"I JM' ) 'IHIIHIHWI’II'HI I'll WI 'i M W 'I lc TH _ EFFECTS CF NUTRETEQNAL DEFECEENCIES EN YGUNQ RATS ON GONADAL RESPONSE TO FREGPQANT MARES' SERUM ‘fl'mais {m the» Degree of M. S. MECHESAN STATE CDLLEGE Krishna fiahari Yadu $950 k2. -ar. rev 1...; “‘7 “"1“ i: .‘I -‘ ‘1 AA): 53’ ‘5‘: i 1 d .n 'f. .- a; , as“. l ,h-' u , J I I £ 1": This is to certify that the thesis entitled Effects of Nutritional Deficiencies in Young Rats on Gonadal Response to Pregnant Mares' Serum presented by Krishna Behari Yadu has been accepted towards fulfillment of the requirements for MOS. degree in Ph2310108y or professor C, Due May 25; 1950 EFFECTS OF NUTRITIONAL DEFICIENCIEB IN YOUNG RATS ON GONADAL mPONSE T0 PREGNANT MAREB' SERUM By Krishna Behari {gdu A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology and Pharmacology 1950 mass ACKNOWLEDGMENT The author wishes to express his sincere gratitude to Dr; Joseph Meites, Associate Professor of Physiology, for his wise guidance throughout the course of this work, for his critical readings of the manuscript and for his generous contributions of time, energy and ad- vice. Thanks are due to Dr. B.'V. Alfredson, Head of the Department of Physiology and Pharmacology, for providing the facilities of the department in conducting this'work. The author also wishes to express his appreciation to Drs. E. P. Reineke, L. F.'Wolterink, and W3 D. Collings for their valuable suggestions. The author is highly grateful to the Government of India and the Government of Central- Provinces and Berar (madhya-Pradesh) for the grant of the scholarship without which it would not have been possible to carry out this project. Acknowledgments are also due to Dr. D. F. Green, of Merck & Co. Inc., for many of the vitamins used in these studies; to Dr. T. H. Jukes of Lederle Laboratories Division for the folic acid antagonist (x-methyl-folic acid); to Dr. R. 0. Roblin, Jr,, of American.Cyana- mid Co., for the biotin-antagonist ( 4-3,4-ureylenecyclohemyl butyric acid); to Dr. C. A. Hoppert of the Chemistry Department, Michigan 'State College, for a generous supply of his rachitogenic diet; and to Dr. C. F. Huffman of the Dairy Department, Michigan State College, for several crystalline vitamins. Last but not least, the author udshes to acknowledge his sincere appreciation to Mr. John Monroe, for his helpfulness in the care of the animals. ssssssstss #**#**** ****** mess st up 6? 5 _‘ (‘9 Gm, #qu '3. TABLE OF CONTENTS Page INTmDUCTIONOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO00...... Oi REVIEW OF IIITERATUREQQOOOOOOOOOOO0.0.0...0.0000000000000000... underIIUtritioneeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 3 F0113 Reideeeeeeeeeeeeeeeeoeeeeeeeeeeeeeeeeeeeeeeeeeee 5 RibthVineeoeoeeeee00000000000000.0000.000.0000000000 7 Thimneeeeeoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 9 Pantothenic aCldeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 10 PyridOXiJleoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 12 vitamin ECOOOOOOOOOOOOOOOOO00....OOOOOOOOOOOOOOOOOOOOO 1-3 Vitamin Aeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 15 Vitamn Deeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 16 PrOtelneeeeeeeeeeeeeeeeeeeesoeeeeeeeeeeeeeeeeeeeeeeeee 17 PROCEDUREeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeo 18 RESULTS...""....................o....o.....................o 20 Effects of reduced caloric or protein intake on semi- nal vesicle response to P.M.S. in Michigan State COlloga mtseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 20 Effects of vitamin deficiencies on seminal vesicle response to PALS. in Michigan State College rats..... 20 Effects of folic acid antagonist and paired-feeding on seminal vesicle response to PALS. in Garworth rats... 23 Effects of folic acid antagonist and paired-feeding on seminal vesicle response to P.M.S. in Camrth rats... 24 Effects of riboflavin deficiency and paired-feeding on seminal vesicle response to PALS. in Carworth rats... 27 DISCUSSIOIIOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0000...OOOOOOOOOOOOOOOO 29 WWEMOOCOOCOOO0....OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOOO... 32 BIBI‘IOGRAPHYOOOOOCOOOOOOOOOOOOOOOO0.0.0.000...OOOOIOOOOOOOOOOO 35 INTRODUCTION A considerable but unresolved literature exists on the relation between nutritional deficiencies and reproductive disorders. Inasmch as the reproductive system is largely under the direct control of the endocrines, it might be expected that the effects of dietary defi- ciencies would be manifested through the hormones. Thus, the secretion of certain hormones may be reduced, the reaction of target organs to these hormones may be decreased or the metabolism of these hormones my be altered. With the conceptions of deficiencies of vitamin E and perhaps A, none of the other vitamin deficiencies have been shown to produce any specific effects on reproductive function. There is ample evidence that starvation depresses reproductive activity, and this is believed to be mediated through a depression of anterior pituitary function. It seems probable that new of the reported deleterious effects of vita- min deficiencies on sexual function were actually due to the concomi- tent inanition produced rather than to any specific vitamin lack per so. In view of the foregoing, it was of particular interest to read the recent work of Hertz (1945, 1948). This investigator and his col- laborators demonstrated that a deficiency of folio acid in chicks, rats and monkeys interfered with the growth-promoting effects of estrogens on the cviducts of these animals. Stimulated by these reports, it was decided to determine whether diets deficient in folic acid or in other factors would interfere with the action of a gonadotrophic hormone on the saninal vesicles and coagulating glands of young male rats. -1- Young mle weanling rats were placed on various deficient diets for a period of twenty-eight days, and during the last four days were injected with a constant dose of pregnant mares' serum. This gonado- trophic substance contains a predominance of follicle stimlating hormone, but also considerable amounts of luteinising (interstitial cell stimulating) hormone. The rats were sacrificed and the combined weights of the seminal vesicles and coagulating glands were used as a measure of gonadotrophic activity. For the most part these studies were largely confined to the effects of B-vitamin deficiencies on gonadotrophic action. Although not all the deitamins deficiencies were induced, it appears on the basis of the data obtained that specific B-vitamin deficiencies did not interfere with the action of pregnant mares' serum except to the ecrtent that they induced decreases in feed intake. The reduced feed consump- tion rather than any specific vitamin deficiency appeared to be re- sponsible for the inhibition of gonadotrophic activity observed in these animals . REVIEW OF LITERATURE This review does not attempt to cover all or even most of the literature available on the interrelationships between nutritional de- ficiencies and reproductive function. Rather, it is an attempt to cover principally such literature as is pertinent to this thesis. Each of the nutritional deficiency-reproduction interrelationships is dealt with under a separate heading, although it is obvious that nutritional factors all influence each other to some extent. Undernutrition Loeb (1921) noted atrophic changes in the ovaries of guinea pig during chronic underfeeding. Narrian and Parkes (1929) noted ovarian atrophy and anestrus during partial inanition in rats. Mbore and Samuels (1931) reported the presence of castrate types of prostates and seminal vesicles during partial inanition in rats. Brencman (1940), Selye and Collip (1936) Kursrok et al., (1940) and Stephens and Allen (1940) all agreed that atrophy of the sex organs and anestrous conditions in.underncurished animals resulted from diminished production of hype- physeal gonadotrophic hormones. Stephens (1941) reported that chronic undernutrition is accompanied by structural and functional alterations in.the hypophysis which result in decreased pituitary activity. He reported that in female guinea pigs, undernutrition of sufficient degree to cause a loss of twenty to thirty percent of body weight in a period of two weeks, resulted in atrophic and retrogressive changes in the ovaries similar in character and degree to those following hypophysectomy. These changes were not affected by -3- administration of vitamin supplements but were reversible with refeed- ing. The ovaries of the undernourished guinea pigs remained responsive to stimulation.by at least one of the gonadotrophic principles (LB) of the anterior pituitary. These observations suggested that changes 4 occurring in the ovaries during inanition may be due partly to inability of the anterior pituitary to continue to produce sufficient gonadotrophic hormone to maintain.the normal structure and function of the ovaries. (Stephens 1941);. Mhlinos and Pomsrantz (1939) reported that adult female rats which were acutely starved or chronically underfed ceased to have estrus when the body weight had dropped about fifteen percent. They found that rate could be brought to estrus by injections of estrogen even though the body weight was reduced by inanition to forty percent of normal. Injec- tions of gonadotrophic hormone also brought these starved female rats to estrus. This indicated that the non-appearance of estrus may have been due to diminished production of gonadotrophic hormone rather than to inability of the ovaries to respond. Mhlinos and Pomerants (1940) also studied the effects of inanition in malerrats. Complete inanition unto death resulted in the destruction of an occasional seminiferous tubule, whereas prolonged chronic inani- tion resulted in severe atrophy of the testes and accessories. This picture resembled exactly that described by Smith (1930) in adult male rats which had been hypophysectomized. The testes and accessories'were only about one fifth the normal weight in both conditions. The production of spermatozoa was abolished after hypophysectomy and diminished or absent -4- during inanition. There was generally no destruction of Sertoli cells or spemtogonia in either condition. 1 Both Mulinos and Pomerantz (1940) and Breneman (1940) presented evidence indicating that the reduction in size of, the gonads and acces- sories during inanition is not due to loss of sensitivity by these organs to hormone stimlation. The former worker showed that injections of gonadotrophins' into starved nle rats reinitiated both spemtogenesis and androgen production. Breneman (1940) reported that injection of testosterone into underfed chicks produced a greater comb response than in noml chicks. Folic acid " Hertz and Sebrell (1944) first noted an impairment in the response of the oviducts of chicks to diethylstilbestrol when fed a diet defi- cient in folic acid. This failure of estrogens to induce oviductal growth in the absence of folic acid has also been observed in the monkey (Hertz, 194s), frog (Goldsmith et al., 1948) and rat (Hertz, 1949). Andrus and Zarrow (1949) noted that estradiol, given to normal chicks, caused a sixty-eight percent increase in oviduct weight but had no effect on the weight of the oviducts of chicks receiving folio acid antagonist. Phosphatase occurred to a marked degree in the oviduots of the control chicks and only (to a slight degree in the birds receiving the antagonist. Hertz (1945) reported a quantitative relationship betwem the stil- bestrol response and dietary folic acid in the chick. Chicks maintained on stock diet showed a forty-fold hypertrophy of the oviduct following stilbestrol administration, *whereas folic acid deficient chicks showed I -5- only a four-fold increase in oviduct weight. In folio acid supplemented chicks, the degree of oviduct-increase varied directly with the level of folic acid ingested. Doses of folic acid considerably in excess of that required for growth and hemopoiesis failed to restore the oviduct response to the level obtained in chicks on a stock diet. Concentrates containing folic acid from yeast and spinach similarly failed to complete- ly replace the stock diet, but liver fractions increased the oviduct weight up to the same levels obtained on the stock diet. Riboflavin, pyridoxine and pantothenic acid deficient chicks exhib- ited optimal oviduct responses, indicating that the failure of the estro- gen response in folio acid deficient chicks was not simply a corollary of arrested body growth and general debility. Hertz (1948) confirmed the above results in monkeys. Of eight sexually inmture monkeys mintained on a folio acid deficient synthetic diet, six failed to show the characteristic nonnal response to estrogen administration. Two control monkeys fed the same diet plus liver, showed typical estrogen responses. Hertz and Tullner (1949) studied the quantitive interference by folic acid antagonist to estrogen-induced tissue growth in rat and chicks. They used eight types of chemically pure folio acid antagonists. Some of the antagonists were relatively more effective in inhibiting the genital tract response to estrogen injections than others. Haque et al., (1949) studied the effects of various vitamin defi- ciencies on the response of the oviducts of chicks to estrogen. They found a nonml response in all of the vitamin deficiencies studied, except in the case of folic acid. The response of the oviducts of the folio acid deficient chicks was less than one half that of the efiducts of the chicks receiving the nornal diet. This confirm the observations of Hertz and Sebrell (1944) and Franklin et al., (1949). Goldsmith et al., (1949) reported on the effects of folic acid deficiency in mice. The seminal vesicles and coagulating glands of testosterone-treated nice on a control stock ration showed the charac— teristic hypertrophy resulting from the androgen treatment. The other group was fed sixty grams of folio acid antagonist for sixty days and showed a reduction in response to testosterone. Therefore these authors concluded that the vitamin antagonist could interfere with the stimula- tion of the male sex accessories by high doses of rule sex hormone. These authors in a private oommication (1949), came to the con- clusion that the reduced growth response of the sale accessories to testosterone noted in their folio acid deficient mice was probably not due to a lack of folic acid Jar so but rather to the concomitant inani- tion. It will be seen later that this view coincides with the results reported in this thesis. Riboflavin Warkany and Schraffenberger (1943) reported on reproduction of fanale rats fed a purified diet without riboflavin. Twenty-one female rats of the Sprague-Dawley strain, reared on an adequate diet and weigh- ing from 170-240 gms. were put at the age of three months, on a purified diet lacking in riboflavin. -7- / The female rats on the purified diet lacking in riboflavin showed regular estrus cycle for several weeks. Nineteen females became preg- nant within three weeks. Eleven pregnancies ended in resorption.while eight females delivered their litters at term. The pregnancies caused such great losses in eleven animals that they were considered unfit for further breeding. Of the sight that remined in the experiment four did not show estrus cycle within eight weeks after the first pregnancy. Four rats underwmt a second pregnancy. One of these resorbed, while three delivered their litters at tenm. They repeated these experiments in several other strains of rats' and noticed the same congenital malfornntions on the same purified diet lacking in riboflavin. About half of the female rats stopped having estrus cycles, lost considerable weight and showed signs of "malnutrition“. They were an- able to "restore" such animals by giving small doses of riboflavin, but these were unable to reproduce especially when their weight was reduced from the normal average of 200 gms. to 175 gm. or lower. Riboflavin administration caused some gain in'weight‘within.twmnty-four hours, but after two or three days there was no further increase in.body weight. These authors concluded that the abnormalities described above'were caused by a prenatal riboflavin deficiency. The mothers apparently had sufficient riboflavin for’naintenance of estrus cycles and for gestation. The fetuses had sufficient riboflavin for growth but not for differen- tiati on. -8- Thiamin bhson.(1929), Evans (1929) and Parkes (1927) studied the effects of various vitamin deficiencies on the mammalian reproductive system. many rats were kept on a diet deficient in the antineuritio factor. They found that the testes of such rats were essentially normal, histologically, but the prostate gland and seminal vesicles were castrate in.type as if the testicles had been removed some weeks before. It was obvious that the male reproductive accessories were incapable of reacting to the testi- cular hormone because of the general decline induced by the deficient diet. Despite the fact that some animals had suffered a weight loss of forty percent, the animals on deficient diet were essentially normal in gross size and appearance. These organs, forche most part, contained histologically normal seminiferous tubules. The majority of the tubules not only showed quantities of mitotic figures but also a normal content of spermatozoa. The majority of rats at death contained spematozoa in the epididymis that were capable of motility when.suspended in normal saline solutions. The interstitial cells appeared somewhat atypical in that the cytoplasm was greatly reduced in amount and nuclei were some- what atypical. An examination of other rats made it apparent that similar conditions were produced on an inanition diet with an excess of vitamin B, as well as on unlimited diets lack ng in vitamin B. In each group the testes were normal in size, and seminiferous tubules were in active spermatogenesis and showed but little or no destruction of germinal epithelium. The prostate gland and seminal vesicles however, were castrate in type in ‘most of the animals under experbmeat. The authors drew the conclusion that a general nutritive deficiency is basically responsible for the peculiar hormone state of these animals rather than any specific effect attributable to the absence of the antineuritio factor. To summarize, the testes of male rats kept upon a diet deficient in.the antineuritic fraction of vitamin B or on insufficient diets con- taining an excess of vitamin B, were found to consist of normal semi- niferous tubules in active spermatogenesis but the reproductive accessor- ies (prostate gland and seminal vesicles) were castrate in'Qype. Daily injections of either testes or anterior pituitary hormone from pregnancy urine, caused the castrate condition to'be replaced by the normal state within a period of ten days. Drill and Burill (1944) noted that the absence of thiamin.in.a diet otherwise adequatelcauses a progressive decrease in food intake, result- ing in loss of body weight and accompanied by cessation of estrus cycles. jf The same loss in body weight and cessation of cycles occurred even when thiamin was given, if the total food intake was reduced to correspond with that of the thiamin-deficient rats. Thus the effect of thiamin defi- ciency on.the ovaries is not one of the thiamin deficiency per se, but rather due to the resulting inanition. Pantothenic acid The importance of pantothenic acid deficiency for normal reproduction and embryonic development in the chick has been stressed by several in- vestigators (Bauernfiend and Norris, 1939; Gillie, Heuser and Norris, 1941: -10- and Pearson et al., 1945). Little is known about the effects of panto- thenic acid deficiency on reproduction in rats. In general, it any be said that in chronic nutritional deficiencies with prolonged survival, the estrus cycle is irregular or absent (Evans and Bishop 1922). In severe, acute deficiency of pantothenic acid it is known that the mjor— ity of animals do not mimre before they die (Figge and Allen 1942). Jukes (1940) mentioned the occurrence of resorptions in part of a group of stock rats placed on pantothenic acid deficient diets at mating. Sure (1941) noted that reproduction on purified diets without the addi- tion of calcium pantothenate was very abnormal, since either “sterility“ of the mother or a high incidence of stillbitths in the young resulted. Nelson and Evans (1946) studied the reproductive phenomena in partial prolonged pantothenic acid deficiency in rate. Normal femle rats, two to five months of age, were placed on pantothenic acid deficient diet on the thirteenth day of gestation. No significant difference in reproductive performance was noticed when compared to control groups. When the female rats were kept on an experimmtal diet starting the first day of gestation, marked upsets in reproductive function, was noticed. Approximately one third of the rats underwent resorpticns instead of littering. This confirmed the findings of Jukes (1940). The reminder of the rats littered but the average weight of the young at birth was significantly decreased. Nelson and Evans (1946) also ran pair-fed control groups. This ea:- periment indicated that in the deficient group three rats failed to implant, four rats resorbed and four rats littered. In the control group -11- restricted in calories (pair-fed), only two rats failed to implant and the remainder of the rats cast litters. The reduction in calories varied from forty-three percent to eighty-four percent. These workers concluded that their experimental data indicated that the disturbance of reproductive phenomena in the deficient ferale rats was due to pantothenic acid deficiency, and not due to inanition. It is of interest that many different conditions have been reported to cause resorption in the pregnant female rat: inanition (Barry, 1920) Vitamin E deficiency (Evans and Bishop, 1922), vitamin A deficiency (Sure, 1928), a deficiency in essential fatty acids (Burr and Burr, 1930), a low protein intake (Guilbert and Goss, 1932), vitamin D deficiency (Demo, 1934), filtrate factor deficiency (pantothenic acid) deficiency (Jukes, 1940) and riboflavin deficiency (Warkany and Nelson, 1942). In some of the studies reported the interpretation of results is difficult because of the concurrent effects of inanition and of one or more speci- fic di etary deficiencies . aridoxine Nelson and Herbert (1946, 1947) reported that when pyridoxine me omitted from a purified diet, starting on the day of breeding in rate there were only slightly adverse effects on reproduction. This was evi- denced principally by the occurrence of resorption in about ten percent of the rats. When the rats were placed on this pyridoxine deficient diet ten to twenty days prior to breeding, marked upsets in reproduction were observed. The percentage of resorptions increased directly with extension of the deficient period. One hundred percent of the rats showed resorption when the pyridoxine deficiency was extended to twenty- two days before breeding. Vitamin E A deficiency of vitamin E in males induces degenerative changes in the germinal epithelium and disappearance of spermatogenesis without apparent morphological alteration of the intertitial cells. The failure of vitamin E deficient female rats to become pregnant is apparently due to disturbance of the implantation process rather than the absence of ovulation; there is no direct proof of ovarian dysfunction (Kaunitz et a1. , 1947; Blandau et al., 1949). Since normal function of the ovaries and testes is depmdant upon the anterior pituitary, several authors have compared the gonadotrophic potency of the anterior pituitary of normal rats with those on diets deficient in vitamin E. In sales, Van Wagenen(1925) found castration changes in the basophils of the gland. Her results were extended and confirmed by Koneff (1939). The anterior pituitary of female E deficient rats showed morphological changes characteristics of spaying, according to Joel (1943). Both Koneff (1939) and Joel (1943) described a decrease in the number of oxyphils (acidophils). Nelson (1931) reported that the increase in the gonadotrophic potency of the pituitary of the deficient E males approached that of the glands of castrates. He detected no difference in the amount of gonadotrophin in the pituitaries of nornnl and E deficient females. However McQueen- Williams (1934) concluded that glands from E deficient female rats -13- contain about twice as such gonadotrophin as those from normal rats. In contrast to the foregoing reports, Biddulph and Meyer (1941) stated that pituitary powder from vitamin E deficient male or female rats produced the same weight change in the ovaries of hypophysectomized rats as pituitary powder from normal rats of the corresponding set. However, they believed that male pituitaries from E deficient rats con- tained an increased amount of luteinising (interstitial cell stimlating) hormone. Rowlands and Singer (1936) found that the pituitaries of E deficient femle rats contained less gonadotrophin than normal rats, as determined by the production of ovulation in estrous rabbits. P' an et a1.,( 1949) noticed that the weight of testes of immature hypophysectomized rats which received injections of anterior pituitary glands from either male or female vitamin E deficient rats was greater than those of rats receiving corresponding doses of the gland from noml male or fmle rats. These findings however, did not indicate whether the increased gonadotrophin content of the anterior pituitary of the vitamin E deficient rats was due to an increased production or to a de- creased secretion or both. Ever since Evans and Bishop (1922) established that rats require vitamin E for successful reproduction, the possible relationship of this factor to breeding troubles has been a subject of much interest to live- stock breeders. There is a vast literature concerning vitamin E in its relation to reproduction in cattle. This has been recently reviewed by Asdell (1949). The information available indicates that it has not been established that cattle and other ruminants need vitamin E for successful -14- reproduction. It is significant that most of the studies reporting im- provement in reproducing ability following vitamin E administration were conducted over relatively short periods with cattle on manual rations (Bay et al., 1934; Gunn, 1941; Jones and Ewalt, 1936). Frequently the treatments followed or occurred simltaneously with other forms of therapy. Gullickson et al., (1949) performed eocperiments in Holstein and grade Jersey cattle to determine whether vitamin E is needed for success- ful reproduction. They fed the animals throughout their lives on rations containing liberal amounts of all known essential nutritional factors except vitamin E. They fed thirty calves of mixed breeding, twenty-two females and eight males. The data showed that reproducing ability of the cattle was not affected adversely by feeding vitamin E poor ration con- tinuously through three generations. However, about a dozen animls died suddenly from the characteristic heart ailment described by Gullickson and Calverlq (1946). Vitamin A The course of pregnancy nay be severely altered by feeding female rats a diet restricted in vitamin A. The severity of the alteration appears to be roughly proportional to the degree of vitamin deprivation. Death and resorption of the fetuses, prolongation of pregnancy and deliv- ery of stillborn young were reported by Mason (1935) and Cannon (1940), who employed deficient diets with little or no supplanent of vitamin A or carotene. Warkany amiSdiraffenberger (1944, 1946), using similar diets supple- mented with smll amounts of carotene, also noted a high incidence of -15.. fetal resorption in rats. By interrupting pregnancy at various times from the thirteenth day to term, they were able to obtain a number of living and recently dead fetuses suitable for histologic studies. The eyes of these offsprings were found to bear several developmental anom- lies, notably: folding and eversion of the retina, persistence of the choroidal fissure, absence of the ciliary both, and postlenticular fibro- plasia. Thus deficiency of vitamin A in the diet of female rats prior to and during pregnancy not only reduces fecundity by causing fetal death and resorption, but also causes developmental anomalies in new of the fetuses that escape intrauterine death. Vitamin D Haque et al.,(1949) used chicks in experiments to find out whether vitamin deficiencies would interfere with the response to rule and female see: hormones. The vitamin deficiencies were induced by giving low levels of a particular vitamin for a period of about six weeks. Male and female sex hormones were injected every alternate day during the last twelve days of the experiments. Testosterone propionate and alpha-estradiol were used as the sex hormones. Forty-eight hours after the last injection the birds were killed, and the combs and testes of mles and oviducts of females were removed and weighed. The results indicated that comb-growth by testosterone propionate was not prevented by am of the vitamin deficiencies except in the case of vitamin D. These authors suggested that the slightly smller response in comb growth from testosterone propionate in) actions in the absence of vitamin D indicates that this vitamin is necessary to secure full andro- genie stimulation. -15- Protein Yamamoto and Chow (1950) studied the effects of low'protein and poor protein diets on the ability of the gonads of rats to react to in- jections of gonadotrophic hormone. They found that the degree of hyper- trophy of the sex organs was related to the nutritive value of the protein and to the amount of protein intake. Low protein or poor quality protein intakes resulted in poor gonadal responses to gonadotrophic hormone. It will be seen that these results, reported only in abstract form, are similar to the findings in the rats on low protein diets reported in this th“1.e -17- PROCEDURE The experiments reported were performed on weanling mle rats of the Michigan State College and Carworth strains. In each experiment uniform groups of rats were placed on special diets for a period of twanty-eight days. During the last four days of the experimental period, all except the control rats were each injected with one-half Cartland- Nelson unit (ten International Units) of pregnant mares' serum (P.M.S.). The animals were sacrificed on the twenty-eighth day, and body weights as well as wbights of the seminal vesicles and coagulating glands were recorded. The testes weights were not recorded, since previous work (Meites and Chandrashakar, 1949) had indicated that P.M.S. had little effect on testes size. A semi-synthetic diet was used in all except a few preliminary ex- periments inwhioh it was desired to determine the effects of a commer- cial diet (Purina laboratory Chow). The composition of the semi-synthe- tic diet was as follows: Cerelose ....................... 62 gm. Alcohol washed casein........... 25 " Corn oil........................ 5 " Salt Mix. HO 2.................. 4 : COd liver Oilseeeeeeeeeeeeeeeeeo 5 Choline".e....................e 100.0 ms Ca Pantothenate.....e........... 2.8 I Niacineeeeeeeeeeeeeeeeeeeeeeeeee 1.0 R RibOfIQVInseeeeeeeeeeeessseeeeee 005 N Thimmeeeeeseeeeeeeeeeeeeeeeeee 0.2 ' Pyrldoxinou..................ee 002 0 2,Me,1,4 Napthoquinone.......... 0.04 " The effects of low calorie intake on the response of the seminal . J. vesicles and coagulating glands to P.M.S. were determined by giving -18.. experimental groups of rats either 3/4 or 1/2 of the amount of feed con- sumed daily by ad~libitum~fed controls. The effects of low protein in- take were produced by reducing the casein content of the semi-synthetic .2 diet from twenty-five to either fifteen or ten grams per 100 gram of diet. Vitamin deficiencies were induced by omitting a particular vita- min from the diet, or by adding sulfasuccidine or antagonists of folic acid and biotin. All animals were housed in an air conditioned room at a constant temperature of 75 degrees Fahrenheit. Fresh rater was available at all times. The data relative to the effects of P.M.S. on the combined weights of the seminal vesicles and coagulating glands (henceforth referred to as 'seminal vesicles') were treated statistically. The standard error of the mean was determined by the following formla: , 2 3.3, as Z d n ( n-lf Significant differences between means were determined by the following formula: ' RESULTS Effects of reduced caloric or protein intake on seminal vesicle response to P.M.S. in Michigan State College rats. The results of this experiment are given in Table I. The first four groups of rats were fed Purina laboratory chow. Group 2, which received P.M.S., showed approximately a 100 percent increase (110.0t13.4 mg.) in the weight of the seminal vesicles as compared to group 1, which receiv- ed no P.M.S. (48.1t3.4 mg.). Groups 3 and 4, which received respectively, only 3/4 and 1/2 of the feed consumed by the ad elibitum fed, rats of group 1, showed decreases in body weight and seminal vesicle response. However, on a 100 gm. body weight basis, the seminal vesicle response was the same as in the ad-libitum fed rats. The effects of the semi-synthetic diet on the reaction of the semi- nal vesicles to P.M.S. were similar to those obtained by feeding Purina laboratory chow. When caloric intake was reduced (groups 7 and‘B), the body weights and seminal vesicle weights were correspondingly reduced, but the response of the latter to P.M.S. remained the same on a 100 gm. body weight basis. The reduction in protein (casein) intake also produced decreases in body growth and seminal vesicle response to P.M.S., but again the response of the latter to the gonadotrophin was the same on a 100 gm. body basis. Effects of vitamin defigienciesjn seminal vesicle response to P.}{.S. in Michigan State College rats. These data are given in Table II. Each of the vitamin deficiencies produced a decrease in growth, with the exception of group 9 which TABLE I EFFECTS OF REDUCED CALORIC OR PROTEIN INTAKE ON SEMINAL VEBICLE RESPONSE TO P.M.S. IN MICHIGAN STATE COLLEGE RATS Group No. Diet fed Was Average Average Average Average of P.M.S. initial final seminal seminal rats given? body body vesicle vesicle weight weight weight weight a. em- ,3. I00 sent _1_ ms. 1 Purina chow' no 43.7 166.0 61.6 4B.lts.4 * z " " Yes 43.9 151.0 160.9 110.0t 13 4 3 3/4 . " " 43.2 127.0 125.6 96.6+ 17 2 I! I! II 4 1/t 43.9 64.9 91.7 107.3t 6.2 5 Semi-synthetic No 43.0 166.0 65.5 41.6, 2 0 6 I! Yes 43.0 154.0 15604 9902* 12.0 7 3/4 " n 43.2 113.0 102.3 90.1* 10 9 6 l/t " " 43.4 69.7 91.6 102.64, 12 4 9 15% Casein " 43.7 134.9 138.1 103.1+ . - 12.9 10 10% n N 43.7 66.3 67.6 95.4: 14 4 " Standard error of mean. TABLE II P.M.S. IN IHCHIGAN STATE COLLEGE RATS EFFECTS OF VITAMIN DEFICIENCIE OH SEMINAL VESICLE RESPONSE TO Group No. Diet fed Was Average Average Average Average of P.M.S. initial final seminal seminal rats given? body body vesicle 'vesicle weight weight weight lei git gm. gnu mg. 100 gm. body weight mg. 1 7 Semi-synthetic No 43.0 156.0 65.5 41.6:2.o ‘ 2 9 * Yes 43.6 150.0 160.2 107.0210.5 3 B No Vitamin.A " 43.5 63.9 135.7 157.2tlo.9 x 4 6 No Vitamin D r 43.0 73.0 61.5 107.6t16.2 x 5 6 No Thiamin " 43.3 76.7 57.6 71.6213.1 6 4 No Riboflavin 0 43.3 54.0 26.6 42.etlo.9 X 7 9 No Pyridoxine " 42.6 91.9 65.2 93.0: 7.1 X 6 6 No Pantothenic " 43.9 90.7 72.7 60.3: 4.5 X Acid ‘ 9 9 No Niacin " 43.7 161.7 150.2 90.7: 6.0 10 9 Biotin.Antag. " 43.7 117.6 103.1 86.6t 6.9 X 1% ll 10 Folic Acid Antag." 43.0 92.4 60.3 64.4: 7.2 x 1% 12 B Sulfasuccidine " 43.9 100.0 65.6 65.6: 6.2 X . Standard error of moan. X. Significant differences: group! 2&3’2009 group 2&8'2034 groups 2&5-2.09 group zen-3.35 groups 2&6-4.25 group 2&10-1.62 groups 2&7-1.lo group zen-3.39 -23- received no niacin. Inasmch as this group, like the others in the ex- periment, received twenty-five percent casein, it seems doubtful that a niacin deficiency was produced. Tryptophan which is present in large quantities in casein, has been shown to be converted to niacin in the body. The emission of vitamin A from the semi-synthetic diet evoked only a small decrease in the response of the seminal vesicles to P.M.S. (group 3). On a 100 gm. body weight basis, it appeared to be responsible for a significant increase in the response to P.M.S. ‘ The omission of thiamin and panthothenic acid (groups 5 and 6) or riboflavin (group 6) from the diet, or the induction of folic acid defi- ciency by feeding one percent of folio acid antagonist (group 10) or sulfasuccidine (group 12), all elicited significant decreases in the re- action of the seminal vesicles to PALS. The greatest inhibition in a seminal vesicle response to P.M.S. was produced in the riboflavin defi- cient rats. On a 100 gm. body weight basis, these rats appeared to show no response at all to P.M.S. The folic acid deficient rats showed the next greatest decrease in seminal vesicle response, while the effects of thismin and panthothenic acid deficiencies were just on the borderline of significance. Effects of folic acid antagonist and paired-feeding on a seminal vesicle response toT.M.S. Tn Carworth rats. The previous experiment demonstrated that folic acid and riboflavin deficiencies were particularly effective in inhibiting the response of seminal vesicles to P.M.S. This and the following two experiments were designed to determine whether deficiencies of these vitamins per se or J /. J the concomitant 2 inanition evoked by their absence were responsible for the observed inhibition of seminal vesicle response to P.M.S. Inasmuch as Michigan State College rats were no longer available for these ex- periments, due to a failure in the animal room breeding program, Carworth rats were purchased and used. Folio acid deficiency was induced by feeding the antagonist. The same amount of feed consumed daily by the vitamin deficient rats was fed ’ to controls, but without folio acid antagonist or sulfasuccidine. The results are shown in Table III. The weight of the seminal vesicles of the controls (group 1) was 123.1t14.0 mg. on a 100 gm. body weight basis, while the seminal vesicles of the P.M.S. injected rats weighed 253.1tl4.2 mg. on a 100 gm. body weight basis. The rats which received 1/2 percent of the folic acid antagonist showed a marked decrease in seminal vesicle response to P.H.8. (group 3). However, the pair-fed rats (group 4) showed a similar re- duction in seminal vesicle response, indicating that the decrease in feed intake alone could account for these results. Effects of folic acid antagonist and paired-feeding on seminal vesicle response to P.M.S. in Carworth rats. This experiment (Table IV) represents an sxtention and confirmation of the data obtained in the previous experiment. The folic acid antagon- ist (group 3) appeared to completely inhibit the response of the seminal vesicles to P.M.S. The pair-fed rats (group 4) showed a small response to P.1‘!.S. which was just on the border line of significance. -24- V . TABLE III EFFECTS OF FOLIC ACID ANTAGONIST, AND PM RED-FEEDING ON SEREINAL VESICLB RESPONSE TO P.M.S. IN CARWORTH RATS Group No. Diet fed The Average Average Average average of P.M.S. initial final seminal seminal rats given? body body vesicle 'vesicle wei ght weight weight weight gm. gm. mg. I50 gnu body weight _ mg. I 1 6 Semi-synthetic No 52.3 154.7 183.9 123.1t14.0 2 8 " Yes 51.9 158.1 401.4 255.1t14.2 3 10 Folic Acid 52.8 93.9 148.8 157.430.? Airbag. 005% 4 10 Pair-fed as 51.5 98.5 179.4 1'79.o!:1'7.:5X above group * Standard error of mean. X Significant differences: groups 3 and 4 -- 1.06 -25- TABLE IV EFFECTS OF FOLIO ACID ANTAGONIST AND PAIRED-FEEDING 0N SEMINAL VESICLE RESPONSE TO P.M.S. IN CARWORTH RATS Group No. Diet fed Was Average Average Average Average of P. M. S . init ial final s eminal s eminal rats given? body . body vesicle vesicle wei ght wei ght weight weight gm. gm. mg. 00 gm. body weight mg. 1 8 Semi-synthetic No 52.8 152.7 170.0 125.5t12.5' 2 7 ' Yes 52.8 155.4 509.8 202.5t12.8 5 8 Folic Acid " 52.1 101.6 121.0 117.7t4.5 Antag. 005% 4 10 Pair-fed as " 51.1 99.4 158.4 159.0t9.7 above group 5 8 Folic Acid N 52.5 95.5 124.0 127.5i9.5 Antag. 0.5% 6 9 Pair-fed as 7 52.5 99.4 148.5 149.8t10.8 above group, plus 10-fold increase in B vitamins I Standard error of mean. Significant differences: groups 3 and 4 -- 2.00 groups 5 and 6 -- 1.65 -25- In order to insure that pai r-fed rats received an adequate B vita- min intake despite reduced caloric intake, these vitamins were increased in the semi-synthetic diet ten-fold above the amounts ordinarily given (see Procedure) and fed to group 6. Although the response in this group was slightly greater than in folic acid deficient rats (group 5), there was no statistically significant difference between the two groups. Effects of riboflavin deficiency and paired-feeding on seminal vesicle response to P.M.S. inTarworth rats. This aperiment was performed in order to determine to what extent inanition was responsible for the reduced response to P.M.S. previously noted in riboflavin deficient rats. The results are given in Table V. It can be seen that when riboflavin was omitted from the diet (group 3) I! j I there was a significant reduction in the body weight and in the response of the animal vesicles to P.M.S. However, the pair-fed rats (group 4) showed a comparable response to PALS. on a 100 gm. body weight basis. The effects of riboflavin deficiency and paired-feeding were further compared (groups 5 and 6), except that in the pair-fed nits the B vitamins were increased ten-fold to insure an adequate vitamin intake. The re- sponse of the seminal vesicles to P.M.S. in the tvm groups of rats was the same on a 100 gm. body weight basis. -27- TABLE V EFFECTS OF RIBOFLAVIN DEFICIENCY AND PAIRED-FEEDING 0N SEMINAL mICLE RESPONSE TO P.M.S. IN CARWORTH RATS Group No. Diet fed Was Average Average .Average Average of P.M.S. init ial final s sminal seminal rats given? body body vesicle vesicle wei ght weight weight weight gm. gm. mg. gm. ody wei ght n45 * 1 8 Semi-synthetic No 52.8 152.7 170.0 121.5112.5 2 7 ' Yes 52.8 155.4 509.8 202.5128.1 5 10 No Riboflavin " 51.1 85.2 126.9 148.7i15.5 4 10 Pair-fed as 7 52.2 99.0 146.2 147.2t14.1 above group 5 9 . No Riboflavin " 52.8 80.2 158.7 167.5t14.4 6 9 Pair-fed as " 52.8 91.2 152.8 166.52 7.5 above group, plus lO-fold increase in B vitandns * Standard error of mean. -23- DI S CUSS I ON In these experiments it was found that a reduction in feed allowance, or omission of individual vitamins which resulted in reduced feed intake, both caused a depression in the rate of body growth. With the exception of deficiencies of folic acid and riboflavin (and probably thiamin and ~/ pantOth‘enio 4 acid) the decreases in the response of the seminal vesicles to P.M.S. were in direct proportion to the reduced body growth of the rats. In the rats made deficient in folic acid and riboflavin, the de- creases in the response of the seminal vesicles to P.M.S. could not be accounted for solely on the basis of reduced body growth. On a 100 gm. body weight basis, the response of these rats was significantly lower ./ than in the normlly-fed rats. However, by using the paired-feeding technique, it was shown that the reduction in caloric intake was primarily responsible for the adverse effects of the folic acid and riboflavin defi- ciencies on gonadotrophic action. Insofar as the effects of folic acid deficiency on P.M.S. action is concerned, these results would seen to be in agreement with the report of Goldsmith et al., (1949, private oommnication) that inanition rather than folic acid deficiency caused the reduced response of the male acces- sories to testosterone in their mouse experimmt. Apparently the male accessories, unlike the fennle oviduct, do not need folic acid in order to respond to their specific growth hormone (androgen). It is unfortunate that the same strain of rate could not be used throughout these experiments. The Michigan State College and Carworth strains were not strictly comparable in their reactions to the various -29- vitamin deficiencies and P.M.S. administration. Thus, in the case of the Michigan State College strain, a one-half reduction in caloric in- take decreased the gain in body growth (to 91.5 grams instead of 154.0- 156.0 grams) and produced a corresponding reduction in the reaction of the seminal vesicles to 13.158. The body growth of the Carworth strain on folic acid or riboflavin deficiency and in the pair-fed groups was similarly reduced (to 95-100 grams instead of 155.0-159.0 grams), but the seminal vesicle response to P.M.S. was depressed below that which could be accounted for on the basis of body weight alone. This only further emphasizes the importance of using uniform strains of rats in such experiments. It seems well established that reduced caloric intake results in a decrease in anterior pituitary function. However, in these experi- ments it seem unlikely that the pituitary could have been an important factor, since it has been demonstrated that the target organs of under- / nourished animals do not decrease in responsiveness to inaected hormones. It must be recognized that the action of PALS. on the saninal vesicles is not a direct one, but is mediated through stimulating the production of androgen by the interstitial tissue of the testes. It cannot be said that the question of whether vitamin or other nutritional deficiencies can affect gonadotrophic action in sale rate has been definitely answered by these experiments. Except in the case J of thiamin, riboflavin and vitamin D, extreme deficiencies were not pro- duced as judged by the decreases in bochr weight gains. Also the age of the animals and the length of time they were on deficient diets mist be -30- considered. Alterations in any of these factors could have led to dif- ferent results from those-reported here. It does not seem unreasonable to suppose that tissues may need certain vitamins in addition to sufficient caloric intake in order to respond to endogenous or exogenous hormones. Although it appears that inanition rather than any B-vitamin deficiency was responsible for the decreased response of the seminal vesicles to P.l{.S., the fact remains that the deficiencies of B vitamins were responsible for the inanition. In short, these animals were unable to obtain the full energy present in the carbohydrates, fats and proteins of the diet without the aid of the B vitamins. Thus, it would seem reasonable to conclude that, al- though B vitamins may not be needed for specific reproductive functions (with the possible exception of folic acid for estrogen action on the oviduct), they probably are needed by the body as a whole in order to obtain the calories from the energy producing foodstuffs. 1. 2. SUMMARY The effects of nutritional deficiencies on the response of seminal vesicles and coagulating glands to pregnant mares' semm were studied in weanling male rats of the Michigan State College and Carworth strains. These animals were placed on various caloric, protein or vitamin deficient diets for a period of twenty-eight days, and during the last four days they were injected with a constant dose of preg- nant nares' serum (P.M.S.). The combined weights of the seminal vesicles and coagulating glands were used' as a measure of gonado- trophic action. In the first experiment the effects of caloric and protein deficiencies , were determined. The ad- libidum fed controls showed approximately a 3. 4. 100 percent increase in the response of the seminal vesicles and co- agulating glands to P.M.S. The rats which were fed either 3/4 or 1/2 of the amount of feed consumed by the ad-ulibidum fed controls showed a reduced response to P.1J.S. However, when computed on 100 gm. body weight basis, the response of the male accessories to P.M.S. in these rats was the same as in the ad- libidum fed controls. A reduction in protein (casein) intake from twenty-five to either fifteen or ten percent resulted in a reduced response to P.M.S. However, on a 100 gm. body weight basis, these animals responded as well to P.M.S. as the controls which received twenty-five percent protein. In another experiment the effects of various vitamin deficiencies were determined. It was found that deficiencies of the following 5. 6. 7. vitamins induced a decrease in the rate of body growth and a corres- ponding decrease in the response of male accessories to P.M.S.x vitamins A, D, biotin and pyridoxine. Deficiencies of riboflavin and folic acid (and possibly thiamin and pantothenic; acid) produced reductions in.the response of the male accessories which could not be accounted for'by the reduction in.body weight alone. In.two experiments it was decided to determine whether the reduced response to P.M.S. obtained by'a folic acid deficiency could be accounted for on the basis of the concomitant inanition. A folic acid antagonist (x-methyl-folic acid) was used to produce the folic acid deficiency. The rats which were restricted to the same caloric intake as the folic acid deficient rats, responded almost the same to the P.M.S. In two of the three paired-feeding experiments there was no significant differences in the response of the seminal vesicles to P.M.S., while in the third paired-feeding experiment the folic acid deficient rats showed a lower response. Increasing the deitamin intake tenpfold in one of the pair-fed groups did not increase the response to P.M.S. The effects of riboflavin deficiency and paired-feeding were compared in four groups of rats. The response of the seminal vesicles to P.M.S. under both nutritional treatments was the same. Increasing the B-vitamin intake ten-fold in one of the pairbfed groups did not increase the response to P.M.S. It is concluded that none of the nutritional deficiencies tested in the above experiments induced a significant reduction in the response -33- of the seminal vesicles and coagulating glands to pregnant mares' serum when computed on a 100 gm.'body weight basis, with the exception of folic acid and riboflavin deficiencies. 'When rats were pair-fed in accordance with the daily feed intake of the folic acid and riboflavin deficient animals, the response of the seminal vesicles and coagulating glands to P.M;S. of the two groups was essentially similar. Thus the effects of deficiencies of these two vitamins on.the seminal vesicle response to P.M.S. could be attri- buted to the concomitant inanition. -34.. BIBLIOGRAPHY Andrus, M., and Zarrow, ML K. Amount of alkaline phosphatase in the 1949 oviduct of folic acid deficient chicks. Proc. Soc. Expt. Biol. 5 Mod.‘ 72:714-716. Asdell, S. 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K. he influence of vitamin E deficiency on the endocrine glands of rats, particularly on the gonadotrophin content of the pituitary gland. Am. J. Physiol. 152.259-271.). P'an, S. Y., VanDyke, H. E., Kaunitz, H., and Slanetz, C. A. Effects 1949 . of vitamin E deficiency on amount of gonadotrophin in the anterior pituitary of rats. Proc. Soc. Exp}. Biol. 8: Med. 72:523-526. Pearson, P. B., Molass, V. H., and Sherwood, R. M. The effect of panto- 1945 thenic acid content of eggs on the amount in newly hatched chicks. Arch. Bioch. 7:353-356. (Cited in Nelson, 11. M. and Evans, H. II. Pantothenic acid deficiency and reproduction in the rat. J. Nutrition 31:189-203. Parkes, A. 8. Effect of X-ray sterilization on development of accessory ' 1927 organs of reproduction in mouse. J. Roy. Micro. Soc. 47:95- 106. (Cited in Moore, c. R. and Saxmels, L. T. To action of testes hormone in correcting changes induced in the rat pros- tate and seminal vesicles by vitamin B deficiency or partial -‘.n:'niticn. Am. J. Physiol. 96:278-288.). Rowlands, I. W. and Singer, E. Gcnadotrophin activity of pituitaries 1936 of vitamin E deficient rats. J. Physiol. 36:323-326. (Cited in P'an, S. Y., VenDyke, H. E., Kaunitz, H. and Slantez, C. A. Effect of vitamin E deficiency on amount of gonadotrophin in the anterior pituitary of rats. Proc. Soc. Expt. Biol. & Med. 72:523-526.). Selye, H. and Collip, J. B. Fundamental factors in interpretation of 1936 stimuli influencing endocrine glands. Endocrinology 20:667-672. (Cited in Stephens, D. J. Anorexia nervosa. Endocrine factors in.undernutrition. J. Clin. Endocrinology l:257-268.). Smith, P. E. Hypophysectomy and replacement therapy in rat. Am. J. Anat. 1930 45:205-273. (Cited in Mhlinos, M. G. and Pomerantz, L. Pseudo- hypophysectomy. A condition resembling hypophysectomy produced by malnutrition. J. 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Congenital malformations induced 1944 in rats by maternal nutritional deficiency. VI. The preven- tive factor. J. Nutrition 27:477-484. Warakny, J., and Schraffenberger, E. Congenital malformations induced 1946 in rats by maternal vitamin A deficiency, defects of eye. Arch. cphth. 35:150-169. Yamamoto, R. S., and Chow, B. F. Role of dietary protein in development 1950 of young rate under stimulus of a gonadotrophin. Federation Proceedingg_(Abstract)9:250. THESIS ABSTRACT EFFECTS OF NUTRITIONAL DEFICIENCIES IN YOUNG RATS ON GONADAL RESPONSE TO PREGNANT MARES' SERUM By Krishna Behari Yadu The effects of nutritional deficiencies on the response of seminal vesicles and coagulating glands to pregnant mares' serum were studied in weanling male rats of the Michigan State College and Carworth strains. These aninals were placed on various caloric, protein or vitamin defi- cient diets for a periof of twenty-eight days, and during the last four days they were injected with a constant dose of pregnant mares' serum (P.M.S.). The combined weights of the seminal vesicles and coagulating glands were used as a measure of gonadotrophic action. In the first experiment the effects of caloric and protein deficien- cies were determined. The ad -libidum fed controls showed approximtely a 100 percent increase in the response of the seminal vesicles and coagu- lating glands to P.M.S. The rats which were fed either 3/4 or 1/2 of the amount of feed consumed by the ad libidum fed controls showed a re- duced response to P.M.S. However, when computed on 100 gm. body weight basis, the response of the male accessories to P.M.S. in these rats was the same as in the ag libigum fed controls. A reduction in protein (casein) intake from twenty-five to either fifteen or ten percent resulted in a reduced response to P.‘M.S. However -1- Krishna Beha ri Yadu on a 100 gm. body weight basis, these animals responded as well to P.M.S. as the controls which received twenty-five percent protein. In another experiment the effects of various vitamin deficialcies were determined. It was found that deficiencies of the following vitamins induced a decrease in the rate of body growth and a corresponding decrease in the response of nale accessories to P.M.S.: vitamins A, D, biotin and pyridoxine. Deficiencies of riboflavin and folic acid (and possibly thiamin and pantothenic: acid) produced reductions in the response of the / mle accessories which could not be accounted for by the reduction in body weight alone. In two experiments it was decided to determine whether the reduced response to P.M.S. obtained by a folic acid deficimcy would be accounted for on the basis of the concomitant inanition. A folic acid antagonist (Ix-methyl-folic acid) was used to produce the folic acid deficiency. The rats which were restricted to the same caloric intake as the folio acid deficient rats, responded almost the same to the P.M.S. In two of the / three paired-feeding experiments there we no significant differences in the response of the seminal vesicles to P.M.S., while in the third paired- feeding experimmt the folioacid deficient rats showed a lower response. Increasing the B-vitamin intake ten-fold in one of the pair-fed groups did not increase the response to P.M.S. The effects of riboflavin deficiency and paired-feeding were compared in four groups of rats. The response of the seminal vesicles to P.M.S. / under both nutritional treatments was the same. Increasing the B-vitamin intake ten-fold in one of the pair-fed groups did not increase the re- sponse to P.M.S. Krishna Behari Yadu It is concluded that none of the nutritional deficiencies tested in the above experiments induced a significant reduction in the response of the seminal vesicles and coagulating glands to pregnant mares' serum when computed on a 100 gm. body weight basis, with the exception of foli; acid and riboflavin deficiencies. When rats were pair-fed in accordance with the daily feed intake of the folic acid and riboflavin deficient animals, the response of the seminal vesicles and coagulating glands to P.M.S. of the two groups was essentially similar. Thus the effects of deficiencies of these two vitamins on the seminal vesicle response to P.M.S. could be attributed to the concomitant inanition. -3- RDUM USE GNU! M - ‘- ”'WI’I‘I'INLHINI‘ifi'tilijfg’l‘iitflmifyfiifi"s 293 03