CRYSTALLINE VITAMIN B±2 REQUIREMENT OF THE YOUNG DAIRY CALF I. II. Development of a Vitamin Deficient Synthetic Milk The Crystalline Vitamin Requirement of the Young Dairy Calf t>y CHARLES A . LASSITER A Thesis Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1952 ProQuest Number: 10008361 All rights reserved INFORM ATION TO ALL USERS The quality o f this reproduction is dependent upon the quality o f the copy submitted. In the unlikely event that the author did not send a com plete m anuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008361 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This w ork is protected against unauthorized copying under Title 17, United States Code M icroform Edition © ProQuest LLC. ProQ uest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 VITA \ Charles Albert Lassiter was born in Murray, Kentucky February 20, 1927, He received his secondary education in the public schools of Calloway County, Kentucky. He was on active duty in the United States Naval Reserve from April, 1945 until August, 1946. He attended Murray State College and was awarded the degree of Bachelor or Science in Agriculture by the University of Kentucky in 1949. He was awarded a Graduate Teaching Assistantship in the Animal Husban dry Department of the University of Kentucky and received the degree of Master of Science in Agriculture from that institution in 1950. At that time he was awarded a Graduate Research Assistantship in the Dairy Department of Michigan State College. He held this appointment for two years while partially completing the requirements for the degree of Doctor of Philosophy. In June 1952, he accepted a position in the Dairy Section of the University of Kentucky as Assistant Professor of Dairying and Assistant Dairy Husbandman. ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Doctor C. F. Huffman, Research Professor of Dairying, Doctor George M. Ward, Assistant Professor of Dairying, and for their counsel and timely suggestions throughout this study and for the critical reading of the manuscript; .Earl Weaver, Professor of Dairying, to Doctor for the award of the Graduate Assistantship and for the provision of the facilities required in conducting this study. The author is indebted to Mr. C. W. Duncan, Resea rch Professor of Agricultural Chemistry, and his associates for the chemical analyses used herein; Instructor in Animal Pathology, mortem examinations used herein; to Doctor H. D* Webster, for performing the p o s t ­ and to unnamed members of the Dairy Department for their many suggestions regarding the conduct of the study and the preparation of the m a n u ­ script. The author is indebted to the Dow Chemical Company for the methionine; to Merck and Company for the vitamin Bqg; and to the Sheffield Farms Company, lactose used in this Incorporated for the study. The author wishes to extend gratitude to his wife, Robbie R. Lassiter, been invaluable. whose never-ending encouragement has CRYSTALLINE VITAMIN B 12 REQUIREMENT OF THE YOUNG DAIRY CALF I. II. Development of a Vitamin Deficient Synthetic Milk The Crystalline Vit amin Requirement of the Young Dairy Calf By Charles A. Lassiter A N ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1952 Approved Charles A. Lassiter Crystalline Vi tamin Bqg Requirement of the Young Dairy Calf Fourteen newborn dairy calves were used as experimental subjects to develop a synthetic milk deficient in vitamin The synthetic milk was composed of "alpha" protein, d, 1 - m e t h i o n i n e , glucose, lactose, lard, lecithin, and salts and was supplemented with all known required vitamins with the exception of vitamin Bqg. The components were ho mo­ genized into a concentrate containing 26.3 percent dry matter. The vitamin B^g deficient ration supported life but did not promote growth. However, if supplemented with either an A P F concentrate or crystalline vitamin B]_g normal or slightly sub-normal growth was promoted. It was found that calves fed such a synthetic milk required d,1-methionine in addition to that contained in the "alpha" protein. dairy calf was The d ,1-methionine requirement of the found to be more than 0.15 percent but less than 1.0 percent of the ration. Twenty-three dairy calves were fed the vitamin B-j_g deficient ration supplemented with 0, 10, 20, 40, and SO micrograms of crystalline vitamin B]_g per kilogram of dry matter consumed for the 6-week period from 3 to 45 days of age in an attempt to establish the crystalline vitamin B 12 requirement of the young dairy calf. The average daily gain - 2 - Charles A. Lassiter of Groups I, II, III, and IV was -0.10, 0.20, 0.20, and 0.65 pound per day, respectively. on Group V. min Incomplete data were obtained It was found that the you ng calf required v ita­ The principal symptoms of a vitamin B^g deficiency observed were growth cessation, lack of appetite, poor condition, muscular weakness, kidney condition. general and a white-spotted Calves fed a ration deficient in vitamin B^g tended to have greater concentrations of hemoglobin, blood cell volume, and red blood cell counts in the blood but the lack of vitamin B-^g on plasma calcium, red appear to have any effect inorganic phosphorus, magnesium, or ascor­ bic acid. Preliminary results indicated that the crystalline vitamin B^g requirement of the dairy calf was more than 20 micrograms but not more than 40 micrograms of vitamin B^g per kilogram of dry matter consumed. Evidence was obtained which indicated that some batches of "alpha" protein were not satisfactory sources of protein for the young dairy calf. Results were obtained which in­ dicated that some batches of "alpha" protein were deficient in one or more required nutrients or contained a toxic factor. Calves fed rations containing this protein con­ sistently died within the first two weeks of life. Such protein was not found to be deficient in the amino acids methionine or lysine. The lecithin content of the synthetic - 3 - Charles A. Lassiter milk was not observed to be the toxic or deficiency factor encountered when feeding such rations. A rat growth study showed that the toxic or deficiency factor was definitely associated with the "alpha" protein in the synthetic ration. TABLE OF CONTENTS Page I N T R O D U C T I O N ........................................... 1 PART I R E VIEW OF LITERATURE Adaptability of Soybeans as a Source of Protein for Animal Consumption ....................... 4 Purified Rations -- Soybean Type........ .......... 14 EXPERIMENTAL PROCEDURE Selection of Animals Feeding and Management . . .......................... 18 ............................ 18 Preparation of F e e d ............................... 20 Criteria for Evaluation of the Nutritive Value of the R a t i o n s ................................. 23 R E S U L T S ................................................. 25 D I S C U S S I O N ........... 28 S U M M A R Y ................................................. 36 PART II REVIEW OF LITERATURE Isolation and Characterization of Vitamin B^g . . 39 Vitamin B^g -- Clinical Aspects 50 .................. The Role of Vitamin B^g in Animal Nutrit ion . • • Metabolism and Mode of Action of Vitamin B^g Vitamin Bl2 Reo^uirement of Various Species . . . . . 55 66 72 TABLE OF CONTENTS (Cont.) Page EXPERIMENTAL PROCEDURE Selection of Ani mal s ................................ Feeding and Management .............................. 75 76 Preparation of F e e d ................................... 77 Criteria for Establishing the Crystalline Vitamin B 12 Requirement of the Dairy C a l f ............ 78 Comparative Nutritive Value of Two Batches of "Alpha" Protein asDetermined by Rat Growth 79 . R E S U L T S ........................................................ 81 D I S C U S S I O N .................................................. 101 S U M M A R Y .......................................................108 LITERATURE C I T E D ........................................... 109 APPENDIX 137 LIST OF TABLES Page Table 1 Assignment of A n i m a l s ........................ 19 Table 2 Components of Rations F e d ................... 21 Table 3 Growth Response of Calves Fed the Various Experimental Rations ....................... 25 Average Blood Analysis of the Calves by R a t i o n s ................................ 27 Table 4 . . Table 5 Vitamin Requirement of Various Species 73 Table 6 Composition of Experimental Groups • • • • 75 Table 7 Basal R a t i o n ................................. 77 Table 8 Composition of Experimental Rat Groups. 80 Table Formulae of Rat R a t i o n s ..................... 80 Table 10 Chemical Analyses of Ingredients of Ration 81 Table 11 Chemical Analysis of Basal Rat ion Table 12 Growth and Feed Utilization Table 13 Weekly Analyses of Blood Plasma Constituents 96 Table 14 Gain and Feed Utilization -- Rat Experiment 98 Table 15 Growth Data — Table 16 Growth Data -- Part I I ........................ 141 Table 17 Gro wth Data of Calves C-865, C-866, C-867, and C-868 9 Part I . . .......... . . . . . . . 81 82 ........................ 138 143 Table 18 Rat Growth Data Table 19 Weekly Blood Analyses -- Experimental Calves Table Feed Consumption of the Experimental Rat G r o u p s .......................................... 150 20 ...................... 143 145 LIST OF FIGURES Page Figure I Growth curves — Figure II Growth curves -- Group Figure III Growth curves -- Group 3 ................. 85 Figure IV Growth curves — Group 4 ................. 86 Figure V Growth curves -- all g r o u p s ............ 87 Figure VI Calf C-841 — B12Ag* Figure VII Figure VIII Figure IX Figure X Group I ................. Group I 2. . . . . . . — . 83 84 0 ug. vitamin ........................... 93 Calf C-844 -- Group II — 10 ug. vitamin Bi2/kg. D.M. • • ............. 94 Calf C-854 -- Group IV -- 40 ug. vitamin Big/kg. D.M. • • • • • • • • • • • • • 95 Changes in some blood constituents of the experimental c a l v e s ................ 97 Growth curves of rats fed two batches of "alpha" p r o t e i n ..................... 99 INTRODUCTION The development of a satisfactory milk replacement for the raising of future herd replacements is a matter of utmost economic importance to dairymen. Many attempts to develop a calf ration which would equal or surpass milk in its ability to support growth of young calves have been made but few have resulted in even moderately satisfactory calf growth* Many of the more successful milk replacements have included milk by-products in relatively large proportions, thus replacing less milk than commonly believed. More complete knowledge of the nutritive requirements of the yo ung calf might pave the way for the use of less expensive ingredients in milk replacements. Synthetic milks have been formulated but, due to the high cost of purified compounds, they are far from being the solution for the economic problem* The synthetic milks have been used very successfully in ascertaining certain of the fundamental nutritional requirements of the young calf. These rations have usually contained casein or other milk protein. Workers in various fields have found that casein is a good source of vitamin B^g, thus rendering synthetic milks containing casein useless for the study of vitamin B 12 defi ci en c y . - 2 - This investigation was initiated in an attempt to for mu­ late a synthetic milk, without the use of protein of animal origin, which would be nutritionally adequate for the young calf and to use the ration formulated in the determination of the vitamin requirement of the young calf. PART I DEVELOPMENT OF A VITAMIN B 12 DEFICIENT SYNTHETIC MILK REVIEW OF LITERATURE Ada ptability of Soybeans as a Source of Protein for Animal Consumption Osborne and Mendel (1917) were the first workers to show that raw soybeans when fed as the sole source of protein in the ration of the rat were unsatisfactory. When raw soybeans were used as the only protein source the rats grew very little and presented rather poor condition. However, these same workers were able to show that if the soybeans were heated in an electric oven at 110° Centigrade for four hours a few of the rats showed increases in growth, while showed no increase. These workers the others attributed this difference to greater consumption of the heated meal by the rats which grew on this ration. In an attempt to further increase the nutritive value of soybeans these workers cooked the soybeans for three hours and found that soybeans processed in this manner increased the growth rate of all the rats. It was further shown that soybeans were relatively poor source of both calcium and chlorine. Robison (1930) showed that the cooking of raw soybeans improved their feeding value for pigs and also stated that the cooking process slightly increased the digestibility of the protein. Vestal and Shrewsbury (1932) confirmed the - 5 - earlier observations of Robison concerning the effect of cooking on raw soybeans for both swine and rats* Mitchell and Smuts (1932) reported that raw soybeans were deficient in cystine for the white rat* When the soy­ beans were supplemented with cystine a marked increase in growth occurred* Other workers observed that methionine, (Jackson and Block, 1932) like cystine, was capable of stimu­ lating growth in rats subsisting on a basal ration poor in cystine. These workers further suggested that cystine and methionine make up a freely interconvertible system of which only one member is necessary and that each could substitute for the other to some degree. Shrewsbury et^ al_. (1932) demonstrated that the addition of 2.25 to 5.0 percent crude casein to a corn-soybean ration improved the growth rate of both rats and swine. Cooked soybeans were definitely superior to raw soybeans although cooked soybeans had slightly less nutritive value than an equivalent amount of crude casein. These same workers found that the addition of 3.0 percent dried yeast to a corn-soybean ration did not improve it. Shrewsbury and Bratzler (1933) in an attempt to explain the beneficial effects of cooking soybeans showed that soybeans were deficient in cystine and, since both cooking and the addition of cystine to this ration improved its nutritive value, suggested that cooking might serve to make the cystine more available. - 6 - Hayward et al, (1936a, 1937) found that a soybean oil meal of the expeller type, one which was processed at low temperature for a long period of time, contained protein of low nutritive value and was similar in nutritive value to raw soybeans. Soybean oil meals prepared at medium or high temperature for short periods of time contained proteins which had about twice the nutritive value of raw soybeans. These workers expressed the same belief as Shrewsbury and Bratzler (1933) that heating causes some essential protein factor to become available which before was unavailable. Later these same workers (Hayward et a l . , 1936b) showed that either the addition of 0.3 percent cystine or the application of heat practically doubled the nutritive value of soybean protein, but the addition of cystine to heated soybeans did not further improve the feeding value of the protein for rats. These observations confirmed the earlier beliefs of Mitchell and Smuts (1932). Johnson et_ a l . (1939) in an attempt to confirm the earlier work of Ha yward ejt a l . (1936b) conducted sulfur and nitrogen balances on rats fed heated soybeans. It was shown that the raw soybeans contained a sulfur-nitrogen complex which was absorbed but could not be used for tissue building. It was also found that treating the soybeans with the proper heat treatment made this complex available to the animal. - 7 - Rose et_ al_. (1936) showed in rat studies that of the sulfur-containing amino acids that methionine was the i n ­ dispensable one rather than cystine. Methionine alone stimulated growth of rats fed a raw soybean ration low in cystine but when cystine was substituted for methionine the rats failed to grow or did so at a reduced rate. These workers further suggested that cystine might be able to sub­ stitute for methionine to a limited extent. (1941) Hayward and Hafner presented similar results with chicks. It was found that 0.3 percent cystine supplemented soybeans to a limited extent, but that 0.3 percent methionine was a better supple­ ment for soybeans. A combination of both cystine and methionine produced no better results than methionine alone. These workers concluded that raw soybeans were deficient in available cystine and contained a suboptimal amount of methionine. It was further thought that the heat treatment of soybeans improved the protein as a whole as well as making cystine available. Klose and Almq uist (1941) showed that methionine was essential for the growth of the chick. homocystine alone could replace of methionine. However, could replace methionine Ne ither cystine nor the growth-promoting effects homocystine and choline together in the ration of the chick. These observations were made on chicks fed a purified ration with the protein arachin from peanuts as the source of protein. - 8 - Marvel et_ a l . (1944) confirmed the choline-methionine relationship proposed by Klose using a basal diet of corn and soybean oil meal* Almquist ejb a l * (1942) reported that even heat-treated soybean protein fed at a 20 percent level in the diet was still slightly deficient in methionine growing chicks. for As the result of many years of work on the amino acid requirements of chicks, Almquist (1947) was able to establish the quantitative requirement for sulfur-containing amino acids for the chick. requirement It was found that the chick's for sulfur-containing amino acids was 0.9 percent of the ration. The methionine requirement of the chick was found to be 0.9 percent of the ration in the absence of cystine and 0.5 percent in the presence of 0.4 percent cystine. After 25 years of investigation workers in various fields had been able to improve the nutritive value of raw soybeans by two principal methods, and Mendel, treating them with heat (Osborne 1917) and supplementing them with methionine (Rose et a l . . 1936). However, a growth-inhibiting substance was shown to be present in soybeans These workers (Ham and Sandstedt, 1944). isolated a substance in unheated soybeans which greatly retarded the activity of trypsin iji v i t r o . The trypsin inhibitor was destroyed by autoclaving the soybean oil meal. A factor very similar to this one, if not identical with it, caused a reduction in chick growth when raw soybeans were fed. The existence of such a substance in raw soybeans - 9 - was confirmed by Bowman (1945). Alm qu ist and Merritt (1952) showed that as little as five percent raw soybeans caused maximum anti-trypsin growth reduction in chicks. The inh ib i­ tion was overcome by adding 0.1 percent trypsin to the ration or by cooking the soybeans. Since cooking or treating raw soybeans with heat improved their nutritive value the problem arose as to the effect of overheating soybeans (Clandinin et_ al^. , 1946). These workers showed that the heating of soybean flakes in an autoclave at 15 pounds pressure for more than 3 3/4 minutes had adverse effects on the nutritive value of the meal. however, These effects, could be corrected by the addition of known vitamins and amino acids. These results showed an apparent destruction of vitamins and amino acids during such heat treatment. same workers later These (1947) showed that overheated soybean oil meal was deficient in available lysine and methionine. Slight destruction of both arginine and tryptophane was also de mon ­ strated. Riese n et a l . (1947) showed that trypsin inhibitor was not the only factor involved when comparing the nutritive value of raw and properly cooked soybeans. It was concluded that a decrease in nutritive value was associated with o v e r ­ heated soybeans because there was decreased liberation of the essential amino acids. after Clandinin et a l . (1948) concluded several years work on the proper heat treatment of raw - 10 - soybeans for chicks that maximum nutritive value could be obtained either by heating at 15 pounds pressure for four minutes or by heating for 45 minutes at four pounds pressure. Byerly et a l . (1937) observed seasonal variation in the hatchability of eggs from hens fed rations containing no animal protein. It was observed that eggs produced from hens fed such a ration consistently had a lower hatchability percentage during the winter months. However, eggs produced from hens exposed to sunlight had a higher hatchability percentage during these months. Bird ejb a l . (1946) observed that the eggs from hens fed a ration which contained 30 percent soybean oil meal as the sole source of protein hatched poorly, thus confirming the earlier findings of Byerly These workers found that over a 43-week (1937). period only 66 percent of the fertile eggs produced by soy­ bean oil meal fed hens hatched, while 84 percent of the eggs from hens fed a sardine meal ration hatched. There was a high rate of mortality even in the hatched chicks from the soybean oil meal fed hens. with the earlier These results were in agreement findings of Heuser (1946). These workers further observed that if the soybean oil meal rations were supplemented with five percent cow (or steer) manure, 10 percent sardine meal, or 10 percent dried milk that the low hatchability condition was corrected. - 11 - Bird and Marvel (1943) observed that if hens on a low riboflavin ration were fed the feces of hens fed a riboflavin supplemented ration a marked increase in the hatchability of the eggs from the low riboflavin groups occurred. (1942) Hammond observed that the growth of chicks being fed a high plant-protein ration could be increased if a small amount of cow manure were added to their rations. was collected and dried at 45° Centigrade The cow manure for 24 hours. Heuser e_b a l . (1946) reported that the inclusion of three percent fish meal into a chick starting ration containing soybean oil meal as the sole source of protein increased growth, decreased mortality, and increased feed efficiency. This beneficial effect appeared to be additive rather than supplementary, thus indicating that it was not due chiefly to the addition of essential amino acids. Rubin and Bird (1946a) started investigations in an attempt to purify the hatchability factor. It was observed that incubated cow manure was definitely a carrier of the factor. These workers found that the cow manure not identical with the Lactobacillus casei liver, yeast, or fermentation residues), factor factor was (from factors U, R, or S, vitamins Bio or B n , synthetic folic acid, or pyracin lactone. Cary et a l . (1946) presented evidence factor for an unidentified (X) which was required for rat growth when rats were - 12 - fed soybean oil meal as the sole source of protein. It was found that commercial casein, Sherman Vitam in A-Free casein, SMA Labco casein, and casein prepared by centrifugation from milk contained va rying amounts of the unidentified factor However, if casein were (X). subjected to 10 six-hour extractions with hot alcohol the factor was removed. It was further found that liver extract was also a good source. Zucker et^ a l ♦ (1948) reported a nutritional factor necessary for rat growth which was associated with animal protein sources. These workers found that the deficiency revealed itself most strikingly after the normal lactation period and it was characterized by a marked growth reduction, high mortality, count. high blood urea, and a low white blood cell These workers were of the opinion that the factor was very similar, if not the same, as the factor X" of Cary and the cow manure Bird. "nutritional factor of Rubi n and Zucker e_t a l . (1948) proposed the name of "zoopherin" for this unidentified nutritional factor for rats. Krider et^ a l . (1943) showed that the addition of six B-complex vitamins pantothenic acid, (thiamine, riboflavin, pyrodoxine, and choline) to a corn-soybean oil meal ration for pigs improved survival, cell counts, niacin, and hemoglobin values. growth rate, However, red blood pigs fed this supplemented ration still gained weight at a suboptimal rate as compared to pigs fed a similar ration containing animal - 13 - protein. The addition of 1.5 percent of an AB liver extract (Lac toba cil lus casei factor) produced an increase in growth in addition to that produced by the six water-soluble v i t a ­ mins. The addition of crystalline pteroylglutamic acid did not produce a growth response. Therefore the A B liver extract contained some factor essential of pigs other than the Lactobacillus Rickes et_ a l . (1948a) and Smith for maximum growth casei factor. (1948a) announced almost simultaneously the isolation of the active principal concerned in the treatment of Addison*s pernicious anemia. Rickes et a l » (1948a) proposed the name of vitamin B^g for this new substance. Ott et^ a l . (1948) vitamin B^g possessed APF soon afterward showed that (Animal Protein Factor) activity when added to a 40 to 70 percent soybean oil meal ration for chicks raised from hens fed an all-plant ration. These workers postulated that vitamin B^g might be the same as the APF factor. Lillie et_ a l . (1948) showed that a soybean oil meal type ration supplemented either with vitamin B^g or cow manure gave essentially the same growth response in chicks. These workers assumed then that the active principal in the cow manure factor was vitamin B\2* Linstrom et_ a l . (1949) reported that the addition of 1.0 percent of a vitamin Bpg concentrate gave as good growth in chicks as the addition of 3.0 percent fish solubles. These workers also reported - 14 - that vitamin B^g produced a beneficial effect on the hatchability of eggs produced by hens being fed rations containing soybean oil meal as the sole source of protein. Hale and Lyman (1949) reported that the addition of a vitamin B^g concentrate to a basal ration composed mainly of corn and soybean oil meal increased growth gains in pigs 31 percent over the unsupplemented group. The vitamin B^g supplement also increased efficiency of feed utilization. Neuma nn ejt a l . (1949) showed that the addition of vitamin B 12 to a soybean protein "synthetic milk" improved growth, hematopoesis, and the general well-being of the pigs. addition of a manure factor produced Purified Rations — The similar results. Soybean Type Johnson et_ a l . (1940) reported the use of a purified ration in studying the growth requirements of calves. This ration contained casein and lactalbumin as the sources of protein. When this ration was fed to young calves subnormal growth resulted. The cause of the slow growth was attributed to the calves * lack of appetite for the ration and periods of digestive upsets. Wiese et^ a l . (1947a) and Johnson ej^ a l . (1947) developed a synthetic milk ration which apparently supported normal growth of calves until the calves were 12 weeks of age. These rations contained casein as the source of protein as did the earlier rations of Johnson and associates (1940). - 15 - Wiese et a l . (1947b) produced a riboflavin deficiency and Johnson et a l . (1948) produced a thiamin deficiency in the calf using a synthetic milk developed by Wiese e^ a l . (1947a). Draper and Johnson (1952a) found the quantitative requirement of the calf for riboflavin to be 1.05 micrograms per gram of dry matter using a synthetic milk which contained casein as the source of protein. In addition to these in ­ vestigations the Illinois workers (Wiese et_ al.. , 1946) have found that the calf requires biotin, (Johnson et a l .. 1950b) demonstrated that pyridoxine is required by the calf and (Johnson et a l . , 1947) showed that pantothenic acid is required by the calf. these The synthetic milks used in all of studies consisted essentially of c e r e l o s e , casein, lard, minerals, vitamins, and water. Flipse et_ a l . (1948) confirmed the earlier work of 'Wiese (1946) that the new-born calf requires biotin. These workers further showed that an interrelationship exists between biotin and potassium in the treatment of paralysis in calves due to the lack of biotin in the calf's ration. Flipse (1948) com­ pared the relative nutritive value of corn starch, dextrin, and corn sugar as sources of carbohydrates in synthetic milks for calves. Flipse et. al.. (1950a, 1950b) further investigated carbohydrate sources for the calf by studying the separate nutritive values of glucose, starch, corn syrup, and lactose and the influence of lactose on the nutritive value of starch - 16 - and corn syrup in synthetic milks. These workers showed that the inclusion of a small amount of lactose in such rations was very beneficial. It was also shown that starch as the principal source of carbohydrate constituted a poor nutrient for the calf. Glucose and corn syrup yielded fair results but were not as beneficial as lactose in the ration of the new-born calf. All of these investigations by Flipse and Flipse ejt al^. (1948) (1948, 1950a, and 1950b) employed synthetic milks of which the protein source was crude casein. Neum ann et^ a l . (1948) developed a synthetic milk for baby pigs containing an isolated protein of the soybean, "alpha” protein, which was shown to be deficient in vitamin Big for the baby pig. The addition to the ration of an antipernicious anemia liver extract produced a growth r e s ­ ponse. Joh nson and Neumann (1949) later showed that vitamin and an antipernicious anemia liver extract produced the same growth response in pigs maintained on the vitamin deficient ration. the symptoms of vitamin Neuma nn et al. "alpha" protein, (1950) described deficiency in the baby pig fed an "alpha" protein synthetic milk. These workers also established the quantitative requirement of the baby pig for vitamin B j_2 when fed as a concentrate. used in these The solids in the synthetic milk studies were composed of 29.4 percent "alpha" protein, 0.6 percent d , 1 - m e t h i o n i n e , 30.9 percent glucose, 30.8 percent lard, and 8.3 percent mineral salts. These - 17 - materials were homogenized with water into a synthetic milk containing 13.0 percent solids and 4.0 percent fat. In addition the ration was supplemented with all the known fat and water-soluble vitamins except vitamin Joh nson (1950) and Nesheim et_ al_. Nesheim and (1950) established the quantitative crystalline vitamin B]_2 requirement of the baby pig. Johnson et a l . (1951) reported the use of an "alpha" protein synthetic milk in producing vitamin B 12 deficiency in the calf. Apparently the composition of the ration was similar to the synthetic-vitamin B^g deficient ration used with pigs as reported by Neumann et^ a l . (1948). Draper ^ejt a l . (1952b) described vitamin B^2 deficiency in the calf utilizing a ration identical to that used by Neumann e^t a l . (1948) in producing vitamin B^g deficiency in the baby pig. For a more comprehensive review of the literature on the general subject of vitamin B^g, Part II of this manuscript. the reader is referred to EXPERIMENTAL PROCEDURE Selection of Animals The animals fed the experimental rations are recorded in Table 1. All of the 14 experimental calves were males with the exception of two females, one each fed Rations 7 and 10. A system of random allotment was used in assigning calves to the rations as the calves were born into the College experi­ mental herd. Calves were also obtained from the main College herd and from a local farmer. These calves followed the same system of allotment to rations as the calves from the College experimental herd. The only prerequisite to assignment was normal health and appearance. Feeding and Management Because of necessity, calves were ment in all seasons of the year. started on the ex per i­ Any possible differences due to prenatal nutrition were minimized by the allotment of calves from the various herds to all of the experimental rations whenever possible. Calves were permitted to remain with their dams for 48 hours following parturition. calf was then placed in an individual pen, hours, weighed, Each fasted for 24 and started on the synthetic milk rations. The calves were fed twice daily by nipple pail at a rate - 19 - TABLE 1 ASSIGNMENT OF ANIMALS Ration No. of calves Breed distribution and herd no. 1 2 C-783 C-784 Ayrshire Holstein 2 2 C-788 C-791 Brown Swiss Holste in 3 1 C-798 Holste in 4 1 C-803 Holstein 5 1 C-809 Hoi stein 6 1 C-810 Hols tei n 7 1 C-814 Holstein (Female) 8 1 C-816 Ayrshire 9 1 C-817 Holstein 10 3 A-95 Holstein (Female) Holstein Hols tei n C-820 C-821 - 20 - calculated to meet the recommended nutrient allowance of the National Research Council (1945)* The constituents of each of the experimental rations fed are listed in Table each calf received 2. In addition to the components listed, (a) at the time it was placed on experiment and at weekly intervals thereafter, a capsule containing 70,000 I. U* of vitamin A (Shark liver oil) and 10,000 I. U. of vitamin D and (viosterol), (b) a daily dosage of 20 milligrams of thiamin hydrochloride, pantothenate, 20 milligrams of calcium 20 milligrams of riboflavin, pyridoxine hydrochloride, 10 milligrams of vitamin K n a p t h o q u i n o n e ), 0*04 milligram of biotin, inositol, and 3 grams of choline chloride* were prepared in a stock solution, under refrigeration, 20 milligrams of (2 methyl 200 milligrams of These vitamins stored in amber glass and administered orally to the calves once daily. The calf pens were bedded with wood shavings. No hay or dry feed was fed* Preparation of Feed The synthetic milk was prepared by a modification of the procedure of Wiese et^ a l . (1947a) with the exception of the rations which contained the 50 percent-protein soybean oil meal. Due to the limited refrigeration facilities available, the synthetic milk was prepared once or twice weekly and stored - 21 - o cr> o p • 1 1 o r-H O o O O O O * « • • • • • 1 1 O C M LO LO O C O C M LO C M 1 1 1 1 P 1 1 C Q a a 3 3 P P ^ C MC M P 3 73 O in LO 00 • 1 CD rH O O O O o o • * ♦ • • • • 1 1 O 1 C M LO LO O M LO C OC C M 1 1 1 H 1 1 1 C Q 3 0 * g 3 o bO 3 to **3 S 3 OJ C M pH *>P CC) 73 P 3 O cd P a* O lO t 00 r• CM O O O O O O • • • • • • • 1 1 O 1 «■ lO C M LO LO o 00 C M LO C M 1 1 1 C Q C MC M P • 1 c- 1 co o o O O o o ............................................. 1 • • 1 1 1 O 1 I C M M LO 00 C LO LO o 'r7> G ■rl ft P cd*o G C M o 1 P 0 1 C Q C MC M P 3 o • • o 73 LO LO ■rl O 1 1 1 /-N I p 73 73 75 73 G 3 3 3 3 a a 3 3 3 cd 3 O P a )c d< h 01 .c < D — ' 0,00 3 O • P 03 C T > X — s LO 4-3 C J CO 1 1 a> LO O p G 3 lo in o • oo o 1 1 1 C M LO 1 1 1 r-H C Q C MC M P 3 W a. > — / LO o o • 1 o 1 P 1 1 LO • 1 o • 1 • o • o • lO LO o C M cd O • • O O • • 00 cm in C M 1 1 1 1 1 1 rH (Q LO O • • 1 1 O O O O O O • • • • • in in o oo C M LO C M 1 1 1 1 I rH CQ 1 lo 1 1 o • 1 o * in C M O O O o o • • • • • lo o C O Ch co P a p 3 o P O 3 P co C M LO 1 P 01 1 o I 3 3 P a 3 O o P > o o ft ft 3 3 P P P P 3 3 a a >> ft ft 73 73 P p • O O a 3 a 3 ft ft a fc tO bO 3 o P co O - O o O P C O 3 - p C D O P ft ft O 3 3 C MC M P o •<— i P LO P • • 1 OQ o a^ O OF • O C O P cd 3 C MC M p LO 1 to U G O Q« JC j P (D O -h cS C MC M P C M COMPONENTS p O H C O 3 ft 3 P 3 x! O p RATIONS FED o O LO o o o • • • • * t C M O O • • 3 G bO P 3 o p r — 1 ■ fH P O O O p 3 g LO • C M LO LO O O O O O in in • • • • • • • • • 1 1 1 1 O to O’ o O 00 C M tO O H i— 1 • 1 O 1 o o o 73 3 P 1 in in o o o O O in 1 • • • • • • • ■ 1 1 o o> o to P o oo cm in 73 o JG p 1 1 1 C D 1 1 1 C O Q-,-G C M P O 3 o 0 a 3 3 3 3 x> >3 o 3 G *fH 3 P 3 C 3 c c 3 P 3 P C 3 P 73 3 G P 3 P O tio c p p 3 3 s *1 -1 o lO ft CL, s JG C M ft -r-1 3 3 3 XI o p o 3 JG o P 3 >> 3 3 3 3 3 S O Oh-3 P 3 < ft O 1 P a. ' ss O 3 - P -d O i— I P p 3 3 ft P P 3 73 ft ft O O 3 3 O o o O G p ■rl 3 J n 4 3 G a 3 Oh a p ft C M P 3 P p 3 p 3 ■rH 3 ,p 3 p ft >i > ft £** 3 Cft P 3 P G ai a 3 P •rH P i> > p O 3 P P 3 0) P P o O C 3 3 ft 3 s a 3 a 3 3 3 ft ft bD bO O o 3 ft ft 3 O O a P O P Qu, 3 P 3 o P Oh LO 3 3 P 3 X 3 3 73 3 a p i—i a G •rl p 3 P G 3 J>5P CO P 3 a* 3 LO 3 O 3 CQ 73 3 3 C >»p Oh O 3 P 3 C o a 3 rH C Q 3 3 C M C Q •,—1 ft P to C M i— 1 O o a 3 p C 3 Pu P o G P O p ft ft O 3 Oh a x: 01 & LO O, o Oh O a ft 3 O. a 3 o ft 3 ft P lO • ft 3 p (P C M r*H 3 a • 3 01 3 ft 3 -a t}0'H 73 o P —t G 73 r—I P r 3 X jG C O O o 3 O S -i—I Oh Oh 3 3 p a ■H a 3 ft P o 00 ft D O o 00 G - 22 - as a liquid concentrate. The frequency of preparation d e ­ pended upon the number of calves on trial at any particular time. Rations 1 and 2 were prepared as follows: 81 grams of sodium bicarbonate and 27 grams of calcium hydroxide were dissolved in 39.6 pounds of water at about 60° Centigrade. A heavy duty electric stirrer was used and 6.2 pounds of soybean oil meal and 40 grams of d ,1-methionine were added with constant agitation. Stirring was continued for 15 to 20 minutes to insure complete mixing. Near the end of the agitation period 1.4 pounds of lard and 164 grams of lecithin were added to the solution. ration, The other ingredients of the 3.5 pounds of glucose, 1.8 pounds of lactose, 3.6 pounds of corn syrup, and 0.8 pounds of salts, were added to the soybean oil meal-lard suspension and agitation was continued until complete mixing occurred. Since the particle size of this solution would not permit homogenization an attempt was made to reduce the particle size of the mixture by agitating it in a Waring Blendor for a period of 2 to 3 minutes. Rations 3 to 10 were prepared as follows: of 4.5 pounds "alpha" protein were washed with tap water twice to remove an anti-vitamin of thiamin. Hot water (60°C.) was then added to bring the water content of the milk up to the required amount. The other ingredients were then added as previously described. The entire liquid concentrate was then homogenized at 3,000 pounds pressure. The synthetic milks thus prepared, dry matter and were contained 26.3 percent stored under refrigeration in this form. At feeding time one part of the concentrate was added to about 1 1/2 parts of hot water and the milk was fed at 85° to 95° Fahrenheit. Criteria for Evaluation of the Nutritive Value of the Rations Evaluation of the response of the calves to the expe ri­ mental rations was based upon daily observations on the health and general appearance, growth rate, blood analyses, and post­ mortem examinations. Health and general a p p e a r a n c e . Observations were made at least once per day with regard to the general appetite, condition, consistency of the feces, and the general reaction of the animals. Growth r a t e . was maintained. 11;00 A.M. A record of feed consumption and refusal Each calf was weighed between the hours of and 1:00 P.M. on the day it was placed on expe ri­ ment and on the third, twenty-first, trial. seventh, twenty-fourth, tenth, fourteenth, seventeenth, and twenty-eighth days of the Feed adjustments were made once per week according to the changes in body weight. - 24 - Blood a n a l y s e s . Two test tubes of blood were collected from the jugular vein of each calf once weekly. One tube contained lithium citrate and the other contained potassium oxalate as anticoagulant* volume Determinations for red blood cell (hematocrit) by the method of Wintrobe and hemoglobin by the method of Sanford the whole blood. (1933) were made on The blood plasma was analyzed for calcium by the method of Shohl (1922), inorganic phosphorus by the method of Fiske and SubbaRow of Briggs (1942, p. 201), (1925), magnesium by the method (1922) as modified by Duncan et a l ♦ (1935), and ascorbic acid by the method of Mi ndl in and Butler Post-mortem ex ami n a t i o n s . (1938). All animals which died during the trial or were killed at the end of the experimental period were subjected to gross post-mortem examinations. Histological sections were made of selected organs from representative animals and of any organ or tissue which appeared abnormal on gross inspection. RESULTS The growth response of the calves to the 10 experimental rations is shown in Table 3. As is shown in Table 3 the growth rate of the calves fed Ration 1 was entirely unsati s­ factory. The growth rates of calves fed Rations 2, 3, 4, 6, and 9 might be considered normal for calves fed synthetic milk rations. TABLE 3 GROWTH RESPONSE OF CALVES FED THE VARIOUS EXPERIMENTAL RATIONS Rati on Average starti ng wei ght lb. 1 2 3 4 5 6 7 8 9 10 93.0 98.5 115.0 102.0 87.0 100.5 84.0 90.5 86.0 92.5 Average gain over 28 day period Average daily gain lb. lb. -1.7 -0.06 0.64 18.0 0.79 22.0 0.75 21.0 0.09 2.5 19.0 0.68 10.5 0.38 Died at 13 days of age 0.71 20.0 7.2 0.26 Increase over start­ ing weight % -1.80 18.27 19.13 20.58 2. 87 18.91 12.50 23.26 7.78 The calves fed the vitamin B^2 deficient ration, Rati on 1 did not increase respects. in weight but appeared to be normal in other As is shown in Table 3 the addition of an APF suppl - 26 - ment and streptomycin to Rat ion 1 caused a marked increase the growth rate of the calves fed Rat io n 2. and general in The growth rate appearance of the calves fed Ration 3 indicated that the 50 percent protein soybean oil meal could be satis­ factorily replaced by "alpha" protein as a source of protein in the ration of the dairy calf. The growth rate of the calf fed Rat ion 4 showed that the observed increase in the growth rate of the calf fed Ra tion 3 was due partly, if not entirely, to the vitamin B^g content of the AP F supplement. As was shown by the growth rate and general appearance of the calf fed Ration 5 the basal ration did not appear to be deficient in d,1-methionine. The basal ration did not appear to be deficient in any unidentified factor that might be supplied by crude casein as is shown by the growth data of the calf fed Ration 6. The intramuscular injection of crystalline vitamin B^g appeared to depress the growth rate of the calf fed Ration 7. The growth rate and general appearance of the calves fed Rations 8, 9, and 10 indicated that d ,1-methionine was not depressing the growth rate of the calves during the early days of life. The average blood analyses of the calves which were fed the different experimental rations are shown in Table 4. The blood data for all of the experimental calves appeared to be within normal limits. - 27 - TABLE 4 AVERA GE BLOOD ANALYSIS OF THE CALVES BY RATIONS Ration 1 2 3 4 5 6 7 8 9 10 Hemo­ globin RBCV Ca Inorg. P mg. ^ mg. % 6.83 7.41 7.95 6.89 6.47 6.94 6.96 8.83 6.68 7.29 2.33 2.47 2. 82 2.12 2.27 2.13 2.28 3.27 2.39 2.31 gra. % fo mg. % 11.76 13.64 12*28 9.97 11.41 13.58 11.68 10.99 12.46 10.92 27.9 34.5 33.5 26.8 29.9 37.5 31.9 30.5 34.7 29.8 10.3 10.2 9.6 10.0 10.7 10.4 10.1 11.9 10.4 9.9 Mg Ascorbic acid mg. 0.373 0.404 0.303 0.205 0.264 0.162 0.184 0.347 0.376 0.215 DISCUSSION A n inspection of the growth data of the calves which were fed Rat ion 1 as noted in Table 3 indicates that this ration was not suitable for new-born calves* Both calves appeared to be normal and ate well throughout the experimental period* The feces of both calves tended to be slightly loose when compared to feces of calves receiving whole milk* Al­ though the calves fed Ration 1 did not increase in body weight, they exhibited exceptional vigor, dition. appetite, and general con­ Rat ion 1 was nutritionally complete as far as was known with the exception of vitamin B-^g. Rati o n 2 was the same as Rati on 1 with the exception that it was supplemented with 1.5 percent of an APF supplement and 0.1 percent of crystalline streptomycin. The A P F supple­ ment contained 3.5 milligrams of vitamin B-^g per pound of supplement. It should be noted that either the APF supplement or streptomycin produced a sharp increase in the growth rate of the calves. Both of these calves exhibited good growth and condition throughout the 28-day experimental period. These calves gained an average of 0.68 pound per day which was still below the Ragsdale standard (Ragsdale, 1934) but it probably could be considered good growth for calves being fed synthetic-milk rations. - 29 - "Alpha" protein was substituted for the 50 percent p r o ­ tein soybean oil meal in Rations 3 through 10. It was believed that the use of this protein would further identify the nutrients in the synthetic milk rations. Since only 50 percent of the soybean oil meal was protein, it was possible that the other portion of the soybean oil meal could be furnishing a factor or factors required by the young calf. The use of the "alpha" protein enabled the homogenization of the complete ration which improved the physical nature of the milk. It was found that "alpha" protein contained a factor which, when not removed by washing with water, acted as an anti-vitamin of thiamin in the calf. the Calf C-798 was the first calf fed "alpha" protein synthetic milk. The protein was not washed and within three days the calf exhibited typical v i t amin deficiency symptoms of general muscular weakness and head retraction. This calf was given 100 milligrams of thiamin hydrochloride subcutaneously and two grams per day orally for three days. The calf was able to stand within two hours after the thiamine was injected and appeared to be normal within two days. The "alpha" protein was washed twice with tap water throughout the remainder of the trial and there was no further evidence of the anti-vitamin activity of the protein. The sodium content of the unwashed "alpha" protein was determined with the Perkin-Elmer Flame Photometer and was - 30 - found to contain 520 milligrams of sodium per 100 grams of "alp h a ” protein. The excessive quantities of sodium or of a compound containing sodium might have been acting as the anti-vitamin. The feces of calf C-798 and all of the other calves receiving rations with "alpha" protein as the source of protein were very fluid and black in color. This diarrhea did not seem to be infectious or to be injurious to the calves in any manner. Beginn ing wit h the calf fed Ration 4 all of the vitamin Big was supplied by crystalline vitamin rather than by the AP F supplement. The purpose of Ration 4 was to determine whether the increase in growth rate of calves fed Rations 2 and 3 was due to the vitamin B^g content of the APF supplement or the antibiotic streptomycin supplementation of these rations. The growth rate of the calf fed Ration 4 was about the same or slightly faster than the growth rate of the calf fed Rati on 3 which indicated that the vitamin content of the A P F supplement rather than streptomycin was causing the in­ creased growth rate of the calves. Calf C-S03 at the end of the experimental period appeared to be normal in appetite, health, and general condition. The feces of this calf were loose and black in color as were the feces of the calf fed Rati on 3. The average daily gain of Rations 2, 3, and 4 could all be considered normal or slightly below the Ragsdale standard - 31 - (Ragsdale, 1934). of these groups However, an inspection of the weekly gains (Appendix, Table 15) will indicate that the calves gained in weight slightly for the first week on trial, remained constant for about 10 days, and then gained normally or slightly faster than normal for the last 10 to 14 days of the trial. It was believed that possibly the ration lacked some factor(s) which was stored at birth and which was also synthesized by the rumen bacteria after the rumen started functioning at about three to four weeks of age. an attempt to correct this condition calves fed Rations In 5, 6, 7, 8, 9, and 10 were subjected to various treatments. Calves fed Rati on 5 received the basal ration plus one percent d ,1 - m e t h i o n i n e . As is shown in Table 3 the growth rate of the calves fed Ration 5 was very poor which might have indicated that the extra methionine was exhibiting an inhibitory effect on the growth rate of the calf. At the end of the experimental period Calf C-809 exhibited very poor condition and had very severe diarrhea. It was established wit h this ration that the basal ration was not deficient in d ,1- m e t hionine• Rati on 6 (Calf C-810) was the same as the basal ration with the exception that five percent of the "alpha” protein was replaced by five percent of crude casein. It was thought that this amount of crude casein would be enough to supply any unidentified factors without changing the amino acid - 32 - balance of the ration significantly. However, the growth curve of the calf fed this ration was very similar to those of calves fed rations previously discussed and it was con­ cluded that the casein did not supply the calf with any unidentified factors not contained in "alpha" protein. Ration 7 was devised to test the affect of adding additional vitamin B^g to the ration. For this reason calf C-814 received daily intramuscular injections of 80 m i c r o ­ grams of vitamin B^g per kilogram of solids consumed. Nesheim and Johnson (1950) showed that swine utilize vita- min B^g about 50 percent more efficiently when it was in­ jected intramuscularly than when administered orally. As is shown in Table 3 the average daily gain of this calf was not as great as the daily gains of Rations 2, 3, 4, or 6. After about two weeks of the injections the calf became very irritable and nervous. Toward the end of the ex peri ­ mental period this condition improved but the animal exhibited a rough hair coat and poor general condition. Un fort unately only one calf was fed this ration and it c a n ­ not be stated with certainty that the growth rate of this calf would be typical of the reaction to this ration. ever, How­ since the extra vitamin B^g did not produce a growth stimulus it was assumed that the lag in growth between the seventh and eighteenth days was not due to the lack of vitam in B]_g. - 33 - Since the supplementation of Ration 5 with 0.5 percent of extra d,1-methionine apparently produced a depression of growth it was believed that d ,1-methionine might be inhibiting growth during the early stages of life. Rations Because of this, 8, 9, and 10 contained 0.25, 0.15, and 0.10 percent d , 1 - m e t h i o n i n e , respectively, replacing the original 0.5 percent. Calf C-816, Ration 8, was fed a ration containing 0.25 percent d,1-methionine. This calf grew normally for the first seven days of the experimental period, became very weak, min thin, and drowsy. but on the eighth day The calf received v i t a ­ therapy, but failed to respond and finally died on the ninth day of the experimental period. Post-mortem examination revealed a patent foramen ovale and although the ductus arteriosus was somewhat constricted it was still patent. The liver had a slight orange tinge and the contents of the gall bladder were very thick and cohesive. The death of the animal was attributed to circulatory disturbances probably not produced by the ration. Ration 9 contained 0.15 percent d ,1 - m e t h i o n i n e . Inspec­ tion of the growth data in Table 3 indicates that the calf grew at about the same rate as the calves which received 0.5 percent d,1-methionine in their rations. However, this calf was maintained on the ration six weeks instead of four weeks to follow the growth rate beyond the regular experimental period. The growth of the calf from the fourth to the sixth - 34 - week was rather poor which might have indicated that the body stores of methionine were depleted at about four weeks of age and that the ration did not contain enough methionine to meet the calf's requirement for that amino acid. The calves fed Ration 10 were supplied with an even smaller amount of d , 1 - m e t h i o n i n e , 0.10 percent of the ration, for the first two weeks of the experimental period. At the end of the two week period it became evident that the ration did not contain sufficient methionine because the calves lost an average of approximately 10 pounds in body weight during this period. After two weeks on this ration the d,1-methionine content of the ration was increased from 0.10 percent to 0.5 percent of the ration. The calves began to increase in body weight and to improve in health and general condition. During the next four weeks the calves gained about 0.75 pounds per day. Based upon the results of Rations 9 and 10 it was evident that the calf's requirement for d,1-methionine under the conditions of this experiment was more than 0.15 percent of the rat io n and an allowance of 0.5 percent of the ration was used in additional research reported in Part II of this m a n u ­ script . The lag in the growth curve during the first ten days of life of calves fed the was still unexplained. "al pha ” protein-synthetic ration However, calves were weighed on the first, beginning with Ration 5 the second, fourth, seventh, - 35 - tenth, and fourteenth days of the experimental period* An examination of the growth data for calves fed these rations (Appendix--Table 15) indicates that the calves consistently weighed four to five pounds more on the second day than on the first day of experiment. These calves made very little gain for the first 14 days of the experimental period, but thereafter gained normally when the ration was nutritionally adequate* Most of the increase in weight was probably due to increased fill rather than growth since the calves were fasted 24 hours before being weighed and placed on the experi mental rations. Assuming this to be true it appeared that the calves increased in weight very little, if any, during the first 14 days on trial but gained normally thereafter. A n inspection of the blood data in Table 4 indicates no significant differences among the various experimental ratio n s . Since Ra tion 1 did not promote growth, but normal growth was promoted when this ration was supplemented with either an A P F supplement or crystalline vitamin Bqg in the presence of adequate d ,1 - m e t h i o n i n e , it was considered to be vitamin b 12 deficient. Therefore, it was postulated that this ration would be suitable to establish the crystalline vitamin B^g requirements of the young dairy calf. SUMMARY Fourteen new-born dairy calves were allotted to ten experimental rations and fed various synthetic milks u t i l i z ­ ing a 50 percent protein soybean oil meal or "alpha” protein as the source of protein in these rations. The calves were placed on these experimental rations at 3 days of age and retained for a 28-day feeding trial. These rations supported life when supplemented with d ,1 - m e t h i o n i n e , but did not promote growth. The supplementation of these rations with either an A P F supplement containing vitamin B^g or crystalline vitamin B-^g promoted sub-normal to normal growth of the calves. The predominant symptom of vitamin B^g deficiency in the dairy calf appeared to be the lack of growth. The lack of vitamin B^g had no effect on hemoglobin content or red blood cell volume of whole blood or the calcium, phosphorus, inorganic magnesium or ascorbic acid contents of the blood plasma. The d,1-methionine requirement of the dairy calf under the conditions of this experiment appeared to be more than 0.15 percent, but less than 1.0 percent of the ration. An allowance of 0.5 percent of the ration appeared to be suffi­ cient to promote normal growth and general health of the dairy calf when the ration was supplemented with 80 m i c r o ­ grams of vitamin B ^ Z P er kilogram of solids consumed. - 37 - Since crystalline vitamin B^g supplementation promoted good growth of calves fed the vitamin B^g deficient rations it was postulated that these rations could be used in estab­ lishing the crystalline vitamin B^g requirements of the young dairy calf* PART II THE CRYSTALLINE VITAMIN B lg REQUIREMENT OF THE YOUNG DAIRY CALF REVIEW OF LITERATURE Isolation and Characterization of Vitamin EL Q J.& Whipple and Robscheit-Robbins (1925) reported the deve lop ­ ment of an experimental anemia in dogs which was curable by the feeding of beef liver. Mur phy This work inspired Minot and (1926) to test such a diet on the treatment of patients suffering from Addiso n*s pernicious anemia, a disease des­ cribed by Addison (1849) more than 70 years previously. These workers treated 45 patients with a special diet high in pro­ tein, iron, and liver. All of the patients showed a decrease in severity of the pernicious anemia symptoms. workers later These same (1927) showed that liver itself was the active principal in the diets. Mammalian liver (200 grams per day) was given in 105 cases of pernicious anemia for varying lengths of time with beneficial effects. cell count resulted in every case. An increase in red blood Chon et^ a l . (1928) showed that the active pernicious anemia principal in liver could be extracted with water, preferably at a faintly acid pH and that purification from proteins and other substances could be brought about by adding ethanol up to a concentration of 70 percent. This procedure resulted in " C h o n ^ fraction G" which was active in pernicious anemia patients when injected. These early findings led to the inevitable conclusion that an active anti-pernicious anemia factor existed in liver. - 40 - Since 1926 numerous chemists, (1936) and SubbaRow and Jacobson including Dakin et a l . (1936), have attempted to isolate and identify the active principal in liver responsible for the cure of pernicious anemia* Castle and Townsend (1929) set forth their well-known theory of the ’’intri nsi c" and "extrinsic" factors concerned in pernicious anemia. workers proposed that these factors, These one present in normal gastric secretions and the other in certain foods, of which liver was an outstanding example, interacted to form a sub­ stance which was essential for the development of mature eryth r o c y t e s ♦ Castle et_ a l . (1944) showed that crude casein was a carrier of the "extrinsic" factor. Extraction of casein with hot alcohol removed the factor. The alcohol extracted casein plus all the members of the vitamin Bcomplex did not reconstitute the in the crude casein. that the "extrinsic" factor found These workers at this time proposed "extrinsic" factor was a thermostable component of the vitamin B-complex as yet unidentified. Shorb (1947) reported that Lactobacillus lactis Dorner (ATCC 8000) failed to grow in an amino acid medium containing all the synthetic B-complex vitamins, or when supplied with either clarified tomato juice or certain liver extracts. But growth did occur when tomato juice plus the liver ex­ tracts were added to the media. The liver factor (LLD) was found in high concentrations in refined liver extracts but - 41 - low ill such products as Wilson Liver fraction L, brewer*s yeast, tomato juice, and yeast. It was also reported that crude casein was inactive in this regard. Shorb suggested that the L L D factor might be related to the animal growth factor. In the meantime investigators in the animal nutrition field were accumulating evidence for the existence of a factor or factors closely associated with animal proteins which was needed for reproduction and growth of rats, pigs, and chickens. Byerly ejt al^. (1937) reported that there was a seasonal varia tio n in the hatchability of eggs produced by hens fed an all-plant ration. The hatchability dropped to a rather low value in the winter months and increased as the warmer seasons approached. Bird and Marvel (1943) ob­ served that if hens on a low riboflavin ration were fed the feces of a riboflavin supplemented group the hatchability of the eggs produced by the low riboflavin group increased. Hammond (1942) reported that dried cow manure added to an all-plant riboflavin-deficient ration increased the growth of chicks. Heuser e_t a l . (1946) showed that the inclusion of three percent fis h meal into an all-plant ration increased the growth response to soybean oil meal. Bird and associates (1946) observed that the hatchability of eggs produced by soybean oil meal fed hens was lower than eggs produced by hens fed sardine meal. This condition was corrected by the - 42 - inclusion of five percent cow (or steer) manure, 10 percent sardine meal or 10 percent dried milk in the ration. workers found kidney damage, These uratic deposits in the ureters, and distended gall bladders on post-mortem examination. Rubin and Bird (1946a) stated in their first report of a chick growth factor in cow manure that the factor was not identical with any of the previously reported growth factors such as the folic acid complex. It was shown that folic acid prevented anemia in the chicks but did not promote good growth, whereas the cow manure factor did promote good growth. These workers also showed that solubilized liver was a good source of the factor but butyl fermentation solubles were not. R u bin and Bird (1946b) showed that the cow manure factor was stable to heat in the dry state at 100° Centigrade for one hour, would not dialyze through cellophane, was moderately soluble in water and ethyl alcohol, ether and chloroform. workers but was insoluble in It was shown further by these same (1947) that the factor was transmitted from the hen to the chick through the egg. The egg yolk and was acetone insoluble. showed that the cow manure factor was present in the Rubin et a l . (1947) factor improved hatchability as well as growth of chicks produced from hens fed an all-plant ration. These workers expressed the belief that the same dietary factor in cow manure affected both hatchability and growth. It was thought that fish meal contained the same - 43 - factor as did cow manure. Bird et_ a l . (1948) further chemi­ cally characterized the chick growth factor in cow manure. It was found that it was soluble in water at pH 3.0 if the protein was previously removed, soluble in 80 percent acetone and was extractable by aramoniacal ethanol. The factor was stable when autoclaved for two hours in neutral solutions but was destroyed by autoclaving for one hour with 2N acid. There was some evidence of destruction by standing in slightly alkaline solutions. McGinnis et_ al^, (1947) showed that an unidentified chick growth factor was produced in the feces of hens after the feces were incubated for 72 hours at 30° Centigrade. However, if the feces were frozen immediately after excre­ tion there was little or none of the factor present. These results indicated that the production of this factor in the feces of hens occurred after the feces were voided by the hen and not to any extent in the digestive tract. and Carver McGinnis (1947) showed that chicks hatched from hens fed a ration low in the growth factor and the chicks themselves fed the ration grew poorly and showed excessively high mortality. When this ration was supplemented with fish meal, growth was promoted and the mortality was prevented. Evidence was also presented which showed that the factor was trans­ mitted from the hen to the egg, thus confirming the earlier work of R u b i n and Bird (1947). The feeding of degydrated alfalfa did not permit the storage of the unidentified factor or factors in the egg. Cary et a l . (1946) reported the existence of an uniden­ tified factor (X) necessary for rat growth when the rats were fed a rati on containing soybean oil meal as the sole source of protein. Crude and Labco casein and liver contained v a r y ­ ing amounts of the factor. However, the factor could be re­ moved from casein by extraction with hot alcohol. and Cary Ha rtman (1946) reported that milk, cheese, beef and pork muscle, and egg yolks contained Cary's unidentified factor (X). It was also found that the potency of egg yolks seemed to vary with the ration of the hen. reported that the unidentified factor Hartman e^t a l . (194 9b) (X) for rats might be synthesized by the microorganisms of the digestive the rat. tract of These workers found that the addition of a single dose of rat feces to rats being fed a ration deficient in the unidentified nutritional factor (X) promoted growth, thus indicating intestinal synthesis. Zucker et^ a l . (1948) reported that rats required a nutritional factor which was associated with animal proteins. These workers called the factor zoopherin and stated that it appeared to be very similar to the nutritional factor of Cary and the cow manure factor of Rubin and Bird. Zucker and Zucker (X) Later (1948a) found that alfalfa leaf meal, dried grass, and young fresh grass did not contain animal protein - 45 - factor activity. These same workers (1948b) also reported that zoopherin was widely distributed among various lower animal forms. Krider et_ a l . (1948) found that the addition of six crystalline B vitamins to a basal ration of corn and soybean oil meal for pigs increased survival, cell counts, and hemoglobin values. of 1.5 percent of an AB liver extract factor) increased growth even more. the increase growth rate, However, red blood the addition (Lactobacillus casei These workers attributed in growth to some factor other than the L a c t o ­ bacillus casei factor in the liver extract because crystalline folic acid did not produce the growth response. Schaefer et_ al^. (1948b) reported that fox require an unidentified factor essential for growth and hemoglobin pro­ duction. These workers (1948a) also reported that mink required an unidentified factor for normal nutrition. Fresh raw liver and whole milk corrected the deficiency in both fox and mink. On April 16, 1948 Rickes ejb a l . (1948a) announced the isolation in minute amounts of a biologically active, pure, crystalline material clinically active in the treatment of pernicious anemia from a clinically active liver concentrate. This material which crystallized in the form of small red needles was tentatively named vitamin B\2* On April 24, 1948, just eight days after the announcement by Rickes et al. (1948a), - 46 - Smith (1948a) reported the purification of two amorphous forms of the anti-pernicious anemia factor from liver. While this material was not as pure as the crystalline material of Rickes et a l . (1948a) it exhibited many of the same physical and chemical properties. Smith used four tons of ox liver to isolate about one gram of the active pernicious anemia p r i n ­ ciple. Smith et al. (1948) later stated that Smith's (1948a) original product was the same as the one described by Rickes et a l . (1948a) and also suggested the name, vitamin B^g. Shorb (1948) working in conjunction with Rickes et^ a l . showed that vitamin was either wholly or partly re s­ ponsible for the LLD growth activity observed for liver ex­ tracts. Rickes et_ al_. (1948b) reported that vitamin contained cobalt and that the cobalt-complex nature of vitamin B ± 2 was an outstanding property of the new vitamin. was confirmed by Smith This finding (1948b) and it was shown that about four percent of the vitamin molecule was cobalt. stated that if each molecule of vitamin Smith also contained one atom of cobalt that the minimum molecular weight of vitamin B 1 g would be about 1600. Smith found the molecular weight to be 1550-1750 by X-ray crystallography and about 3,000 by diffusion methods. It was also found that the molecule con­ tained three atoms of phosphorus. ported that vit ami n Rickes et a l . (1948c) r e ­ was obtainable from a new source, - 47 - Streptomyces griseus fermentation* liver it contained cobalt, Like vitamin B^g from phosphorus, had comparable activity for growth of Lactobacillus l a c t i s . and had APF activity for chicks# Brink et a l # (1949) found that each molecule of vitamin B-^g contained one atom of cobalt, one atom of phos­ phorus, and was l e v o r otatory. The molecule was not a peptide since hydrolysis of the vitamin did not liberate alpha amino acids# Alkali fusion of vitamin B^g indicated the presence of certain cyclic five membered nitrogen-containing compounds including pyrrols# Other forms of vitamin B^g have been isolated# Kaczka et a l * (1949) demonstrated the existence of vitamin B^ga and Lick t man at a l * (1949), the existence of vitamin B^g^# How­ ever, Kaczka et a l * (1951) demonstrated that vitamin B^ga and vitamin B^g^ were identical and contained a hydroxyl group in place of a cyanide radical in the vitamin Big m o l e ­ cule# These workers suggested the name of cyanocobalamin for vitamin B^g and hydroxocobalamin for vitamins B^ga and B 12b* Buchanan at aJU (1950) isolated vitamin B-^2c from culture broths of Streptomyces grieseus* These workers showed that vitamin B^ga , B-^g^, and B^gc were equally as active as vitamin B^g when measured by animal growth response. Smith (1951) found that vitamin B 12c differed from vitamin Big only in having a nitrite group in place of the cyano group. Smith also showed that if the nitrite group was re­ moved from vitamin B i g c that vitamin B i g d was formed. Lewis - 48 - et a l . (1952) showed the presence of another active form of vitamin B^g in rat feces. The compound had growth-promoting properties for Lactobacillus leichmannii and the chick but not for the rat. When inorganic cobalt was fed to rats an increased production of the factor occurred in the intestinal tract of the rat. The new active form of vitamin B^g and vitamin B-^g appeared separate on a paper chromatogram and had a different absorption spectrum. Tove et_ a l . (1950) reported that mink required a methanol soluble factor of the for growth. Fish solubles were a good source factor but dried distillers solubles or dried whey were not. These workers found that vitamin B^g and the methanol soluble factor had many of the same properties, but the methanol soluble factor seemed to contain another factor or factors in addition to vitamin B^g. Groschke et al. (1950b) found that incubated pig manure contained large quantities of vitamin B^g whereas fresh pig manure did not. Miller and Groschke (1950) showed that incubated horse manure was another potent source of vitamin b 12 * Bickoff e^t al,. (1950) showed the existence of vitamin Big like growth factors for Lactobacillus leichmannii alfalfa. in However, when the vitamin B 12 was destroyed by alkali digestion the results showed that 85 percent or more of the original activity was due to factors other than - 49 - vitamin B^g. These factors did not replace vitamin B^g for chick growth. Wright et a l . (1948) showed that thymidine was able to replace vitamin B 12 for Lactobacillus lactis (ATCC 8000). Thymine under the same conditions was inactive. These workers proposed that vitamin B^g acts in a co-enzyme system in carry­ ing out reactions concerned with the conversion of thymine to thymidine because in the presence of thymidine vitamin B^g was no longer required by Lactobacillus l a c t i s . These workers expressed the belief also that one of the troubles in perni­ cious anemia might be the inability to synthesize certain nucleosides. Folic acid was found to increase thymine, thus explaining the value of folic acid in some cases of pernicious anemia. The effects of large amounts of thymine in pernicious anemia might be explained on the same basis. Kitany et^ al. (1949) showed that the desoxyribosides of a number of purines and pyrimidines were found to be active in replacing the vitamin B^g requirement for Lactobacillus leic h m a n n i i ♦ Folkers (1950) demonstrated that 5,6-dimethyl- benzimidazole was a degradation product of vitamin B^g. Ribo­ flavin has a structure similar to this compound. Trenner _et al^. (1950) reported that vitamin B 12 and ascorbic acid were incompatable in the same solution. It was found that when these two vitamins were in solution together that there was some destruction of vitamin B^g activity, par- - 50 - ticularly vitamin B 12a.* ^ competitive antagonist of vitamin B 12 was reported by Beiler et, a l . (1951). The antagonist of vitamin B-±q was produced by treating the vitamin with strong acid with hydrogen peroxide. Indications were that the cyanide- cobalt complex was attacked. Vitamin B^g — Clinical Aspects A ddis onn ian pernicious anemia was first described by Addiso n (1849). It was found that it was a type of anemia characterized by a reduced number of red blood cells of abnormally high hemoglobin content. themselves were large, The red blood cells immature forms. Degeneration of the spinal cord nerve trunks was frequently associated with the disease. Little advancement was made in the treatment of pernicious anemia until Minot and Murphy (1926) reported that large amounts of liver relieved the symptoms. From this time until the isolation of vitamin B^g in 1948 it was mostly a problem of concentration of the active principal in liver as has already been described. Since folic acid was known to be important in red blood cell formation and function some workers thought that the anti-pernicious anemia factor and folic acid might be the same. However, Wilkins on (1946) stated that they were not the same and Spies and Stone (1947) showed that synthetic folic acid neither prevented or relieved subacute combined - 51 - degeneration of the cord in pernicious anemia, whereas certain types of liver extracts did both. West Rickes (1948) showed that the crystalline vitamin B^g of (1948a) produced positive hematological activity in three cases of pernicious anemia# West obtained increases in reticulocyte counts, red blood cell counts, and hemoglobin. Ungl ey (1948) reported similar results utilizing large doses of liver or one of Smith's (1948a) highly purified extracts# Berk et a l # (1948a, 1948b) reported that vitamin B]_g when injected was effective in relieving both the nervous symptoms and the hematological symptoms of pernicious anemia. There were no allergic reactions as had been found when liver ex­ tracts were used, thus showing that vitamin B^g was not the cause of the allergic reactions. One patient had acquired the nervous symptoms while being treated with folic acid. Vitamin B-^g therapy quickly relieved this condition thus show­ ing again the ineffectiveness of folic acid in curing the nervous phase of the disease. Berk et a l , (1948b) showed that vit amin B-^g was actually the "extrinsicw factor con­ cerned with pernicious anemia as had been proposed by Castle (1929) earlier. These workers showed that 5 micrograms of vitamin B 12 given orally had little if any effect on reticulocytosis. However, when the same amount of vitamin B^g was given with 125 to 150 milliliters of normal the reticulocyte rise was then present. dicated that gastric juice gastric juice These results in­ (intrinsic factor) was necessary - 52 - for the optional utilization of the relatively small amount of vi tamin B^g, These workers further suggested that the function of the intrinsic factor of normal gastric juice was to facilitate the absorption by the intestine of vitamin B-^g, rather than to react with the extrinsic factor as hitherto had been assumed. Spies >T0 i —I— ■ o o 03 TO a * 1 C * ,H S X i —11flj|O o —l a) § i bOCQ XI Is <=8 *~D 01 o s a 0 3O S i —I s dt ps 0 30 3 O XI o 03 in PS 03 • G H i —I a p| 0 3 | o 8 °3 >> c in O S J~ v—' P l^ o a o o i—I TO 03 LO O G TO ^ ccS O TO TO b.o rHI031 U oj in a s l 0 3JG 03 t01 T O TO O rH P |jG ta -^ 031 O 0 308 a *H V < a; > p p p j s : 0 30 30 3 • ■ r l« HH C Q T OT OT 3 • • * •b O b Db Ob O£4 p p 0 30 3 • r l* i — f T OT O p 0 3 • H T O • • b Db D • b O \ W • • * *b D b Ob Ob OP S P SP SP S I D O O O • H i n 03 o w • • b Db D P SP S • b O P 3 i ni —i i —1r H r l H i—i o PQ 0* CO CO 13 o ! —1 PS < > TABLE in < 1 3 TO O fit P h P S P-t XI p 5 o = = u o gJ pH o SS e* P #= ^ t pq PS H g CD s 03 u S3 r/ C O *H Pi PS PS >» p •H 4G p & x> O «S S-, ,C a C 3 P PS oH 0 3 I— I CQ p p p p p 0 30 3 0 30 30 30 3 ’ H * H' H* H • Hr l T 3T O, ! * JT OT OT OT O 0 3 • • • • • *03 Ob Db Db D b Ob O£ b M X X X w •w w • •b D • • ♦ • Db Db Db D b Db Dp 3b P S3 3 P S p SP S C O o m •r l0 0^ O C OH O i —1 i —1 S << E-« • G ' O• r l o & j Gto C S p C 0 3 O r l C C S U ~ O T O 0 3 P O 0 3r H • i “ oa j G U= s = n O C Q 0 3 •i— I O 0 3 &t CO • r lS T bo ■rl xl e x . o o T O 0 3 P O 0 3 • * T > ~ = = G 1 —1 r H C C S G O - 74 - by Neumann et a l . (1950), Nesheim and Johnson Nesheim et_ al_. synthetic milk. to the vitamin (1950), and (1950) were made utilizing an "alpha” protein The author was able to find no reference requirement of the calf. experimental procedure Selection of Animals The composition of the experimental groups is shown in Table 6. All of the S3 experimental calves were Holstein males with the exception of one Holstein female in Group II and one Brown Swiss male in Group I. The selection of the experimental animals and their allotment to the five experi­ mental groups was accomplished by the same procedure as was followed in Part I • Calves were placed on experiment three days after birth and retained for a 42 day feeding trial* All calves that died during the experimental period and other selected animals were subjected to post-mortem exami­ nation* TABLE 6 COMPOSITION OF EXPERIMENTAL GROUPS Group No • of animals Breed di stribution 2 Holstein 1 Brown Swiss Average starting weight lbs • 98.0 II 3 3 Holstein 98*0 III 3 3 Holstein 99.0 IV 4 4 Holstein 94.0 V 10 10 Holstein 100.5 - 76 - Feeding and Management The experimental animals were fed and managed essentially the same as described in Part I* The calves were permitted to remain with their dams for 48 hours following parturition, but were not fasted prior to being placed on the experimental rations. The experimental trial period was not started un­ til the calves were three days of age. The calves received two feedings of the synthetic milk to insure the obtaining of the true weight of a calf before starting the experimental period, All the calves were given three intramuscular injections of 500,000 units of procaine penicillin in oil during the first week of the experimental period. These injections were given on the second, fourth, and sixth days after birth. The incidence of pneumonia in the dairy experimental barn had been quite severe just prior to the start of this trial and because of this all calves were treated with penicillin in an attempt to protect the calves during the first few days of life. The formula for the basal ration is noted in Table 7. The five experimental rations are identical with the excep­ tion of the amount of crystalline vitamin varied. which was Groups I, II, III, IV, and V received 0, 10, 20, 40, and 80 micrograras of crystalline vitamin respectively, - 77 - TABLE 7 BASAL RATION % "Alpha" protein^d,1-Methionine Glucose (Cerelose) Corn syrup Lactose Soya lecithin Lard Salts** 29*5 0.5 25.0 20.0 10.0 2.0 8.0 5.0 ^ "Alpha" protein was washed twice with tap water to remove the anti-vitamin of thiamine. ^ Salt mixture was the same as fed in Part I. per kilogram of dry matter consumed. The vitamin was added to the water-soluble vitamins and administered orally from a test tube once each day following the morning feeding. In addition to the fat soluble vitamins fed in Part I, 150 milligrams of mixed tocopherols were given to each calf each week. The calves received the same vitamins of the B-complex and in the same amounts as outlined in Part I with the addi­ tion of 20 milligrams of thiamine hydrochloride and 416 micrograms of crystalline folic acid per day. Preparation of Feed The synthetic milk was prepared as outlined in Part I for the "alpha" protein rations. The synthetic milk contained 26.3 percent dry matter and was fed as previously stated. - 78 - Criteria for Establishing the Crystalline Vitamin B12 Requirement of the Dairy Calf Evaluation of the vitamin B^g requirement of the dairy calf was based upon daily observations on the health and general appearance, utilization, growth rate and efficiency of feed blood analyses, and post-mortem examinations» Health and general appea ranc e. Observations were made at least once per day with regard to the appetite, general condition, consistency of the feces, and the general reaction of the animals. Growth rate and efficiency of feed utiliza tion . An accurate record of the feed consumption and refusal was main ­ tained. Each calf was weighed between the hours of 11:00 A.M. and 1:00 P.M. on the day it was placed on experiment and on the first, first, third, twenty-fourth, seventh, tenth, seventeenth, twenty- twenty -eig hth , thirty-first, thirty- fifth, thirty-eighth, and forty-second days of the experi­ mental period. Feed adjustments were made once per week according to the changes in body weight. Blood a n a l y s e s . The blood was analyzed for the same constituents as outlined in Part I with the exception that red blood cell counts by the method of Wintrobe (1942, p. 211) were made once weekly. Post-mortem examinations. All animals which died during the experimental period or were sacrificed at the end of the - 79 - trial were subjected to gross post-mortem examination* Histological sections were made of selected organs from representative animals and of any organ or tissue which appeared abnormal upon gross inspection* Comparative Nutritive Value of Two Batches of "Alpha" Protein as Determined by Hat Growth The nutritive value of two batches of "alpha" protein was determined by a rat growth study. The purpose of this study is outlined in other parts of this manuscript* The composition of the two experimental groups is shown in Table 8 and the formulae for the rations used are presented in Table 9. The rats were maintained on the experimental rations for six weeks. These animals were housed two per cage and weighed three times weekly* 80 - TABLE 8 COMPOSITION OF EXPERIMENTAL RAT GROUPS Group No. of rats Average starting weight A 4 grams 69.0 B 4 69.0 TABLE 9 FORMULAE OF RAT RATIONS Ration Ingredients B A i % "Alpha" protein^ "Alpha" protein^ d ,1-Methionine Glucose (Cerelose) Lactose Lard Salts3 Cod liver oil Vit amin mixture^ 29.7 --0.3 45.0 10.0 5.0 5.0 5.0 — 29.7 0.3 45.0 10.0 5.0 5.0 5.0 ^ "Alpha" protein from batch used in Ration A was from the same batch as was fed in the first experiment (Part I) and as fed to Groups I, II, III, and IV of this experiment. 2 "Alpha" protein from batch that was fed to Group V (Part II). 3 Salts as listed in Table 2 of Part I. 4 Vitamin mixture composed of 2.0 grams of choline chloride, 56 mg. of calcium pantothenate, 20 mg. of nicotinic acid, 10 mg. of riboflavin, 8 mg. thiamine hydrochloride, 4 mg. pyridoxine hydrochloride, 0.8 mg. vitamin K (2—methyl— n a p t h o q u i n o n e ), and 141 ug. of crystalline vitamin P©r kilogram of ration. RESULTS Tne chemical analyses of the ingredients used in the experimental rations are presented in Table 10 and that of the basal ration (liquid concentrate) in Table 11, TABLE 10 CHEMICAL ANALYSES OF INGREDIENTS OF RATION Ingredients "Alpha «• protein Lactose Cerelose Corn syrup Water Ash i % 9.05 1.53 9.10 26.44 1.57 0.12 0.05 0.81 Crude Ether fiber extract i 0.00 0.00 0.00 0.00 i 0.17 0.58 1.35 2.15 Protein N-free extract % £ 88.50 0 •66 0.31 0.18 TABLE 11 CHEMICAL ANALYSIS OF BASAL RATION Constituent Percent Moi sture Protein Crude fiber Ether extract Ash Nitrogen-free extract 73.69 8.13 0.00 2.75 1.67 13.76 Ca P Mg K Mn (Mg/kg.) Co (Mg/kg.) Fe (Mg/kg.) Cu (Mg/kg.) 0.247 0.220 0.736 0.238 3.44 2.45 86.30 6.15 0.71 97.11 89.19 70.42 - 82 - As is shown in Table 6 the starting weights of Groups I, I I » III, and V were nearly the same. The average starting weight of the calves in Group IV was slightly less than that of the other four groups. The average growth rate and efficiency of feed utiliza­ tion of the five experimental groups are shown in Table 12. • o to o The average daily gains of Groups I , I I , III, and IV were -0.10, 0.20, and 0.65 pound per day, respectively. Incomplete data were obtained on Group V due either to a deficiency or toxic factor existing in the '‘alpha " protein which was fed to this group* This was the only group fed this batch of pro- tein because the calves in this group were started on experi- ment after those in the other four groups had been completed. TABLE 12 GROWTH AN D FEED UTILIZATION Group Average starting weight Average daily gain Increase over Gain/D.M. starting weight consumed I lb. 98.0 lb. -0.10 -9.80 II 98.0 0.20 8.48 0.124 III 99.0 0.20 8.40 0.132 IV 94.0 0.65 28.84 0.399 V 100.0 ib./lb. Incomplete data The growth curves of calves in Groups I, II, III, and IV are presented in Figures I, II, III, and IV, respectively. The average growth curves of all groups are shown in Figure V by 10-day periods. - S3 - C-834 C-835 C- 8 4 1 140 BODY W EIGHT-LBS. 130 20 O 100 DIED 90 80 \ DIED 10 20 DAYS 30 ON EXPERIMENT FIG .X .G R O W TH C U R V E S - NO VITAMIN 40 B , 2 / KG. D. GROUP I M. 50 84 - 4~96 C-832 C -844 140 W EIG H T-LB S. 130 120 BODY 100 90 90 O 10 20 40 30 50 DAYS ON EXPERIMENT FIG. H . GROWTH CURVES - - GROUP 1 0 ^ 0 . VITAMIN B i 2 /K G . D. M. 2 ~ 85 - C- 825 C-837 C-843 140 BODY WEIGHT - LBS. 130 120 - no DIED 100 - 90 80 C lO 20 DAYS 30 ON 40 EXPERIMENT FIG.JK. GROWTH C U R V E S --G R O U P 3 20 a G. VITAMIN B 12/KG.D.M. 50 - 86 C- 828 C *-831 C “847 C —854 BODY W EIGHT-LBS. I4C|- 134I20r no DIED locfcC 80 o 10 20 DAYS 30 ON 40 EXPERIMENT FIG.EC. GROWTH CURVES - - GROUP 4 4 0 ^*G. VITAMIN B |2/fc. Chem. and Ind. 22:426. ± Burns, M. M. , and J. M. McKibbin. 1951. The lipotropic effect of vitamin B 12 in the dog. J. Nutr. 44:487-500. Burnside, J. E. , T. J. Cunha, H. G. A. LaMar, A. M. Pearson, 1950. Response of the pig Expt. Biol, and Med. Proc. Byerly, T. C . , H. 1937. Effects supplements on tion. Poultry M. Edwards, G. B. Meadows, and R. S. Glasscock. to A P F , Bio and Biov,. Soc. 74:173-174. W. Titus, N. R. Ellis, and R. B. Nestles. of light, soybean and other diet seasonal hatchability and egg produc­ Sci. 16:322-330. Carlson, C. W . , R. F. Miller, H. T. Peeler, L. C. Norris, and G. F. Heuser. 1949. The complex nature of the animal protein factor. Poultry Sci. 28:750-752. Cartwright, G. E. , and M. Wintrobe. 1949. Experimental production of a nutritional macrocytic anemia in swine. Fed. Proc. 8:351-352. Carver, J. S., and J. McGinnis. 1950. Effect of vitamin B 12 and supplements on hatchability of chicken eggs. Poultry Sci. 29:752. Cary, C. A., A. M. Hartman, L. P. Dryden, and G. D. Likely. 1946. A n unidentified factor essential for rat growth. Fed. Proc. 5:128. Castle, W. B., J. B. Ross, C. S. Davidson, J. H. Burchenal, H. J. Fox, and T. H. Ham. 1944. Extrinsic factor in pernicious anemia: Ineffectiveness of purified casein and of identified components of the vitamin B complex. Science 100:81-83. - 113 - Castle, W. B., and «V. C. Townsend* 1929* Observations on etiologic relationship of achy1 ia gastrica to perni­ cious anemia: The effect of the administration to patients with pernicious anemia of beef muscle after the incubation with normal human gastric juice. Am. J. Med. Sci. 178:764. C&tron, V* , and C. C. Culbertson. 1949. with A P F • Iowa Farm Sci. 3:3-6. Faster gains Catron, D. V., V. C. Speer, H. M. Maddock, and R. L. Vohs. 1950* Effect of different levels of aureomycin with and without vitamin B-. ? on growing-fattening swine. J. Anim. Sci. 9:652. Charkey, L. W. , H. S. Wilgus, A. R. Patton, and F. X. Gassner. 1950. Vitamin B,g in amino acid metabolism. Soc. Expt. Biol, and led. Proc. 73:21-24. Chow, B. F. 1951. Sequelae to the administrati on of vitamin B^g to humans. J. Nutr. 43:323-343. Chow, B. F . , L. Barrows, and C. Lang. 1950. The micro­ biological activity of vitamin B12 urine of normal rats following the oral and subcutaneous administration of this vitamin. J. Nutr. 42:405-414. Chow, B. F . , L. Barrows, and C. A. Ling. 1951. The di s­ tribution of radioactivity in the organs of the fetus or of young rats born by mothers injected with vitamin b 12 containing C o 6 0 . Arch. Biochem. and Biophys. 34:151-157. Clandinin, D(. R. , W. W. Cravens, C. A. Elvehjem, and J. G. Halpin. 1946. Deficiencies in overheated soybean oil meal. Poultry Sci. 25:399. Clandinin, D. R . , W. W. Cravens, C. A. Elvehjem, and J. G. Halpin. 1947. Deficiencies in overheated soybean oil meal. Poultry Sci. 26:150-156. Clandinin, D. R. , V/. W. Cravens, C. A. Elvehjem, and J. G. Halpin. 1948. The relationship between time and temperature to the nutritive value of soybean oil meal. Poultry Sci. 27:370-371. Cohn, E. J. , G. R. Minot, G. A. Allen, and W. T. Salter. 1928. The nature of the material in liver effective in pernicious anemia. II. J • Biol. Chem. 77:325-358. - 114 - C o l b y , R. W. , and M. E. Ensminger. 1950a. Effect of vitamin B 12 on the growing pig. J. Anim. Sci. 9:90-93. Colby, R. W. , F. A. R a u , and J. R. Couch. 1950b. Effect of feeding an ,fanimal protein factor'1 concentrate to young lambs. Am. J. Physiol. 163:418-421. Collins, R. A., A. E. Harper, M. Sc hreib er, and C. A. Elvehjem. 1951. The folic acid and vitamin Big content of the milk of various species. J. Nutr. 43:313-321. Cooperman, J. M. , B. Tabenkin, and R. Drucker. 1952. Growth response and vitamin B-,g tissue levels in vitamin Big-deficient rats ana chicks fed riboflavin, 5 , 6-dimethylbenzimidazole and related compounds. J. Nutr. 46:467-478. Couch, J. R . , and 0. Olcese. 1950a. The vitamin B content of chick tissues as influenced by the diet. J. Nutr. 42:337-346. Couch, J. R. , 0. Olcese, and H. L. German. 1950b. 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Effect of APF supplement on efficiency of feed utilization for the pig. J- Anim. Sci. 9:615-618. - 115 - Cunha, T. J. , H. H. Hopper, J. E. Burnside, A. M. Pearson, R. S. Glasscock, and A. L. Shealy. 1949b. Effect of vit amin B^g and APF supplements on methionine needs of the pig. Arch. Biochem. 23:510-512. Cuthbertson, W. F. J., and D. N. Thornton. 1951. The effect of dietary lactose on the response of the rat to vitamin B 1 2 . Brit. J. Nutr. 5:xii. Dakin, H. D . , C. C. Ungley, and R. West. 1936. Further observations on the chemical nature of a hematopoeitic substance occurring in liver. J. Biol. Chem. 115: 771-791. Dameshek, W. 1 9 4 9 ......... And now B 1 2 .f Blood 4:76-78. Davis, R. L . , and B. F. Chow. 1951. Content of radioactive vitamin B ^ 2 in the feces of rats fed Co^O and aureomycin. Soc. Expt. Biol, and Med. Proc. 77:218-221. Dedichen, J. , and P. Laland. 1949. Anti-pernicious-anemia factor and white-cell count. Lancet 257:282-283. Dietrich, L. S., W. J. 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The effect of feeding aureomycin on the vitamin B^g requirement of the chick* Arch. Biochem. 29:334-338. Ott, W. H., E. L. Rickes, and T. R. Wood. 1948. Activity of crystalline vitamin B 12 for chick growth. J. Biol. Chem.. 174:1047. Osborne, T. B. , and L. B. Mendel. 1917. as food. J* Biol. Chem* 32:369-387. The use of soybean Palmer, L. S., W. S. Cunningham, and C. H. Eckles. 1930. Normal variations in the inorganic phosphorus of the blood of dairy cattle. J. Dairy Sci. 13:174-195. - 128 - Patel, J. C. 1948. Crystalline anti-pernicious-anemia factor in treatment of two cases of tropical macro­ cytic anemia. Brit. Med. J. 2:934-935. Patrick, H. 1950. Growth.-promoting effect of methionine and vitamin B^g on chicks. Poultry Sci. 24:923-924. Peeler, H. T. , R. F. Miller, C. W. Carlson, L. C. Norris, and G. F. Heuser. 1951. Studies of the effect of vitamin B 12 on hatchability. Poultry Sci. 30:11-17. Petersen, C. F., C. E. Lampman, and A. C. Wiese. 1950a. Role of vitamin and other APF factors for repro­ duction and chick growth. Poultry Sci. 29:775. Petersen, C. F., A. C. Wiese, C. E. Lampman, and R. V. Dahlstrom. 1950b. Role of crystalline vitamin B^2 for hat ch ab il it y. Poultry Sci. 29:618-619. Powick, W. C . , N. R. Ellis, C. N. Dale, and M. R. Zinober. 1951. Effect of nicotinic acid, vitamin B^2 and aureomycin on growth of pigs and on resistance to artificial infection with salmonella choleraesuis. J. Anim. Sci. 10:617-623. Ragsdale, A. C. 1934. Growth standards for dairy cattle. Mo. Agr. Expt. Sta. Bull. 336:1-12. Reece, R, P. 1950. The influence of thyroprotein feeding on gains in body weight of dairy calves. J. Dairy Sci. 33:387. Reed, J. R . , Jr., and J. R. Couch. 1950a. Vitamin B ^ 2 and A P F concentrate in the nutrition of the growing chick. Poultry Sci. 29:776-777. Reed, J. R., Jr., and J. R. Couch. 1950b. The efficacy of different APF concentrates for chicks. Poultry Sci. 29:897-902. Register, U. D. , U. J. Lewis, H. T. Thompson, and C. A. Elvehjem. 1949. Variations in the vitamin B 12 content of selected samples of pork and beef muscle. Soc. Expt. Biol, and Med. Proc. 70:167-168. Register, U. D. , and H. P. Sarett. 1951. Urinary excretion of vitamin B 1 2 , folio aoid, and citrovorum factor in human subjects on various diets. Soc. Expt. Biol, and Med. Proc. 77:837-839. - 129 - Richardson, D . , D. V. Ca,tron, L. A. Underkofler, and H. M. Maddock. 1950* Vitamin B-, p requirement of weanling pigs fed a semi-synthetic ration, J. Anim. Sci. 9 :665-666 . Richardson, D. , D. V. Catron, L. A. Underkofler, H. M. Maddock, and W. C. Friedland. 1951. Vitamin B-. P requirement of male weanling pigs. J. Nutr. 44: 371-381. Richardson, L. R . , and L, G. Blaylock. 1950. Vitamin B 12 and amino acids as supplements to soybean oil meal and cottonseed oil meal for growing chicks. J. Nutr. 40:169-176. Richardson, L. R. , L. G. Blaylock, H. L. German, and R. M. Sherwood. 1949. Amino acids and vitamin Big as supplements to plant proteins for growing chicks. Fed. Proc. 8:393. Richardson, L. R . , P. W. Witten, and J. R. Couch. 1951. Diet of mother and vitamin B 12 content of tissues of infant rats. Soc. Expt. Biol, and Med. Proc. 76: 265-267. Rickes, E. L*, N. G. Brink, F. R. Koniuszy, T. R. Wood, and K. Folkers. 1948a. Crystalline vitamin B 1 2 . Science 107:396-397. Rickes, E. L., N. G. Brink, F. R. Koniuszy, T. R. Wood, and K. Folkers* 1948b. Vitamin A cobalt complex. Science 108:134. Rickes, E. L. , N. G. Brink, F. R. Koniuszy, T. R. Wood, and K. Folkers. 1948c. Comparative data on vitamin b 12 from l*-ver and from a new source, Streptomyces g r i s e u s * Science 108:634-635. Riesen, W. H. , D. R. Clandinin, C. A. Elvehjem, and W. W. Cravens. 1947. Liberation o f essential amino acids from raw, p r o p e r l y - h e a t e d , and over-heated soybean oil meal. ay ex 0 3 5 7 10 12 14 17 19 21 24 26 28 Rat N o . 1 gms. 77.0 74.0 95.0 102.0 103.0 120.0 127.0 142.0 128.0 136.0 152.0 147.0 158.0 2 gms. 74.0 74.0 80.0 88.0 92.0 107.0 112.0 128.0 118.0 128.0 143.0 137.0 152.0 3 gms. 63.0 62.0 65.0 71.0 77.0 80.0 75.0 92.0 98.0 108.0 115.0 123.0 127.0 4 gms. 60.0 66.0 70.0 75.0 81.0 86.0 82.0 106.0 106.0 120.0 128.0 140.0 139.0 - 144 - TABLE 18 (Coat.) Group A (cont.) Days on expt. 31 33 35 38 40 42 Group B 0 3 5 7 10 12 14 17 19 21 24 26 28 31 33 35 38 40 42 Rat No. 1 gras. 172.0 170.0 175.0 173.0 188.0 197.0 2 gms. 169.0 165.0 156.0 145.0 164.0 166.0 3 gms. 137.0 142.0 150,0 141.0 163.0 162.0 4 gms. 152.0 156.0 158.0 144.0 164.0 160.0 5 6 7 8 78.0 82.0 89.0 91.0 99.0 108.0 116.0 114.0 111.0 121.0 127.0 127.0 135.0 132.0 126.0 138.0 139.0 150.0 134.0 75.0 75.0 77.0 80.0 88.0 101.0 108.0 111.0 121.0 129.0 143.0 144.0 150.0 144.0 142.0 152.0 148.0 170.0 138.0 64.0 63.0 62.0 69.0 72.0 78.0 82.0 91.0 101.0 101.0 110.0 114.0 114.0 123.0 130.0 128.0 126.0 136.0 128.0 58.0 61.0 68.0 73.0 67.0 80.0 89.0 99.0 104.0 104.0 112.0 116.0 114.0 126.0 133.0 133,0 139.0 144.0 136.0 - 145 - TABLE 19 WEEKLY BLOOD ANALYSES — Calf no • Week Whole Blood Hb RBCC RBCV loVmr6 --------------------- gm.^ 7.73 8.67 8.90 8.97 9.03 21.0 23.0 23.0 23.0 23.0 % EXPERIMENTAL CALVES Plasma Ca Asc or ­ Inorg. Mg bic acid P mg. % mg. % mg. % mg. % 0.497 6.82 11.3 2.77 0.440 2.72 11.0 7.86 0.488 10.3 6.82 2.03 0.667 9.5 6.82 2.07 0.693 11.2 6.82 2.40 C-783 1 2 3 4 5 C-7 84 1 2 3 4 ---------— 15.50 15.50 14.45 13.95 42.5 43.0 37.5 39.5 0.257 0.192 0.101 0.200 10.5 9.4 9.9 9.5 7.06 7.43 5.85 6.17 2.84 2.07 2.14 2.00 C-7 88 1 2 3 4 —_ ... ... 13.70 13.00 13.70 12.90 34.5 32.0 33.0 32.0 0.515 0.277 0.326 0.320 10.2 9.9 —9.5 8.17 6.82 6.59 6.90 3.03 2.77 2.62 2.82 C-791 1 2 3 4 ... ... ... 13.70 14.37 15.00 12.83 36.0 38.5 36.0 34.0 0.434 0.420 0.371 0.567 10.0 10.4 11.5 10.3 7.64 6.70 9.02 7.43 2.31 2.00 1.86 2.29 C-798 1 2 3 4 ... ... 13.43 11.23 11.67 12.77 38.5 31.5 32.0 32.0 0.294 0.442 0.197 0.279 9.9 9.2 9.6 9.8 9.02 8. 54 6.74 7.52 3.12 1.52 1.95 4.70 mmM --- C-803 1 2 3 4 5 10,93 10.43 10.13 9.40 8.97 29.0 29.5 26.0 25.0 24.5 0.289 0.142 0.184 0.220 0.188 10.4 10.5 9.5 10.0 9.5 7.95 6.59 6.63 7.22 6.06 2.52 1.93 2.08 2.04 2.05 C-809 1 2 3 4 11.60 11.15 11.60 11.30 30.5 30.0 32.0 27.0 0.479 0.212 0.182 0.182 11.9 10.5 9.9 10.4 9.26 5.53 5.03 6 •06 2.44 2.13 2.05 2.44 C-810 1 2 3 4 14.55 13.25 13.25 13.25 38.5 38.5 39.0 34.0 0.126 0.083 0.182 0.255 11.1 10.5 10.0 9.8 6. 82 6.44 7.18 7.31 2.51 2.05 1.80 2.17 - 146 - TABLE 19 (Cont.) Whole Blood RBCC Hb RBCV .% 1 0 b/M M ° gm Asc or ­ bic acid mg. % 0.223 0.186 0.173 0.154 Plasma Ca Inorg. Mg P mg. % mg.$J mg.$? ' 9.6 6.25 2.60 10.4 7.02 1.91 10.3 6.63 2.00 9.9 7.95 2.59 1 2 3 4 12.60 12.20 11.43 10.50 33.5 34.0 31.5 28.5 1 2 10.13 11.05 27.0 34.0 0.712 12.7 9.02 2.52 ------ 13.17 12.70 12.83 12.45 11.15 9.83 9.90 32.5 37.5 37.0 36.0 30.5 26.0 26.5 0.538 0.381 0.208 0.343 0.309 0.328 0.355 11.5 10.0 10.6 10.1 9.9 9.6 9.7 6.06 5.88 6.25 7.02 8.17 6.82 6.90 2.44 2.02 2.00 2.29 3.21 2.75 1.86 3 4 5 6 ... ... ... ... ... ... 10.50 10.93 11.23 11.43 11.23 10.63 28.5 33.5 28.0 29.5 29.5 29.5 0.253 0.328 0.184 0.305 0.216 0.222 11.8 11.4 10.4 9.1 9.8 10.8 7.90 8.04 8.77 8.26 7.95 8.97 2.08 2.59 2.70 2.23 2.14 3.34 1 2 3 4 5 6 ... ... ... ... ... 10.07 11.50 11.30 10.80 9.77 10.20 25.0 33.0 30.5 29.0 27.0 27.0 0.278 0.292 0.225 0.241 0.231 0.193 9.5 9.8 9.2 9.0 9.3 10.1 6.40 6.82 6.51 8.08 6.44 7.68 2.22 2.05 2.22 2.31 2.51 2.34 « •— 10.57 10.93 11.23 10.50 9.77 9.77 29.5 31.5 30.5 29.0 27.0 26.5 0.247 0.206 0.164 0.099 0.161 0.115 9.8 9.6 9.2 9.7 9.2 9.8 7.22 6.21 6.06 7.26 5.60 7.47 2.16 2.00 2.22 2.16 1.93 2.14 13.87 11.67 14.27 13.14 9.99 16.90 16.40 17.00 16.00 15.90 47.0 44.0 47.5 41.0 43.0 0.309 0.182 0.337 0.162 0.054 10.9 8.2 9.2 8. 5 8. 8 8.30 6.25 7.86 7.14 7.73 3.16 2.60 2.14 2.22 1.65 1 2 3 4 5 6 7 1 2 1 2 3 4 5 6 1 2 3 4 5 -------------— ------- ... ... ___ mm - 147 - TABLE 19 1 2 (Coat,) __________ Plasma_____ Whole Blood HBCC Hb RBCV AscorCa Iaorg, ______ _______________ bic acid__________ P 10 6/ M M 3 gm. # % mg, # mg.# mg.# 6.02 8.47 23.0 0.453 9.7 6.82 7.88 8.13 23.5 0.320 10.5 7.64 1 2 3 4 5 6 7 6.47 8.16 7.71 8.05 8.91 7.72 8.32 10.50 10.43 10.50 10.43 10.30 9.77 9.10 25.5 27.5 25.0 26.0 26.0 24.5 25.5 0.604 0.340 0.325 0.208 0.238 0.247 0.246 11.1 9.7 10.4 9.0 9.8 9.7 9.2 5.36 7.03 7.52 6.98 7.02 6.25 6.86 1 2 3 4 5 6 7 --- 11.00 11.60 10.50 12.00 11.60 10.57 12.45 29.0 32.0 30.0 33.0 33.0 29.0 34.0 0.245 0.243 0.220 0.209 0.259 0.258 0.316 11.1 9.6 10.2 10.8 10.3 10.2 9. 8 7.43 6.25 6.06 8.30 7.68 7.86 7.90 9.8 9.4 10.4 9.5 9.6 9.8 7.18 7.18 7.82 6.25 7.73 5.53 7.47 8.87 9.17 9.73 8.33 10.55 1 2 3 4 5 6 12.04 15.00 45.0 12.77 10.55 11.44 11.03 13.70 12.90 14.45 12.27 0.175 0.137 39.5 0.251 36.5 0.179 41.0 0.142 53.0 0.125 1 2 3 4 5 6 7 7.40 9.44 10.04 8.11 7.55 6.33 7.17 7.33 9.33 8.75 8.67 8.53 8.07 8.55 20.5 28.0 26.5 25.5 24.5 23.5 25.5 0.445 0.224 0.222 0.491 0.246 0.179 0.124 11.0 9.8 10.2 9.1 9.4 8.6 9.5 8.49 8.04 8.30 9.73 8.77 6.44 _7.22_ 10.3 11.67 10.63 10.70 9.40 8.90 26.5 29.0 35.5 26.0 24.5 22.5 0.197 0.161 0.167 0.277 0.247 0.137 9.7 9.2 9.6 8.9 9.5 9.4 7.02 5. 88 6.58 6. 66 8.04 5.92 1 2 3 4 5 6 __m 8.77 8.23 8.13 - 148 - TABLE 19 Whole Blood HBCC Hb RBCV 1 2 3 4 5 6/ m m 3 6.70 7.77 7.90 6.71 6.70 7.78 ___________ Plasma_____ AscorCa Inorg. blc acid P mg. % mg. % mg. % 0.262 11.4 9.17 0.233 9.3 7.64 0.144 8.9 5.95 0.212 8.7 6.59 0.221 9.0 7.68 9.5 0.186 6.06 gm. % 9.70 10.07 10.63 10.27 10.87 8.67 26.0 29.0 28.0 25.5 25.0 21.0 7.00 8.00 8.87 7.41 10.26 8.27 9.10 10.57 9.20 10.43 20.5 24.5 28.0 25.0 27.5 0.437 0.337 0.490 0.223 0.381 10.5 10.0 10.5 9.3 9.2 9.07 7.06 7.43 5.85 8.35 --- 10.43 10.50 10.50 29.0 29.0 29.5 10.0 9.9 10.2 9.7 9.6 8.6 7.22 6.63 6.63 8.44 7.43 5.40 io 1 2 3 4 5 6_ (Cont.) % --- --- --- 7.42 6 .61 8. 97 8.00 27.0 24.0 0.228 0.251 0.157 0.173 0.251 0.179 1 2 3 4 5 6 9.11 8.50 7.56 6.25 8.04 5.45 11.15 11.60 10.50 10.13 9.20 9.77 33.5 31.0 29.0 26.5 24.5 25.5 0.247 0.341 0.277 0.197 0.125 0.160 9.5 10.3 10.2 9.0 9.8 9.4 6.82 7.35 7.22 5.71 7.02 6.82 1 2 3 4 5 6 6.07 7.57 6.75 6.96 6.91 6.89 7.50 8.27 7.67 8.75 9.33 8.75 22.5 23.0 21.0 26.0 25.0 25.0 0.230 0.223 0.254 0.297 0.187 0.194 9.7 9.0 9.4 9.0 9.0 9.1 8.77 6.66 7.02 7.06 7.02 6.29 1 2 3 4 5 6.96 8.70 7.58 9.47 9.03 9.77 10.00 9.27 8.33 9.07 27.0 26.5 25.5 26.5 25.5 24.0 28.0 0.253 0.154 0.114 0.223 0.207 0.174 0.269 11.2 9.6 9.2 9.3 9.9 9.2 9.8 7. 82 6.44 6.51 6.74 7.02 6.32 7.57 1 2 3 4 5 6 6 7.44 8.68 _ _ — 7.85 7.71 8.98 - 149 - TABLE 19 (Cont.) Calf no. Week Whole Blood RBCC Hb RBCV 106/MM^ gm. % 9.49 13.10 Plasma Ascor­ Ca Inorg. Mg bic acid P mg. % mg. % mg. % mg. % % 36.0 0.107 9.0 6.44 2.60 C-840 1 C-845 1 7.99 11.60 30.0 0.571 11.5 7.43 2.69 C-S46 1 2 7.95 6.59 12.00 10.87 32.0 31.0 0.355 0.249 11.3 9.6 8.54 5.88 2.70 2.14 C-848 1 2 9.71 9.82 13.7 14.45 35.0 38.0 0.310 0.429 10.7 9.2 6.21 7.64 2.22 3.10 C-849 1 2 9.51 9.57 12.35 12.35 34.0 34.0 0.294 0.311 11.1 9.6 7.77 5.88 2.69 5.08 C-850 1 2 6.42 8.46 9.47 10.13 29.5 34.5 0.310 0.258 9.6 9.4 7.06 6. 82 2.49 2.40 C-851 1 2 6.86 7.43 9.77 10.07 26.5 31.0 0.238 0.250 9.6 9.3 7.02 6.82 2.67 2.07 C-855 1 11.37 15.10 48.0 0.258 10.2 9.02 2.52 C-856 1 9.92 16.30 48.0 0.328 9.1 7.64 2.60 C-858 1 2 7.66 11.65 8.33 12.63 24.5 35.0 0.210 0.458 9. 5 10.0 9.47 12.39 2.13 4.41 C-865 No data C-866 1 2 3 12.10 10.43 10.20 34.5 29.5 29.0 0. 255 0.184 0. 266 9.2 9.6 9.3 7.47 8.26 8.73 2.13 2.14 2.22 C-867 1 13.25 39.0 0.402 8.5 6.44 2.69 C-868 1 12.33 33.5 0.274 9.0 6.82 7.79 « mm« _ ~ ** — --- - 150 - TABLE 20 FEED CONSUMPTION OF THE EXPERIMENTAL RAT GROUPS Week Group A Group B Lot 1 Lot 2 Lot 1 Lot 2 1 2 3 4 5 6 gms * 117.0 156.0 182.0 180.0 177.0 155.0 gms. 100.0 129.0 162.0 176.0 189.0 185.0 gms. 103.0 137.0 143.0 171.0 178.0 158.0 gms • 95.0 116.0 138.0 138.0 136.0 122.0 Total 967.0 941.0 890.0 745.0 4