sorbed and thus, a large prOportion is eliminated in the feces. Harp and Secular (1952) reported that young women absorb 73% to 97% of their dietary Co (5 to 8/ug/day). An average of 67% of the total daily intake of Co appeared in the urine in these studies. Paley and Sussman (1963) found that the absorption of Co was generally diminished when the Co was administered after a meal pretagged to protein or administered carrier free, wherein binding to residual protein in the digestive tract may be presumed to have occurred. They concluded from this that the absorption of Co takes place in the GI tract above the region of protein digestion. In contrast to the previously cited paper, only 16% of the cobalt load was recovered in the urine in these studies. Henderickx (1964) found evidence that 57Cowas absorbed from the large intestine of swine. III. EXPERIMENTAL PROCEDURE A. Introduction Four experiments were carried out to study the effects of level and source of protein and level of vitamin D on mineral utilization by the baby pig. These experiments were: Experiment I. Effect of protein level on mineral utilization by the baby pig. Experiment II. Effect of level of protein and vitamin D on mineral utilization by the baby pig. Experiment III. Effect of level of protein and vitamin D on mineral utilization by the baby pig. Experiment IV. Effect of source and level of protein on mineral utilization by the baby pig. Experiment I was carried out in an effort to determine if higher levels of protein in the diet were more rachitogenic than lower levels. The subsequent two experiments were conducted to determine if additional vitamin D would overcome the effects seen in Experiment 1. Experiment IV was conducted to determine if those differences observed in the pre- vious experiments were due to source of protein as well as to the level of protein in the diet. B. General Conduct of Experiments Baby pigs were taken from their dams at 3 days of age and bottle- fed homogenized non-fortified cows milk 4 times daily during the first few days. A purified diet in the form of a dry meal (table 1) was also placed in small feeders and intake was encouraged by placing small amounts in the animal's mouth after liquid consumption. The pigs were 32 33 .mH xHocomm< mom N .mHocHHHH .owu4 .mcmmEoo nauseoum ouoo .oHoumz o .wH Nchoamd mom m .mHoaHHHH .owud .zoomaou muosvoum cuoc .omoHouoU q .owooHso .hcwmfioo cecum .oon oxHom m .ocoHo>oHU .GOHumuomuoo mHouHEocoOHm HdcoHuHuuoz .aHmmou ooHMIcHEoqu N .mHHoaomccHzn.hommEoo ocmHoHZTmHoHcontnonuu<..oHououm honed Hno sum H + + + + + .+ NououxHE oHEduH> H H H H H H oHHo upon 0 o o c o o mousuxHa Houocfiz me me mm mm no mo donoode m m m m m m momOHsHHou iv“ m m m m m m oqu m.o m.o m.o oaHoochozTHn oq om ON Nfiwmmmo N.mm N.mN N.mH Hhom N N N N N N :Homoo :Hmuoum mom aHomwu cHououn hoe cHomoo :Hououa mow menace :Hououm cHououmtooauo Nun :Hououn\ov:uu Nam :Hououm dunno NmH Ho>oH cHououm .mubae smamausa no aoHuHmomsoo .H oHnoH 34 consuming the dry diet very well by one week of age. Liquid milk feed- ing was discontinued as soon as the dry diet was being consumed volun- tarily. At one week of age pigs were randomly allotted to treatment after equalizing for sex, litter and weight. Pigs were housed in wire bottomed metal cages in a room in which all windows were painted to pre- vent the entrance of ultraviolet rays. Room temperature was held con- stant at 20° C and infrared heat lamps were utilized to maintain a cage temperature of 30° C during the first 2 weeks of the trial. Pigs were ad-libitum fed and had free access to fresh water during the experimental period. All feed was weighed into the feeders and pigs were individually weighed‘weekly. Blood was withdrawn from the anterior vena cava on 3 occasions (initial, 3 weeks, and final) during each experiment for determination of serum constituents. IMineral balance studies were conducted following the fourth or fifth week of each experiment. Pigs were individually fed 3 times daily an amount of food and water'which they would consume within a 5 to 10 minute period. After a 3 day adjustment period, they were placed in individual metabolism cages for urine and fecal collection. Pigs were removed 3 times daily for feeding after which their mouths were wiped clean and then returned to their cages. Constant daily feed intakes ‘were maintained throughout the 3 day collection period. Feces were collected separately from urine by means of a fine screen placed above the collection tray. Total fecal collections were dried in a low temperature oven, weighed, finely ground and stored in air tight plastic containers. Total urine collections were acidified with 6 N HCl to a pH of between 1 and 2, 35 the volume accurately measured and 100 m1 samples stored in polyethylene bottles. At the conclusion of each trial pigs were killed and various bones, organs and glands were removed and weighed. 1. Experiment I. Effect of level of_protein on mineral utiliza- tion by baby pigs. Twelve baby pigs were adapted to the dry diet (see table 1) and at one week of age were allotted to diets consisting of 20%, 30% or 40% of isolated soybean protein. These diets analyzed by the Kjeldahl method 16%, 24% and 32% crude protein, respectively. All diets in the experiment were supplemented with only 6.25/ug of ergocalciferol/kg of diet in an attempt to show the rachitogenicity of high levels of soy protein, if they were, indeed, rachitogenic. Two pigs per treatment were killed to obtain bone and organ weight data. The remaining 6 pigs were returned to the University herd. 2. .Experiment II. Effect of level of protein and vitamin D on minergl utilization by baby pigs. Eight baby pigs were adapted to the dry diet and at one week of age were allotted to treatments of 30% soy protein with 6.25 or 12.50/ug of ergocalciferol/kg of diet or 40% soy protein with 6.25 or 12.50/ug of ergocalciferol/kg of diet. Kjeldahl analyses showed these diets to contain 24% or 32% crude protein. 3. Experiment III. Effect of level of protein and vitamin D on mineral utilization by baby pigs. Sixteen baby pigs were adapted to the dry diet and at one 36 week of age were allotted to dietary treatments of 20% soy pro- tein with 6.25 or 12.50/ug/kg of ergocalciferol or 40% soy pro- tein with 6.25 or 12.50/ug/kg of ergocalciferol. Analyzed crude protein in these diets was 16% and 32%. In this experiment 3 pigs per treatment were killed for bone and organ weight data. The remaining pig per treatment was re- turned to the University herd. 4. ‘Egperiment IV. Effect of source and level of ppotein. Sixteen baby pigs were adapted to the dry diet and at one week of age were allotted to dietary treatments of 20% soy or casein protein or 40% soy or casein protein. All treatments in this experiment, as in Experiment I, received 6.25/4g ergocal- ciferol/kg of diet. Kjeldahl N x 6.25 showed these diets to con- tain 16% and 32% crude protein. In this experiment pigs were fed 0.5% chromic oxide in the diet for a period of 4 days prior to beginning the balance study and were maintained on this feeding regime until they were killed following the collection period. Pigs were then killed one and one-half hours or three hours post feeding. The alimentary tract was quickly exposed, the sections tied to prevent movement of chyme and the tract removed and separated into stomach, cranial small intestine, caudal small intestine, cecum and colon. Digesta was cleaned semi-quantitatively from each section, weighed, pH determined on a small portion and the remainder thoroughly mixed in a Waring blender. If the digests was too thick for proper mixing, it was appropriately diluted with deionized distilled 37 water. A 20 g portion of this material was then placed in a plastic bag and frozen for later analysis. The indicator method for determining digestibility was adopted to measure absorption and/or secretion of nutrients along the GI tract of pigs. The following equation, % diges- tibility = 100 _ (% indicator in feed X % nutrient in feces) 100 , % indicator in feces % nutrient in food as given on page 303 of the textbook by Maynard and Loosli (1962) ‘was used. In order to measure the degree of absorption and se- cretion in the different sections of the GI tract as the digesta moved posteriorly, two consecutive sections of the tract were used in relation to the above equation. The digesta in a given organ was considered to be the feed for the next posterior organ. The digesta in the second organ would be equivalent to the feces for the calculation using the ratio technique equation. Using 2 consecutive organs in relation to the above equation the degree of digestion in the following alimentary sections was calculated. a. Stomach -- using feed as fed and stomach digesta as feces; b. Cranial small intestine (cranial s.i.) -- using stomach digesta as the feed and cranial s.i. digesta as feces; c. Caudal small intestine (caudal s.i.) -- using cranial s.i. digesta as the feed and caudal s.i. digesta as feces; d. Cecum -- using caudal s.i. digesta as the feed and cecal digesta as the feces; e. Colon -- using cecal digesta as the feed and digesta in the colon as the feces; f. Rectum -- using the digesta in the colon as the feed and the feces voided as the feces. With this method, a positive value indicates net absorption from 38 the GI tract and a negative value would indicate net secretion into that section of the tract. C. Chemical Analyses l. §g£gm, After the blood was drawn from the animals, it was put into acid washed test tubes. After the blood had coagulated, the clot was removed and the serum spun in an international centrifuge at 1500 RPM for 15 minutes. The cell free serum was then poured into acid washed vials. Serum alkaline phosphatase and serum protein were determined within 24 hours of serum collection. The remaining serum was frozen at -10° C for future analysis. a. Calcium In Experiments I and II serum calcium was determined by the compleximetric method of Mori (1959). A Jarrell-Ash atomic absorption spectrophotometer with a hydrogen-air flame was used for Ca determinations in Experiments III and IV. The Ca absorption line at 4227 Ap'was used to determine concentration of Ca in standards and unknowns. 10,000 ppm Sr was added to all samples to suppress any phosphate interferences (Appendix 1). b. Phosphorus A11 serum P analyses were determined by the method of Gomori (1942) and read on a Bausch and Lomb spectronic 20. c. Magnesium Serum Mg in Experiments I and II was determined by the colorimetric method of Orange and Rhein (1951). In 39 ExperimentsIII and IV the Jarrell-Ash atomic absorption spectrophotometer was used to make these determinations. A wave length of 2852 A0 was used to determine absorption, and 10,000 ppm of Sr was added to suppress phosphate interference (Appendix 1). d. Alkaline phosphatase All serum alkaline phosphatase values were determined by the Sigma method (Sigma, 1963). e. Total protein Serum total protein values were determined by the method of Waddell (1956) and read on a Beckman DU spectrophotometer. 2. Feed, feces gnd digesta. In Experiments I and II, one g of oven dried fecal material was weighed into a 50 g acid washed crucible and ashed in a muffle furnace by slowly raising the temperature to 5500 C over a 6 hour period and allowing to re- main at 5500 C for an additional 6 hours. This resulted in a clean ash which was then taken up to 5 ml 6 N HCl and diluted to 100 ml with deionized distilled water in an air tight polyethylene bottle which had been acid washed. .A wet ashing procedure was used in Experiments III and IV. A 0.5 g sample was placed in a 250 ml extraction flask and 20 ml of concentrated HNO3 were added. After allowing to stand for 15 minutes, the sample was heated to near dryness on a hot plate. The nearly dry sample was then cooled, 7 m1 of perchloric acid were added and digestion continued over the hot plate. A watch 40 glass over the flask acted to reflux the moisture and thus mini- mize losses. As the sample became clear the flask was removed from the heat, cooled and diluted to 100 ml with deionized dis- tilled water and stored in an air tight acid washed polyethylene bottle. Standards and blanks were prepared in the same manner as the unknowns. a. Calcium In Experiments I and II feed and fecal Ca was deter- mined by the method of Mori (1959). A micro spatula tip full of ascorbic acid added to the samples to be titrated resulted in a sharper end point. Calcium in the feed and feces samples prepared in Experiments III and IV was de- termined as described for serum Ca using atomic absorp- tion (Appendix 1). b. Phosphorus All P determinations were made by the colorimetric method of Gomori (1942). c. Magnesium Feed and fecal Mg in Experiments I and II was deter- mined on a Perkin Elmer 303 atomic absorption unit. A Jarrell-Ash atomic absorption spectrophotometer was used for these determinations in Experiments III and IV. d. Sodium, Potassium and Chromium In Experiment IV, Na, K and Or were determined by flame emission spectrophotometry. Samples were wet ashed 41 as described and were diluted and read as shown in Appendix 1. e. Iron, Zinc, Manganese, Copper and Cobalt These minerals were determined only in Experiment IV and were analyzed from a single wet ashing of material (Appendix 1). .Manganese gave a sharper response when 10,000 ppm Sr was added; therefore, this was used to suppress interferences when Mn was determined. f. Nitrogen All N determinations were carried out on oven dried samples using the semi-micro Kjeldahl technique. g. Energy Energy was determined on feed and feces in Experiment IV by the use of a Parr adiabatic oxygen bomb calorimeter. h. Phytin Phosphorus In Experiment IV a modification of the method of Barley (1944) was used to determine the amount of dietary P that is bound as phytin phosphorus. One g of feed or feces was extracted with 20 ml of 0.15 N HCl containing 10% NaZSO4, for 2 hours on the shaking machine. The acid extract was centrifuged and then filtered through a filter paper (Watman #5) using suction. Fifteen ml of ferric chloride solution, which was prepared in 0.07 N HCl and contained about 0.2% Fe, was then slowly added to the supernatant. The container was rotated gently until the ferric phytate formed. The ferric phytate was allowed to 42 stand over night before it was centrifuged and the super- natant poured off. The precipitated ferris phytate was washed with cold distilled water, recentrifuged and then taken up in 20 m1 of 15.8 N HNO3. The washed precipitate ‘was then subjected to the perchloric acid digestion de- scribed above. Inorganic P was then determined as described previously. 3. 9312;. With the exception of Ca, all determinations were carried out as described for the elements in feed or feces. Urinary Ca was determined on 2 ml of urine after the Ca had been precipitated at a pH of 4.0 over night with 1 ml of satur- ated sodium oxalate. The precipitate was spun down, the super- natant poured off, and the Ca taken up with 2 ml diluted nitric acid. The Ca determinations were then carried out as described previously. Urinary energy was calculated using 5.41 KCal/g N (Kleiber, 1961). Urinary N was determined by the Kjehldahl method. 4. Bone analysis. Femurs and the 8th rib from the right side were taken from each pig. The bones were scraped free of all flesh and the periosteum was trimmed from the bone. The cleaned bones were placed in an air tight plastic bag and stored at 5° C until analyzed. a. Specific gravity Both femurs and the rib from each pig were weighed in air and water and the specific gravity of the bones calculated. 43 b. Strength tests Breaking tests were made on each femur using a Tinius- Olsen universal testing device. Strength characteristics were calculated as described by Miller E; a1. (1962). c. Bone ash After the femur was broken in the strength tests, it , was sawed into small pieces, the water and fat were ex- tracted in a Soxhlet extractor and the dried bone ground through a 40 mesh screen. A 2 g sample of the finely ground sample was then ashed in a muffle furnace. Ash was calcu- 1ated and Ca, P, and Mg were determined by methods described for feed and feces. D. Statistical Analyses Statistical analyses were carried out by analysis of variance and treatment differences were determined by application of the multiple range test of Duncan (1955). In Experiment IV, in order to analyze statistically for the effects of time after feeding, as well as the effects of litter, sex, protein level and protein source, the digesti- bility data were subjected to least squares analysis. Duncan's multiple range test was then applied to determine mean differences. IV. RESULTS AND DISCUSSION A. Experiment I. Effect of protein level on mineral utilization by the baby pig. The growth and serum data from Experiment I are presented in table 2. Figs receiving the higher levels of dietary protein (24% and 32%) tended to gain as well or better than pigs receiving the lower level of protein (16%). Feed intake, however, was lower at the high protein con- centration which resulted in.improved feed utilization at the higher levels of protein intake. Serum inorganic P values were higher in pigs receiving 16% crude protein than in pigs receiving higher levels. The higher levels of serum alkaline phosphatase values shown in pigs on high protein diets, along with the lowered serum inorganic P could be indi- cative of the onset of rickets. Data from the balance trial (table 3) show that maximal mineral retention occurred in pigs receiving 24% protein diets. Feeding high levels of protein resulted in decreased retention of nitrogen by the pig. Apparent digestibility of nitrogenous matter, however, was not affected by level of dietary intake. I Relatively constant amounts of feed intake between lots and the ‘wide differences in mineral intake were due to analyzed values being used rather than calculated values. 'Mineral composition of all diets ‘was calculated to be equal, however, table 3 shows that this was not accomplished. Throughout the remainder of this report all values re- ported are as analyzed, unless stated otherwise. Table 4 shows that density, mineral content and strength of bone ‘were depressed by increasing levels of dietary protein. Lowered serum 44 45 Table 2. Growth and serum analyses of baby pigs fed different levels of protein. Dietary protein, % 16 24 32 No. of pigs 4 4 4 ISE1 Initial wt, kg 2.84 2.85 2.86 0.14 Daily gain, kg 0.19 0.25 0.19 0.02 Daily food intake, kg 0.39 0.40 0.34 Gain/food 0.49 0.62 0.56 Serum Ca, mg/100 ml Initial 10.9 11.3 11.3 0.3 3 weeks 10.2 11.5 10.4 0.3 5 weeks 10.6 10.5 11.4 0.3 Serum P, mg/100 ml Initial 7.3 b 7.8 7.1 0.4 3 weeks 8.533' 5.5 4.5 0.6 5 weeks 6.0aa.b 4 s 3.9 0.3 Serum Mg, mg/100 ml Initial 2.6 2.2 1.8 0.1 5 weeks 2.7 2.5 2.3 0.1 Serum alkaline phosphatase, Sigma units Initial 13.9 12.1 15.7 1.1 3 weeks 7.9 8.9 10.3 1.1 5 weeks 8.0 7.2 14.iaa:b 1.1 Serum protein, g/100 ml Initial 5.3 5.1 5.1 0.2 5 weeks 4.6 4.9 4.7 0.1 1 Standard error of the mean. a Significantly greater than least value (P<0.05); 33 P <0.01. Significantly greater than least two values (P‘<:0.05). Table 3. 46 Daily calcium, phosphorus, magnesium and nitrogen excretion and retention as affected by level of dietary protein. Dietary protein, % 16 24 32 No. of collections 3 3 3 fSE1 Daily food intake, g 372 375 372 2 Daily water intake, m1 840 897 824 5 Daily feces, g 27.8 27.2 31.2 1.3 Daily urine, m1 384 429 438 23 Ca balance, daily Ca intake, g 2.91 3.01 3.40 0.06 Fecal Ca, g 1.15 1.07 1.24 0.06 Urinary Ca, mg 18 33 24 3 Ca retention, g 1.75 1.91 2.14 0.07 Ga retention, % 60 63 63 2 Ca apparent digest., % 61 65 64 2 P balance, daily P intake, g 2.31 2.46 2.51 0.03 Fecal P, g 0.99 0.82 0.94 0.05 Urinary P, g 0.10 0.10 0.01 0.02 P retention, g 1.22 1.54 1.56 0.07 P retention, % 53 63 62 3 P apparent digest., % 57 67 62 3 Mg balance, daily Mg intake, mg 189 210 232 4 Fecal Mg, mg 100 93 125 6 Urinary Mg, mg 18 ll 5 3 IMg retention, mg 71 106 102 8 IMg retention, % 38 51 44 4 ng apparent digest., % 47 56 46 4 N balance, daily N intake, g 9.07 14.175la 18.51bb 1.18 Fecal N, g 0.49 0.48 0.79bb 0.04 Urinary N, g 2.21 5.5488 8.841“) 0.83 N retention, g 6.37 8.1588 8.88aa 0.34 N retention, % 70 58 48 4 N apparent digest., % 95 97 96 0 1 Standard error of the mean. aa Significantly greater than least value (P-<:0.01). b Significantly greater than least two values (P<H .95 .a one»; 77 .nauuoum huuuogv mo nau>oa uaoummmuu osu um ufiuuoum hon no aamnmo vow omen mo uouuu Ho 0nu macaw aafiv0n mo ubamma and usamaH In .>H .muu .OH ouawum 78 .aamuoun huuuoqv mo ufim>ma uauuowwav 03» an :«uuoum how no :«ouuu cum omwn mo uomuu He may macaw auamuuuom mo Nuammm was xuamaH u: .bH .nxm .HH unamam ...... 7 . . . gun-=- . g _ . , . . . , . . . , , u ‘ . . . . “ ‘ V » _ . . . . . -- w .m a ,r _r L. 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W fifii >3 $21!. 3.3 2 2 85 length of fast prior to slaughter (Appendix 3) had a significant effect on the pH (Appendix 4) and the absorption of nutrients within the stomach and cranial small intestine. Graham gt El- (1959) showed that 28Mg absorption began within an hour of ingestion in man. Smith (1959) states that samples taken from the small intestine of a calf close to the cecal junction between 2 and 6 hours after feeding contained 70% to 802 of the residue from the feed. Casein diets appear to promote secretions into the cranial small intestine,‘whereas the soy diets promote absorption of dry matter within that section of the GI tract. Within the caudal small intestine, how- ever, dry matter from casein'was readily absorbed. There was a highly significant difference between the rates of absorption of the dry matter due to protein source in this section of the tract. Figure 8 shows that Ca movement within the GI tract was variable. Absorption of Ca from the casein diets was greater than Ca from the soy diets in the caudal small intestine. Phosphorus was secreted into the cranial small intestine and ab- sorbed from the caudal small intestine to a greater extent when the casein diet was fed than when soy protein was in the diet. Figure 9 shows that by the method used about 60% of the dietary P is absorbed from the stomach. Sodium and K followed each other rather closely during passage through the GI tract. Figures 10 and 11 show that the only dietary effect on the assimilation of Na and K into the body was a large influx of both nutrients into the cranial small intestine when casein diets were fed . iMagnesium influx or efflux along the GI tract closely paralleled A.“ '8 .» .s '3‘“,- 86 that of P (see Figures 9 and 12). The magnitude oijg movement, however, was generally not as great as that of P. Statistical analysis showed that Mg influx into the cranial small intestine was greater in pigs fed casein than in pigs fed soy protein diets.(P.<:0.05). Efflux of Mg from the caudal small intestine was also greater in pigs fed casein diets. Iron absorption from the caudal small intestine was significantly greater in casein fed pigs than in those receiving soy protein diets (P< 0.05) (Figure 13). Zinc, Mn and Cu showed the same response to dietary treatment as observed in iron movement (Figures 14, 15, 16). In each case the influx of the respective mineral into the cranial small intestine was enhanced when casein was the source of dietary protein. As a consequence of the greater influx of minerals into the cranial small intestine, there was more mineral available to be absorbed in the caudal small intestine. Iron, Zn, Mn and Cu all show a greater magnitude of efflux from the caudal small intestine when casein was the protein source. Level of protein in the diet did not greatly affect the absorp- tion or secretion of any of these minerals. Results of the Go flux studies are varied CFigure 17). Cobalt appears to differ from tne other minerals studied in that it was ab- sorbed from the cecum when isolated soy was the source of protein but not when casein was fed. Figure 18 summarizes the absorption and secretion of nutrients along the GI tract as determined by the chromic oxide ratio technique. Due to the high degree of variation of absorption and secretion, only minerals showing net absorption or secretion of greater than 10% are shown in Figure 18. Any absorption from the cranial small intestine is 87 .353 32: 5033 on» .3 sodas—opus as nuan— hnun mo uucuu nguuougouuuum on» no 233000 596 gamma no «935.. £8.33: no: mo hug-5m .... .>H .g .3 mus—mam Z :2 6: 00 .5 :U :0 CN CN on on 2n 2“ CU. C¢< u¢¢ .zd .6 2 an 8 z z a an :0 :N 8 cm 5 x :2 x a oz a: :2 x 2 as. a as. 02 x no cu m 22 oz .2: .2... .26 82.9.: o n v a a . m0... >¢02 88 masked by the large influx of nutrients via the duodenal, pancreatic and biliary secretions. What may be indicated by these data, rather than abSorption within the stomach and an influx into the cranial small intestine, is a difference in the rates of nutrient passage from one section of the tract to another. Ali (1967) reported that 450a moves out of the stomach of rats at the same rate as chromic oxide. Figure 8 shows that Ca is not absorbed in the stomach nor is there a great influx of Ca into the cranial small intestine. Phosphorus and the other minerals studied, however, appeared to be largely absorbed in the stomach and resecreted into the cranial small intestine. If these nutrients were to pass out of the stomach more rapidly than the indi- cator, this same pattern of absorption and secretion would be observed. Gorrill, 25 El- (1967) have shown that volume of pancreatic se— cretion of calves fed milk diets was significantly greater than that of calves fed soy protein diets. The magnitude of the difference in the two diets appeared to increase with age. If these data can be applied to pigs, it would partly explain the greater influx of nutrients into the cranial small intestine when the casein diet was fed. V. SUMMARY Four experiments, involving a total of 52 baby pigs, were conducted to study the effects of level of protein (16%, 24%, 32%), level of ergocalciferol (6.25 or 12.50/ug/kg diet) and source of protein (C-l isolated soy protein or vitamin-free casein) on mineral utilization by the baby pig. 'Experiment I Twelve baby pigs were assigned to 3 levels of dietary protein, with 4 pigs per treatment. Diets contained 20%, 30%, or 40% isolated soy protein and 6.2fi/4g/kg ergocalciferol. Growth response was the same for all treatments. Serum inorganic P and alkaline phosphatase concen- tration indicated superior P utilization occurred in pigs receiving only 16%.crude protein in the diet. Femur mineral and strength data showed that bones of pigs from the low protein lot contained more mineral and were stronger than those from pigs fed either 24% or 32% crude pro- tein in the diet. A 72-hour mineral balance study did not bear this out 9 Experiment II Eight baby pigs were assigned, 2 per lot, to the following dietary treatments! 24% crude protein with 6.25/“g ergocalciferol/kg diet, 32% crude protein with 6.21xlg ergocalciferol/kg diet, 24% crude protein ‘with 12.59/ug ergocalciferol/kg diet or 32% crude protein with 12.5Q/Ag ergocalciferol/kg diet. The protein source in all diets was isolated soy protein. Performance was similar in all pigs irrespective of dietary treatment. Neither serum analyses nor mineral balance studies revealed 89 90 any treatment effects. Femur breaking strength appeared to be reduced at the higher level of vitamin D intake. However, when breaking strength was placed on the basis of bone cross sectional area, or correcting for bone size, it was shown that vitamin D had little effect but the higher levels of protein intake tended to reduce the femur strength. Organ.weight data showed that kidneys and liver were hypertrophied in direct proportion to dietary protein intake. Urinary N excretion ‘was also reflected in the relative size of these organs. Experiment III Sixteen 7-day old pigs were allotted in lots of 4. Dietary treat- ments were similar to Experiment II except that 16% crude protein was the low level of protein intake rather than 24%. Feed intake and feed efficiency was 8% to 10% higher in pigs fed 32% crude protein diets. Dietary intake of vitamin D had no effect on performance. Mineral balance studies showed a slightly reduced retention of minerals when high levels of isolated soy were fed in the diet. Vitamin D treatment had no effect on mineral retention. Bone analyses showed that mineral deposition, and consequently bone strength, were reduced when 40% of the diet was made up of isolated soy protein. High levels of ergocal- ciferol did not improve mineral deposition in the bones of pigs fed the high protein diets. Weights of the kidneys, pancreas and thymus were increased in pigs fed the high protein diets. Spleen weight, in this experiment, was inversely related to level of protein intake. Experiment IV Four lots of 4 baby pigs each were assigned to dietary treatments 91 of 20% or 40% casein or 20% or 40% isolated soy. Crude protein content was 16% and 32% in the low and high protein diets, respectively. Feed intake was 20% lower when high levels of protein were fed. Growth was not affected by dietary treatment. Serum analyses showed that serum inorganic P was reduced and alkaline phosphatase correspondingly in- creased when high levels of soy protein were fed. Mineral balance studies showed that Ca and Mg retention were greater when casein was the source of dietary protein. Increasing casein content of the diet enhanced the utilization of Ca, P and.Mg. Increasing the amount of isolated soy protein in the diet, however, was found to suppress the utilization of these minerals. Differences in retention and excretion of Na, K, Fe, Zn, Mn, Cu, Co or Cr could not be attributed to dietary treatment. Optimum bone mineralization did not occur when soy protein was fed at 40% of the diet. Twenty percent isolated soy protein in the diet, however, allowed proper utilization of mineral for compact bone forma- tion as did casein diets at either level of protein intake. Weight of kidneys, liver and pancreas increased as protein level in the diet increased. Protein source did not appear to affect the weight of any of the organs or glands that were studied. When studying the absorption and secretion of nutrients along the alimentary tract by the indicator ratio technique, it was found that casein in the diet, regardless of level, results in an increased in- flux of minerals into the cranial small intestine and a compensatory efflux of minerals from the caudal small intestine. Due to a high de- gree of variation within treatment groups, specific conclusions per- taining to individual minerals were difficult to make. VI. CONCLUSIONS Within the limits of the experimental conditions employed, the results of this study have led the author to make the following con- clusions! 1. When dietary P is held constant, increasing the level of isolated soy protein in the diet results in poorer utili- zation of Ca, P and.Mg in the diet. Increasing ergocalciferol in the diet from 6.25 to 12.50 g/kg diet will not overcome the suppressing effects of high levels of isolated soy protein on mineral utilization. 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Abst. and Rev. 34:262. Woodman, H. E., R. E. Evans and W. G. Turpitt. 1937 The nutrition of the bacon pig. J. Agr. Sci. 27:569-583. Worker, N. A. and B. B. Migicovsky. 1961a Effect of vitamin D on the utilization of beryllium, magnesium, calcium, strontium and barium in the chick. J. Nutr. 74:490-494. Worker, N. A. and B. B. Migicovsky. 1961b Effect of vitamin D on the utilization of zinc, cadmium and mercury in the chick. J. Nutr. 75:222-224. Yang, M. G. and J. W. Thomas. 1965 Absorption and secretion of some organic and inorganic constituents and the distribution of these constituents throughout the alimentary tract of young calves. J. Nutr. 87:444-458. 109 Appendix Table 1. Dilutions used for analysesl. Mineral Serumé Feed Feces Urine Strontium5 X6 Ca 5 500 4000 0 + 4227 22 10 100 1000 10 - 7000 ‘Mg 20 500 4000 10 + 2852 Na3 - ‘600 1000 200 - 5890 K3 - 600 1000 200 - 7665 Fe - 100 400 0 - 2483 Zn - 100 400 0 - 2139 Mn - 100 100 0 + 2795 Cu - 100 100 0 - 3248 C0 - 100 100 0 - 2407 0:3 - 400 2000 0 - 4254 l Diluted with deionized distilled water. 2 Determined spectrophotometrically. Determined by flame emission. Dilution after precipitation of 1 m1 of serum with 4 m1’12.5% T.C.A. Added at 10,000 ppm Sr +-1Z.NaC1 to overcome interferences. Wave length at which absorption or emission was read. (Robinson, 1966) 110 Appendix Table 2. Intake and excreta of pigs during balance trial, Exp. IV.1 - Treatment Pig No. Pig wt, kg Feed, g 'Water, ml Feces, g2 'Urine, m1 16% Casein 8-2 12.66 1022 2700 73.3 1460 8-8 11.94 1022 2700 73.5 1100 9-3 11.36 702 1881 30.2 9-12 11.48 822 2250 58.2 1400 32% Casein 8-4 11.86 1021 2675 54.4 1410 8-9 13.00 1021 2675 34.5 1170 9—1 10.78 1025 2700 137.0 1885 9-8 8.34 1025 2700 69.8 1920 16% Soy 8-5 13.26 1032 2700 38.5 1850 8-7 12.12 1027 2675 75.1 1440 9-2 10.80 742 1846 43.6 1180 9-10 11.24 825 2250 48.6 1225 32% Soy 8-3 11.40 1041 2700 95.3 1560 8-6 8.98 808 2176 62.1 1460 9-6 13.20 1040 2700 75.6 1920 9-9 12.28 1040 2700 77.8 1830 1 Intake or excreta/72 hrs. 2 Oven dry weight. 111 Appendix Table 3. Slaughter sequence of pigs from Exp. IV. Day 1 2 3 4 .AIM 1% hrs post feeding 9-121 9-8 8-5 8-6 3 hrs post feeding 9-1 9-10 8-3 8-8 P'M 1% hrs post feeding 9-2 9-9 8-2 8-9 3 hrs post feeding 9-6 9-3 8-4 8-7 1 Pig identification number. Appendix Table 4. pH within the GI tract of baby pigs, Exp. IV. Pig Cranial Caudal Treatment No. Stomach SI SI Cecum Colon 16% Casein 8-2 F1 5.3 5.3 6.9 6.4 7.5 8-8M 4.1 5.5 6.2 6.5 6.6 9-3 F 3.6 5.2 7.0 6.8 7.3 9-12 M. 4.6 5.1 6.8 6.9 7.1 32% Casein 8-4 F 4.4 5.8 6.6 6.6 7.2 8-9 M 4.8 5.7 6.7 7.3 6.3 9-1 F 4.5 5.5 5.6 6.5 6.4 9-8 M1 4.6 5.7 6.5 6.4 6.4 16% Soy 8-5 F 6.0 5.6 6.9 6.9 6.8 8-7 MI 3.0 5.4 6.5 6.2 7.2 9-2 F 4.6 5.9 6.1 6.5 6.5 9-10 M. 4.1 5.6 6.4 6.5 6.1 32%.Soy 8-3 F 4.0 5.5 6.9 6.6 6.5 8-6 MI 5.2 5.5 6.5 6.3 6.5 9-6 F 5.1 5.5 6.3 5.0 6.7 9—9 M1 4.8 5.3 7.1 6.6 6.6 y 1 F =- female; M =- male. 112 Appendix Table 5. Dry matter copcentration within the GI tract of baby pigs, Exp. IV . Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon 16% Casein 8-2 26.9 19.1 4.8 13.2 27.6 40.0 8-8 26.9 19.4 3.1 3.6 10.7 32.1 9-3 26.1 17.2 10.9 16.2 22.4 41.6 9-12 26.1 14.4 7.5 12 4 20.2 34.2 32% Casein 8-4 26.8 23.7 14.9 14.0 18.3 32.2 8-9 26.8 22.8 6.4 12.8 39.2 25.2 9-1 26.8 28 3 6.1 3.6 6.4 20.2 9-8 26.8 24 5 6.3 7.1 12.4 22.2 16% Soy 8-5 27.0 16.2 6 7 12.6 19.5 42.8 8-7 27.0 20.3 4 5 9.5 18.4 40.8 9-2 27.0 15.6 4 5 5.3 12.9 24.8 9-10 26.2 21.0 6 7 7.0 23.7 36.8 32% Soy 8-3 27.2 14.6 8.3 14.7 24.0 35.6 8-6 26.4 24 6 3.6 10.4 25.8 30.4 9-6 27.2 19 9 8.5 8.0 16.8 34.5 9-9 27.2 23 2 5.0 6.9 10.0 14.8 Expressed as a percent of material within the portion of GI tract. 113 Appendix Table 6. Calcium conceniration within the GI tract of baby pigs, Exp. IV. Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon Feces 16% Casein 8-2 0.83 1.52 0.33 1.58 2.74 2.38 3.87 8-8 0.83 1.54 0.39 1.81 2.71 2.72 3.16 9-3 0.83 1.50 0.50 1.68 2.26 2.93 2.59 9-12 0.83 1.71 0.57 1.53 2.22 2.25 3.17 32% Casein 8-4 0.82 1.34 0.15 1.12 1.67 2.90 2.31 8-9 0.82 1.36 0.31 0.45 1.49 1.24 1.66 9-1 0.82 1.11 0.86 0.49 1.28 2.83 1.90 9-8 0.82 0.89 0.62 0.79 1.66 2.53 2.36 16% Soy 8-5 0.81 1.42 1.13 1.73 2.05 3.90 5.81 8-7 0.81 1.84 1.14 2.03 3.69 3.92 6.21 9-2 0.81 1.19 0.89 1.11 3.29 4.02 5.37 9-12 0.81 1.58 0.95 0.48 2.29 3.07 4.13 32% Soy 8-3 0.84 1.41 0.90 2.12 1.65 4.12 5.73 8-6 0.84 1.44 0.78 1.80 2.82 3.55 4.91 9-6 0.84 1.39 0.87 1.37 3.32 4.51 6.03 9-9 0.84 1.33 0.95 0.81 2.37 3.57 5.70 1 Expressed as a percent of dry matter within portion of GI tract. 114 Appendix Table 7. Phosphorus concentration within the GI tract of baby pigs, Exp. IV.1 Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon Feces 16% Casein 8-2 0.65 0.40 0.82 1.23 1.59 1.04 2.21 8-8 0.65 0.45 0.51 1.24 1.61 1.62 1.97 9-3 0.65 0.37 1.02 1.24 1.15 1.35 1.62 9-12 0.65 0.46 1.00 1.49 1.31 1.30 1.90 32% Casein 8-4 0.63 0.38 1.06 0.94 0.61 1.05 1.03 8-9 0.63 0.35 0.98 0.91 0.62 0.39 0.85 9-1 0.63 0.50 0.81 0.91 1.18 1.31 0.89 9-8 0.63 0.34 1.00 0.97 1.32 1.51 1.14 16% Soy 8-5 0.60 0.35 1.17 1.54 1.46 2.29 3.38 8-7 0.60 0.50 0.92 1.54 2.37 2.48 3.36 9-2 0.60 0.39 0.91 1.28 2.34 2.48 3.11 9-10 0.60 0.38 0.88 0.81 1.70 2.01 2.74 32% Soy 8-3 0.55 0.38 1.04 1.53 1.11 2.13 2.90 8-6 0.55 0.31 0.68 1.26 1.92 2.05 2.88 9-6 0.55 0.32 0.85 1.15 2.06 2.35 2.86 9-9 0.55 0.34 0.84 1.08 1.68 2.00 2.89 1 Expressed as a percent of dry matter within the portion of GI tract. 115 Appendix Table 8. Sodium concentration within the GI tract of baby pigs, Exp. IV.1 Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon Feces 16% Casein 8-2 0.42 0.56 2.93 1.92 0.60 0.54 0.45 8-8 0.42 0.35 5.05 6.22 0.54 0.50 0.45 9-3 0.42 0.58 1.98 1.58 1.04 0.41 0.17 9-12 0.42 0.59 1.57 1.83 1.19 0.64 0.31 32% Casein 8-4 0.46 0.17 1.10 1.62 1.50 0.47 0.31 8-9 0.46 0.36 2.46 1.74 0.33 0.64 0.41 9-1 0.46 0.35 2.98 6.53 3.87 0.99 0.32 9-8 0.46 0.39 3.07 3.82 2.16 0.68 0.43 16% Soy 8-5 0.48 0.70 2.59 2.13 1.33 0.39 0.44 8-7 0.48 0.42 3.24 2.32 1.15 0.58 0.37 9-2 0.48 0.63 4.59 5.13 2.09 0.91 0.26 9-10 0.48 0.36 2.54 3.40 1.10 0.49 0.29 32% Soy 8-3 0.49 0.29 2.22 1.79 1.23 0.46 0.30 8-6 0.49 0.65 4.43 2.31 0.95 0.71 0.38 9-6 0.49 0.71 1.84 3.27 1.95 0.69 0.22 9-9 0.49 0.38 3 1.90 0.51 .78 3.94 3.01 1 Expressed as a percent of dry matter within the portion of GI tract. 116 Appendix Table 9. Potassium concentration within the GI tract of baby pigs, Exp. IV.1 Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon Feces 16% Casein 8-2 0.29 0.40 2.02 0.71 0.24 0.20 0.22 8-8 0.29 0.35 1.37 1.31 0.59 0.61 0.14 9-3 0.29 0.41 1.69 0.61 0.32 0.35 0.49 9-12 0.29 0.44 1.63 1.13 0.43 0.32 0.33 32% Casein 8-4 0.33 0.24 1.43 1.06 0.31 0.25 0.21 8-9 0.33 0.31 1.91 1.22 0.54 0.46 0.14 9-1 0.33 0.18 1.25 1.62 1.14 0.83 0.30 9-8 0.33 0.26 1.67 0.86 1.07 1.18 0.39 16% Soy 8-5 0.30 0.41 1.61 0.94 0.40 0.29 0.19 8-7 0.30 0.34 1.40 0.72 0.31 0.22 0.21 9-2 0.30 0.43 1.34 1.23 0.56 0.41 0.29 9-10 0.30 0.28 0.78 0.89 0.36 0.40 0.25 32% Soy 8-3 0.29 0.40 1.50 0.74 0.40 0.65 0.28 8-6 0.29 0.33 1.97 0.91 0.47 0.53 0.79 9-6 0.29 0.35 1.29 1.00 0.37 0.43 0.34 9-9 0.29 0.30 1.54 1.48 0.56 0.56 0.19 1 Expressed as a percent of dry matter within the portion of GI tract. 117 Appendix Table 10. Magnesium concentration within the GI tract of baby pigs, Exp. IV.1 Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon Feces 16% Casein 8-2 0.037 0.045 0.170 0.142 0.352 0.220 0.178 8-8 0.037 0.035 0.115 0.171 0.270 0.280 0.186 9-3 0.037 0.037 0.149 0.182 0.239 0.171 0.165 9-12 0.037 0.038 0.107 0.207 0.153 0.194 0.202 32% Casein 0.035 0.030 0.100 0.156 0.110 0.147 0.103 0.035 0.037 0.159 0.134 0.153 0.174 0.138 0.035 0.020 0.113 0.159 0.155 0.241 0.108 0.035 0.034 0.151 0.103 0.209 0.324 0.150 0.038 0.054 0.132 0.152 0.193 0.270 0.237 0.038 0.059 0.142 0.236 0.463 0.328 0.223 0.038 0.045 0.111 0.114 0.250 0.265 0.247 0.038 0.041 0.083 0.156 0.243 0.239 0.215 0.040’ 0.041 0.166 0.185 0.142 0.222 0.236 0.040 0.054 0.189 0.200 0.203 0.230 0.224 0.040 0.044 0.117 0.128 0.266 0.261 0.216 0.040 0.043 0.186 0.142 0.133 0.164 0.213 1 Expressed as a percent of dry matter within the portion of GI tract. 118 Appendix Table 11. Iron concentration within the GI tract of baby pigs, Exp. IV.1 Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon Feces 16% Casein 8-2 0.0111 0.0117 0.0101 0.0527 0.0784 0.1295 0.0852 8-8 0.0111 0.0126 0.0134 0.0397 0.1178 0.1202 0.0888 9-3 0.0111 0.0096 0.0270 0.0608 0.1072 0.1295 0.0588 9-12 0.0111 0.0139 0.0250 0.0366 0.0745 0.0952 0.0707 32% Casein 8-4 0.0106 0.0180 0.0118 0.0570 0.1288 0.1644 0.1024 8-9 0.0106 0.0115 0.0236 0.0298 0.1197 0.1151 0.0991 9-1 0.0106 0.0097 0.0188 0.0253 0.0366 0.0834 0.0934 9-8 0.0106 0.0077 0.0174 0.0229 0.0494 0.0944 0.0851 16% Soy 8-5 0.0143 0.0149 0.0319 0.0376 0.1016 0.1483 0.0973 8-7 0.0143 0.0207 0.0294 0.0725 0.1286 0.1383 0.1084 9-2 0.0143 0.0142 0.0297 0.0360 0.1147 0.1726 0.0939 9-10 0.0143 0.0242 0.0431 0.0613 0.1495 0.2118 0.1117 32% Soy 8-3 0.0194 0.0192 0.0390 0.0668 0.0845 0.1576 0.1298 8-6 0.0194 0.0191 0.0241 0.0428 0.1367 0.1507 0.1078 9-6 0.0194 0.0200 0.0271 0.0379 0.1143 0.1519 0.1311 9-9 0.0194 0.0166 0.0260 0.0268 0.0899 0.1325 0.1163 1 Expressed as a percent of dry matter within the portion of GI tract. 119 Appendix Table 12. Zinc concentration within the GI tract of baby pigs, Exp. IV. Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon Feces 16% Casein 0.0090 0.0218 0.0266 0.0856 0.0976 0.1346 0.0736 0.0090 0.0126 0.0156 0.0513 0.1387 0.1534 0.0840 0.0090 0.0111 0.0548 0.0819 0.0925 0.1112 0.0704 0.0090 0.0109 0.0131 0.0575 0.0716 0.0780 0.0768 32% Casein 0.0102 0.0173 0.0242 0.0557 0.0793 0.1082 0.1021 0.0102 0.0204 0.0192 0.0813 0.1005 0.1044 0.0990 0.0102 0.0076 0.0130 0.0273 0.0489 0.0876 0.1202 0.0102 0.0085 0.0104 0.0209 0.0545 0.0664 0.0978 0.0092 0.0104 0.0170 0.0241 0.0613 0.0920 0.0832 0.0092 0.0173 0.0189 0.0400 0.0606 0.0793 0.0764 0.0092 0.0111 0.0138 0.0312 0.0873 0.1144 0.0968 0.0092 0.0142 0.0117 0.0381 0.1068 0.1352 0.0808 0.0094 0.0103 0.0233 0.0387 0.0423 0.0683 0.0642 0.0094 0.0106 0.0177 0.0331 0.0631 0.0720 0.0652 0.0094 0.0104 0.0107 0.0232 0.0638 0.1039 0.0702 0.0094 0.0166 0.0129 0.0197 0.0661 0.0703 0.0788 1 Expressed as a percent of dry matter within the portion of GI tract. 120 Appendix Table 13. Manganese concentration within the GI tract of baby pigs, Exp. Iv.1 Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon Feces 16% Casein 0.0025 0.0026 0.0023 0.0116 0.0302 0.0299 0.0296 0.0025 0.0026 0.0041 0.0053 0.0419 0.0439 0.0285 0.0025 0.0034 0.0061 0.0136 0.0302 0.0316 0.0289 0.0025 0.0028 0.0026 0.0069 0.0230 0.0324 0.0318 32% Casein 0.0025 0.0050 0.0060 0.0268 0.0283 0.0357 0.0414 0.0025 0.0028 0.0027 0.0124 0.0359 0.0260 0.0517 0.0025 0.0007 0.0026 0.0041 0.0123 0.0186 0.0530 0.0025 0.0043 0.0048 0.0073 0.0084 0.0222 0.0365 0.0025 0.0026 0.0029 0.0051 0.0218 0.0422 0.0298 0.0025 0.0038 0.0022 0.0103 0.0220 0.0223 0.0303 0.0025 0.0034 0.0047 0.0063 0.0241 0.0246 0.0336 0.0025 0.0033 0.0032 0.0106 0.0251 0.0253 0.0370 0.0025 0.0021 0.0049 0.0092 0.0123 0.0241 0.0273 0.0025 0.0026 0.0011 0.0053 0.0194 0.0216 0.0335 0.0025 0.0060 0,0030 0.0084 0.0225 0.0470 0.0394 0.0025 0.0021 0.0046 0.0034 0.0151 0.0222 0.0355 Expressed as a percent of dry matter within the portion of GI tract. Appendix Table 14. 121 Copper concentration within the GI tract of baby pigs, Exp. IV.1 Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon Feces 16% Casein 8-2 0.0022 0.0030 0.0014 0.0102 0.0209 0.0277 0.0275 8-8 0.0022 0.0048 0.0025 0.0085 0.0258 0.0268 0.0273 9-3 0.0022 0.0029 0.0082 0.0160 0.0261 0.0293 0.0231 9-12 0.0022 0.0035 0.0029 0.0134 0.0224 0.0261 0.0235 32% Casein 8-4 0.0020 0.0023 0.0023 0.0098 0.0222 0.0264 0.0301 8-9 0.0020 0.0030 0.0030 0.0120 0.0250 0.0179 0.0285 9-1 0.0020 0.0026 0.0026 0.0053 0.0127 0.0160 0.0265 9-8 0.0020 0.0014 0.0014 0.0057 0.0135 0.0155 0.0220 16% Soy 8-5 0.0024 0.0032 0.0032 0.0083 0.0221 0.0290 0.0302 8-7 0.0024 0.0040 0.0040 0.0146 0.0246 0.0259 0.0286 9-2 0.0024 0.0049 0.0049 0.0069 0.0212 0.0269 0.0225 9-10 0.0024 0.0062 0.0062 0.0120 0.0226 0.0284 0.0275 32% Soy 8-3 0.0026 0.0040 0.0040 0.0094 0.0156 0.0250 0.0269 8-6 0.0026 0.0041 0.0041 0.0077 0.0263 0.0239 0.0273 9-6 0.0026 0.0027 0.0027 0.0058 0.0202 0.0279 0.0266 9-9 0.0026 0.0039 0.0039 0.0044 0.0161 0.0231 0.0228 1 Expressed as a percent of dry matter within the portion of GI tract. Appendix Table 15. 122 pigs, Exp. IV.1 Cobalt concentration within the GI tract of baby Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon Feces 16% Casein 8-2 0.0018 0.0028 0.0006 0.0072 0.0113 0.0172 0.0180 8-8 0.0018 0.0022 0.0016 0.0069 0.0174 0.0175 0.0217 9-3 0.0018 0.0027 0.0032 0.0081 0.0161 0.0173 0.0188 9-12 0.0018 0.0029 0.0012 0.0047 0.0119 0.0145 0.0191 32% Casein 8-4 0.0017 0.0026 0.0016 0.0079 0.0154 0.0207 0.0181 8-9 0.0017 0.0022 0.0010 0.0054 0.0177 0.0159 0.0215 9-1 0.0017 0.0013 0.0021 0.0056 0.0055 0.0129 0.0219 9-8 0.0017 0.0012 0.0022 0.0022 0.0061 0.0099 0.0211 16% Soy 8-5 0.0016 0.0022 0.0023 0.0042 0.0089 0.0144 0.0127 8-7 0.0016 0.0036 0.0026 0.0080 0.0125 0.0135 0.0193 9-2 0.0016 0.0021 0.0009 0.0038 0.0129 0.0172 0.0179 9-10 0.0016 0.0027 0.0027 0.0042 0.0132 0.0160 0.0173 32% Soy 8-3 0.0018 0.0020 0.0033 0.0063 0.0005 0.0128 0.0137 8-6 0.0018 0.0028 0.0027 0.0059 0.0104 0.0113 0.0120 9-6 0.0018 0.0026 0.0017 0.0029 0.0094 0.0116 0.0142 9-9 0.0018 0.0021 0.0031 0.0024 0.0068 0.0107 0.0141 1 Expressed as a percent of dry matter within the portion of GI tract. Appendix Table 14. 121 pigs, Exp. IV.1 Copper concentration within the GI tract of baby Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon Feces 16% Casein 8-2 0.0022 0.0030 0.0014 0.0102 0.0209 0.0277 0.0275 8-8 0.0022 0.0048 0.0025 0.0085 0.0258 0.0268 0.0273 9-3 0.0022 0.0029 0.0082 0.0160 0.0261 0.0293 0.0231 9-12 0.0022 0.0035 0.0029 0.0134 0.0224 0.0261 0.0235 32% Casein 8-4 0.0020 0.0023 0.0023 0.0098 0.0222 0.0264 0.0301 8-9 0.0020 0.0030 0.0030 0.0120 0.0250 0.0179 0.0285 9-1 0.0020 0.0026 0.0026 0.0053 0.0127 0.0160 0.0265 9-8 0.0020 0.0014 0.0014 0.0057 0.0135 0.0155 0.0220 16% Soy 8-5 0.0024 0.0032 0.0032 0.0083 0.0221 0.0290 0.0302 8-7 0.0024 0.0040 0.0040 0.0146 0.0246 0.0259 0.0286 9-2 0.0024 0.0049 0.0049 0.0069 0.0212 0.0269 0.0225 9-10 0.0024 0.0062 0.0062 0.0120 0.0226 0.0284 0.0275 32% Soy 8-3 0.0026 0.0040 0.0040 0.0094 0.0156 0.0250 '0.0269 8-6 0.0026 0.0041 0.0041 0.0077 0.0263 0.0239 0.0273 9-6 0.0026 0.0027 0.0027 0.0058 0.0202 0.0279 0.0266 9-9 0.0026 0.0039 0.0039 0.0044 0.0161 0.0231 0.0228 1 Expressed as a percent of dry matter within the portion of GI tract. 123 Appendix Table 16. Chromium concentration within the GI tract of baby pigs, Exp. IV.1 Pig Cranial Caudal Treatment No. Feed Stomach SI SI Cecum Colon Feces 16% Casein 8-2 0.33 0.48 0.10 2.24 4.62 4.12 4.22 8-8 0.33 1.01 0.16 1.31 3.26 3.32 4.36 9-3 0.33 0.41 0.58 2.56 2.56 4.40 4.46 9-12 0.33 0.55 0.18 0.90 2.72 4.15 4.29 32% Casein 8-4 0.36 0.57 0.07 1.14 3.55 3.78 4.52 8-9 0.36 0.46 0.15 1.45 1.44 3.38 5.12 9-1 0.36 0.82 0.29 0.45 1.92 2.38 5.08 9-8 0.36 0.64 0.17 0.89 1.79 2.28 4.11 16% Soy 8-5 0.33 0.41 0.44 1.03 3.98 3.29 3.42 8-7 0.33 0.67 0.49 1.68 4.27 2.90 3.84 9-2 0.33 0.70 0.57 0.76 6.91 3.07 3.66 9-10 0.33 0.87 0.73 0.77 2.04 3.53 4.60 32% Soy 8-3 0.33 0.54 0.39 1.24 2.86 2.73 3.30 8-6 0.33 0.44 0.25 0.75 3.35 2.81 3.45 9-6 0.33 0.46 0.35 0.69 1.79 3.03 3.34 9-9 0.33 0.60 0.41 0.44 2.34 2.56 3.78 1 Expressed as a percent of dry matter within the portion of GI tract. Appendix Table 17. 124 Comparison of indicator and balance trial methods for determining apparent digestibility of nutrients, Exp. IV. Nutrient Method 16% Casein 32% Casein 167. Soy 327. Soy Ca Indicator 70 80 42 37 Balance 75 82 62 47 P Indicator 77 88 54 50 Balance 81 89 70 59 Na Indicator 94 94 94 93 Balance 95 94 96 94 K Indicator 92 94 93 91 Balance 95 94 97 91 Mg Indicator 62 72 47 47 Balance 65 73 62 49 Fe Indicator 47 34 37 37 Balance 55 35 58 51 Zn Indicator 35 20 18 27 Balance 45 29 47 49 Mn Indicator -14 -74 -41 -69 Balance 33 -35 25 - 6 Cu Indicator 3 10 7 - 4 Balance 24 4 36 21 Co Indicator 18 7 10 29 Balance 29 11 42 42 N Indicator 97 98 96 97 Balance 98 98 98 97 125 Appendix Table 18. Mineral mixtures used in purified diets. 16% crude 24% crude 32% crude protein protein protein 2 z 2 KCl 10 10 10 K1 0.002 0.002 0.002 FeSO4-2H20 0,7 0.7 0.7 CuSO4 0.1 0.1 0.1 C0003 0.1 0.1 0.1 iMnSO4-H20 0.1 0.1 0.1 “504-1120 0.4 0.4 0.4 Mam3 zo 2o 26 NaHCO3 25.0 25.0 25.0 CaHPOi-ZHZOI 42.4 36.0 29.6 CaCO3 8.8 12.5 16.2 Glucose1 10.4 13.1 15.8 1 Adjusted to keep phosphorus level constant in all diets. Appendix Table 19. 126 Vitamin mixture used in purified diets. Thiamine°mononitrate Riboflavin Nicotinamide Calcium pantothenate Pyridoxine-hydrochloride p-Amino benzoic acid Ascorbic acid a-Tocopheryl acetate Inositol Choline chloride Pteroylglutamic acid Biotin Cyanocobalamin 2-Methyl-1,4-naphthoquinone Retinyl palmitate Ergocalciferol mg/kg diet 3 6 40 30 2 13 80 10 130 1300 flg/kg diet 260 50 100 40 600 6.25 or 12.50 1 Added at 6.25 or 12.50/ug/kg for each diet.