THE B VITAMIN REQiJIRET’ IENT OP THE BABY PIG I. II. III. RIBOFLAVIN THIAMINE PYRIDOXIHE by Elwyn R. Miller Submitted to the School of Graduate Studies of Michi State University of Agriculture and Applied Scienc In partial fulfillment of the requirements for the degree of DOCTOR OP PHILOSOPHY Department of Animal Husbandry Year Approved 9 • <$. W ^ G ’ 19^6 CD 0 9 AN ABSTRACT Elwyn Miller 'The work presented is an attempt to establish the re­ quirement of the baby pig for riboflavin, thiamine and pyridoxine and to obtain more information regarding the clinical and subclinical changes which occur when these vitamins are deficient in the diet. In all of the experiments the baby pigs were taken from the sow at three to five days of age. The basal diet was a synthetic milk containing If?-1 5 percent solids of which casein composed 30 percent, lard 10 percent, percent and minerals 6 percent. cerelose 514- Adequate supplementation of all the known essential fat soluble and water soluble vitamins other than that vitamin being studied were supplied to the basal diet. Fifty-five baby pigs were used in three trials to de­ termine the riboflavin requirement. Following a depletion- adjustment period on a riboflavin-free, synthetic milk diet the pigs were individually or group fed various levels of riboflavin in the diet. Analysis of the data on individual growth response and dietary intake indicates that the ribo­ flavin requirement of the baby pig for optimum growth and feed efficiency approximates 3 - 0 mg per kilogram of solids In the diet. External, gross and microscopic lesions were present only in those animals receiving less than 2 .0 mg of riboflavin per kilogram of solids. Deficiency symptoms 2 Elwyn Miller could be alleviated by riboflavin supplementation. Fifty-five baby pigs were used in a triplicated experi­ ment to determine the thiamine requirement. Following a de- pletion-adjustment period on a thiamine-free, synthetic milk diet, the pigs were individually fed diets containing 0 , 0 .5 * 1 .0 , 1 . 5 and 2 . 0 mg of thiamine per kilogram of solids. Analysis of the data on individual growth response and diet­ ary intake indicates that the minimum thiamine requirement of the baby pig for optimum growth rate and feed efficiency approximates 1.5 mg per kilogram of dietary solids intake. External, gross and microscopic lesions were present in all pigs receiving less than 1 .0 mg per kilogram of solids. Blood thiamine levels were positively related to dietary thi­ amine intake. G-ood gaining ability was rapidly restored to deficient animals which received thiamine treatment. oixty-five baby pigs were used in three trials to deter­ mine the pyridoxine requirement. An analysis of the growth and feed consumption data indicates that the pyridoxine r e ­ quirement of the baby pig does not exceed 0 . 5 mg per kilo­ gram of dietary solids. However, blood hemoglobin data and urine xanthurenic acid data are presented which indicate that the minimum requirement, when a more nearly total con­ sideration of the pig's well-being is recognized, is not less than 1.0 mg of pyridoxine per kilogram of solids. Class­ ical deficiency symptoms were observed in those pigs receiv­ 3 Elwyn Mi H e r ing no pyridoxine, the performance of paired fed controls in­ dicated that the reduced blood hemoglobin level and the in­ creased urine xanthurenic acid concentration in deficient pigs were due specifically to a lack of pyridoxine and not an effect of inanition. therapeutically treated, pyridoxine- deficient pigs recovered rapidly. THE B VITAMIN REQUIREMENT OF THE BABY PIG I. II. III. RIBOFLAVIN THIAMINE PYRIDOXINE by Elwyn R. Miller Submitted to the Graduate School of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 1956 ProQuest Number: 10008674 All rights reserved INFORMATION TO ALL USERS The quality o f this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008674 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6 Elwyn R. Miller candidate for the degree of Doctor of Philosophy Pinal Examination, May 1$, 1956, 1:15 P.M., 201 Agr*l.' Hall Dissertation: The B Vitamin Requirement of the Baby Pig. I• II. III• Riboflavin Thiamine Pyri doxine Outline of Studies Major subject: Animal Husbandry Minor subjects: Biochemistry, Physiology Biographical Items Born, December 10, 1923, Edon, Ohio Undergraduate Studies, Tri State College Michigan State College, I9I4-6-I948 Graduate Studies, Michigan State University, 1951-1956 Experience: Aviation Electronics Technician, United States Navy, 19l|-5-li-6, Instructor of Vocational Agriculture, W. K. Kellogg High School, 19l|-8-5l* Instructor, Department of Vocational Education, Michigan State College, 1951-52, Graduate Assist­ ant, Department of Animal Husbandry, Michigan State University, 1952-56 Member of Phi Kappa Phi, Society of the Sigma Xi, Kappa Delta Pi, Chi Epsilon, Alpha Zeta, American Society of Animal Production ACKI'IOiVLEDGEMEN TS The author wishes to express his sincere thanks to Dr. J. A. Hoefer and Dr. R. W. Luecke for their constant personal interest and continual guidance. Their encouragement and stimulation like that of Dr. F. Thorp, Jr. before his u n ­ timely death have been invaluable. Thanks are due to Dr. Hoefer and Dr. Luecke also for their valuable suggestions in the preparation of this thesis. The writer is greatly Indebted to Dr. D. A. Schmidt and Dr. R. L. Johnston for their pathological work in this study and gratefully acknowledges their Interest and suggestions. He expresses appreciation to Dr. 3. C. Stothers and Mr. Howard Stowe for their assistance in carrying out the experi­ ments and for their helpful suggestions. Thanks are due to Dr. W. D. Collings for his assistance in the interpretation of the electrocardiographic data. Technicians Mrs. Schwartz and Mrs. Van Lue rendered valuable service in the pathological studies, their -work is greatly appreciated. Sincere thanks are due to Miss Betty Baltzer and Mrs. Kennolly for their assistance with micro­ biological assays and chemical determinations. The author wishes to express deep appreciation for the financial support of Michigan State University through an Assistantship and is grateful to Dr. R. H. Nelson, Dr. B. V. Alfredson, Dr. G. G. Morrill and Dr. E. J. -Miller for the use of the facilities of the Animal husbandry, Physiology, Animal Pathology and Agricultural Chemistry Departments. Above all, the author is indebted to his wife Mildred for her constant interest and encouragement and for her assistance In the preparation of this thesis. Finally, the author is grateful to his three daughters Kim, Pam and Jill who were born during the course of this study for their perserverance. TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF LITERATURE Part I - - - - Riboflavin Part II - - - 'Tliiamine Part III - - - - 5 17 Pyridoxine 31 Part I - - - - Riboflavin 55 Part II 69 EXPERIMENTAL PROCEDURE RESULTS - - - Thiamine Part III - -- Pyridoxine 99 CONCLUSIONS lUj- BIBLIOGRAPHY 116 INTRODUCTION For generations animal husbandmen have been concerned with the proper feeding of farm animals. This concern has become increasingly greater as the science of animal nutri­ tion has advanced and has brought about a demand for a quali­ tative knowledge of all the nutrient elements which are re­ quired by economically Important animal species at all stages of their growth and productive life. Further, a quantitative knowledge of the required nutritional essentials is no less indispensable. Probably in no area of animal nutrition endeavor has the determination of kind and amount of essential nutrients been more difficult than In the acquiring of such Information for the very young of mammals during the suckling period. To ob­ tain such qualitative and quantitative Information the re­ search worker must place the young animals on a practically simulated regimen which will produce normal physical and physiological development. Furthermore it requires the feed­ ing of a diet in which exact amounts of all elementary ingre­ dients are knov/n to the experimenter. Such are the basic problems Involved in the determination of the B-vitamin re­ quirements of the baby pig. An early Impetus to the study was given by WIntrobe (l9 3 9 o.) when he was able with a synthetic diet to obtain posi- tive, although admittedly poor growth in early weaned pigs. The composition of this diet was based on the equivalent values of nutrients in sow’s milk as reported by Newlander and Jones (1935)* Hughes and Hart (1935) and Abderhalden (1 8 9 9 ) together with such pig requirement determinations as were reported previously by Thompson (1932), Newlander and Jones (1935)# Dunlop (1935a*b) Aubel ejb aJL. (193&)* Mitchell et al. (1937), Guilbert et_ al. c) and Hughes (1938). (1937), Chick _et al. (1938a,b, Using this diet Wintrobe was able to de­ termine crude requirements of a few of the B-vitamins for very young pigs. McRoberts and Hogan (I9ljip) obtained somewhat better gains and Anderson and Hogan (19^4-7» 1950) good gains in pigs to wean­ ing age in one experiment but only fair growth rate in two other trials using a simplified synthetic diet. Bustad et a l . (I9 I4-8 ) reported having no success in raising pigs from birth on a synthetic milk diet containing all known vitamins unless they received some colostrum or colostrum substitute. By providing either serum or plasma as a colostrum substitute they were able to obtain survival of the pigs for a maximum of 22 days. A serious diarrhea was present which could not be controlled by any of the then commonly used antibiotics. Green et. al. (19^4-7) made a very practical contribution to the more satisfactory rearing of baby pigs under artificial con ditions by studying the pigs eating habits and providing speci­ fications for equipment and environmental conditions. 3 Johnson <5t ah. (194-^a) then were able to obtain an essentially normal growth rate in pigs to weaning age on a synthetic milk diet similar to that which, they had propared Tor dairy calves and reported by Wiese et, al, (19^4-7) • How­ ever, these pigs remained on the sow Tor a minimum of four days before going on the synthetic diet, Lehrer jet al, (19^9), using a similar diet, successfully raised 2 day old pigs to weaning age. Urinary excretion of B-vitamins indi­ cated that the pigs were receiving adequate amounts of these vitamins. et al. The diets used by Johnson et_ al* (19l4-3a), Wiese (l9l|-7a) and Lehrer _et al. (19^4-9) then became the basis for many experimental and commercial sow milk replacers which have been much used since. The formulation of adequate synthetic milk, baby pig diets have been abetted by the work on older pigs of Cunha and coworkers (19^, 19^-6a,b, 19^7> 19^-3, 19^-9&*b, 1950; Ross et a l . 19^1; Snsminger et. al. 1914-7; Heinemann ejfc aR. 19I4-6 ), Ellis et al . (l9lj-3> 19Mt> VanEtten et ah. I9I4-O, Powick oib al. 19lp7a, b, 19^ 8, Miller and Ellis 1951), Hughes et al. b, 19ip2a,b, c, d, 19^-3) and Luecke et_ ah. (1939, 19^0a, (I9^j-7a, I9I4.8 , 19^-9a, b, 1950a, 1 951, 1 9 5 2 ) together with the studies of Wintrobe et al. (1939b, I9I4.O, 19^4-2 a,b,c, 19^ 3a,b, c, I9I4J-I-, 19I4-6) and the less apparent contribution of many others. With an adequate synthetic milk diet established Johnson ot a l . (1914.8 a,b) then studied pteroylglutamic acid and choline deficiency in the baby pig and Lehrer et ah. (1951, 1952a,b) k and Wiese jet al. (1951 ) produced experimental pyridoxine, pantothenic acid, biotin and riboflavin deficiencies. Nesheim et al. (19I4.8 ) and IJeumann et al# (I9ip9 a*lh 1950 ) determined the requirement of baby pigs for vitamin Bq_2 and. choline. Forbes and Haines (1952 ) reported a study on the riboflavin require­ ment of the baby pig and Stothers (1952, 195^!-* 1955) then made a study of the pantothenic acid and niacin requirements of the baby pig and reported on the pathology of these vitamin deficiencies. The reports of Hiller ejb al. (I95ip» 1955) on the thiamine and riboflavin requirements of the baby pig com­ prise a portion of this thesis. The work to be presented in this thesis is a study of the requirement of the baby pig for thiamine, riboflavin and pyri­ doxine. Various physiological, biochemical and pathological studies were made in addition to collecting growth and diet­ ary intake data in order to provide more criterial bases for determining requirement levels of these B-vitannins. REVIEW OF LITERATURE Part I— Riboflavin After Goldberger (1926) showed that there was.a heat stable water soluble factor, an official committee in England (1 9 2 7 ) named this component (which they assumed to be a sin­ gle substance) vitamin B£. Many workers then started working with this fraction in an attempt to identify it. The first recognized vitamin Isolated from the B 2 com­ ponent was riboflavin. This was accomplished in 1933 when Ruhn, Gydrgy and Wagner-Jauregg showed it 3 growth promoting activity for rats. Warburg and Christian (1932) had dis­ covered the "yellow enzyme,T and had described lumiflavin (a degradation product of riboflavin) which was valuable In the elucidation of the chemical structure of riboflavin by Kuhn and his coworkers. The synthesis of riboflavin was accom­ plished In 1935 hy Kuhn and coworkers in Heidelberg and Karrer and his coworkers in Zdrich. Riboflavin crystallizes into fine orange-yellow needles. Its melting and decomposition point is 278 to 282°C. odorless and has a bitter taste. It is Riboflavin is soluble in water to an extent of 13 mg percent and in ethanol to Ip. 5 mg percent. Its chemical name Is 6 ,7“bimethyl-9” (D,1 1-ribityl) isoalloxazine. Its richest feed sources are dried brewers yeast and dried milk products. 6 Theorell (l93i-j-) demonstrated that Warburg's enzyme con­ tained one molecule of phosphate and Kuhn ot al. (193&) gave proof that the constitution of this prosthetic group was riboflavin-J-phosphate (FIvlK). Warburg and Christian (1938) showed that there was another riboflavin containing co-enzyme, flavin adenine dinucleotide (FAD). The enzymatic phosphorylation of riboflavin by an enzyme in yeast named flavokinasc has been re­ ported by Kearney and knglard (1951). is : RiboflavintATP hl^vpJjina This catalyzed reaction ribof lavin-5-phosphate+ADP. The mechanism of transformation of FMXT to FAD is not known, but it has been shown to occur in human blood cells by Klein, and hohn (l9 lj-0 ). Both FAD and FMN act as prosthetic groups or coenzymes with apoenzymes to form flavoproteins or metalloflavoproteins and as such provide hydrogen transfer in the oxidative systems of the cell. Mahler (1951-1-) has diagrammed their activity as shown below. (other acceptor) ^pyridine nucleotides -►f lavoprotein ---- 1 Substrate--------------------- ». f lavoprot ein->* c y t o c h r o m e s 0 £ _______________________ flavoprotein___________ f Following the successful synthesis of riboflavin and its general availability a vast amount of research has been done to determine the symptoms manifest by a riboflavin deficien­ cy in many of the animal species. Further, work has been done to determine the requirement of riboflavin in the diet of these species, especially of the young growing animals. Lepkovsky and Jukes (1935) found the adsorbate of liver 7 extract to be essential in producing normal growth in chicks. Phillips and Engel (1936) demonstrated that the presence of insufficient riboflavin in chick diets caused two types of de­ ficiencies. The first produces a rapidly acute paralysis characterized as ncuromalacia and the second, a more slowly developing form called ”curled too 11 paralysis. Both could be prevented by supplementing the basal diet with riboflavin. Day et_ al. (1938 ) found that lens cataracts developed in a high percentage of rats receiving a riboflavin deficient diet. Decreased growth rate, alopecia and keratitis were other symptoms present. Dydrgy (1935) and Bossoy and Wolbach (1 9 3 9 ) observed only rare cases of lens cataracts, however, the latter observed corneal vascularization in all rats kept on the riboflavin diet for Ip weeks or more. They considered vascularization of the cornea, in the absence of antecedent pathology, to be specific and the most reliable criterion of riboflavin deficiency and present the hypothesis that the vas­ cularization is a response to asphyxia of the tunica propria. Lippincott and Morris (19 I4.2 ) associated riboflavin deficiency in the mouse with greatly impaired growth rate, loss of hair, an atrophic and hyperkeratotic dermatosis associated with fissuring of the skin, keratitis, infrequently cataract, as well as ill-defined myelin degeneration in the spinal cord ahd the sciatic nerves. Street and Cowgill (1939) produced in dogs an acute riboflavin deficiency as evidenced by collapse after 102 to 8 llj.0 days on a riboflavin deficient diet, Axelrod the higher requirement value has been chosen In this translation. 17 Part IX— Thiamin© Takaki (1885) reported curing beriberi among seamen in the Japanese Navy by making certain changes in the m e n ’s diet. He concluded that the cure was achieved as a result of in­ crease in protein intake. Eijkman (I89O, 1892) demonstrated that fowl fed polished rice developed a polyneuritis which soon ended fatally. could be cured by feeding raw rice. It Later (1896) he proved that beriberi wae caused by eating polished rice as the chief food. He believed, however, that polished rice contained some toxin which was causing the disease. Eijkman’s collaborator, G-rijns (19OI) was the first to state clearly that beriberi was due solely to a dietary deficiency. He called this dietary de­ ficient substance "protective substance". Punk (1911) extracted and partially purified the antineuritic factor in rice bran to the extent that 50 mg of the extract rapidly cured a severely polyneuritic pigeon. In 1912 he used the term 1vitamin© 1 in ref©ring to this and other factors needed In small amounts in the diet. Hopkins (1912) noting growth factors from small amounts of milk called them "accessory factors of the diet". McCollum and Davis (1915 a, b) found essential growth factors in both the water soluble and the fat soluble portions of the diet. They refered to these as "fat-soluble A" and "water-soluble B". Since no evidence had been forthcoming to support Punic’s original idea that these indispensable constituents were amines, 18 Drummond (1 9 2 0 ) suggested that the terminal n-en be dropped. He further suggested that McCollum’s (fat-soluble A and watersoluble B) be dropped and these substances be refered to as vitamin A, B, etc. Both of these suggestions were adopted. Later Jansen and Donath (1 9 2 6 ) succeeded in isolating the anti­ neurit ic or antiberiberi vitamin in the pure crystalline state. In 1 926 Goldberger and coworkers showed "'that pellagra was associated with the lack of a vitamin which had a distri­ bution similar to that of the antiberiberi factor but was more heat stable. In 1927 an official committee in England revised the nomenclature and the antiberiberi or antineuritic vitamin was named vitamin Bg. Jansen (1935) proposed the name "aneurine" for this substance, a name extensively adopted in Europe while in America the name "thiamine 11 has been consis­ tently used. The structural formula of thiamine then became establish­ ed and the synthesis was accomplished by Williams and Cline (1936), Grewe (193&)» Todd and Bergel (1937) and Andersag and Westphal (1937)• Thiamine is composed of both a pyrimidine and a thiasole portion and Its chemical name is 3-(k~amino-2methylpyrinidyl- 5 -methyl)-ip-methyl-5- b eta-hydroxyethyl thiazolium chloride hydrochloride. Thiamine chloride hydrochlor­ ide is.a white crystalline substance which melts and decom­ poses at 2l^8-250°C., is soluble in water and alcohol and Is stable in acid solution. in alkaline solutions. Thiamine is very labile to heat and Thiamine has a widespread distribu- 19 tion in human foods and animal feeds. Brewer's yeast is an especially rich source. Lohman and Schuster (1937) succeeded in isolating cocarboxylase in a pure, crystalline state and showed that it was the pyrophosphoric ester of thiamine. This coenzyme plus yeast carboxylase plus magnesium ions decarboxylated pyruvic acid. Thiamine is present in the animal body mainly in the pyrophosphate form. Jansen (19^4-9> 1954) ably reviewed the re­ sults of many studies and lists 25 separate reactions in which thiamine pyrophosphate has been shown to act in a coenzymal ca­ pacity. Later reviews by Cheldelin and King (1954)> Johnson (1 9 5 5 ) and We Inhouse (1 9 5 4 ) Lave clarified the dual action of thiamine in oxidative decarboxylation reactions. Gunsalus (1953) and Seaman (1953) have shown that while lipoic acid is not necessary for the direct decarboxylation of pyruvate to acetate, it is required for the oxidative decarboxylation re­ action in which pyruvate enters into acetyl CoA formation. Reed (1953) and DeBusk (1953) presented evidence that the over­ all conversion of pyruvate to acetyl GoA may be expressed In these three steps: 1. Pyruvate S Acetyl-S^ I^ L T P P —^ .LTPP + Cog S HS Acetyl-S JTLTPP ¥ 2. HS GoASH IIS.____ .LTPP + Acetyl S GoA HS 20 HS 3 S ^ L T P P f DPN— *■ ^ L T P P + DPNH f H + s HS^ in vmich LTPP is lipothiamide pyrophosphate and has this structural formula: ^ O H 3 C-C c-ch 2 I I \ N - CH \ \ s s CHo I „0 ,0 = G - CHo - CHp - O - P - O - P ' - O H +N I I S I OH 0 - H Soon after the isolation, identification and synthesis of thiamine, riboflavin and niacin and their recognition as B-vItamin components, Chick (1938a), Hughes (1938) and Win- trobe (1 9 3 8 ) demonstrated thiamine to be beneficial in the growth of weanling pigs on purified diets. Hughes (1939) found thiamine deficient pigs had greatly reduced appetites and leg weakness* Two deficient pigs died suddenly but flabby hearts were not in evidence in these pigs. Supplementation of other thiamine deficient pigs with cry­ stalline thiamine rapidly restored their appetite and activity. This experiment gave evidence that the requirement of the wean­ ling pig for thiamine was less than 6 mg per 100 pounds of pig daily. The following year, Hughes (19 ^-0 ) reported an experi­ ment with weanling pigs on a purified diet in which rate of growth and muscle thiamine assay were the criteria for ade­ quacy of thiamine supplementation. The results indicated the 21 requirement to be about 1 mg per 100 pounds of growing pig daily. VanEtten and coworkers (I9 lp0 ) reported a study with, young pigs. Three week-old pigs were placed on a sodium sul­ fite-sulfur dioxide treated diet containing 10 $ fat and supple­ mented with different levels of thiamine. The pigs receiving no thiamine in the diet consistently developed symptoms as f ollows: almost complete refusal of food with occasional vomiting, extreme emaciation and marked lowering of body temper­ ature. When thiamine was not added, death usually occurred within 5 weeks. Upon autopsy of these animals very flabby hearts were noted. The intestinal tract was always almost com­ pletely empty but pathological changes were not always noted. In some cases liver damage was noted. Thiamine administration rapidly alleviated the anorexic and growth depressed condition of the deficient pigs. For pigs under these conditions the requirement for thiamine appeared to be 106 to 120 meg per 1 0 0 gm of carbohydrate and protein consumed in the diet. V/Introbe et al. (I9lp2c) studied thiamine deficiency In young pigs on a purified diet in which crystalline vitamins supplied the chief source of the S-vitamins. The chief symp­ toms of thiamine deficiency again were anorexia, vomiting, dyspnea, cyanosis and great weakness. The symptoms of cardiac failure appeared suddenly and unless thiamine was given re­ sulted promptly in death. Impairment of growth was not a pro­ minent sign in pigs dying early of acute thiamine deficiency. 22 In all animals dying of thiamine deficiency, focal necroses of the myocardium were observed. No neurological symptoms were observed nor were any degenerative changes in the nervous system found even in animals in which thiamine deficiency of long duration was produced. 'These authors found that blood pyruvic acid level was found to be positively correlated with the onset and severity of symptoms of thiamine deficiency and is a useful test. More useful than a single determination of blood pyruvate, however, was comparison of the value before and after the administra­ tion of a measured amount of glucose. Follis and coworkers (l9lp3) found that cardiac dilatation without hypertrophy, and focal and difuse myocardial necrosis were the characteristic heart findings in thiamine deficient pigs. In most of these pigs necrotic lesions were found in both the auricles and the ventricles. These lesions were in­ filtrated with polymorphonuclear and mononuclear cells. In older lesions there were connective tissue cells as well. The initial change seemed to be a loss of striation accompanied by vacuolization and hyalinization of the fiber. no intact muscle fibers could be found. In older lesions The interventricular septum was more severely damaged than the walls of the ventri­ cles. Fat stains revealed foci of fatty infIltration of the muscle fibers in the fresh lesions. The increase of heart weight as a percent of total body weight was due more to loss of body weight rather than to cardiac hypertrophy. 23 There was usually lung edema present with fluid and red blood cells in the pulmonary alveoli. Histological examina­ tion of the nervous tissues revealed no lesions in any of the pigs. None of the cardiac or the pulmonary lesions were found in any of the animals In which inanition alone was produced. Miller et al. (194 -3 ) found that weanling pigs receiving 3 * 5 mg thiamine per pound of feed did not gain any more rapid­ ly or efficiently than pigs receiving 1 .3 mg thiamine per pound of feed but that these pigs receiving the higher level deposit­ ed twice as much thiamine in the muscle. This may be of signi­ ficance since pork has long been considered (Hughes, 19^1 &hd Waisman and Elvehjem, 194-1) a good source of thiamine in the human diet. The workers at Washington State, Ensminger et_ al. (194-3) and Heinemann _et al. (194-6) also reported that a positive re­ lation exists between thiamine intake and the deposition of this vitamin. Symptoms observed on the thiamine deficient pigs were vomiting, - anorexia, slight staggering, cyanosis and a re­ duction In rectal temperature, heart rate and respiratory rate late In the experiment. Enlarged hearts were obtained from the pigs on the thiamine deficient ration. 'Ihese authors showed that the pig can store thiamine and can utilize stored thiamine over a considerable period of time. In addition, it was found that .1 9 Eig thiamine daily per kg live weight supported normal rate of gain and feed efficiency of pigs on a purified diet. 21+ Ellis and Madsen (1944-) showed that the thiamine require­ ment of pigs Is reduced by increasing the level of fat in the They fed pigs on three levels of fat (2$, 11$ and 2 8 $) diet. in the diet. As indicated by failure in appetite and cessa­ tion of growth, the animals on the low level of fat showed evi­ dence of thiamine depletion on the average in 25 days, those on the medium level in 28 days and those on the high level in 33 days. Lack of thiamine resulted in weakening of the heart, de­ crease in body temperature and emaciation. Thiamine adminis­ tration rapidly restored appetite, growth and general well being. It was found that the level of thiamine required to produce a maximum rate of growth and otherwise maintain good health fell within the range of 125 to ll|JL meg per 100 gm car­ bohydrates and protein In the diet on the medium level of fat. However, these amounts were insufficient to promote the normal amount of storage of thiamine in the meat tissue such as is found in commercial pork cuts. Such a sparing action of fat on thiamine as noted in this experiment had previously been re­ ported by Evans and Lepkovsky (1 9 2 9 ) in the rat and by Arnold and Elvehjem (1939) the dog. Electrocardiograms (ECG-) were taken on four pigs late In the experiment. Electrocardiographic studies with pigs have been extreme­ ly few and limited from the standpoint of both normal animals and animals v/hich have been subjected to nutrient deficiencies. Fortunately, some of the electrocardiographic studies that have been made in swine have been concerned with thiamine de- 25 ficiency also. Lepeschkin (1951) reports that the EGG- of the normal pig usually shows marked Sjx and Sjjl and often an inverted Tp, however. ic. Tjx and Till are usually positive and rarely diphas­ The apex lead almost invariably had a positive T wave according to the work of V/lntrobe et; al. (1943c). voltage usually decroased with the heart rate. The QRS The rhythm was sino-auricular with slight or moderate sinus arrhythmia. Nor­ mal heart rates of pigs used in Wintrobe’s work were between 130 and 150 beats per minute (bpm), averaging l^-O. In these experiments electrocardiograms were not taken until the pigs were to 4 months of age. The P-R interval ranged between .0 6 and .12 seconds, usually .0 9 seconds. usually .04 to .0 6 seconds in duration. The P waves were The duration of QRS was . 0 4 to .0 6 seconds. Ellis and Madsen (1944) book electrocardiograms on only one normal pig. was made. The pig was 71 days old when the ECG study The heart rate v/as 138 bpm, the P-R Interval was .10 seconds, the Q,-T Interval was .21 seconds and the constant for relating systolic duration to length of cycle (K= was .34Moustgaard (1953) reporting electrocardiogram data from two normal pigs in h i 3 thiamine work found heart rate to be l 6 o bpm, P-R interval .0 6 seconds and S-T interval .08 seconds. Thiamine deficiency produces greatly different electro- 26 cardiographic effects in different species. ficient dogs Swank et al* In thiamine de­ (19)pl) found tachycardia, elevation of P, inversion of T and increase of relative Q-T duration re­ versible after thiamine injection. Dogs showing an elevated S-T also had myocardial necroses. In thiamine-deficient cats Toman el; al. (194-5) reported that bradycardia was more common. There was increase In QRS duration and the systolic portion of the cycle was relatively Increased. T u was often Inverted. In thiamine-deficient pigeons, bradycardia, increase of P-R and inversion of T was found by Swank and Bessey (194-2). Brady­ cardia did not appear if starvation were prevented by tube feeding. Thiamine Injection rapidly restored the birds 1 heart manifestations to normal. In rice-fed pigeons Carter and Drury (1929) noted bradycardia and heart-block. In thiamine-deficient rats, many investigators, Vanlieerswynghels (194-5)* Weiss et al_. (1938), Zoll and Weiss Weiss (1936), Haynes and (194-0) and Hundley _et al. (1945) have observed brady­ cardia, lengthened P-R, QRS and relative Q-T duration and T wave changes all of which could be normalized by thiamine ad­ ministration. Weiss et al. (1938) was not able to abolish cardiac slowing or electrocardiographic changes by the admin­ istration of atropine or by vagal section. Hundley et. al. In addition, (194-5) noted sinus arrhythmia, auricular fi­ brillation, A-V nodal rhythms, sinus arrest, shifting pace­ maker, first degree A-V block and auricular, A-V nodal and ventricular ectopic beats. These wore partially or completely 27 corrected by thiamine therapy. The heart lesion was essenti­ ally a necrosis of muscle fiber followed by cellular infiltra­ tion, fibroblast proliferation and varying degrees of fibrosis. King and Sebrell (194-6) found similar symptoms. In the latter study, heart rates in thiamine-deficient rats dropped from a normal of 4-56 to 369 beats per minute. 'Hie accumulation of pyruvic acid in thiamine deficiency has been shown by Haynes and Weiss (194-°) and Wilkins et a l . (1939) to cause bradycardia and increase of Q-T in rats. However, VanHeerswynghels (194-5) found that this only occurr­ ed when the concentration of pyruvic acid became extremely high. Handles els al. (1947) reported that T wave changes appeared in dogs only when pyruvic acid began to be liberated by the heart. In human beriberi, Weiss and Wilkins (1937) found tachy­ cardia, bounding arterial pulsation, arterial pistol sounds, engorged veins, increased venous pressure, increased velocity of blood flow and decreased peripheral utilization of arteri­ al oxygen. Sturkie (1954) reported that chickens fed a diet com­ pletely devoid of thiamine developed abnormal electrocardio­ grams In 7 to 10 days. The abnormalities included bradycardia, depression of S-T segment, sinus arrythmia, sinus arrest, and auriculoventricular dissociation with nodal rhythm. The most extensive work on electrocardiographic changes in thiamine-deficient pigs has been reported by Wintrobe jet a l . 28 (I9^3c). Their experimental animals were 36 to 38 days of age when placed on a diet low in thiamine and were maintained in a chronic to severe thiamine deficiency for up to 258 days before death occurred. The thiamine deficiency changes mani­ fested by the electrocardiograms were bradycardia, prolonged P-R Interval, second degree auriculo-ventricular block, slight to pronounced sinus arrhythmia., nodal and ventricular premature beats, auriculo-ventricular dissociation with variable ventri­ cular foci, complete block with ectopic ventricular rhythm as well as bigeminal rhythm and auricular fibrillation. Pjj be­ came abnormally high and broad and was sometimes notched. One noted a great similarity between these changes and those later reported by Hundley _et af. (194-5) bn thiamine-deficient rats. The injection of atropine sulfate into two pigs soon re­ sulted In increased heart rate and shortening of P-R interval. The QRS complex was no longer dropped and there was a decrease in the frequency of premature ventricular beats. This would tend to Indicate that the bradycardia was due to vagal overaction since atror)ine is inhibitory to vagal action. These workers found that thiamine deficiency produced in pigs a much more Intense bradycardia than that observed in pigs suffer­ ing from inanition due to riboflavin, niacin, pyridoxin© or pantothenic acid deficiency. Jakobsen et al. (1 9 5 0 ) concluded that the thiamine deficiency causes partly a disturbance of the regulation of the transmission frequency of the sino-auricular node as a consequence of imbalance of the vagus action 29 and partly a disturbance of the normal course of the impulse through the cardiac neuromuscular system. Both Ellis and Madsen (1944) an(b Houstgaard (1953) ob­ served bradycardia and increased P-R and Q-T or S-T intervals. In the former work an Increase in the relative systolic por­ tion of the cycle was reported. Both studies dealt with pigs beyond weaning age and dealt with a minimum of experimental animals. koustgaard (1 9 5 3 ) reported deficiency symptoms similar to those previously reported except that no signs of disturbances of movements or intestinal disorders occurred. Furthermore, (1953, 1954) be reported that no rise In blood pyruvate level occurs if the animals have been trained to lie still for 4 ^ bo 60 minutes before taking the blood sample. If excited, normal animals showed elevated blood pyruvate and lactate. vated blood pyruvates found at times The ele­ (WIntrobe, 1 9 4 3 c) in thiamine-deficient pigs are possibly attributable to their greater excitability. He reported a thiamine requirement of 75 meg per kilogram of body weight daily to support maximum growth.. When Ensminger (1947) deleted thiamine from the ration of gilts a low storage of thiamine occurred In both the gilts and the offspring. Premature parturition occurred in several of the gilts and there was a high birth mortality in litters farrowed. The pigs which survived were usually weak and un­ thrifty and attained subnormal weaning weights. 30 She thiamine level in sow’s colostrum has been reported by Luecke et al. (I9li-7b), Braude ejb al. (19 )4-6 ) and Davis et al. (1951) as being .5 to 1.0 meg per milliliter. al. Davis et_ (1 9 5 1 ) reported tliat the thiamine concentration remains quite constant throughout the lactation period. Their re­ ported mean value for sows in advanced lactation v/as .6 meg per milliliter. This is equivalent to approximately 3 mg of thiamine per kilogram of solids in the milk. In reviewing the literature one notes a great diversity In the reported thiamine requirements. These are condensed and presented in the following table. Thiamine requirement of the growing pig Reported requirement ^m^/kg^"dlet^n Investigator 0. Iplp Hughes (19 ^4-0 ) 106 to 120 mcg/l 0 0 gm carbohydrate+protein in diet. O .96 VanEtten _et al. (19I4.O) 123 to llplmcg/lOOgm carbohydrate+protein in diet. 1.12 Ellis & Madsen (I9 I4I4-) 1 mg/100 lb. body wt./day 75 mcg/kg body wt./day. 1.50 Moustgaard (1953) -«-In translating the reported requirements in terms of mg Bq per kilogram of diet, it has been assumed that the animals con­ sume daily an amount of diet constituting 5 % of their body weight and that the carbohydrate and protein portion con­ stitutes 80 / of the diet. Where a range in requirement has been given, the higher requirement value has been chosen In this translation. The thiamine requirement listed by the National Research Council (Beeson et al. 1953) is *5 Big per pound of diet or 1 . 1 mg per kilogram of diet. 31 Part III--Pyridoxine Soon after riboflavin became recognized as a vitamin, G-ydrgy (193 )4-) identified a second component of the vitamin B£ complex in an experiment with rats. This was first called vitamin B£, later renamed adermin and now is generally known as pyridoxine and the related amine and aldehyde. Birch and Gy#rgy (1 9 3 6 ) definitely established the apparent and specific chemical properties of vitamin trates. as present in crude concen­ Lepkovsky (1938)> Keresztesy and Stevens (1933), GyBrgy (1938), Kuhn and Wendt (1938) and Ichiba and Michi (1938) succeeded in isolating pyridoxine from various natural materials. The structure of pyridoxine was established by Stiller ufc al_. (1939) 3-n(i Kuhn et a l . (1939)* in the same year Kuhn et_ ah. (l939a,b,c) (1939) and Karris and Eolkers completed snythesis of the vitamin. Vitamin B3 is now used as a class name and includes pyri­ doxine: 2 -methyl- 3 “hydroxy-)4_, 5 -dihy functions as a metal chelate formed from the amino acid and pyridoxal and refered to as the Schiff base. The scheme is particularly well presented by the last cited reviewers. Since Sherman (1950) and Sebrell and Harris (195^) have so ably reviewed the literature anent the effects of pyridox­ ine deficiency and the requirements for pyridoxine in various species, the present writer will limit the remainder of the review to include predominantly only that which appertains to swine. Hughes (1938) fed a semi-purified, diet- to weanling pigs and found that supplying thiamine, riboflavin and nicotinic acid did not support as rapid growth as when the filtrate factor in the form of rice bran filtrate was supplied. This factor included both pyridoxine and pantothenic acid. Chick and her coworkers (1 9 3 8 c) found that optimum growth could be obtained in pigs on a purified diet if yeast were added. Replacing yeast with thiamine, riboflavin and nicotinic acid did not support growth. When the eluate 3k fraction was absent from the diet the pigs showed slow growth, epileptiform seizures, and microcytic hypochromic anemia. Administration of the eluate, which was known to contain pyri­ doxine, relieved the anemia and caused the fits to cease. Wintrobe (1 9 3 9 a) fed early weaned pigs on a synthetic milk * diet supplied with yeast as a source of B-vitamins. When the yeast was withdrawn, prostration developed and convulsions occurred. Again, these could not be corrected by supplying thiamine, riboflavin and nicotinic acid but could be avoided by feeding yeast as .3% of the diet. Wintrobe et_ al. (1939HJ had also observed anemia in pigs receiving thiamine, riboflavin and nicotinic acid but no yeast. Further, these authors (19^-0) showed that degenerative changes in the nervous system of the pig occurs when only these three B vitamins were supplied. Wintrobe and coworkers (l9l|-2a) also showed that anti-pernicious anemia liver extract did not prevent convulsions and anemia from developing on this diet. Wintrobe et_ al. (I9lf2b) when feeding a diet more complete­ ly supplied with B vitamins found that whenever either pyri­ doxine or pantothenic acid was omitted from the diet, the pigs developed an abnormal gait and showed degenerative changes in the peripheral nerves, the posterior root ganglion, the pos­ terior roots and the posterior funiculi of the spinal cord. When pyridoxine was absent epileptiform fits were observed and anemia appeared. These promptly disappeared with administra­ tion of pyridoxine. Fatty infiltration of the liver occurred 35 when either choline or pyridoxine was omitted from the diet for a prolonged period. Wintrobe and his coworkers (19^-3a*) studied uncomplicated pyridoxine deficiency and found that a severe anemia developed which differed from pernicious anemia and iron-deficient ane­ mia. This anemia was characterized by microcytosis, an in­ crease of polychromatophilia, reticulocytes and nucleated red cells in the blood, a rise in serum iron, bone marrow hyper­ plasia and hemosiderosis in the spleen, liver and bone marrow. Administration of pyridoxine orally at a level of JLpO meg per kilogram body weight daily was followed by a maximal reticulo­ cyte response and rapid regeneration of blood with restoration of the normal size of the red corpuscles. Mobilization of iron from the tissues and its utilization in blood formation was indicated by the disappearance of hemosiderosis and a fall in the serum iron. This indicated that pyridoxine was related to the utilization of iron. Epileptiform seizures were also observed in this study and fatty infiltration of the liver was apparent when prolonged pyridoxine deficiency had taken place. Follis and Wintrobe (I9lf5) observed that pyridoxine de­ ficient pigs showed an abnormal gait characterized by swaying and tv/is ting of the legs. This gait differed from the ’goose stepping’ characteristic of pantothenic acid deficiency. In a further study on the anemia which develops in pyridoxine-deficient pigs, Cartv/right et al. (l9Mi-) found that the condition was not due to an increase in hemolysis but due to a 36 diminution in hemoglobin formation* Vitamin B£> anemia was found to be similar to pernicious anemia in that both condi­ tions were characterized by increased serum iron, hemosidero­ sis of the tissues, hyperplastic bone marrow and neurological lesions. Both differed from iron-deficient anemia in which neither hemosiderosis nor ferremia occurred. Limiting the iron intake in pyridoxine anemia, while abolishing hemosidero­ sis and ferremia, ataxia, did not, however, prevent the development of convulsions, neurological lesions and fatty livers. Evidence presented indicated that the continued absorption or decreased excretion of Iron prevailed even when the hemoglobin formation was at a minimum and when the tissues had an abun­ dant content of iron. These workers (l9l{-5) found that the anemia produced In pigs caused by a lack of dietary tryptophan was quite different from vitamin B£> anemia. deficiency anemia there was no hemosiderosis. level was low. anemia. In tryptophanThe serum iron It was essentially a normocytic, normochromic A reduction of tryptophan in the diet diminished the severity of B£> deficiency. Cartwright and Wintrobe (19^ 8 a) suggested that the funda­ mental disturbance in pyridoxine-deficient anemia in swine is a failure to synthesize protoporphyrin. They determined ery­ throcyte protoporphyrin level in normal and pyridoxine defici­ ent pigs and found that the mean for pyridoxine-deficient pigs \vas Ip7il3.6 meg per 100 ml of red cells as compared to ll8i meg per 100 ml of red cells in normal pigs. Plasma iron 37 levels at the same time rose from a normal of 1 6 9 -3 8 *8 meg percent to [1.6 8 * 1 6 6 .6 meg percent In the B^-deficient pigs. On the otherhand, plasma copper levels dropped from a normal of 206+26.3 meg percent to 1 60 * 3 8 .8 meg percent. Urinary copro- porphvrin levels appeared unaffected. Hughes and Squibb (l9lj-2d) used two groups of weanling pigs fed a purified diet In which the positive controls re­ ceived 5 mg of pyridoxine per 100 pounds daily while the other group received no supplemental pyridoxine. The pyridoxlne-de- ficient pigs became unsteady on their feet and showed reduced appetites within a month after the start of the experiment. Later the pigs ceased to gain and occasionally an epileptic­ like fit was observed. The author’s description of a typical epileptiform seizure is presented as follows: nA pig would be walking about the pen when suddenly he would stop,- become unsteady on his feet and fall on his side, struggling violent­ ly, and screaming with a high-pitched voice, with mouth open and legs kicking out in all directions, then his body would stiffen with head back and with open starry eyes. This would be followed by a comatose stage when the pig appeared dead. Respiration was first deep, then shallow, then deep again. In about a minute or two, the pig would regain partial conscious­ ness, would arise and stagger in any direction. Finally, in a few seconds, he would become conscious and would walk to the trough and drink water and would appear normal though somewhat weakened11. Blood studies showed the characteristic microcytic 38 hypochromic anemia in the deficient pigs. Administration of pyridoxine to these pigs rapidly corrected all of the observ­ ed symptoms. None of these symptoms were observed in the control pigs. Emsbo and associates (19lp9) in Denmark observed similar deficiency symptoms in pigs. In their trials reduction of appetite occurred during the second week of deficiency and growth stopped after 3 weeks. the diet lacking pyridoxine. in the experiment. Pigs died after two months on Convulsions were frequent late Nitrogen balance studies showed a reduc­ tion In nitrogen retention in B^ deficiency. This was appar­ ent before the external manifestations and the reduction in­ creased as the severity of B£> deficiency advanced. Miller (1951) performing complete balance studies with human babies and with pigs on all known B£> compounds found that elimination of was greater than the Intake. This suggested limited intestinal bacterial synthesis which, how­ ever, was inadequate to meet the growing p i g s ’ needs. Lehrer et, al_. (1 9 5 1 ) studied pyridoxine deficiency in baby pigs taken from the sow at 2 days of age and placed on a synthetic milk diet (Lehrer et al•, 19^-9) • Dn these experi­ ments pyridoxine deficiency was characterized by poor appetite, vomiting, inco-ordination cf the muscles, spastic gait, poor growth, epileptiform fits, comas, rough haircoat, brown exu­ date around the eyes and impairment of eyesight. All defici­ ency symptoms with the exception of impaired eyesight could be 39 cured by adequate pyridoxine supplementation. The excretion of xanthurenic acid was detected in the B^-deficient animals and these animals excreted low levels of pyridoxine. Xanthurenic acid was first identified In the urine of pyridoxine-deficient pigs by Cartwright et al. (I9i|li-) although a green pigment-producing substance was earlier reported by Wintrobe ejb al^. (I9ip3)* The presence of this green pigment- producing substance was first reported In the urine of pyri­ doxine- deficient rats by Lepkovsky and Nielsen (l9lp2). Later Fouts and Lepkovsky (19lp2) found this substance In the urine of pyridoxine-deficient dogs. Still later Lepkovsky et, al . (19 lp3 ) showed this substance to be xanthuranic acid, a meta­ bolic product of tryptophan. Moustgaard and coworkers (1952* 1953* 195^4-) have made the most recent study of the requirement of the pig for pyridoxine. At the time these studies were made the function of pyridoxine was more fully understood and a greater number of criterial measures could be employed In determining this requirement. Their studies showed that the daily requirement value of IpO meg of pyridoxine per kilogram of body weight found by Win­ trobe at al. (l9 ^-3 a), while adequate to prevent the occurrence of clinical deficiency symptoms, was inadequate to support maximum growth and nitrogen deposition in weanling pigs. To acquire maximum protein utilization it was necessary that they supply twice this amount of pyridoxine. Several determinations reported by Moustgaard (1953) have b-o added to the evidence of poor protein utilization in B^-deficient pigs in addition to reduced growth rate and nitrogen re­ tention. Urine studies shov/ed a greatly increased excretion of xanthurenic acid and kynurenic acid In the urine of these pigs which paralleled the fall in protein utilization. When these pigs were given an oral dose of L-tryptophan, the dis­ turbance of the enzymatic transformation of tryptophan caused by the pyridoxine deficiency was revealed through an increas­ ed urinary xanthurenic acid excretion long before any clinical symptoms of pyridoxine deficiency could be demonstrated. The simultaneous reduction of blood hemoglobin and the increase of serum iron probably results from its liberation from the ferri­ tin iron of the tissues as hemoglobin formation is reduced. Moustgaard (1953) explains the occurrences of epilepti­ form seizures in pyridoxine-deficient pigs as an excitation re­ lease. The pigs are on the verge of seizures and their thres­ hold of excitation is lowered. The disturbances of the protein metabolism also gave rise to impairment of the p i g fs ability to produce the antibody-carrying serum protein, gamma globulin. The gamma globulin fraction of total serum protein in the pyridoxine-deficient pigs constituted only 5 percent. This frac­ tion normally comprises 15 to 20 percent of the serum protein. Moustgaard (1953) f«els that since this fraction is especially rich in tryptophan, this may account for a reduced defense mechanism. It should be stated here that pyridoxine has been shown to function In enzyme systems concerned with most if not ljl now all or the essential amino acids (viz* reviews cited)* It would seem wise to consider this pyridoxine-antibody response relation further as applied to other experimental animals. Stoerk (19l{-6a) and Agnew and Cook (19^4-9) found a greatly reduced thymus weight in pyridoxine-deficient rats. The lymph nodes were greatly depleted of lymphocytes and there was a general atrophy of all lymphoid tissue. Dougher­ ty eJi al. (I9 I1J4-) had previously demonstrated In mice the presence of antibodies in lymphocytes and substantiated the production of antibodies in lymph nodes* A significant por­ tion of the antibody protein was contained in lymphocyte ele­ ments of immunized animals. These authors later (191+5) show­ ed that adrenal cortical mediation is essential for control of the release of antibodies from lymphocytes. Stoerck and Eisen (I9 I4-6 ) and Agnew and Cook (191+9) immun­ ized pyridoxine-deficient rats v/ith washed sheep erythrocytes* The development of antibody response in the serum of these animals was greatly reduced as compared to that developed in either Inanition controls or full controls. Axelrod ejb a l * (191+7) made similar observations In hemagglutinin production in response to human erythrocyte inoculation in both pyridox­ in© and pantothenic acid-deficient rats. Ludovici _et al* (I9 5 l) observed, however, that if the immunization were made while the rats were receiving a complete diet, the serum anti­ body titers remained high even during protracted periods of either pantothenic acid or pyridoxine deficiency. Bosse and 1+2 Axelrod (I9 I+8 ) noted marked impairment of rate and quality of healing in pyridoxine or riboflavin-deficient rats. Axel­ rod (1952a, b) has v/ritten two reviews on the role of the B vitamins in antibody synthesis. Olsen and Mart indale (1951+b) have reported that there was an increase in the wet weight of the adrenal, ventricular mass, liver and kidney of pyridoxine-deficient rats. There was a 30% decrease in the oxygen consumption of the broken­ cell preparations of hepatic tissue as studied In the Warburg apparatus. Renal tissue preparations showed a 20% decrease in 0 2 consumption. Agnew (1955) also found heavier wet and dry weights of heart, liver and kidneys in pyridoxine-deficient rats. Olsen and MartIndale (1951+a) observed an elevation in systolic blood pressure in B^-deficient rats. No such ob­ servations as these have been reported for the pyridoxine-de­ ficient pigs. This writer believes that while many enlightening and de­ tailed studies have been made in relation to various clinical and subclinical conditions arising in a pyridoxine deficiency in the pig, the determination studies on the requirement of the pig for this vitamin have been inadequate, for the baby pig. especially so The pyridoxine requirement listed by the National Research Council (Beeson jet al. > 1953) for the w e a n ­ ling pig is .6 mg per pound of diet. 1.3 mg per kilogram of diet. This is equivalent to The few reported requirements are presented in the table below. k3 Pyridoxine requirement of the growing pig Reported requirement . Equivalent m mg/ kg diet*«* Investigator ipO meg per kg body wt. daily .8 5 mg per 100 lb. body wt. daily 2 .2 Hughe s and Squibb (19lj2d) 80 meg per kg body wt. daily 1 .6 Moustgaard (1953) Wintrobe ei: al. (19 l4-3 a *In translating the reported requirements in terms of mg pyridoxine per kilogram of diet, it has been assumed that the animals consume daily an amount of diet constituting %% of their body weight. Experimental Procedure I. Riboflavin The riboflavin experiments were conducted first. consisted of three trials involving 55 baby pigs. This The first of these trials was a group feeding trial in which 15 purebred Duroc Jersey pigs were used. These pigs were taken from the sow when they were three days old and were lotted as uniform­ ly as possible according to sex, size and litter into five groups of three pigs each. They were reared In *terralactors* These were commercially made metal cages with wire-mesh bottoms that were designed for the battery raising of early weaned baby pigs on sow milk replacers. The diet fed was basically that used by Lehrer _et al. (19^-9)» Wiese et al. Il9^7aj and Johnson et al. (I9^8a) and identical to that used by Stothers (1952) with the exceptions that the pantothenic acid was supplied at a level of 2 mg per kilogram of synthetic milk and riboflavin was added at graded levels of 0 , 2 , Ip, 6 and 8 mg per kilogram of solids in the milk. Stothers (1952) has adequately described the prepara­ tion of the milk. The baby pigs readily learned to drink the milk from a shallow trough when it was witheld from them for the first eight hours. To encourage its consumption milk was warmed somewhat for the first few days. The pigs were fed ad libitum Feedings were made three or four times daily to prevent sour­ ing of the milk in the trough. fully measured. The amount of milk was care­ Troughs were scrubbed and rinsed once daily. Room temperature averaged about 7 2 °F. and heating units within the cages supplied additional heat as needed during the first two weeks of the experiment. Pig weights were taken and recorded every fourth day. The pigs remained on the experimental feeding period for 32 days. The deficient pigs remained on their diet until death or until autopsy was made when death appeared Imminent'. Post mortem studies were made on the three pigs receiving no riboflavin. A measure of the hemoglobin and white blood cell levels was made on one of these pigs. Samples of the diet of each lot were assayed microbiologically for riboflavin at frequent Intervals to verify the prepared concentration. Determinations were made using the method of Snell and Strong (1939) and samples were prepared according to the directions of Strong and Carpenter (l9lj-2). In the second riboflavin trial the pigs were fed individ­ ually. This was made possible by inserting two removable partitions Into each cage. This provided for the Isolation of each of three pigs within a cage. Accurate records of in­ dividual pig consumption were then possible. A lp-day deple- tIon-adjustment period was also introduced Into this trial and was followed in subsequent trials. In this Ip-day period, all pigs in the trial were fed a riboflavin-free diet for the pur­ pose of reducing riboflavin stores within the pigs and to allow for an adjustment by the pigs to the cages, to the diet and to the feeding procedure. At the end of this depletion- adjustment period, the pigs were assigned to lots on the bases of sex, size, litter and general appearance. The assign­ ed lots then were placed on different levels of dietary ribo­ flavin intake. These levels were 0, 1 , 2, 3> 4 and 5 mg of riboflavin per kilogram of solids in the milk consumed. ■ Other­ wise, the feeding procedures and the environmental conditions were similar to those described for the first trial. All pigs used in this trial were purebred Yorkshires. Work by Mitchell ejfc al. (1950) suggested that blood studies might reveal a more sensitive test for adequacy of riboflavin intake. Consequently, blood samples were taken ft’om a laige ear vein bi-weekly to determine blood hemoglobin levels, white cell counts and white cell differentiation*. Blood samples were taken from the anterior vena cava during the late stages of the trial for blood riboflavin assay. The pigs in this trial were on the experimental diets for 28 days. Again, as in the former trial, post mortem examinations were made on all pigs which died or were sacrificed. During the second trial three other pigs were placed on a commercial sow milk replacer to which In addition to the Bvitamins which it contained was added the same supplementation of B-vitamins used in the synthetic milk. These three pigs were all males and were heavier than most of the other pigs started. Further, they were placed in newly designed Individ- b-7 ual cages which, became available at this time. Microbiologi­ cal assay revealed that the diet of these pigs contained lip mg of riboflavin per kilogram of solids. This diet contained about ten percent solids. From the experience gained from these two trials and from a related study on the bacterial count in the milk as affected by frequency of feeding and cleaning of troughs and from some of the experiences of Stothers (1 9 5 2 ), it became apparent that a standardization of experimental procedures for future experimental work of this nature was necessary in order to obtain more valid results with a lesser degree of individual variation within lots (experimental error). These procedures are listed below. Standard procedures for experimentation with baby pigs on synthetic milk diet 1. Selection - Be able to select from 1%0% of the num­ ber of pigs required in the experiment. Since the pigs are being individually fed It is not necessary to start the pigs all at once. 2. This is a practical aid in selection. Starting age and weight - Minimum of 3 days of age and 3 pounds body weight and maximum of 5 days of age and 5 pounds body weight. 3* Starvation period - 8 hours. Ip. Depletion-adjustment period - Ip days. 5. Temperature of milk fed to pigs - ip8 (a) 80° to 100°F for first week. (b) 50° to 70°F for second week. (c) temperature of stored milk thereafter. 6. Milk storage temperature - 3°to ip°G. 7* Temperature of water for making milk - 65° to 70°C. 8. Amount of water per can of milk - 28 liters. 9* Amount of total solids per can of milk - 5 kilograms. (a) casein - 30$ - 1500 gm. (b) lard - 10$ - 500 gm. (c) salts - 6$ - 300 gm. (d) cerelose - 5^$ - 2700 gm. This makes a total of 33 kilograms of milk per can. The milk contains l5.l5$ solids. 10. Amount of vitamins per kilogram of milk Thiamine Riboflavin Niacin Pyridoxine Calcium pantothenate Para-aminobenzoic acid Inositol Choline Biotin Pteroylglutamic acid Ascorbic acid Alpha-tocopherol acetate 2-methyl-l, Ip-naphthoquinone Vitamin A Vitamin D 0.65 mg 0.65 11 2.50 u 0.65 ,r 3»00 2.60 Tr 26.00 ft 260.00 " 0.01 ,T 0.0 5 2 fr 16.00 ,f 1.00 " 0.28 mg 2,000 I.U. 200 I.U. Samples of the diet should be assayed periodically for the particular vitamin in study to verify prepared levels. 11. Feeding schedule (a) First 2 weeks - 6 times daily 14-9 (b) Thereafter - 5 times daily 12. Cleaning schedule (a) Troughs - Rinse with hot water after each feeding. (b) Cages - Scrub and rinse 2 or 3 times weekly. 13. Environmental temperature - Room temperature of 7 0 °F. plus infra-red heat lamps to give proper temperature for the pig. 1 I4.. Hemoglobin level precaution - Give several drops of saturated ferrous sulfate solution to pigs when they are placed on experiment; thereafter give an Fe-Cu-Co tablet to each pig weekly. 15. Blood studies - Make bi-weekly cell count and hemo­ globin determinations. 16 . Relative humidity - Keep as uniform as possible; measure and record occasionally. 17. Recording data - Keep accurate individual records of all measures and observations. 18. Closing the experiment (a) Make arrangements for blood assays. (b) Make arrangements for autopsies. (c) Make arrangements for pictures. (d) Thoroughly clean all cages and utensils. (e) Thoroughly clean and steam the room twi c e annu ally. These procedures have been followed with but few modi­ fications in subsequent trials. 50 In the third riboflavin trial seven Chester White-Duroc Jersey crossbred pigs and 12 Chester White-Yorkshire Cross­ bred pigs from two litters were used. It was quite clear f r o m the two previous experiments as well as from reports of other workers under a variety of conditions that the minimum riboflavin requirement of the baby pig for normal growth was not in excess of Ip mg of riboflavin per kilogram of solids consumed. Consequently, this level was chosen as the positive control level in this experiment. Three pigs were placed on the basal diet containing no riboflavin and l\. pigs were p lac­ ed 011 each of Ip diets containing the following levels of ribo­ flavin, In mg per kilogram of solids: 1.0, 2.0, 3.0 and lp.0. All pigs were fed regularly 6 times a day an amount of diet which they wo uld consume in the Ip-hour period. diet was limited only when scouring appeared. The amount of Scouring was not a particular problem after the first week of experiment­ al feeding. The experimental feeding period was 28 days in duration. II. Thiamine The thiamine work was embodied in a triplicated experi­ ment involving 55 baby pigs of which 25 were Chester WhiteYorkshire crossbred pigs, 13 were purebred Chester White pigs, 7 were B erkshire-Yorkshire crossbred pigs and 10 were pure­ bred Duroc Jersey pigs. cedures were followed. The previously stated standard p r o ­ 5i On the basis of the reported work with older pigs, the minimum thiamine requirement of the baby pig was judged not to exceed 2 mg of thiamine per kilogram of dietary solids in­ take. Consequently, this level was chosen as the positive control level in this experiment and fed concurrently with other lots receiving levels of 0 , 0 .5 , 1*0 and 1.5 mg of thi­ amine per kilogram of solids consumed. Samples of each diet were assayed frequently by the thiochrome method as describ­ ed in the methods of vitamin assay of the A. V. C. (1951) to verify the prepared concentration of thiamine In the diet. Electrocardiographic studies were made weekly on all pigs throughout the second and third replications. In order to obtain valid electrocardiographic data the pigs were sus­ pended In a canvas carrier device shown in figure 1. With some training the pigs learned to lie still for a considera­ ble period of time. No recordings were made until the pigs had remained still for sometime. The recording machine used was a ij. channel, model 67 Sanborn. Five leads were connected to electrodes situated on the four legs and the chest. The leg electrodes were situat­ ed just above the knees in the fore legs and a little above the hocks in the hind legs. The chest electrode was situated slightly to the left of the sternum. The hair was clipped from these areas and electrode paste was applied in order to establish Intimate contact between electrode and skin. 52 Fig. 1 - Apparatus used in obtaining electrocardiograms. The pig is suspended in a canvas sling in which holes were cut to allow the legs to hang freely. The recording device is a i). channel, model 67 Sanborn. Blood samples were taken from the anterior vena cava during the late stages of the experimental period for blood thiamine assay. The experimental feeding period lasted 32 days. Post mortem examinations were made on all pigs that died during the experiment and on those pigs which were sacrificed while in a semimoribund condition. Several positive control pigs were sacrificed at the end of the experiment to serve as comparative standards in these examinations. Heart, adrenal 53 and thyroid weights were taken and recorded. tissues were taken and fixed in formal saline. Blocks of Hematoxylin- eosin was used on all sections and myelin sheaths were stain­ ed using W e i l ’s (1945) method. Nissl bodies were stained by a thionin technique described by Fletcher (1947). Ill. Pyridoxine Three pyridoxine trials have been conducted involving the use of 65 experimental animals. In the first trial, 4 lots of 4 pigs each were individually fed diets containing 0 , 1 .0 , 2.0 and i|*0 mg of pyridoxine per kilogram of solids in the diet. In addition, two other pigs received the positive control diet but were pair fed with two pigs In the negative control lot. The experimental feeding period lasted 32 days and all 18 pigs used were Chester White-Yorkshire crossbreds. Blood samples were taken weekly for red and white cell counts and hemoglobin determination. Twenty-four purebred Duroc Jersey pigs were used In the second trial. Five levels of pyridoxine supplementation were used in this trial. These levels were 0, 0*5> 0*75, 1*0 and 2.0 mg of pyridoxine per kilogram of solids In the diet. Four pigs were individually fed on each diet and in addition four other pigs were fed the positive control diet but were pair fed with pigs in the lot receiving no pyridoxine. Complica­ tions developed in this experiment which rendered a consider­ ation of the data obtained inadvisable. A description of some 5k of the results of this experiment, however, will appear later in the thesis. Twenty-three pigs in all were used In the third trial. Fourteen of these were purebred Chester White pigs, 4 were Chester White-Yorkshire crossbred pigs and 5 were Chester White-Duroc crossbred pigs. These pigs were placed on the same levels of pyridoxine supplementation used In the second trial. Complicating factors unrelated to level of pyridoxine supplementation developed in the case of three pigs whereby it was deemed inadvisable to use the data obtained from them. Blood samples again were taken weekly from an ear vein to determine R B C and W B C counts and hemoglobin level. Urine collections were taken at weekly intervals and xanthur­ enic acid excretion determinations were made. Prior to the urine collection, the pigs received an oral dose of DL-tryptophan amounting to 100 mg per pound of body weight. Urine xanthurenic acid concentrations were determined using the method of Wachstein and Gudaitis (1952). Blood samples were taken from the anterior vena cava during the last week of the experiment to determine the serum euglobulin fraction. As in previous experiments, post mortem studies were made and various organ weights recorded. RESULTS Part I--Riboflavin Studies Experiment I The results of the first trial pertaining to pig growth and feed consumption are presented In table 1. Statistical treatment was given to these data and to the data of subse­ quent trials, unless otherwise specified, using the method of Snedecor (1946) analyzing single classification variance. These results show that all lots receiving riboflavin gained significantly faster at the 10% level than the lot receiving no riboflavin. Pigs from the lots receiving riboflavin also gained 25% to 34% more efficiently than pigs from the lot re­ ceiving no riboflavin. The rate of pig growth was not particularly good in any of the lots and there was a great deal of individual varia­ tion within all of the lots. However, all of the pigs receiv ing riboflavin appeared much thriftier after about two weeks of the experiment and throughout the remainder of the trial than the deficient pigs. Near the end of the trial the hair- coats of the deficient pigs became quite rough, a heavy se­ baceous exudate accumulated about the eye and a dried some­ what caked exudate collected on the skin. There was a consid erable amount of loss of pigment from the skin of these pigs 00 OA CM oa o p W rH bD S3 •H «H ft > cd > > rH X> f t ed O . f t X> •H ■ ft f t © ft ft o ft ft © O rH ft © £50 > © f t rH O »>> © ft © cd s3 p o © f t »H « *d © P3 oa 1A • -d H © oA -d • o M \ nO © rH ft O ft •iH ft © ft p -d OA CM rH CM • O +1 f t oa • CM © P CM -d • o d \ oA 0 rH d +5 S3 i •H ft © X* P & © •s S3 •H o o CM • rH o rH bQ bo a a* o ' o • o +1 f t OA • O \A oA • fA f t o • o +1 CM OA • O O O ' • H +1 -d O • oA rH rH © > © S3 o 0A -d • O • rH i—I 0 o of H S ft -P CM OA CM oa ft O tA CM • O +1 f t o • OA rH CO • o a CM O • o +1 o OA • O OA f t « CM rH © »H O OA 1 CM OA rH CM o> CM © o -H 1A CM • OA rH CM * O -H OA C— • 00 nQ O -d • O 0A • rH CM CO • rH H 0A • O £ TO § d ft CM P S3 © bO •H P © ft © P ft O S3 o ft © © ft 3 *25 fid P> 0A ----• * > * ft H rH T 3 ~ f t S3 •H • fid © bO <3 £ 0 O 0 • *H S3ft ■§ oS *H © d £ S bD O *H © ft TO O S 'H O ft © ft ,__. © • rH f t XI 1—1 fid rH •H v— ' rH — P fid •H p S3 f t •H f t S3 f t •H tkO f t tsO •H •H • © • © © % © £ > t> ■5s -p S3 © i—1 ,__„ 0 © O r*— oA O » O +1 E"i—1 • O cd e* ft rH rH © > *s ft ♦ © ft © rH H — l^ rH *H «d ft • © 'S j if t © S • X> rH r x • ft X) © H ft— p ® s3 o o © 73 « rt rH O GQ S3 *h fid bO O S3 f t f t *H ft a © bo u © ft p «d o no riboflavin p S3 © ft © ft ft «H n © ft *H rH O © rH -d • O +1 rH rH • xl i—I © S3 bO «d H «H -P H Cft CQ <3 57 and diarrhea was mono frequently a problem in these pigs than in those pigs receiving riboflavin in the diet. No di­ fferences were apparent between any of the lots receiving riboflavin. All three of the pigs receiving no riboflavin died or were sacrificed within a few days after the close of the experimental feeding period. Extensive fatty degeneration was apparent in the livers of all these pigs and pneumonia was observed in one animal. Experiment II In the second trial considerable growth variation was observed within lots. The growth rate and feed consumption data are presented in table 2. Analysis of the data indi­ cated that maximum growth rate and greatest feed efficiency were obtained when the pig's diet contained 3 Ij_ mg of riboflavin per kilogram of solids. The blood studies made during this trial indicate that the level of blood hemoglobin was not affected by the level of riboflavin in the diet. This is shown in table 3 . There appeared to be an increase in the total leucocyte count and the percentage of these which are polymorphonuclear when diet­ ary riboflavin was reduced. However, the individual varia­ tion within lots was so extensive that these differences at the end of the experiment were not statistically significant. Mitchell et al. (1950) reported that the percent of neutro- (A tA • O +1 1 ca • P t © o • o o +1 CO OA • f t CM • CM ft ft • CO +• • oo 'LA • rH O © o O O o bO o • 1A M \ bp OA ft • O H-l ft \A • ft CM rH • o o +1 OA OA • O +i co • o * P 0 CO 'LA • O +1 CA O * OA CO CM 3 (A O • 'LA oa • OA +1 OA CO * OA 'L A o rH * O +1 CM -ft" • O o +« rH OA • O O CM 0 o Fl ft cd AJ rH ft O XI *H Ft cA OA CO CM i—1 C\J © > OA CO CM rH rH ft rH +1 GO -d - o co ft • -cf• o f t O rH • O -H AA0 OA 0 0 CM © +1 oA OA CM o 'LA A• n± rH +1 rH o OA O O • • CM ft rH CM O O * P © , • rH f t S{ rH ft F3 S3 © © f t '— ' ft P ft P ft O Fl 3 Fl S3 0 ft O © P O ft 3 {St cd Q • ft i—1 rH cd S3 P FJ X I f t X I b O f t to ft ft • © © £ > OA <—** • l»ft rH f t f t -—' Gd ft 0 > < cd b0 <1 © XI p O S3 f t © Fl ft ft ft o CO ft cd bD Ft © P © O ft © © Fi oq o cd e Ft ft 0 r^rH rH P SS cd o o ft •H ft ft ft f t S3 S3 to bO ft cd 09 f t O ft 03 09 0 Xt Ft © ft FI o ft © £ cd v O Ft b O o ft F t l A F! 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The average percentage neutrophiles for all pigs reported in table 3 was )±y/o at one week of age and at 5 weeks of age. The mean values for blood riboflavin level determined for some of the pigs at the close of the experiment did in­ dicate that these values were related to dietary riboflavin intake* These mean values expressed in micrograms per 100 milliliters of blood for lots 1 through 6 were 13 .0 , 21 .0 , 1 6 *5 , 34*0 , 27.0 and 38-0 respectively. Experiment I I I _ The most important results of the third trial are pre­ sented in table ij.* Growth curves showing the average lot weights throughout the feeding period are shown in figure 2 . All of the pigs on the basal diet containing no ribo­ flavin and all of the pigs receiving 1 mg of riboflavin per kilogram of solids showed gross external symptoms of ribo­ flavin deficiency. These pigs exhibited a rough haircoat, a heavy sebaceous exudate about the eye and ear and a dried, somewhat caked exudate on the skin (fig. 3). These pigs be­ came weak and thin and had a higher incidence of diarrhea O' • o +1 c*o• vO ca IfN - d I4. - Response of baby pigs to synthetic milk diets containing different levels of riboflavin 03 CO CM •H H O 63 bC cd iH u\ O •H CM - d CO CM O -d H o +1 CM o +1 oA CO CO • O• O +1 O ' CM • O • o +1 CA tr\ • O -H r1A oA rH CM o o +1 U\ bO -d 1—I O CM • o +1 o td vO o OA CM rH O vO sO n j-ao -d o CM CM CA O • o +1 o rH CM * CA O• O drH CM O• o oA OA •o CM rH © > © t-3 1A C • rH r^ O O CM O +1 vQ -d ♦ • o +1 vO co VO -d o o• o +1 I—I o 1A O o o +1 _ d t>s o *H rH A 5 d 03 -p *H CM CM -P © i—1 \ A Ch to © O S A a •«H £ •H £ •H © •H -P i—I GO £ £ >» •H o £ > o 3 © p rH d © © o 'd A to o £ •rl w > vO • o 1—1 +1 +1 o - CO vO CO • • +1 CM rH ■tfv t 'rH rH CO TA CM • • o CM +1 +1 at co © CM A CO • • + 1 + 1 CM CM O i—1 i—1 d 1A vO • C\J CM • o CM +1 +1 © rH Pi £ • P £ © S £ © • P»P X £ © © o £ o © a P O £ © £h •H A cd £ © o £ © Pi rH rH © rH cj 2 P S O £ P 50 o O •H 4h £ o o A £ *£ o © S 50 © © £ cd £ © S © P •H £ © Pi £ © d £ rH 50 © o © © TJ £ W £ £ £ © w •H *H © P i © 03 £ A £ © 50 © O £ £ © A © © •H © P © © £ © £ £ £ A © © £ Pi © rH £ © ro © © © rH © © lA rH £ O © o £ o © A P £ © © S rH •H © © © P £ rH © £ © > -d - rH A fid rH + 1 1A U\ CO C —1 • P h P CO rH © © > o © A in • P £ £ O o © © © © £ Pi X © t> £ Pi £ X *H © > © © rH © i—l • O CM +1 ON UN C" * -d ’ UN ON • i—1 +1 -d CM • vO i—1 -d* O • O +1 MO on • 1 1 1 1 ON ON ON O O • • o O +1 +1 on i—1 vO CM • • rH •H P ft) •rH d a •H 4) £ *H O • ON -d* rH ON O O • • o o +1 +1 o ON UN -d * * • rH o -d •H • CM P UN • CM -d* 1 o P t +i O UN o UN r-H A ft) > 00 ft) rH £1 i — 1 O o • rH -d- 1 +1 1 CO o r— • -d f 1 t 1 1 1 1 1 1 1 1 1 UN ♦ £0 bO d P rH ,,_, >• -—- P P • i—1 •rH >_> P "w- 05 eg • rH d r-'- bO — •H • -P ■ p • • •H rH P £ P eg O rH P « vD C\J d tQ - p P rH © rH P 9& 85 bo Ert iH P rH «H •♦H ft) P £ ro P 3 £ P O £1 O P ft) P P S2J 05 eg P •rH £ i—1 Cg bO >* rH to — t>*d rH ft) rH P ro P< P •H •rH B P P •rH eft «ft p O •H

<1 ft) > CD O t> rH O CO bO O O ft) P P rCl i-| P P 05 }>sCh bO P O r t O Ih N rH *»H P CM < -P Eh -I OCM ft) © •r-j P +i 05 71 All deficient pigs which died or were sacrificed show­ ed gross symptoms of thiamine deficiency. In animals dying naturally, there was marked cyanosis noticeable in the skin, nos© and mucous membranes. The most common gross finding was a pale yellowish-gray mottled heart. The left ventricle was usually contracted and the right ventricle was quite flabby. The whole heart was less firm than normal and in several in­ stances it was rounded and resembled a myxedematous heart (fig. 15) . There was always an excess of pericardial fluid and in many cases the peritoneal and pleural cavities also contained excessive fluid. In most cases, there was congestion of the liver and the serosa of the small intestine was reddened. teric vessels were usually injected. The mesen­ In a few of the animals, the liver showed a yellowish mottling suggestive of fat. There was inflammation of the cecum or colon varying from a mild reddening to a severe necrotic enteritis with ulcers and caseous masses adhering to the intestinal wall. On microscopic examination the hearts of thiamine-de­ ficient pigs showed congestion, focal fragmentation and some necrosis of the muscle fibers. The cells in the inflamed area were primarily macrophages although some lymphocytes and Anitschkow cells were also present. This Inflammation was more pronounced in the hearts of pigs from lots 2 and 3 and there was an increase in collagenous fibers as demonstrat­ ed by Mallory*s aniline blue stain. Sudan IV stains showed 72 extensive fatty degeneration in the heart tissue of all de­ ficient pigs but the fat droplets were small and tell-tale vacuoles were not readily observed by the heraatoxylin-eosin stain. All portions of the myocardium were affected but lesions were more marked near the epicardium. The left ven­ tricle was involved to a greater extent than the right. Heart lesions were not noted In pigs from lots i|. and 5 nor in deficient pigs that had recovered after parenteral treatment with thiamine. by Follis et al. These findings are similar to those described (I9if3) and WIntrobe _et al. (l9il-2) except that fatty changes in the heart were more marked in this series and the neutrophilic exudate described by Follis e_t al. (19U3) was not observed in this experiment. Congestions of the mucosa of the gastro-intestinal tract and mucoid degeneration of the cecum or colon were observed In pigs from lots 1 and 2. No changes were noted in the central nervous system nor in the sciatic nerves. Experiment II The second trial was carried out during the summer months when daytime temperatures were often higher than the controlled temperature of other trials. It is possible that the increased temperature may have had somewhat of a sparing action on the thiamine requirement of the pigs. It is apparent from an analysis of the data presented in table 7 that 1.0 mg of thiamine per kilogram of solids in the diet of SP d ft d ft Cti -P d O o 01 •P © ft ft pH b o ft -P © d © ■p d d *h i>» S 01 © ft O 2 -P -P «0 ft ft rH O 00 cA CA c a CA • • o rH O • l a _ d CM + 1 -H CM > © > d > © © C ■ft rH f t +3 o d © © d 00 «i d ft O ft ft ft 00 f t © K -d • -d o • o +1 l> -d • bO jy "LA • "b i rH - d - d B o o • ca -d CM CM • o CM + 1 CA CA CA • -d CM C*CA • O rH rH - d 1 +1 1 CM rH • "LA 1 C© rH ft •* EH 00 to »H •P f t 00 © Oh +3 O d o d © 1 1 • >—* •' ft 1--1 f t i—1 • ■P • £ +3 rH £ ft ■P •rH d rH © d • ft to © ■p o p 0$ > <1 d Q • © d © ft 9 d ft ft ft > "LA O • O -H ft • ft 00 rH •w ft ^ ft • d ft rH f t al 0 rH bO 01 v— 1>> }> *ft i—I rH © ft ft B d al p f t 00 ft d © • o © © o > < t> < 1 1 • • d ft © rH ft— © ft *h rH O CQ d tH d bO 1 d ft CM +3 o rH © d • d CM © d 43 43 O rH rH © d > © © d h 43 R 3 •h d +3 ft © > ft © o © d rH © > © rH ft © d O rH © d 43 O 43 rH © d o © >> d «H 43 43 d 43 © © ft O d ft © ft +3 f t n © © ft © d d © © ^ rH B d 43 ft d >» © d rH O 43 d • ft d * rH ft © ft © ft o © ft d ft S )ft ft d ft 43 fo)t f£t bO ft © • © d d ft ft 4B © 01 d 43 © d _ rH ft B f t f t d © O *P f t b0 d ft O ft O f—I d LA © © d bO O ft B o © d d la d d d d ft ft 43 © -d d csl to J>>ft b O d d •» ft d c A -d a oi f t d C 93 91 rH O © 43 43 rH f t P> O O < !-P W J J rH OCM c A -d ft © o ft ft ft d O al © s © •r-3 7k the p i g is adequate to promote normal rate and efficiency of gain. The pigs receiving no thiamine in their diet did not show symptoms of a deficiency as early in this trial as in the former trial. One of these pigs died on the 2 8 th day of experimental feeding. Another pig from this lot receiv­ ed therapeutic thiamine injections beginning on the 30th day and was recovered. The other tino pigs from this lot were in a very weak condition at the end of the trial and were sacrificed. Post mortem examination of these pigs show­ ed evidences of thiamine deficiency similar to those pre­ viously described. Two of the lot 2 pigs died a few days after the end of the experimental feeding period and exhibit­ ed these deficiency symptoms also. One pig from each of the upper three levels was also sacrificed and none of these ex­ hibited any thiamine deficiency symptoms. Experiment III A third trial was conducted to aid in determining a re­ solution of the somewhat differing results obtained in the two previous trials. A summary of the growth and feed con­ sumption data is presented in table 8. There were no signi­ ficant differences in mean values of rate or efficiency of gain between any two of the three lots receiving 1.0 mg or more of thiamine per kilogram of solids in the diet. How­ ever, vomiting occurred occasionally in pigs on the 1.0 mg CO © £ S o • eg © ft d P © © •H d to \ o c a eg rg _d- c— o eg \r\ c a bp s © rH © > © rH £ ft c a o 1A • rH o © P ftd © bO © £ ft ft -do o eg o • • o • o o -H _d +1 rH © © -P ft £ © # o O ' vO -d o o rH -d I— I " jifst ct ft £ £ © •H © £ i a rH o CA * O • rH CA CA • • o +1 eA 00 H d -d CO • • eg o eg O o -H -d o eg +| CA 1A • o -H CO -d • eg o • o +i o eg eg i i c a rH >* © d OS © d d CA vO eg ft © © « d rH o X © bO £ © £ (DH !n d d H £ £ © n p 1 1 £ O • • •H ctf £ ft ft P o P>> © • d p < • © a <3 O I 1 © t i „h f t d do© rH ft d s o bO d rH £ H i—I f t a f d P P ft £ d d eg d© ft P r H S ft w al i © ft — 1 ft O © O d rH ft © ft d rH _• - CO O ft ft d © -p ©x -_< P £ « © © £ £ • ^—s i £ £ -p ft CA _d -P d © rH o o• 1— 1 C A 1 +i I o o © © £ ft © O X! © • £ © © ^ d ft o +i o cA -d © O • ft © © ft eg CA o © i— 1 d « EH (—i • * -=f ca o ft IA• p d p O o o cA d O O • *H bO o £ £ + 1 +1 -d 1A 1A O d- CA CO +1 +1 - H cA O'— CT' tft •* P © ft d r- • © £ £ ft © ft © ft £ ft • H d e ft * d eg ft © bO *H ft eg \A o o +i +i +• p d P £ o • eg ft o 1A CO ca • •d in aS P O -d- • d rH '■—^ £ os bO prop © d p P cti O • bO r l rH d O rH d © - rH !>> > > d rH rH © ft ft s as ai P d d © £ • • o © o © > > C £ ft £ H © £ t o bO © *H d © O £ d cti © p d bD d £ ft ft £ ft O d a © d «* £ O ft «=tj O f t C O rH H eg p c a © o CO ft © d 76 level and two of these pigs showed a somewhat reduced appetite late in the experiment. One of these pigs was sacrificed and a fine yellow speckling of the left ventricle of the heart was observed. Thus it would seem that while 1.0 mg of thiamine per kilo­ gram of solids may be adequate to produce normal rate of gain under the conditions of this experiment, it may be inaufficiend to prevent the onset of subclinical pathological conditions in the animal. Post mortem studies on all of the pigs in lots 1 and 2 revealed varying degrees of the thiamine deficiency syndrome previously described. A summary of the pig growth and feed consumption data for all three trials is presented in table 9. Statistical treat­ ment of this data was given, using the method of Snedecor (1946) for analyzing multiple classification variance permitt­ ing the removal of replication error. An analysis of these composited data indicates that under the conditions of these experiments the minimum thiamine requirement of the baby pig lies between 1.0 and 1.5 mg per kilogram of solids in the diet and further suggests that the 1.5 mg level should be con­ sidered a practical minimum thiamine concentration in the baby p i g Ts diet. Since the daily dietary solids intake of each baby pig constituted approximately %% of its body weight throughout the experiments, this requirement value is equiva­ lent to that of 75 meg per kilogram body weight daily report­ ed by Moustgaard (1953) for older pigs. d o o GO p © P Ti M P 4|—1 s O •H P © Pi © P PI PiP > to*§ a) •H o& p p Wp bO o *r4 ft © rH © X> > cd © rOP P O P! © © Pi © © PiP O cp ft*H © *d © P i ca © i —i A at EH p « > © a o• i —1 P P o CA P CM• CM O t +1 I o1P • -d" ■ —• p -—-• p © bo P P ft CO © P. © P p ,Q O Pi 3 u o d © co p 'S t >9 c d o id *23 Q p a H P P Pi P • © > > OP ©•>—' © P © p s ed p T> « d •o © o > <1 -p © ' d as ci at © *d cp C C w w O O aJ P P p P O O P P oO PI K OCA-GhA o © level than lot 3 . The pigs in the second were significantly more efficient in food pigs in the first replication. t(D d •H Pi •H v\ CM « o O • V\ i —1 C\J + • •£H 03 O ct* O £ 3 3 3 pi 9 P a 3»B s: 9 9 9 £ M P ri 9 VA 4=- V*J ro 3* § P 9 O' 3 9 c+ £ 3 3 3 Ct* Cfl 3 Pi O P B 3 P* ct* 9 Pi P B 9 CO 3 P* ct* 3 B 9 *d O QQ P P 3 3 3 9 O 9 9 O B 3 B P> ct* ct* 3 3 ct* t r 9 ct* H * t r 9 O C T 9 9 CO O 9 CO P« 3 P CO Pi S H* p 9 9 B €-»• P P i P P 3 B ct* O 9 H * • p 9 B O ' B ctP» ct* 3 * o ' 9 9 9 CO 9 P B 3 ct* ca P * ct* U) 3 § *< a " d ct* • o 9 9 3 3 H^O 3 -0 & H> P* 9 P * ca *d 3 3 B § pi O' 9 9 c+ 3 9 9 g p H* »d 3 •3 P» M B ^ OS P ct* P» B* CO 9 O H> »-3 P )q 9 gP* 9 ro -o 4? -o ro ro H or M P» I- 1 *o O' »+ 1 + 1 + HP* P» - o P* O VA H P» ro •~0 ro -o id O • O i+ p> ro u> t-t 9 9 P M O p* '— ^—>. »>“-v -0 -0 -o ---- "— M p* H OD vO vO ro - 0 VA 1 + i + 1 + HvO P» P* ro o M -O -J ro —' P« ro P» CD • u>, o Pi p» 9 ctw i+ U) B -o ro P 1 p * p * ro H O v O v O J r* • v£> ro ro VA 03 VA 1 + i + i + H- 1 + s O H p* CD -B~ Os o o H -o -s i > M 03 CD ro v O 1+ H* 1 + P* *4 H P1 O P1 OO p 0s Qq N 15?* oq ca O P* H* Pi 9 **-+. .«—•. —J - 0 ---- B H* V -o ct* B* p* p 3 p* B 9 ro M ro ro 0 3 \A • VA o 1 + H* P* o P* VA H> 9 3 9 B c+ P* 9 < 9 P> 9 83 The pigs receiving no thiamine did not indicate a pro­ nounced bradycardia until after the characteristic symptoms of anorexia, vomiting and reduced rate of gain appeared. This usually occurred when the pigs had been on no thiamine for two weeks and were hence about three weeks old. In each instance the heart rate was reduced further as the degree of thiamine deficiency progressed. The lowest recorded heart rate was 100 for one of the deficient pigs several hours b e ­ fore sudden d eath occurred. Three negative control pigs had died before ECGs could be taken in the final week. The PR interval in the heart cycle is a measure of the time relation between auricular and ventricular contrac­ tions. A lengthening of the PR interval of more then 5>0 percent above normal denotes a delay in conduction from the auricles to the ventricles. culoventricular block. This is first degree auri- The absence of the ventricular phase in a cycle is termed second degree auriculoventricular block. A summary of the PR interval data is presented in table 13 and figure 5* The PR interval was lengthened in most of the deficient pigs. recorded was The longest PR interval .II4. seconds in two of the deficient pigs, and indicates first degree auriculoventricular block. The average PR Interval of the negative control pigs during the final week was 25 percent longer than the average PR interval of each of the other lots. o o o p± p± o o O o o • • • • eg +1 -M +1 to vO A- Ao o •d O • • • *H rH O 03 rA eg o o O t£ o o o IA o • • • • (— i +1 +1 +i NO A- As o O O • • • p •H -p P 0 P <3 * ,0 © <* p •° Tl g p 8 •H *H & 0 P to P

A o o o rH © rH rH <* > |>» 1—1 0 P P 0 eg > A 0 o P P o 0 iP o • • •H «iH P 'd eg rH O o O o * • +i + 1 CO oo o o • • o PP PH i 1 A o o A O O • ♦ +1 +1 vO A- r— o O o • • • AO O o 1—1 o • • +1 + 1 CA o H O • • • TO 'd P o o 0 TO P •H 'd 0 TO TO 0 P S' K 0 TO •H rH Cti > P 0 P P »H PS PH Pi O H 0 rH P 0 fciO ctf o *H P rO at EH Pi O 0 0 & 1— 1 eg A Pi" A P r-i r~ 9 rH 85 Lot 1 10 PH interval (Sec. x 10-2) 1 2 3 k 5 Age in weeks Pig. 5 - PR interval as affected by age and dietary level of thiamine. The interval between the initial and final ventricular complexes is known as the ST segment. This segment should be isoelectric and a great deviation from this indicates anoxia in the myocardium. A great elevation of this segment was found in the ECG of only one deficient animal. apparent in the ECG of pig in figure 13. This is The ST inter­ val represents the period during which depolarization per­ sists during ventricular systole. A lengthening of the ST interval will be noted in deficient pigs in table lij. and figure 6. £ © £ © ft ft •H IA O O O • • CM + 1 60 sO *d o ♦ •H rH O CO o feO rH "LA o -c** • • fc£) rH +1 g vO o • £ *rH rA A- I A I A O O o o O o O o • • • • +1 +1 +1 CO 00 O o o O i—t rH • « • • AO O O O • * +1 +1 • o o i—! 1—1 W • • £ o o rQ O -P o -d -d U \ r - © c* £ © CO o o o o o £> *d •rH O• o • o © o• o • o • £ rH +1 +1 + i +1 +1 •rH £ * vD CO CO o •H »H £ o o o o rH 1—I s •iH • © • * © * © -p co 03 © eo £ ft £ O O •H © o -d -d CO CA £ ft H 60 03 n o o o O o © rH •rH \A o o o o o K • • • © * * • £ © r£ + 1 -P O > +1 +1 + 1 +1 O CM C xO 00 CA CM H © rH rH f t o o O H rH •H • • • • • C* o rH fc> >> ai 1 — 1 £ £ © ctf © > 'LA -d CM O -P -P > £ O rH rH © o o £ © © •H *H id o o o O O O -P • £ • • * « • o +1 + 1 + 1 + 1 + 1 EH vO CO CA CM vO CO o• o O• rH• rH Eh • CO • 1 ft _d O i—i O -P 03 to •H ft © J— \ & <* Eh CA IA O O O O • • +1 +1 CO CO o o • * CM © to of ft o X © © £ o •tH -P 1—I CM CA -d IA £j Q H 87 Lot 1 16 14 ST Interval (Sac. x 10*2) 12 Lot 2 10 Lota 3, U & S 8 6 1 2 3 Age in weeks Pig. - 6 - ST interval as affejcted by age and dietary level of thiamine. The QRS complex occurs at the start of ventricular systole. The length of this interval is prolonged in cases in which excitation spreads by abnormal routes. In these experiments there seemed to be no lengthening of this inter­ val due to thiamine deficiency and it was consistently .03 or •01+. seconds in duration. The QRST Interval Is known as the electrical systole. The duration of this complex is approximately the same as 88 that of mechanical ventricular systele. A lengthening of this period is due to a delaying of the bundle-branch spread of excitation and to a lessening in the repolarizing ability of the myocardium. That this interval is lengthened in animals which are thiamine deficient is shown in table 1$ and figure 7 * 30 Lot 1 26 QRST interval (Sec. x 10r~2 ) Lot 2 22 Lots 3* 1+ and 5 18 1 2 3 1 + $ Age in weeks Pig. 7 _ QjRST interval as affected by age and dietary level of thiamine. The T wave results from a repolarization of the ventri­ cular septum and apical portions of the heart. The end of the T wave coincides with the end of ventricular systole. Thiamine deficiency in the pigs in this experiment resulted in a moderate lengthening of the duration of the T wave p £ © £ © > & d £ •H * -P © © £ £ *H •H g Cl £ © »H •H W r£ £ -P © O £ ftPt •H m o s © d £ w •H rH d rH © P rH t> al © Pf t> i —I O £ • >■> rH P £ © £ d > *«H P © © EH *H CO TJ Cd & o I—1 O o • • C\J +1 -d i—1 • o C\l IA o • • rH +1 -d rH • IA A- AO O O O O O • • • + 1 +1 +1 CA GO rH l — 1 CM H • • • -d o o • +1 o CM • 'LA vO O o o o• • +1 +1 CO CA i—i rH • • AO O • +1 o CM © cA O O • +1 rH CM • r— © rH ,Q d EH £ O o © w IA O o o • • rH +1 -d i—1 • CO CO O o o O • • • +i +1 +1 ( —1 CA CO i—! i—1 CM « • • CO O £ #r-f O * +1 d i —I © CM ca • © © £ IA O 'LA o • • O +1 -d i—i • O -d IA rH o O o O o • • • +i +1 +1 CA CA OO — 1 CM rH 1 • • • A© O O • TO 4*1 •H cA CM rH • d o o > o -d o o o o • • • o +» + 1 -d CA rH rH • » rH CA O o • +1 CA iH • Ai —1 O ■ -d CM • -A rH O • +1 r— CM • 1 IA rH • © £ © p £ •H EH CO 5 Q rH 90 suggestive of a decreased ability of the thiamine deficient pig to bring about a restitution of the polarized state of the myocardium. This effect of thiamine deficiency upon T wave duration is shown in table 16. Table 16 - T wave duration! response in baby pigs to differ­ ent dietary levels of thiamine Level of thiamine in diet, in mg/kg diet 0*0 0.5 1 .06*.005 2 1.5 2.0 .O6t.O05 1.0 .06*.000 .06*.005 .06*.005 .07-.002 .06*.002 .07*.002 .06*.002 .07*.003 3 .07*.00* .07*.003 .06*.00* .06*.003 .06*.002 k • O 1+ • o o -F* Week of age .07*.002; .07*.002 .07*.002 .07*.002 .07*.006 .06*.002 .06*.00* 5 .08*.009 ^Duration of T wave is expressed in seconds. .06*.003 The systolic portion of the cycle increases as thiamine deficiency occurs. This is shown in table 17 and figure 8. B azett’s constant (K; Table 1? - : = JS!) response in baby pigs to different dietary ^RR levels of thiamine Level of thiamine in diet, in mg/kg solids Week of age 0.0 0.5 1.0 1.5 2.0 1 .30*.015 .31*.010 .31*.005 .29*.015 .29*.010 2 •33-.012 .32*.008 .33*.009 .32*.010 .32*.007 3 .33*.005 .33*.007 .33*.012 .3**.009 .33*.010 k .38*.025 .38*.015 .37*.012 .35*.005 .35*.009 5 .39*.020 .39*.011 .36*.013 .35*.009 .35*.008 91 Lots 1 and 2 QfiST -Lots 3, Ij. and 5 vRR 30 ,28 1 2 3 Age in weeks Pig# 8 - Bazett*s constant as affected by age and dietary level of thiamine. It appears tenable from the data presented that elec­ trocardiographic recordings are not the most sensitive test for measuring dietary thiamine adequacy for the baby pig. Both rate of growth and efficiency of feed utilization seem to be more sensitive. However, the ECO does supply infor­ mation which makes possible a more complete knowledge of the thiamine deficiency syndrome in the baby pig. Electrocardio­ graphic recordings from several of the positive and negative control pigs are shown In figures 9, 10, 11, 12 and 13. Pig* 9 - The seven lead recordings made on all electro­ cardiograms. Leads I, II and III are the standard leads. Lead V is the unipolar chest lead and the AVR, AVL and AVF leads are unipolar leads from the right front, left front and the left hind legs respectively. This ECO was taken from positive control pig 15-1 late in the experiment. Heart cycle length Is uniform and waves P, R and T are of positive potential in all leads except the AVR lead in which all these potentials are in a negative direction. Heart rate is 190 bpm, PR time is .08 seconds and QRST duration Is .20 seconds. P and T are barely perceptible in lead III. 92 fir r «L 4 4 1 r:rSm M* $ 4; I z fH: u -AA-i—■— • A s■ —+_ 1 m* 4 1"' W ■ri-‘' .ri '.:I 1 -i— — h —»— I ! 4' L1 1 , A 1: iLd ■N A S«■MAi.wV 1 td\\ K I W A'^" s4 ::f- {4"K 4 •::. 4' :; :'.' ; ■ '••f' -C, S''1 ;f ■■ Jkt V ' - 1 jL A*J- A A aa 'T JL •N A I& J* A A 1 A Ss M * KS -U: :ttl -■ < —1 —!' ,4 m - V :_r-" T\ 4 4 :r .;*" ,,■ r IP Lfft Yfd’-4 ~~Tt :^ ■ I 4-’ fJ' : > 4 .,-1 L. liid .4 ■ •\\i : .i:: r 4; 1= • '4 -Uh.-4 :i -:■ :4 'i■‘ t-■ 1 I 'i Si' 1 ! , , r' :r,!■-,.r:Sr ;; :'.,' IkL J 1 rr#rr i '1 -4 4 'S.!::1• — 1 Wr ikJ■—■▼• *gg|jme ■y dE ft* -v ¥ w "V !T jT rn 4_ 1 a■; ;'"K:i’ fns — n p't$ I S - / . hr— - 4 t . '- .S'4 i-s;! f 1 Lf ~ ::4 4: '' 4ir 4-S_j 1 fia.9 — Electrocardiogram tro l ! i; i _j r.K jrr;s from positive Con­ Pig. 10 - The seven lead tracings on the ECG of negative control pig 15-11 taken late in the experiment after pronounced external thiamine deficiency symptoms were observed*. Second degree A-V block is the most pro nounced manifestation observed. A moderate sinus arrhy thmia and bradycardia are apparent. PR time is .12 seconds, QRST time is .214. seconds. FiS . 10- Electrocar d tog r a m from pig /5~ / a. siAte of acute, thiamin*, deficiency. oJhi/e in Pig. 11 - The heart rates of the positive control pigs remained relatively constant throughout the experi­ ment. In pig 12-1 the heart rate was 180 to 190 bpm in all recordings. The heart cycles are uniform in length. The PR Interval is .08 seconds, the QRST interval is .18 to .20 seconds and the T wave potential is less than .2 millivolts in lead II. First w e e k of e x p e r i m e n t S e c o n d cueek of e x p e r i m e n t Third cueek of experiment Fourth w e e k of experiment Pig.U — Electroca rd io gr am s of positive Control pij / Z - / taken weekly throvghoot the experiment Fig. 12 - Most of the negative control pigs began to show a slight to marked bradycardia during the third week of the experiment. By the fourth week of the experiment the bradycardia was quite pronounced. The heart rate of pig 12-6 was about 180 bpm during both the first and se­ cond weeks of the experiment. By the third week of the experiment the heart rate was down to llj.0 bpm and in the fourth week was 100 bpm. On the final recording the PR time was .111 seconds (first degree A-V block), the QRST interval duration was *34 seconds and the T wave poten­ tial was .3 to millivolts in lead II. 95 F /r st u/eek o f export m e nt S e c o n d w e e k of e xp eriment Third week of exp erim en t Fourth w e e k of e x p e r i m e n t F/g.f& - S / e c f r o c a r d i o g r a m s of negative Control pij tZ- 6 taken uteekfy throuyhoot the OxpeTimentm Fig. 13 - ECG- findings in these thiamine deficient pigs reveal varying degrees of bradycardia in all pigs. A mark ed sinus arrythmia is exhibited by pigs 16— 15-11* and 15-4* First degree auriculoventricular block is apparent in pig 12-6 and second degree AV block is exhibited by pig 15-11. The duration of the QRST interval is excessive in all pigs except 15-4* The duration of the PR interval is greatly lengthened in pigs 12-6 and 15-11. The ST segment is greatly elevated in pig 15-4* T wave potential was ab­ normally high in pigs 12-6, 15-9 and 15-4* J3 ~ E le c tr o c a r d io g r a m s d e fic ie n t P*S3* taken fr»m thiamine I. 9 Pig. 11\. - Two photographs of thiamin®-deficient pig 15-11. The lower photograph was taken a few minutes after the upper photograph. Sudden death occurred ad interim. This pig had been on the negative control diet for 23 days and weighed 2*68 kilograms. This pig had manifested second degree A-V block on the ECG- taken ^ days previously. 98 2017X Fig. 1~> - Heart of pig This pig died after 5 weeks on a regime of .5> mg thiamine per kilogram of solids. A pal© mottling is present, the whole heart is flabby and re­ sembles a myxedematous heart. Heart weight represents 1*38$ of the total body weight. 99 Part III--Pyridoxin© Studies Experiment I A summary of the results of the first pyridoxin© trial are presented in table 18. On the basis of these experi­ mental results and using the rate of daily gain, daily feed intake and efficiency of gain as criteria for determining pyridoxin© requirement, it seems apparent that the minimum B& requirement of the baby pig does not exceed 1 mg per kilogram of solids in the diet. The growth curves presented in figure 16 support this conclusion. 18 Lots 2, 3 and Ij. - — Lot 1 P f ‘ 1 * 8 * 16 1 1 , 1 — 2k 32 Days on experimental diet Fig. 16 - Growth response of baby pigs to different diet­ ary levels of B^. Table 18 - Response of baby pigs to synthetic milk diets taining different levels of pyridoxin© con­ « d •H rH O n r— r H r H © ft ft • t-3 d rH to ft S*. © ft O Si © -p © © -p q ft S H rt •H ft •H o n ♦ *P •ft t>> © o «S > 1-3 Q «! t H ft •H d © © ft © Si o S rH ft S3 © O ft •H S3 bO S3 d o Si o © rH 43 PQ bO •H 43 • ft >i q rH •rH © ft ft •H q o © *H £ q © •H O *H ft ft © q ftft K *H © q ft n-H o w © S i- * ' © b0 © Si © £ O rH © £ o ^ • ^ II 4 3 © H ft ft q f t *— -d~* h q K bO O O • q d © •H *H © S3 S i Si S3 rH O d i>iH O o © • d* • •H S3 fl d f t X • © H-I d b O O -H H-I H-I © CM - d CO H-l rH «H d S3 bQd E © •H © S3 *H CM o rH CO r H • • • • • ■ © Si Si^ © rH d - d O© »>>© *> l» S3 Si ftft d ft cti q o © © o O 00 bO O r I—I S3 © rH S3 ©o © d £ H ©■— • 50 rH bD o g q rH £ • q o \ •H O *H || *rl S3 * S3 43 I —I d ih q X • X ft w rH > O II o — O > d d -H O d > 5 d p-»d © r-* 50 rH •H © •rH © ft ' —' l —I Si Si • Si o ft o © © ft © (—f 51 d f t q Si f t f t t S q o ft ft « ft 50 S3 bO X bO ft O o o £ rH CJ S3 *H S3 o_ S3 o d d «H d K *r l •H ft * CO C o > •rH ^ rH o d d Si d o d 50 ft •H © f o •H *H >>© 5 rH rH i -p Si O ft O ft i —I < * 5 0 0 1 © O ft O rH !» © ft —1 rH © ft Si o Si £ CsO 00 C fctO O S3 O © m © q 43 t>s f>» U o S3 ft b O ft Jidft of rH rH © £ d O S3 o ft *H S3 ft © o bOi—Id rH S3 d o — o c* cK ft *—i rH \_r\ o o « Q d S3 > ft d •H i—I d rH d O bOO d d ft ft rH O • o •H > d ©O rH <3j OS ft £ II © © © rH o d <*! €Xd © ft S> O i—i © rH <> aj <>; f t —' o Si CO ft ft CO I d rH I d • • CM + 1 + 1 < d CM c d d- d cd ctf 43 -d* rH ft © 43 o d - CM CM cd O CM d - O O O O d - S3 • • • • m -d * • * • H-l c d _ d ~ CM +1 + 1 +1 +1 H -I 43 < d CO CO CO o vO H-l -d * f t d< d -d * O Cd CM • • ♦ © • • • Si r ft I d CM d - © t—f rH ft on <* o CM I d rH rH CM ft CM i d O O O o o • • • • » Id • r>5 • H-l rH C\J _d- CM + 1 H-i H-l H-I + 1 r f t CO -d * Cd O H-l vO f t d- o cd -d - o P— o q ♦ • • • © • • © c d 'd i—i i—1 d - o Si ft ft O 1 © Si rH d i rH *«H ft i—t iH -d * d S3 50 •H ra 101 An analysis of the weekly hemoglobin and R3C count values lends further support to the conclusion that 1 mg per kilogram of solids is adequate. All blood samples taken from the negative control pigs from the third week of experimental feeding until their death (fifth week) showed pale microcytic erythrocytes with anisocytosis. That hypo- chromia develops rapidly in pyridoxin© deficiency is appar­ ent from figure 17* * 114- Blood hemo­ globin level (gm/lOOml) 12 — Lots 2,3 and 4 10 8 Lot 1 1 2 3 4 5 Weeks on experimental diet Fig. 17 - Blood hemoglobin response of baby pigs to differ­ ent dietary levels of pyridoxin©. A concurrent experiment, in which two pigs received the positive control level of pyridoxine supplementation and were pair fed with two of the negative control pigs, indi­ cated that the hypochromic microcytic anemia and its subse­ 102 quent effects was not due to the reduced dietary Intake of the negative control pigs but rather was due specifically to the absence of in the diet as shown in table 19# Table 19 - Comparison of rate of gain, feed efficiency, blood hemoglobin level and red blood cell count of positive control pigs pair fed Level of pyridoxine in diet, In mg/kg solids !(. 0 5 Lot number Number of pigs Days on test 3.1(5*0.20 6.75*0.50 6 2 32 3.01*0.11 9 .214* 0.18 .10*0.01 .20*0.01 .23*0.01 2.21*0.10 .23*0.01 1 .18* 0.02 z 32 Ave* initial wt*(lb.) Ave. final wt.(lb.) Ave. daily gain (lb.) Ave. daily solids con­ sumed (lb •) Solids per lb.gain(lb.) Blood hemoglobin level (gm/100 ml)l 8.3*0.50 Red blood cell counts (mi H i on/mm3) 5.914*1.20 H4.l4iO.l4O 9.11*0.30 week of experiment* At no time during this trial were epileptiform seizures * observed in any of the pigs. The pigs in lot 1 began to manifest anorexia during the second week of the trial and reduced growth rate was a proximal sequel. These pigs be­ came weak and emaciated late in the trial and three of them died on the second day following the close of the experi­ mental feeding period. The fourth pig from this lot died two days later* Post mortem studies of these pigs consis­ tently revealed the presence of generalized anasarca. 103 Experiment II Many of the baby pigs started on the second trial were small and weak* None of the pigs did well on the syhthetic milk diet at first and scouring appeared in every pig. This necessitated a drastic reduction in feed offered for con­ sumption In an effort to stop the scouring. Oral doses of terramyein in the milk and drinking water seemed to help stop the scouring. As a result of the diarrhea and the low caloric intake the pigs were in a weak condition. This weak condition persisted and feed consumption remained low or was arbitrarily reduced because of the susceptibility of the pigs to a recurrence of the morbid dlarrhetie state. During the third week of experimental feeding all pigs were given intraperi tone ally two 10 ml doseS of a multiple B-vitamin solution. The B-vitamins were in a sterile saline solution and consisted of 5 mg niacinamide, £ mg calcium pantothen­ ate, 2 mg riboflavin, 2 mg thiamine, 2^0 meg folic acid and meg B ^ P®r ^ solution. No noticeable improvement followed and feed consumption remained low throughout the trial. Consequently, the growth and feed consumption data obtained in this experiment were considered invalid. In this trial epileptiform seizures were observed In the negative control pigs on 29 recorded occasions. These seizures occurred on every observed occasion either immedi­ ately prior to, during or just after feeding. Although the seizure appeared spontaneously, Its onset seemed to be lollstimulated by the excitement or anxiety of the feeding ex­ perience. The seizures were of varying degrees of severity and duration. On each observed occasion the seizure came suddenly, the pig began to shake and tremble convulsively and occasionally threw himself on his back pawing wildly. The seizure would close with the pig stretching himself prone, apparently dead, with foam appearing at the mouth. Shortly thereafter the pig would relax and still later rise weakly and walk over to the feed trough and consume slowly the milk. On no occasion was the duression sustained for more than two or three minutes. Seizures were observed only in the negative control group. Experiment III A presentation of the general results of the third trial is made in table 20. Statistical analysis of the data pertaining to rate of gain, feed consumption and feed efficiency indicate that the minimum pyridoxine requirement of the baby pig is not in excess of .5 mg of solids in the diet. per kilogram However, the subclinical data ob­ tained, i.e. blood hemoglobin level and urine xanthurenic acid concentration, both Indicate that the .5 mg level and possibly the .7? rag level of pyridoxine administration may be too low for the total well-being of the pig. 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CM -d * -d - CA • m -= t - d XA o • • rH o +1 +» vO CA A - CA • • vO rH CA CA O o O +1 H-* CA sO CA CA • m 1A I> - • ,n • H «._^ • rH ■P m bO H £ O CM sO O • • O O +1 +1 1 A rH O -d • • IsrH o ^ a ? o “ £ £ © o £ cxo O '- ' .*d >sP *50 O © P| § • © w jd © n O £ £ P P £ ^ S o o fid © z* •d o .s d d rH rH O f-i •H TJ - bO © £ M O O H rH rH *50 £ © © rH rH rH O O £ r H 'd rH rH rH •rl *H V t P © O •O *H CO 11 10 Blood hemo­ globin level (gm/100 ml) 9 8 Lot 2 iOt 1 6 1 2 3 *1- S Weeks on experimental diet Fig, 19 - Blood hemoglobin response of baby pigs to different dietary levels of pyridoxine. 108 Differences in blood hemoglobin level and urine xan­ thurenic acid concentration between the lot receiving no pyridoxine and lots receiving pyridoxine were present before two weeks of the trial had elapsed. As the trial progress­ ed, differences developed in both blood hemoglobin level and urine xanthurenic acid concentration between lots receiving the various leyels of dietary pyridoxine. Lot 1 Urine XA concentration (mcg/ml.) 60 Lot 2 20 Lot 3 Lots & 5 Weeks on experimental diet Pig, 20 - Urine xanthurenic acid concentration of baby pigs on different dietary levels of pyridoxine. 109 Only pigs in lot 1 exhibited epileptiform convulsions during the course of the esqperiment. Vomiting with expul­ sion of copious amounts of a thick yellowish-green fluid was an occasional occurrence in the lot 1 pigs as the de­ ficiency developed. One of the pigs from this lot died on the final day of the experimental feeding period and all other pigs receiving no pyridoxine were weak and emaciated at this time. One of the three remaining pigs was autop- sied In this weak condition. Generalized anasarca was evidenced by both these pigs when post mortem examinations were made. There was an excessive amount of peritoneal fluid present and both the liver and heart were somewhat fatty. The remaining two pigs from lot 1 were placed on the positive control diet and one of them received a single intraperitoneal injection of $0 mg of pyridoxine. Within a few days the anorexia subsided and normal gains followed in both pigs. By the end of the 2J+-day recovery period both pigs were making excellent gains. The blood hemoglobin value and RBC count rose to normal within one week in the case of the pig which received the B& injection and within two weeks in the case of the pig receiving only the dietary source of pyridoxine. Neither of the pigs had a recurrence of the epileptiform seizures after treatment commenced and the urine xanthurenic acid excretion dropped sharply. The recovery of the pig receiving only the dietary source of 110 pyridoxine is shown in figures 21 and 22. The pig which re­ ceived the injection was sacrificed at the end of the 2l|-day recovery period. Post mortem examination revealed no ab­ normalities. - Urinary XA ---Body weight ---Blood hemoglobin 60 -- Body weight (pounds) 20 added and Urinary XA concentration Blood hemoglobin level (gm/lOOml) (mcg/ml) 12 v 12 Weeks on experiment Fig. 21 - Response of negative control pig kh-3 to diet­ ary supplementation of pyridoxine. Data are presented in table 21 giving a general com­ parison between pair fed positive and negative dietary pyri' Ill Fig* 22 - Two photographs of negative control pig The upper photo was taken at the end of the 32 day experi­ mental feeding period when the pig weighed 9 pounds* The lower photo was taken after 3 weeks of dietary pyridoxine treatment at which time the pig weighed 22 pounds. 112 doxine control pigs. A statistical analysis of this data was not made because of the small number of animals. How­ ever, the data do show that the limited fed pigs receiving pyridoxine gained 50 percent faster and hence 50 percent more efficiently than those pigs receiving no pyridoxine. Furthermore, the pigs receiving pyridoxine showed normal blood hemoglobin level and RBC count indicating again that the oligocythemic microcytic hypochromic anemia which de­ veloped in the negative control pigs was a specific effect of pyridoxine deficiency and did not result from the ensu­ ing anorexia. Table 21 - Comparison of rate of gain, feed efficiency, blood and urine data of positive control pigs pair fed with negative control pigs Level of B£> in diet, in mg/kg solids 0 Lot number Number of pigs Days on test Ave. initial wt. (lb.) A v e . final w t . (lb.) Ave. daily gain (lb.) Ave. daily solids con­ sumed (lb.) Solids per lb. gain (lb.) Blood hemoglobin level (gm/100 ml)1 Red blood cell counts (million/irai?)1 Urine xanthurenic acid _ concentration (mcg/ml) 7 2 3.35-•09 7.09±.l|-8 .12*.01 .19*.02 1.38±.o5 32 6.l4±.l 5.22*.01 6 2 32 3.37*.00 9.13*. 61+ .18*.02 .19*.02 1.03*.02 12.2*.1 6.86*.20 37*13 ^Blood hemoglobin and RBC count values taken during 5th week of experiment. ^XA excretion values taken during )+th week of experiment 113 Lymphocyte production appeared to be impaired in the acutely deficient pigs in the pyridoxine trials. This im­ pairment of lymphocyte production was not pronounced until the Ipth week of the experiment and occurred only in the ne­ gative control group. This is shown in table 22.. Table 22 - Lymphocytel response in baby pigs receiving _____________different dietary levels of pyridoxins2 Level of B& in diet, Pair o in mg/kg solids _ fed3 0.0 2.0 1+.0 1.0 0.5 0.75 8 8 8 No. of pigs 3 1+ i+ 3 1st week 55*1 59*3 53*6 59*2 59*3 63*5 58*5 2nd week 57*3 5i+*l+ 62*9 57*10 58*3 52*5 1+7*1 57*8 3rd week 52*1; 56*8 51+*5 56*5 56*5 60*1+ 62*5 1+1*5 57*1+ 60*2 52*2 55*3 62*5 55*1+ 5th week 33*6 59*1+ 63*3 60*5 60*5 61+*1+ ^Lymphocytes expressed as percent of total WBC count. ^Trials one and three combined. 3Positive control pigs pair fed with negative controls. ipfch week Conclusions F r o m an examination of the results of this study one may reasonably conclude that under the conditions of these tr i a l s : 1* The minimum riboflavin requirement of the baby pig approximates 3*0 mg per kilogram of dietary solids intake. 2. The riboflavin deficiency syndrome in the baby pig consists of a rough haircoat, a heavy sebaceous exudate on the skin, decreased appetite, decreased growth rate, lens cataracts, cecal and colon mucinous degeneration and rectal hemorrhage. 3. Riboflavin deficiency symptoms in the baby pig may be rapidly alleviated by riboflavin supplementation. Ij.. The minimum thiamine requirement of the baby pig approximates 1.5 mg per kilogram of dietary solids intake. 5. The thiamine deficiency syndrome in the baby pig consists of severe anorexia, vomiting, emaciation, weight, cyanosis of nose and skin and sudden death. loss of Common gross finding are a pale yellowish-gray mottled heart with flabby ventricles and a myxedematous appearance, excess of pericardial fluid and inflammation of the cecum and colon. Microscopic examination reveals necrosis of the cardiac muscle fibers, fatty degeneration in the heart tissue and 115 mucoid degeneration of the cecum and colon. graphic studies reveal bradycardia, Electrocardio­ sinus arrhythmia, auri- culoventricular block and lengthening of the PR and the QRST intervals. 6. Blood thiamine levels are low. Good gaining ability and general well-being may be rapidly restored to thiamine deficient pigs which receive thiamine therapy. 7. The minimum pyridoxine requirement of the baby pig approximates 1.0 mg per kilogram of dietary solids intake. 8. The pyridoxine deficiency syndrome in the baby pig consists of anorexia, reduced growth rate, vomiting and epi­ leptiform seizures. The most common gross autopsy finding is a generalized anasarca. Blood studies reveal a reduced hemoglobin level, microcytosis, anisocytosis, oligocythemia and decreased lymphocyte production. Urine analysis reveals a greatly increased level of xanthurenic acid excretion. 9. 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