‘FHE CLNICOPAIHOLGGIC EFF-ICES OF TWRQIEEWMY GM HMODUCTNE AND QYHER BODY WNCHONS 3N THE MALE GOAT Thurs 5m- i'iu [Dogma of DB. D. MICEEGEK STHE UKFVERSIE'L’Y James Aflfxred Wifliams 1960 r.— -! TH Efz'it‘a LIBRARY Michigan State University THE CLINICOPATHOLOGIC EFFECTS OF THYROIDECTOMY ON REPRODUCTIVE AND OTHER BODY FUNCTIONS IN THE MALE GOAT By JAMES ALFRED WILLIAMS AN ABSTRACT Submitted to the School for Advanced Graduate Studies of ' Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Veterinary Pathology ' ‘ 1-9601 ' , .. f, , 4 ,1 / , Approved: (1. Q 4'42/[1'0 E, u { ( JAMES ALFRED WILLIAMS ABSTRACT The clinical and pathological effects of complete and partial thyroidectomy and thyroidectomy plus iodinated protein therapy on reproductive and other body functions in young goats were studied. Twenty male goats of mixed breeding were surgically thyroidectomized at two weeks to one month of age. Eight were left as controls. Clinical studies included growth rates, libido, semen production (by electrical ejaculation), sperm morphology, hematology; and blood sugar, plasma calcium, and plasma in- organic phosphorus determinations. Post-mortem examinations were conducted on control and experimental goats after 15 to 18 months of age to determine gross and histologic lesions if any. When thyroidectomy was complete typical cretinoid symptoms including lethargy, edema, stiffness, hyperkeratosis, alOpecia, bloating and stunting of growth appeared. The athyroid goats failed to show libido, produced significantly fewer motile spermatozoa, and significantly more abnormal spermatozoa per ejaculate than did the controls. No decrease in testicular size relative to body weight was observed in the athyroid goats, but the mean JAMES ALFRED WILLIAMS testicular weight was less than half that of the controls. Histologically there was more vacuolation and hyaline- like change among secondary spermatocytes and spermatids and more sudanophilia in the seminiferous tubules of the com- pletely thyroidectomized goats than in the controls. Cyto- plasmic atrophy, absence of granulation, and pyknosis of the Leydig cells were more pronounced in the athyroid than in the euthyroid goats. Pronounced stereocilial loss, epithelial vacuolation, and the formation of bodies resembling corpora amylacea in the epithelium of the head of the epididymis were observed in the athyroid goats. Prolonged athyroidism resulted in pronounced de- granulation and vacuolation of the large basophils in the anterior lobe of the pituitary gland. The adrenal glands of cretinoid goats showed decreased cortical depth and in- creased medullary diameter when compared to non-thyroidectomized goats. Marked hyperkeratosis of the epidermis and hair follicles along with vacuolar degeneration of the smooth muscle in the dermis was a constant feature of the untreated thyroidectomized goats. Shallow erosions of the articular cartilages of the tibiotarsal joint occurred in the athyroid goats but not in the controls. Reproductive and other body functions were not JAMES ALFRED WILLIAMS impaired in partially thyroidectomized goats nor in athyroid goats which were fed iodinated protein before cretinoid symptoms developed. Replacement therapy after pronounced cretinoid symptoms developed did not restore normal growth nor reproductive functions but did alleviate other symptoms of cretinism. Accessory thyroid tissue was found in ten of nineteen goats by 1131 uptake studies. It was concluded that this procedure is essential in determining the thyroid status of goats used in athyroid experimentation. Testicular biopsy provided an adequate means of assessing testicular histology but resulted in some local necrosis and calcification along the path of needle entry. It was found that extreme care should be taken to prevent puncture of the epididymis; otherwise a sperm granuloma invariably results. THE CLINICOPATHOLOGIC EFFECTS OF THYROIDECTOMY ON REPRODUCTIVE AND OTHER BODY FUNCTIONS IN THE MALE GOAT By JAMES ALFRED WILLIAMS A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Veterinary Pathology 1960 ACKNOWLEDGMENTS The author is sincerely grateful to the following for their inspiration and assistance in making the completion of this thesis possible: To Dr. C. C. Merrill, Department of Veterinary To To To To To T0 T0 Pathology, for his guidance and valuable sugges- tions in the pathological sepects of the research and preparation of the thesis, and for his patience and encouragement throughout the period of study. Dr. R. F. Langham, Department of Veterinary Pathology, for guidance in histonathology and assistance in making the photomicrographs. Dr. E. P. Reineke, Department of Physiology and Pharmacology, who unstintingly gave assistance and valuable suggestions in the physiological aspects of the research and for making available radioactive iodine I131 and iodinated protein for treatment of the goats. Dr. Harold Hafs, Department of Dairy Science, for assistance in making the statistical analysis of variance of semen data. the staff technicians in the Department of Veterinary Pathology for assistance in preparing tissue sections and performing hematological examinations and biochemical determinations. Dr. c. F. Huffman, Department of Dairy Science, and the late Dr. Frank Thorp whose early inepira- tion led to the initiation of this research by the author. Michigan Artificial Breeders' Cooperative, Inc. whose continuing grant in aid, in part, made this study possible. my wife, Lorene, for her assistance in typing the rough draft of the thesis, and without whose un- failing devotion, patience, and encouragement this undertaking would not have been possible. VITA The author was born in Michigan March 23, 1919, and raised on a farm near Riverdale. High school training was completed at Riverdale High School in 1936. He attended Michigan State College from 1937 to 1941, receiving the B.S. degree in Animal Husbandry and Agricul- tural Education. During World War II he was commissioned a naval aviator and was discharged as a lieutenant in 1945. He re-entered Michigan State College in the School of Veterinary Medicine in 1946 and received the D.V.M. degree in 1950. Upon joining the staff of the Department of Veterinary Pathology in 1950 graduate study undertaken in 1946 was con- tinued and the M. S. degree in Dairy Husbandry received in 1951. Since joining the staff of the Department of Veterinary Pathology he has engaged in research on diseases of infertil- ity, particularly in cattle. iii Dedicated to Kala, Melanie, and Jama iv. TABLE OF CONTENTS Page I 0 INTRODUCTION 0 O O O O O O O O O O O O O O 0 1 II. REVIE'N OF LITERATURE O O O O O O O O O O O O 3 A. Relation of Thyroid Gland to Reproductive Function in the Male . . . . . . . . . 3 B. Effects of Thyroidectomy on Other En- docrine Glands O O O O O O O O O O O O 12 C. Pathological Changes in Extra-endocrine Tissues . . . . . . . . . . . . . . . . 16 D. Thyroid Secretion Rates . . . . . . . . 20 E. Therapy with Thyroactive Compounds in Male Infertility o o o o o o o o o o o 22 III.PROCEDURESOOOOOOOOOOOO0.00. 27 A. Source of Coats . . . . . . . . . . . . 27 B. Thyroidectomy Procedures . . . . . . . 27 C. Organization of Experimental Groups . 29 D. Clinical Observations . . . . . . . . . 32 E. Replacement Therapy . . . . . . . . . . 33 F. Testicular Biopsy . . . . . . . . . . . 3t G. Semen Collection and Evaluation . . . . 35 H. Libido Measurements . . . . . . . . . . 37 I. Post-nurtem Examination . . . . . . . . 38 J. Tissue Procedures . . . . . . . . . . . 38 IVOEOULTS00000000000000. #1 A. Clinical Observations . V TABLE OF CONTENTS Page B. Semen Data . . . . . . . . . . . . . . . 51 C. Libido Measurements . . . . . . . .1. . 56 D. Post-mortem Findings . . . . . . . . . . 57 V. DISCUSSION . . . . . . . . . . . . . . . . . 104 A. Relation of Clinical Symptoms to Thyroid DefiCiency o o o o o o o o o o o o o o 0 10h B. Semen Production and Sperm Morphology . 106 C. PathOIOgical Changes 0 o o o o ... o o o 108 D. Testicular Biopsy as a Tool for Measuring Testicular Function . . . . . . . . . . 114 VI. SUMMARY AND CONCLUSIONS . . . . . . . . . . . 115 VII. REFERENCES CITED 0 O O O O O O O C O O O O O 118 vi I. INTRODUCTION Infertility in livestock constitutes a major economic loss to farmers in Michigan and elsewhere in the nation. The annual Michigan Dairy Herd Improvement Association reports have listed sterility and delayed fertility as the second most frequent reason for removal of cows from dairy herds. Approximately seven percent of the cows in herds participat- ing in the DHIA - IBM program are removed annually because of sterility or delayed fertility. Sterility and delayed fertility result from many causes either individually or collectively. These causes include congenital or hereditary defects of the reproductive organs, infectious disease, hormonal disturbances, nutri- tional imbalances, and faulty management practices. EXperi- ments conducted in recent years have pointed out the need for studying the relationships of non-reproductive hormones to reproductive function. One of the hormones known to affect reproductive function, particularly in the male, is thyroxine, the secretory substance produced by the thyroid gland. Decreased sperm out- put and lowered fertility are known to result when hypothyroid states occur, whether naturally or experimentally induced. It is often difficult to determine whether reduced 8permatozoan production and reduced libido are due to 1 2 gonadotrophic hormone failure per se or whether it may be complicated by a secondary thyroid hormone disturbance. From experimental studies of thyroid inhibition in the rabbit and ram characteristic histologic changes in the testis have suggested the possible relationship of thyroxine to Sperm production. The primary purpose of the study presented in this thesis was to gain information relative to clinical and pathologic effects of supplemented and unsupplemented athyroidism in the male goat, with particular regard to reproductive function. An attempt was also made to deter- mine whether testicular biopsy might be a feasible applied technique in correlating testicular function with the thyroid status. Goats were used in this study because of their immediate availability and their ruminant nature. It was hoped that the information gained would be useful in extend- ing these studies to bulls. The seasonal variation in sperm production in goats appears to compare favorably to that of bulls. II. 'REVIEW or LITERATURE A. Relation of Thyroid.Gland to Reproductive Function in the Male 1. General considerations. Several literature re- views during the late 1940's and early 1950's touch on the role of the thyroid gland and its relation to reproductive function. Beach (1947) reviewed studies relating to sexual behavior in mammals. He concluded that, ”thyroid secretions appear to affect sexual activity primarily if not exclusively by way of systemic changes in metabolic function”. Blaxter 23.51. (1949) and Dyrendahl (1949) reviewing the effects of thyroid materials on fertility in male ruminants concluded on the basis of evidence available up to 1949 that thyroid hormone affected reproduction both directly and indirectly. Maqsood (1952) summarized his eXperiments with rabbits and rams and critically reviewed the literature relating male reproductive function to thyroid secretory status. He has clearly shown that thyroprotein or thyroxine prevents sea- sonal decline in ram sperm production. It was important to administer thyroactive compounds, however, at or near physio- 1OSically optimum levels to obtain maximum reproductive re- sponse. . Evidence has accumulated that thyroid gland activity varies considerably under varying environmental conditions 3 and physiological states. Some of these thyroid changes have also been associated with changes in reproductive function in animals. McKenzie and Berliner (1937) early considered that ”summer sterility" in rams was due to high environmental temperature. It was not known at that time whether the decreased reproductive function was due to re- duced metabolic rate or to high scrotal temperatures. That marked depression of thyroid secretion rates do occur as environmental temperatures increase was shown by Henneman, Reineke, and Griffin (1955). By observing the rate at which injected radioactive iodine (1131) disappears from the thyroid glands of sheep they estimated an approxi- mately fourfold decline in thyroid secretion rate between May and July in Michigan. Brenton _e_§ 9;. (1955) found that there was a highly significant negative correlation between the serum protein bound iodine (FBI) in bulls and the average monthly maximum temperature in Louisiana. Fertility and percent of usable ejaculates declined with the fall in PBI. Under controlled environmental conditions Dutt and Bush (1955) found that in ewes and rams housed at 45-48OF. only 1.9 services per conception were required. On the other hand rams and ewes housed under temperatures simulating summer conditions required 5.3 services per conception. Dutt and Simpson (1957) bred separate groups of ewes with Southdown rams housed at 45-48OF. and rams kept at ordinary Bummer environmental temperatures. Cleavage occurred in 64 and 26 percent of the ova resulting from the treated and control ram matings respectively. In those ewes allowed to complete gestation 50 percent of the treated and 13 percent of the control groups dropped lambs. Although thyroid activities were not measured in these studies they tend to substantiate the earlier observations by Maqsood and Reineke (1950) that 30°C. environmental temperature caused signifi- cant testicular atrophic and degenerative changes in male mice. Thyroprotein in physiologically optimum amounts in the diet prevented these degenerative changes. Thiouracil, an antithyroid compound, produced testicular changes similar to the high environmental temperature, which were also pre- vented by thyroprotein. Age plays a role in thyroid activity and consequently reproductive function. A decrease in body metabolism is known to be associated with advancing age (Brody, 1945). Maqsood(fi§fljflzeported reduced apermatogenic function in aged male rabbits. Reineke (1946) reported that aged bulls re- sponded with improved sperm production and shorter ejacula- tion time following the administration of therprotein. Among farm animals, effects of light on thyroid function in the male have received little study. Puntriano and Meites (1951) found that continuous light decreased and continuous darkness increased thyroidal 1131 uptake in mice. Limited genetic observations between and within animal Species have revealed differences in thyroid activity. Meites and Chandrashaker (1918i finding that injected thyrox- ine increased the response to gonadotrophin, concluded that normal mice are slightly hypothyroid. Silberberg 23.31. (1955) have reported that certain strains of’mice become hypothyroid and characteristically show Joint lesions early in life. Mixner and Upp (1947) observed that thyroxine secretion rates were much higher in hybrid chicks resulting from mating inbred Rhode Island Reds to White Leghorn inbred lines than in single crossed chicks produced by mating two inbred lines of the same breed. These authors postulated that "hybrid vigor” may be due to increased thyroid activity. Level of food intake may affect thyroid activity. Armstrong and Hansel (1956) reported that the pituitary glands from heifers fed 60-70 percent of Morrison's recom- mended total digestible nutrient (TEN) intake had lower thyroid stimulating potency than those fed at 140-160 per- cent of necessary TDN. Recently Sorenson 23 21. (1959) re- porting on another phase of the above experiment found the low level heifers to have significantly smaller thyroid glands than did the high level heifers. In addition the thyroid acinar epithelial cell heights averaged 12.4 microns for high level heifers and 8.7 microns for low level heifers, a statistically significant difference. Similar feeding level trials were reported in bulls by Bratton 23.21. (1959) and Flipse.23l§l. (1953). Although thyroid activities were not observed, onset of puberty was greatly delayed by low TEN intake. Maqsood (1951b) reported a similar delay in the onset of semen production in young rams fed thiouracil continuously. 2. libido. Sexual desire is decreased in hypothyroid animals. Thyroidectomy in young rats results in a failure to develop sexual maturity (Rickey, 1925). Maqsood (l951d) reported that aged buck rabbits showing poor sexual desire responded to thyroxine therapy. Thyroidectomy in a four month old bull did not prevent spermatogenesis but did pre- vent libido (Petersen's; g1., 1941). When thyroxine was fed to this bull at sexual maturity normal libido was re- stored. Maqsood (1950a) also found that thyroxine treated rams showed greater sexual interest than control rams during the warm summer months, when thyroid secretion rates are usually low. Cloufa‘gt 31. (1951) reported that high tempera- ture (90-920F.) either continuously or intermittently, high temperature plus thyroprotein, or low temperatures (55-650 F.) plus thiouracil caused buck rabbits to be slower in their sex drive than control rabbits kept at 55 to 65°F. without other treatment. 3- W- The effects of the thyroid secretion on semen quality, quantity, and fertility vary among Species and environmental conditions. Cloufa gt El. (1951) reported that, in rabbits, temperatures above 90°F. or thiouracil caused a decrease in semen volume, motility, sperm concentration, percent of live sperm and normal sperm. Thyroprotein as .01 percent of the diet given to rabbits kept continuously in a high temperature room de- creased semen quality. However it alleviated to some extent the adverse effect produced when rabbits were alternated between a warm room and one kept at 55°-65°F. Maqsood (1951d) administered thyroxine to male rabbits in amounts. estimated to be necessary to meet physiological requirements. Both libido and semen quality were considerably improved Over that obtained in litter mate controls. Maqsood (1951a) also reported that a peculiar deformity on the head of spermatozoa obtained from rabbits showing poor semen quality, disappeared after thyroxine treatment. In rams, thyroid- ectomy hot summer temperatures, or thiouracil treatment, has decreased semen volume, number of motile sperm, and often increased the number of abnormal spermatozoa, (Berliner and Warbritton, 1937; Bogart and Mayer, 1946; Maqsood, 1952). Dutt and Simpson (1947), as stated previously, found in- creased embryonic death to be associated with high environ- mental temperature in rams. A In the bovine male, Petersen ggflgl. (1941) found that thyroidectomy at four months halted sexual desire, but did not prevent spermatogenesis. Libido was restored by giving thyroxine. Brenton 23 El. (1955) found semen quality to be poorer when PBI values were lowest during the net summer months. Few data are available on actual effect on fertiliz- ing capacity or spermatozoa produced by bulls in a hypo- thyroid or hyperthyroid state. Eaton and Simmons (1952) reported the results of 'monthly collections of semen from Toggenburg and Common American goats during a three year period. Their results appear to be more comprehensive and provide a better basis for average values than those reported by other authors (Mbckel, 1937; Phillips‘gg'21., 1943; Perry, 1945). Average values for all seasons were: semen volume, .65 cc; motility, 1.51 (1 = maximum motility; 6 = no motility); concentration, 2.724 billion spermatozoa per milliliter; total spermatozoa per ejaculate, 1.658 billion; percentage of abnormal sperma- tozoa, 8.46. Variations in values were greater among bucks than for seasons of the year. Highest values for all of the characters listed above except percent of abnormal Sperma- tozoa corresponded to the normal breeding season. Only semen volume and concentration were statistically significantly different. Abnormalities of spermatozoa occurred most frequently in the neck and tail. Heads separated from tails and sharply bent middle pieces constituted the most frequent types of abnormality. 4. Testicular size. Maqsood and Reineke (1950) found that testicular weight in mice increased when thyro- protein was given in physiologic doses. On the other hand, temperatures of 30°C. and thiouracil decreased the testicular size from that recorded for control mice housed at 24°C. Young rats thyroidectomized a day or two after birth had testes which were smaller both absolutely and in proportion to body size than did normal rats of the same age, (Scow and 10 Simpson 1945L Jones, Delfs and Fbot (1946) studied the effect of thiouracil feeding in rats and found that it did not de- crease testicular size. Kirkpatrick (1955) has reported that among wild mammals the testis of the adult fox-squirrel undergoes marked changes in size and histology during the year. The testis weights were greatest during the principal mating seasons, December to March, and June to August. Testes weights ranged from 2.6 - 12.6 gm. (average 6.9) during periods of full apermatogenic activity and between 1.7 and 5.5 (average 3.9) during the redevelopment phase. Maqsood (1950b) has found that thiouracil treatment or thyroidectomy results in significantly lower weights of the sex organs of rabbits. The effects of thyroid inhibition were more marked in the growing animal than in the adult animal. Several investigators have reported a decrease in size of the testes, comb, and wattle, in the cookerels of several breeds of chickens following thyroidectomy. (Green- wood and Chu, 1939; Payne, 1944; Blivaiss, 1947). 5. Histologic chggges in the male_reprodugtiye organs. Histologic changes in the male gonads and accessory sex glands which are associated with thyroid activity have been I‘eported by several investigators in laboratory animals. Unsupplemented thiouracil treated young mice show marked atrophy and degenerative changes in the seminiferous tubules of the testis (Maqsood and Reineke, 1950). MaQSOOG (19509) 11 investigated the effects of prolonged thyroid depression with thiouracil in rabbits. Rabbits starting at four weeks of age received .1 percent thiouracil in their diet for 16 weeks. Control rabbits received thyroxine injected at Opti- mum levels for their weight. The seminiferous tubules from the testes of the thiouracil treated rabbits exhibited ex- tensive degeneration of the spermatogenic cells. Fluid con- taining protoplasmic debris occupied the lumina of many of the tubules. Marked atrophy and degenerative changes along with a decrease in number of interstitial cells also were noted. In the thyroxine treated rabbits there was active spermatogenesis with many maturing spermatids present in the tubules. In addition the epididymal ducts contained masses of normal appearing spermatozoa and tall epithelial cells compared to numerous degenerated spermatogenic cells and a lower epithelial height in the thiouracil treated rabbits. Fiaccavento (1952) reported that testicular changes did not occur in guinea pigs 10 days after starting thiouracil treatment, but after 40 days spermatogenesis was definitely impaired. Leydig cell changes were not consistent even after the longer treatment period. Among wild mammals Kirkpatrick (1955) described pro- found cyclic changes in the testis of the fox-squirrel. Degenerative changes of the spermatogenic cells are most Pronounced during the non-mating season from June to August. The annual cycle of epithelial regeneration corresponds 12 generally to the appearance of the development of the testis from birth to puberty. Though thyroid status was not con- sidered in this study the period of decreased spermatogenesis correSponds closely with that noted in the ram when lowered thyroxine secretion is known to occur. Mossmanlgt'g1. (1955) noted marked epithelial degeneration in the accessory genital organs of the fox-squirrel during the late summer months. Regeneration of the epithelium followed this period. Among farm mammals the sheep has received principal attention in regard to the histologic structure of the gonads in varying thyroid states. Berliner and Warbritten (1937) observed a fall in sperm numbers and an increase in abnormal Spermatozoa in thyroidectomized and partially thyroidectomized lambS. Thyroidectomy was accompanied by testicular edema, atrophy of interstitial tissues, and sloughing and pyknosis in the seminiferous tubules. Again Maqsood (1951c) has re- ported that thiouracil given to young rams for up to twelve months caused progressive impairment to epermatogenesis and decreased Leydig cell activitY- B. Effects of Thyroidectomy on Other Endocrine Glands 1. w. Jubb and McEntee (1955a) ex- tensively reviewed the literature pertaining to the general problem of adenohypophyseal functional cytology. Halmi (1950), using the Gomori (1950) aldehyde-fuchsin stain on sections of pituitaries from rats subjected to various l3 endocrine disturbances, found two distinct baBOphilic cell types which he described as beta and delta cells. The cells he called delta cells become progressively hyperplastic and vacuolated after castration and thyroidectomy. In a later study by Halmi (1952) thyroxine therapy increased the beta cell count and decreased the delta cell count in hypothyroid . rats. Vacuolation of the delta cells disappeared. His find- ings suggested that the beta cells might be the site of . thyrotrophin storage. Griesbach and Purves (1945) found that the depletion of granules from acidophils in the pituitary following thyroidectomy, reported by earlier investigators, only occurred in extreme thyroxine deficiency. When physiologi- cal levels of thyroxine were given to young thyroidectomized rats, acidOphils were present in normal numbers and there were no "thyroidectomy cell" basophils present. Slightly lower than optimal thyroxine levels caused "thyroidectomy cells" to appear without disappearance of acidophils. A thyroxine secretion rate of 2.25 micrograms/100 gm. body weight in young rats was determined by this technique. Purves and Griesbach (1951a, 1951b), employing periodic acid Schiff stain (PAS) to detect glchprotein in the pitui- tary cells of rats, found two types of cells present. A round cell type located adjacent to the pars intermedia and to the lower surface of the pars distalis stained intensely with 2A3, This cell type was inhibited by injected estrogen, 14 which led to the conclusion that these were the "gonadotroph" basophils corresponding to delta cells of Halmi (1952). A second baSOphilic cell type, large polyhedral cells with vesicular nuclei, was inhibited by thyroxine and formed thyroidectomy cells in thyroxine deficiency. The intensity of the PAS stain correlated with the thyrotrophic hormone content of the gland. These "thyroidectomy cells" corre- sponded to the beta cells of Halmi (1952). Griesbach (1951) reported having seen vacuolated basophils in the pituitary glands of rabbits, dogs, and sheep suffering from thyroid hyperplasia. Jubb and McEntee (1955b) reported the histologic architecture of the bovine pituitary in various physiologic and pathologic states. In 14 naturally occurring cases of thyroid epithelial hyper- trOphy or hyperplasia. the pituitary beta cells were largely degranulated and vacuolated. These cells occupied the medullary zone of the anterior lobe primarily, although some were found throughout the anterior lobe. These investi- gators conclusively demonstrated with PAS staining techniques that thyroidectomy of a young bull results in degranulation of the beta basOphils. Six weeks following complete thyroid- ectomy they began to degranulate without any definite pattern. Some areas contained normal granulated acidophils while in other areas the granules were replaced with vacuoles. The delta baSOphils apparently remained unaltered 15 or, at least, retained their PAS staining granules. Pro- longed thyroidectomy and its possible effects on the "gonad- otroph" cells in the bovine were not reported. Reineke §t_gl. (1941) reported lowered gonadotrophic potency in the pituitary glands of thyroidectomized goats than in the controls. Histologic examination of the pitui- tary glands of these goats was not reported. 2. ‘Effect of thyroid secretion on adrena1_gland. Changes in thyroid gland secretion affect the gross and histologic structure of the adrenal gland. Bauman and Marine (1945) noted that the adrenal glands involute to one-half their normal size in rats fed three to four months on thiouracil. The involution was primarily due to shrink- ing of the cortex accompanied by a decrease in lipid stain- ing material. Occasionally hemorrhage and cyst formation was noted in the reticular zone. Several investigators (Wallach and Reineke, 1945 ; Maqsood, 1954) have shown that giving thyroxine or thyroprotein above physiologic levels increases the size of the adrenal glands in labora- tory animals. Demxaand Greep (1947), studying the effects of hypothyroid and hyperthyroid states on adrenal gland histology in rats, found that thyroidectomy or thiouracil decreased sudanophilic ketosteroid substances in the zona fasciculata. At the same time ketosteroids in the zona Slomeruloaa increased. These investigators found that thyroxine given above phySiologic levels stimulated the zona.fascnnaatn at first 16 but later depleted both the.flxmicular and glomerular zones of the adrenal cortex. Maqsood (1950a) fed thyroprotein to mice at different levels in their ration and kept them in environmental temperatures of 24° or 30°C. At 24°C. thyro- protein above .1 percent of the ration decreased adrenal weight while at 30°C., .025 percent thyroprotein was the upper limit which the ration could contain without decreas- ing adrenal weight. Functional studies in rats (Timiras, 1955) and humans (Levin and Daughaday, 1955) have revealed that hypothyroid states decrease the amount of l7-hydroxy-corticosteroid excretion. C. Pathological Changes in Extra-endrocrine Ti sues 1. S512. There have been few detailed reports on gross and histologic changes which occur in the skin of thyroidectomized ruminant animals. Perhaps this is because more attention was paid to growth and to other pathologic changes by investigators in the past. Simpson (192hb) re- ported that wool became coarse and easily pulled out in sheep and horn growth was greatly retarded. Marston and Pierce (1932) noted delayed wool growth in sheep also. Busted gglgl. (1957) report that adult ewes showed pro- nounced loss of hair, especially over the poll and carpus, two months or more after continously feeding 240 or 1800 151 microcuries I per day. Lambs born from ewes receiving (h (J (D 17 high doses of I131 also exhibited variable alopecia. Some showed extreme denudation except for small patches of wool and hair about the face. legs, and ventral midline. De- scription of the skin in experimental thyroprivia of goats was not found in the literature studied. 2. W. Myxedema is a pathologic change commonly noted in hypothyrcidism. Marked subcutaneous edema was noted by Goldberg (1927) in two thyroidectomized male sheep. To a lesser extent edema was present in most of the rest of 16 sheep and three goats which were thyroid- ectomized between eight and 105 days after birth. Bustad 23.21. (1957) noted edema in sheep radiothyroidectomized with 1131, particularly in the neck region around the thy- roid gland. 3. Cardiovascular changes. Goldberg (1927) found aortic calcification in 11 of 17 sheep that lived over 244 days after thyroidectomy. The arteriosclerosis attendant to the calcification was unlike that noted in man. Histo- logically the aorta and pulmonary arteries were thin walled, showed hyaline degeneration of the elastic fibers, and pro- duction of new tissue mostly between the calcified plaques. Three cretinoid goats did not show aortic calcification. Cardiac dilatation was present in both sheep and goats Which was attributed to imperfect or arrested muscular deve10pment. The cardiac muscle was constantly pale and flabby as compared to the controls. Histologically, 18 calcification was not seen in the thyroidectomized goats but cross striations were absent. Bustad egugl. (1957) noted cardiac enlargement and calcific plaQues in the aorta, Jugular vein, and coronary vessels of adult ewes receiving 240 and 1800 millicuries of 1131 per day. Offspring of these radiothyroidectomized ewes also showed calcification of the large vessels close to the heart. 4. Renal pathology. All eXperimental sheep and goat cretins examined by Goldberg (1927) showed renal lesions. Degeneration of tubular epithelial cells and colloid casts were constantly present while hyaline degeneration occurred in 10 of the 21 thyroidectomized sheep and goats. 5. Skeletal muscle. Goldberg and Simpson (1925) reported that both skeletal and cardiac muscle were pale and flabby in thyroidectomized sheep. Striations in the skeletal muscle fibers had almost completely disappeared. In addi- tion the sarcolemmal nuclei were smaller and more numerous than in the control animals. Simpson, (192%fi found muscular changes in thyroki- ectomized sheep. She found that the muscle cell nuclear count remains high in the cretin as compared to that of normal sheep. She concluded that early thyroidectomy pre- vents normal development of the sarcoplasm since the cross sectional area of the fibers is much less than in control sheep. Cross striations remain in the muscle fibers of both the controls and the thyroidectomized animals. In a later 19 communication (1927b) the same author concluded that the lack of muscle development in the athyroid sheep was due to lack of exercise. Millikan and Haines (1953) have found impaired muscular function in human patients who suffer from hyperthyroidism or hypothyroidism separately or in conjunc- tion with other conditions. Muscular atrophy with or with- out other degenerative processes has been found in these cases. 6. Osseous changes. Skeletal changes involving the long bones in sheep and goats have been reported by Goldberg and Simpson (1925). Todd and Wharton (1938) found that the diaphyso-epiphyseal junction of the long bones failed to develop prOperly in athyroid sheep. Obvious pathological changes did not become evident until the sheep were two or more years old. Features of the changes included: a) irregular exuberances resembling proud flesh on the shaft, b) inturned, clawed, trachoma-like epiphyseal margins, and c) poorly modeled, ill fitting epiphysis scarcely covering the shaft end. Failure of the head to grow properly has been observed by Liddel (1925) and Todd and Wharton (1934). More recently Silberberg and Silberberg (1954, 1954a, 1954b, 1955) have reported that non-degenerative Joint lesions occur in radiothyroidectomized mice. The incidence of this lesion is higher in young than old mice, higher in females than males, and higher in some strains of mice than others. 20 D. Thyroid Secretion Rates 1. Bioassay technigues. Thyroid secretion rates in varying metabolic states in normal animals are fundamental to determining when the thyroid activity is abnormal. Basal metabolism rates commonly employed in measuring human thy- roid activity are generally impractical in animals. Reineke and Turner (1945) fed thiouracil to chicks to inhibit the thyroid gland and injected thyroxine in graded doses to determine the thyroid secretion. That weight of thyroid tissue per 100 gm. body weight which corresponded with the control thyroid weight determined the thyroid secretion rate. By this method it was found that thyroid secretion rates in female white Plymouth Rock chicks were higher in all seasons than were the secretion rates in the male. The secretion in the male varied between a low of .75 micrograms d.l. thyroxine per day in March to a high of 2.45 micrograms Per day in October and November. Using the same method in rabbits, Maqsood (1950c) determined that although thyroid secretion rates increase with age there is a decrease in secretion in proportion to the body weight increase. Rabbits four and 48 weeks old secreted 8.4 and 21.9 micrograms of thyroxine respectively daily. He also worked out an equa- tion for determining the secretion rate in rabbits: y z .43 X .48 where y is the number of micrograms of thyroxine and X is the body weight in grams. 21 2. Radio-active iodine (I131) measurements. Henneman (1953) developed a technique for the measurement of thyroid secretion rate in sheep which involved injecting tracer doses of 1131. Counts were made after seven days as the zero time and gradually increasing doses of l-thyroxine were injected daily until no further decrease in counts due to thyroid gland output of thyroxine occurred. The level of thyroxine injection at which 100 percent of the previous thyroid count was obtained determined the thyroxine secretion rate. By this technique Henneman gt‘gl. (1955) determined that the secretion rate during the summer in ewes was .078 mg.per day, which was significantly different from .26 mg.per day, the next higher secretion rate obtained in May. Lactation increased the daily output of thyroxine while pregnancy had no effect. Two-year-old ewes had significantly higher thy- roid secretion rates than four-year-old ewes in all months exceptlJuly. Lodge (1957) and Lewis, Lodge and Reineke (1957) studied the thyroid secretion rate in four dairy breeds of calves. They used the technique of Henneman (1953). but 1131 to the thyroid found that cows are able to recycle gland. To overcome this they gave thiouracil, a goitrogenic agent which prevents uptake of iodine. Daily thyroxine secretion rates on 42 calves was .57 mg.per 100 1b. body Weight. Seasonal variations were not noted but calves were housed inside. 22 A seasonal variation in secretion rate in cows and calves was obtained by;£remachandra‘ggigl. (1957) and Pipes .21.§l- (1957). Using a technique similar to that employed above, thyroxine levels ranged from .2 to .7 mg. per 100 lb. body weight during the winter season with a mean of .5 mg. per 100 lb. The summer level was .1 to .3 mg. with a mean of .17 mg. per 100 lb. body weight. 5. Protein bound iodine. Plasma protein bound iodine (PBI) level has been advocated as a measure of thyroid activ- ity and Lewis (1952) and Lewis and Ralston (1953) found that there is considerable variation in PBI levels in cattle of various ages. Values ranged from a maximum of 18.0 micro- grams percent £Kg%) in calves under 48 hours old to a min- imum of 3.0 gu5%) in cows over two years old. More recent investigations of PBI levels indicate varying plasma levels associated with various physiological states (Lennon and Mixner, 1958; ASplund 33; 91., 1959: Brenton. 1955). E. Therapy with Thyroactive COmpounds in Male Infertility l. Thyggxine. Somewhat conflicting reports have arisen from the use of thyroactive substances in the treat- ment of male infertility. MaQSood (1951d) treated male rabbits that had low sexual desire, poor semen quality and arrested spermatogenesis. Also a peculiar protoplasmic mass was present on the heads of a small percentage of the apermatozoa produced by these rabbits. Thyroxine administered 23 in optimal physiologic doses improved spermatogenesis, libido, and fertility. The sperm abnormality also disappeared in i the treated rabbits. 2. Synthetic iodinated proteins. Reineke (1946) re- ported that thyroprotein given orally at the rate of .5 to 1 gm. per 100 lbs. of body weight improved the libido in 10 of 14 bulls. It also improved the fertility in four bulls which had previously exhibited poor sexual desire or low fertility or both. Schultze and Davis (1946) gave seven bulls used in artificial insemination 1.0 to 1.24 gm. of thyroprotein per 100 lbs. body weight. Fertility increased from 51 percent to 55.7 percent in the treated bulls. Five of the seven bulls showed definite improvement in fertility. Thyroxine restored libido in a male goat which had shown a definite lack of interest in doe goats (Turner ggflgl., 1943). Bogart and Mayer (1946) and Maqsood (1951c) have reported that thyroprotein prevents "summer sterility” in rams. There was marked improvement in libido, normal spermatozoa, and sperm motility. Bogart and Mayer (1946), however, found that therapy did not prevent gradual loss of volume of semen and concentration of spermatozoa. On the other hand, no improvement in semen concentra- tion or abnormal sperm was obtained by Williams (1956) from feeding 10 gm. of therprotein per day to two bulls which produced low quality semen during two successive summers 1131 prior to therapy. Thyroid uptake and decay countsxin 24 these bulls were similar to two bulls of similar age and breed that produced normal semen. Spontaneous recovery of quality had occurred in the autumn in the two years prior to treatment. It also occurred in the autumn following the summer in which treatment was administered. Eaton'gg‘gl, (1948) failed to obtain improvement in ram sperm concentration, motility, volume, or abnormal forms by feeding one gm. or two gm. thyroprotein daily from May to September. In fact two gm.. therprotein per day in- creased the number of abnormal forms significantly from that found in the controls. Aged rams were used in the study however. Warwick 23 a1. (1948) obtained similar results in rams. These investigators found that thyroprotein decreased semen quality when given during April and May. At levels of .5 and 1.5 gm. of thyroprotein per day there was a slight increase in motility and methylene blue reduction time, but not in Sperm concentration. Some rams tolerated six gms. of thyroprotein per day while others showed increased heart rate, lost weight and became lethargic. No beneficial effects to reproductive efficiency resulted from thyropro- tein therapy to rams in work reported by Black 23 a1. (1950). Maqsood (1952) pointed out that treatment of rams should start before the beginning of the seasonal decline and con- tinue through the season. Also, dosage should be adjusted to meet the ram's demands for thyroxine, depending on the environmental temperature. 25 Wilwerth etH§;., (1954) found that thyroprotein in excess of .04 percent of the ration to Rhode Island Red cookerels significantly decreased both semen volume and sperm concentration. It is apparent that treatment of hypo- thyroidism will be dependent on the relative need for thy- roid-active substances, which is in turn dependent on such factors as age, size, environmental factors (such as tempera- ture and light), genetic makeup and, possibly, nutrition. From the review of literature there are several as- pects of the relation of the thyroid gland to reproductive function on which data are still lacking. From the informa- tion available it has been inferred that it is possible to prevent seasonal changes in semen quality by administration of thyroid-active compounds. However in most instances there has not been a clear cut appraisal of the actual thyroid function. Thiouracil has been used to depress thyroid function but some question has arisen as to whether there is complete block of the thyroid gland. Possible side effects of thioura- cil on spermatogenic function have not been adequately in- vestigated. Where thyroidectomy has been employed in studying reproductive function, little or no evidence has been pre- sented as to whether thyroidectomy was complete. For these reasons this eXperiment was set up to determine the effects of complete thyroidectomy on the reproductive functions in 26 the male goat. The extent to which thyroidectomy was complete was carefully checked by measuring thyroid uptake of radio- 131 and by post-mortem examination. active I Survey of the literature indicates that spermatogene- sis or libido or both may be related to the level of thyroid function. In this eXperiment an attempt was made to dif- ferentiate between these two factors by clinical and patho- logic studies. III. PROCEDURES A. Source of Goats Male offspring of mixed breeding, born in 1957 and 1958 and maintained by the Departments of Veterinary Pathology and Physiology and Pharmacology at Michigan State University, were used in these eXperiments. No outside breeding had entered these goat herds since 1954. The bucks were uniform in size. The majority of the goats had horns, only five of thirty being polled. B. Thyroidectomy Procedures 1. Surgica1_procedures. Thyroidectomy was performed between two and four weeks after birth. Modifications of surgical procedures outlined by Reineke gg_gl. (1941) in goats and George et_al.(l957) in sheep were followed in re- moving the thyroid glands. Some difficulty was encountered in providing sufficiently deep anesthesia to permit manipu- lation of the thyroid gland during surgery. Fasting the goats for 12 to 24 hours lessened compli- cations due to regurgitation. It was found that sodium renumnrbital administered intravenously at the rate of one grain per three pounds body weight provided adequate anes- thesia. Occasionally ether was needed to supplement the 27 28 barbiturate anesthesia when tension was placed on the thy- roid gland in separating it from its connective tissue attachments. Two percent procaine hydrochloride was in- jected locally in some goats. The extra fluid made it more difficult to locate the isthmus of the thyroid and occasional strands of accessory thyroid tissue. Following recovery from surgery the goats were re- turned to their dams to nurse or weaned and fed milk until they were at least six weeks of age. All goats were weaned from their dams by two months.of age. A grain ration containing 38.5% corn, 38.5% cats, 19% soybean oil meal, 2% steamed bone meal, and 2% iodized salt was fed along with alfalfa-brome grass hay.' The goats were fed ad libitum twice daily. Some of the goats were allowed to nurse their dams while on pasture prior to weaning in 1957.‘ A heavy helminth infestation resulted and therefore all goats in the 1958 experiment were kept inside after weaning. In order to determine whether thyroidectomy was com- plete, radioactive iodine (1131) was administered. A tracer dose of 30 microcuries 91c) was injected subcutaneously. Forty-eight to 72 hours later, thyroid uptakes of the 1131 were measured with a scintillation tube and a disintegration count rate meter. A number of the goats were found to have thyroid tissue present which went undetected at surgery. The disintegration rate detected in these goats when 29 compared with that of the controls gave the approximate per- cent of thyroid tissue remaining. 2. Radioactive iodine 1131 administration. Approxi- mately 60 percent of the surgically thyroidectomized goats were found to have unremoved accessory thyroid tissue. Seven bucks were injected subcutaneously with eight milli- I131 curies of each to destroy the remaining tissue. This procedure proved effective as determined by subsequent 1131 uptake. 131 Repeat surgery was attempted in two goats using I , a scintillation tube, and count rate meter to determine the location of the accessory thyroid tissue. Because of the diffuse distribution of the tissue in one of these goats it was deemed inadvisable to use this procedure routinely to effect complete thyroidectomy. .r 0. Organization of Experimental Groups In the spring of 1957 thirteen young male goats were available for the experiment and it was planned to thyroid- ectomize nine of these and leave four as controls. All nine of the thyroidectomized goats were to be allowed to develop symptoms of cretinism;then four of them were to be given thyroprotein to determine whether recovery could be effected. The other four were to remain cretinoid as long as they would live or until the experiment was terminated. The original plan could not be followed since two 0f the goats died during surgery. Accessory thyroid tissue, 3O undetected at surgery, remained in five of the seven experi- mental goats, as detected by radioactive iodine uptake measurements. One of the two completely thyroidectomized bucks was injured while still nursing its mother in pasture and had to be destroyed. The other deveIOped such severe abdominal distension and dyspneic symptoms three months following thyroidectomy that supplemental thyroprotein was administered to keep him alive. When it was learned that accessory thyroid tissue re- mained in the other surgically treated goats it was decided to destroy the remaining tissue in three of the five bucks with radioactive iodine 1131. Two were left partially thyroidectomized. In 1958 the eXperiment was repeated and extended. It was planned to have four groups of four male goats each: (1) control group, (2) thyroidectomy plus immediate thyroid replacement therapy, (3) thyroidectomy plus delayed therapy after thyroid symptoms became marked, (4) prolonged athyroid- ism without treatment. Although extreme care was taken to remove all suspected accessory tissue only five of 13 goats Operated proved to be completely thyroidectomized. Of the eight remaining, four were given thyroid destroying doses of 1131, two were reoperated to remove the remaining tissue, while two were left partially thyroidectomized. As indicated in Table 1, six groups of goats evolved as a result of the delay encountered in thyroidectomy at a uniform age. 31 .02 .tho mmmmmx .moHaoomaonmA Hhmaoawo an ooHHaQSm hHome .meHeaoaoaa oHoahSp wnH>m£ :Hcmmo ooemuHUOH mo ocean eosoampoam: AHV .pnoaHaomxo msHaso ooHo upmow was .pzesHaomxo wmaaso ooHo poem He .psoaHsoaxo no one as omHHHx no geese HHpms 0H0 nmfipono mmmHmaom .npnoa H on meow: m ooNHaoaoooHoahna m at: *H h .hHHmo GHouosooamzu .93 hoop .QH OH sea .am so. suHa caveman some weepaehm eHomecao some or omaoHHm .spmos H on meow: m ooNHSOPoooHomana e sen H m .H.ps hoop .9H 0H eon .aw e0.v ooSmHHnmpmo amHoHoshmpm me soon mm “H nHopoaaoahnp so cavemen seem .aHHeonsdm popesceo awn one .owm mo npsos H on once: m oouHsopoooHosmma m m n a .aEOpoooHoaanpOHoma an no mHHwOHmasm oo>oama osmme mo poem use» see we menace w HHams uses lemon» nonpasm usoana cm on omaoHH< .pcomose oHoaama haOmmooom .omm ho apnea H op memos m mouHeopoooHoahme m m **m o .eoNHEOpoo uoHoAhga mHHmesee mm puma .ooeHmsoe mammHa oHopaSp maommcoom .owm no space H cu memo: m ooNHEOpoooHoahsa e m m m .pncapmmap on I macaw Hoapmoo w a e m (7, (P1) . d. mdoaw pmmspmoae Hsuoa mpeomy oz mpeom oz mmswmmH mmubmmH new» fill-iii if; .mopmpm odoshma mSOHam> :H mpmow ho wansoam HmpnoaHAoaxm waHsomm .H mqmma 32 D. Clinical Observations Goats were observed daily for onset of symptoms of hypothyroidism. Notes regarding appetite, horn growth, hair coat, skin condition, edema, gait when walking, bloating, respiratory distress, aggressiveness, riding, scrotal develop- ment, and weakness were recorded. Photographs were made of goats representative of each group during and at the end of the experiment. Weights were recorded weekly during the first three months, biweekly until eight or nine months old, then monthly. Blood was collected at irregular intervals during 1957-58 for detection of possible hematological changes. During 1958-59 monthly blood collections were made for hematological and biochemical study. White blood cell total and differential counts were made. Circulating eosinOphils were stained with .1 percent Phloxine dissolved in 50 percent propylene glycol and counted in a Spiers-Levy.eosin0phil counting chamber. Averages of counts made in each end of the chamber were recorded. Hemoglobin was measured as oxyhemoglobin using a Bausch and Lomb "Spectronic 20" spectrophotometer. Blood sugar was determined by the method of Somogyi (1945) after precipita- tion of the blood proteins with a saturated solution of barium hydroxide and five percent zinc sulphate. Smears of fresh heparinized blood were made and stained with Wrights' stain for differential white blood cell counting. 33 Biochemical determinations of serum calcium, phospho- rous, sodium, and potassium were made infrequently during the latter part of the 1958-59 experiment. A Coleman model 21 flame photometer coupled with a Coleman junior spectro- photometer was used to make the sodium, potassium and calcium determinations. The procedure used in these determinations was that outlined in the manual accompanying the instrument. Serum phosphorus was determined by the method of Picks and Subb‘arrow (1925) . E. Replacement Therapy To determine whether normal growth and reproductive function could be maintained in recently thyroidectomized young goats a thyroactive iodinated casein called "Protamone" was fed by capsule daily to two thyroprival goats. This material contains approximately one percent of thyroxine, and approximately 10 percent of the active principle is ab- sorbed by the ruminant, according to Reineke (1946). On the basis of a thyroxine secretion rate of .4 mg. per 100 lbs. in immature goats reported by Flamboe and Reineke (1957, 1959) a dosage rate of .04 gm. iodinated protein per 10 lbs. bOdy weight per day was determined. The dose was increased by .04 gm. per 10 lb._ as body weight increased. One goat in the 1957-58 group and two goats in the 1958-59 group were given iodinated protein after they had developed severe symptoms of cretinism. Two other 34 thyroidectomized goats in the 1958-59 group were started on iodinated casein as soon as symptoms were noted and it was established that they were free of accessory thyroid tissue. F. Testicular BiOpsy Testicular biopsies were performed on control and experimental goats at intervals during prepuberal and post- puberal development. The procedure followed was that de- scribed by Barker (1949) for use in bulls. In this techni- que the skin over the posterior aspect of the scrotum was cleaned, local anesthetic was injected subcutaneously, and a small incision through the skin made with a scalpel. A Vim-Silverman biOpsy needle was used to obtain the specimen. Knudsen (1954) found that the dorsal surface of the testis lateral to the head of the epididymis has a minimum number of small blood vessels. This was the needle insertion site chosen for testicular biOpsy in the goat. The outer needle and cannula were inserted parallel to the long axis through the tunica albuginea. The cannula was withdrawn and the divided cutting needle inserted through the outer trocar needle. Gentle pressure was exerted on both needles to penetrate the testicular parenchyma approximately two centi- meters. Holding the inner needle in position the tip of the outer needle was pushed past the tip of the cutting needle. With a tubular piece of testicular tissue thus entrapped in the inner cutting needle, both were withdrawn from the 35 testis. The excised tissue was immediately fixed in Bouin's fluid. No significant hemorrhage following biopsy was de- tected. A single suture was used to close the skin wound. G. Semen Collection and Evaluation Thyroidectomized goats showed little or no libido at the outset of the eXperiment. In view of this fact electro- ejaculation was selected as the means of obtaining semen for study. The electroejaculation apparatus and technique used were essentially those of Dzuik §_t_ §_l_. (1954). In order to provide consistent stimulation for ejaculation the number of stimuli necessary to produce a satisfactory ejaculate in the control goats, was selected. Weak stimuli were applied intermittently until erection occurred and seminal fluids appeared. Stronger stimuli were then initiated to obtain ejaculation. A special stand was erected to provide suit- able goat restraint and convenient collecting. Volume of semen was determined by collecting it in a graduated 15 cc. conical centrifuge tube. A plastic test tube containing water at approximately 35°C. was fitted over the collecting test tube to protect the semen from cold shock while collecting. Semen evaluation was carried out immediately after collection for total and progressive straight line motility of spermatozoa. Egg yolk-citrate, (one part egg yolk - one part 2.9 percent sodium citrate), heated whole homogenized r+ ALA 36 milk, and reconstituted skim milk were tried as diluters but these failed to promote survival of goat semen on storage. This prevented the study of the effect of thyroidectomy on the livability of stored goat semen. A stage incubator thermostatically set at 37°C. was placed on the microscope stage prior to microscopic evaluation. A drop of 2.9 per- cent sodium citrate was placed on one end of a clean warmed glass slide with a dropping pipette. To the citrate a drop of semen was added from a clean chromel wire loop. Sufficient dilution was obtained by this technique to evaluate total and progressive sperm motility. On the other end of the slide a drop of raw semen was placed to estimate the amount of swirling and eddying present. A rating of O to 5 was given to the raw semen according to the procedure outlined by Hermann and Madden (1947). A two-millimeter chromel wire 100p was used to lift a drop of raw semen from the collec- tion tube for making a smear of each sample. An eight- millimeter wire lOOpful of distilled water was placed on a clean glass slide prior to mixing the loopful of semen for smearing. The semen was smeared by touching the tip of a second glass slide to the drOp of semen, and with the two Slides at an angle of approximately 30° to each other, a thin film was formed. This procedure allowed better separation of spermatozoa for study of abnormal morphology. No ad- verse effects were noted as a result of using the water- diluted semen when these smears were compared with those of 37 undiluted semen. Slides were air dried and a number one coverslip was mounted with clear fingernail polish over the smear. Examinations for morphological abnormality were made with a phase contrast microscope. Two hundred spermatozoa were counted using the 95X oil immersion objective. A green Kodak no. 58 filter gave better contrast to the cells than.unfiltered illumination. Tabulation of ab- normal forms was done with a hematological differential counter. H. Libido Measurements Mature does were brought into estrus by giving five mg. diethylstilbestrol daily or two to four mg. estradiol (Haver-Lockhart) on two successive days. The latter re- sulted in more certain and prompt appearance of estrus. These does were placed in a pen singly with each buck. In the early trials one half hour per buck was permitted for mounting to occur. However, it developed that if a buck did not mount the estrous doe during the first five minutes of contact he would not mount at all. Thereafter all con- tacts were for five minutes per buck. Notes were made as to sexual interest (e.g. masturbating, riding one another), occurring among bucks penned together also. 38 I. Post-mortem Examination Gross post-mortem examinations were made of several normal young goats which either died during surgery or were killed to obtain normal tissues prior to starting the experi- ment. Goats dying during the experiment were examined as soon as they were found dead. All control and eXperimental goats were killed between 14 and 18 months of age for gross and histOpathologic examination. These goats were killed by electricity. Skin, horns, Joints, skeletal muscles, all visceral organs and endocrine glands were carefully scruti- nized for gross pathological changes. The testes, epididymides, seminal vesicles, bulbo— urethral glands, adrenal glands, thyroid glands, and pitui- tary glands were removed and weighed. A careful search for accessory thyroid tissue was made and, whenever found, its location and weight were recorded. Photographs comparing representative tissues from experimental and control goats were taken. J. Tissue Procedures 1. Fixatives. Ten percent formol-saline or buffered formalin was used as the general fixative. Preliminary ob- Servations in goats and bulls revealed that testicular tissue fixed in formalin showed considerable loss of the spermato- genic elements in the seminiferous tubules. Zenker's fluid and Bouin's fluid were used to fix blocks of testicular tissue in an effort to overcome this deficiency of formalin. 39 Blocks of tissue approximately five millimeters thick were cut from the testes, epididymides, seminal vesicles, and bulbourethral glands, and a block was placed in each of the three fixatives. Adrenal glands, thyroid glands and pituitary glands were fixed in formalin and Bouin's fluid. Pancreatic tissue was fixed in Bouin's fluid. Routinely a block of tissue from the pillar between the dorsal and ven- tral sacs of the rumen was fixed in Zenker's fixative as was a portion of the abomasal wall. Blocks of skin from the neck, back and flank region were fixed in Zenker's fluid and formalin. Other tissues routinely fixed in formalin included lung, kidney, the femoro-tibial Joint, and the tibio-tarsal joint. Tissues for frozen sectioning were preserved in formalin. 2. Staining procedures. Paraffin sections were pre- pared by dehydrating fixed tissues in gradually increasing concentrations of alcohol, clearing in xylene, and impreg- nating with paraffin on an Autotechnicon. Sections were cut at six microns and stained with Harris' hematoxylin and eosin according to procedures out- lined in the Armed Forces Institute of Pathology Manual of Histologic and Special Staining Techniques (1957).l 1Manual'gf Histologic and S ecial Stainin' Techni es. ,1957. Armed ForcesIInstitute of a o ogy, WashéthEn, g. C. 40 Zenker-fixed testicular tissue sections were stained with Heidenhain's modification of Mallory's aniline blue stain to detect collagen in the interstitial tissue. Pitui- tary gland sections fixed in formalin were stained by the periodic acid - Schiff (PAS) technic using orange G as a counter stain as outlined by Jubb and McEntee. (1955b). IV. RESULTS A. Clinical Observations 1. General. Completely thyroidectomized goats developed cretinoid symptoms starting about two weeks after surgery. First the hair coat began to have a harsh appear- ance. The subcutaneous tissues about the face and in the throat soon became puffy. The deficient groups E and F were reluctant to move about in the pen. During the development of cretinism there was no apparent lack of interest in food. As symptoms became more pronounced, particularly severe bloating, the completely thyroidectomized groups B and F ate very mincingly and were slow in coming to the feed rack. Goats in these two groups reclined a much greater part of the time than did those in the control group A. 2. Growth rates. Growth was extremely slow in thyroid- ectomized goats (group F). The growth curves for the 1958- 59 groups of goats are depicted in Figure l. The partially thyroidectomized goats in group C grew at rates comparable to the controls until complete thyroidectomy at about six months of age. Growth in this group then leveled off close to that of the group-F goats. Table 2 shows the mean daily rates of gain for the combined groups for 1958 and 1959. Groups A, B, and D 41 42 messes aw om< ma mH ma m e - . a - . cs M .ow Campoamonzge poppmpme a. fix mm u do hsoeoopdofinsa pmoemmw . s®ww V. o .ew samwoneoahpe oopaspme adubmmmmwvo . om geopomedopase HsHpHeHa ..s.nxwwmms\.. . .\ ms .\ atv:@-:tieaxw :sx» : o 00 _, -.\@.i.-e\1 . .qx ..¥..\..m.‘.m‘.,n \ 4.. . .q. . . .<. . . .d. .\..o_i.1..o\..\ \.\\\.oa\0\\\o mm. i\2.!.§.. \mes \...O‘:\..\... . x . om .\.\o m3 xi ex .\ \n\\ o\\\\0 hQsLmSB oz mEOpompaosmme aassm m .es.AVi. y omH .. .\ \ hmmhmefi. 09mg mSHm keepommfiofinsgm 393.23. . . massage mfiamm mnflm hsopoonfiosmme Q .em-lfe mMH mangoes oz maouompaophae mama o .mm:é. . gametes oz.ewNHeOposefionase aaflsaseem m .as.lv. omH mHoproo < .gm .? mmpmpm neonhne msoHum> cw mumoo Mom mm>hsu unmwmz npzoho .H casmHm spunoa - iqSIeA 43 showed no significant differences in daily rate of gain throughout the eXperiment. Groups 0, E, and F had signifi- cantly (P<;.Ol) lower rates of gain after complete thyroid- ectomy than the controls. Treatment of group E with thyro- protein after prolonged cretinoid symptoms did not bring about a return of normal growth rate. A ”Students" 3 test was used to test the significance of the differences between the mean rates of gain. The relative size and appearance of representative goats from groups A, B, C, and F are shown in Figure 2. Goats representative of groups A, D, and.F are shown in Figure 3. TABLE 2. Rates of Gain in Control and Thyroidectomized Goats. U Rate of Gain Group No. of Goats _» _lb. per da. Ave. S.E. a .245 1 .0155 .226 1. .0187 c 4 (1) . 242 1 .0115 (2) .llO** 1. .0291 D 2 .232 1 .0273 E 3 (3) .086" 1 .0057 (4) .073* i. .0592 p 3 . 101“ _+_ .0157 —__ (1% After partial thyroidectomy. (2 After complete thyroidectomy. '2 J. After complete thyroidectomy but before thyroid treatment. (#) After complete thyroidectomy and thyroid treat- ment. **P < .01 *P< .05 pr‘ “ (I‘D r 44 3. Horn_growth_and skin appearance. Horn growth was markedly stunted in the goats completely thyroidectomized be- fore one month of age. Complete thyroidectomy after two months of age did not result in pronounced horn stunting although growth was less than in the controls. In the thyro- prival goats the horns grew nearly straight up from the head while in the controls horns grew out in widely sweeping curves. Figure 4 shows the gross appearance of the horns of a goat in a prolonged thyroprival state. As mentioned pre- viously some of the goats were polled and therefore a com- parison of horn lengths in the various groups was impossible. The outer curvature of two representative control goats (nos. 135 and 137) measured 13 inches each while the horns from two representative completely thyroidectomized goats (nos. 144 and 158) measured 8.75 and 9.75 inches reapectively. Iodinated protein supplementation after prolonged cretinism improved the texture of the outer horn lamina, but stimulated lengthening only slightly. The skin of thyroidectomized goats underwent marked visible changes as the thyroid deficiency became more pro- nounced. One of the earliest symptoms of cretinism was a harsh dry appearing hair coat. Gradually a10pecia developed until areas on the face and along the back were nearly void 0f hair. Marked "puffiness" about the face and neck accom- panied the development of alopecia. Wrinkling of the skin became so pronounced that folds appeared on the neck of 45 completely thyroidectomized goats. The skin about the face, flanks, and scrotum became hard and dry to the touch. Scurf- ing and peeling of keratinized epidermal layers occurred on the inner surfaces of the thighs,on the scrotum and in the axillary region. The group-C late-thyroidectomized goats showed only harshness of hair coat after the thyroprival condition was established. Administering iodinated protein early in cretinism prevented skin changes. Treatment after pro- nounced alopecia occurred prevented further hair loss but did not stimulate complete replacement of hair. 4. Myxedema. Subcutaneous edema was absent in the control and partially thyroidectomized goats. Pronounced puffiness of the face and neck region suggesting edema was characteristic of the thyroidectomized goats in groups B and F. In group E, iodinated protein alleviated the edema within two weeks after starting replacement therapy. Group-C goats showed varying amounts of edema, principally about the face, although one (goat 158) showed marked wrinkling of the neck also. Group-D goats which received iodinated protein immediately following complete thyroidectomy did not develop edema. 5. Bloating. Between one and two months following complete thyroidectomy those goats in groups E and F became greatly bloated in addition to other cretinoid symptoms. This bloating caused a marked "pot belly" appearance and was accompanied by dyspnea. It was not typical of the 45 chronic bloat seen in cattle since it persisted continuously rather than appearing intermittently. Soon after administer- ing iodinated protein the bloat symptoms disappeared. Tem- porary suspension of therapy was followed by recurrence of bloating. 6. Locomotor disturbances. Two to three months after complete thyroidectomy most of the goats were noted to be walking with a stiff-legged gait. walking or running appear- ed to be painful. When they were led out of the pen for weighing or other purposes they balked and had to be dragged or carried. This reluctance was not noticed in the control or partially thyroidectomized groups. Extreme muscular weakness characterized by "knuckling" of the pasterns de- veloped in one thyroidectomized goat (no. 169). Both the stiffness, which was noted consistently, and the muscular weakness in a single goat were alleviated by administering iodinated protein. 7. 3 ea. A constant observation among the com- pletely thyroidectomized goats was that of difficult breath- ing. Stertorous sounds were present in the trachea. Exer- tion and bloating aggravated the dyspneic symptoms. Ausculta- tion revealed that ventral portions of the lungs in these goats were not receiving air. Though iodinated protein relieved the bloat in these hypothyroid goats, it did not bring about complete remission of difficult respiration. 8. Death prior to termination of egpggiment. In 47 the 1957-58 group of goats three died or were killed before termination of the experiment. One of these suffered un- eXplained damage to the testis and scrotum while nursing its dam in pasture. The other two died from the effects of prolonged hypothyroidism complicated by pneumonia. In the 1958-59 eXperiment one goat each from groups E (no. 681) and F (no. 159) died suddenly soon after they had developed pro- nounced hypothyroid symptoms. Cause of death in each case proved to be the lodging of a calculus in the urethra. The urinary bladder had ruptured in one of these goats (681). 9. Hematological and blood chemical findiggs. No significant differences were noted among the hematological values observed on monthly examination of blood from the control and experimental goats during 1958 and 1959. The average values for total hemoglobin, microhematocrit, total white blood cells, lymphocytes, neutrOphils, monocytes, eosin0phils, and basophils and circulating eosin0phils are given in Table 3. From the table it appears that thyroty. ectomy results in a smaller number of circulating eosin0phils, There were greater individual variations within groups than between the groups, however. Aberrant erythrocyte morphology was present in all Soats sampled at three months of age. Pointed cells with Single elongated processes and poikilocytes were the most frequent abnormalities. Anisocytosis was also noted fre- Quently. These abnormalities disappeared for the most part H.mm ma. mm. Hm. m.om m.we mmm.m m.em e.mH mm m a m.mn oe. mm. an. m.mm m.os wwa.m m.mm m.mH mm m m e.mm am. so. mm. m.mm m.ee HNH.HH s.mn e.aH em m a m.mn Hm. mm. as. m.mm s.He mom.m e.mm m.mH mm m o m.mo mm. mm. mm. m.mm m.ms mme.m m.mm m.mH .mm m m n.0m mm. mm. em. o.mm e.oe mmo.m e.mm m.mH om e a .88 .30 K) I. R. .1, R7 e 71w. .88 .50 & .ooooH madam. mpoow macaw goo madam waded nooks madam mopmo poo page nos needs .02 .camom 10mmm nondmom mono: vonpsoz nomeshq mHHoo soweson .sw tampon messes eooap -oaoae canoes .oz unopao . oaasz Ioaom .mpmow monasopoopaoamns has Hoapsoo pom mosas> Hmoawoaopesom owmso>< .m mqmgs 49 by six months of age except in the completely thyroidectomized untreated group-F goats and in the late-treated group-E goats. Although the proportion of abnormal erythrocytes declined from 90 percent.to approximately 10 percent by six months of age, five to 10 percent of pointed cells persisted inter- mittently in three of four goats in groups E and F. The significance of these observations was not determined. Biochemical determinations for blood sugar were made monthly from three months of age until the end of the 1958-59 eXperiment. Serum calcium, phosphorus, sodium and potassium were determined during the latter part of the 1958-59 eXperi- ment. In part, the latter determinations were made in an ‘ effort to establish whether imbalances existed which could explain the occurrence of the urethral calculi in two thy- roidectomized goats which died earlier in the experiment. Table 4 includes the values for blood sugar, serum calcium, Phosphorus, sodium and potassium for the control and experi- mental groups in the 1958-59 goats. Very little difference existed among the groups with respect to these constituents, with the possible exception of serUm calcium and phosphorus. Here a narrowing Ca to-P ratio for the thyroidectomized goats was observed. The trend toward lower blood sugar values noted in the thyroidectomized groups was offset by marked variation within individuals. 50 E omH ¢.m b mo.H m.m e H.0H a m.me ma m a Hma m.m m am. m.oH q m.oa m ¢.me Hm m m oma a.m m mm. ¢.HH m m.oa a o.me om m a mma. s.m m m.H w.w e w.oa w m.we an m o mea m.m m e.H m.> m m.HH a m.>e Hm m m med s.e HH e.H m.> a m.HH ma m.om we a a panda you. amvmflyhmn H\ ri¢$< .>¢Ir .>« .Hm>fiscm ..Hm>dddo -HHHME -aHHHa mcpwmn oases has me use we me has .02 az asamm M asamm mwso m asacm so asacm as am vocam mpmow macaw ‘l': .mpmow was: caudaopommHOA%ns was mouaaopocoaoamna hafiwapamm .Hoaonoo ma asdwom cam adammmpomxmsaonamosm .ESHono asamm .mesm cocam .¢ mamas 51 B. Semen Data .1. Eglgg2_§§g_gggilitx, Semen samples collected by electrical ejaculation were examined for volume and percent of sperm which were motile immediately following collection. The observations made on 163 ejaculates are presented in Table 5. The average volume and range in volume are given for each group of goats as outlined under procedures. The volume differences between the control group A and thyroid- ectomized group-F goats were not significant. Group E pro- duced an.average volume similar to the group-F goats. Only one goat appeared in group E because the second goat in this group failed to produce sperm. Clear seminal fluid was ob- tained each time that collection was attempted. Volume varied greatly between ejaculates among goats, and within individuals. This variability appears similar to that re- ported for ejaculate volume in bulls (Dziuk, 1954) when semen was collected by electrical ejaculation. The average sperm motility for group-A goats was considerably higher than for the group-F thyroidectomized goats, 57.8 and 40.3 percent respectively. Treatment of cretinoid male goats in group E with iodinated casein failed to increase the average motility above that of the untreated group-F goats. Late thyroidectomy decreased the number of motile Sperm 10.1 percent below that of the controls. 2. Concentration and total motile spermatozoa. Table 5 also shows the mean sperm concentration in billions i 8. Var. } Ema .. 9: $0 .. o a firm .. H; ma. .+. 1%. 2d mas HA ma m a Amm.n - Ho.v Ame - cal ho.m - m.v ‘ E. .+. 3%. no; «.3 1H mm m m Amo.¢ a mm.v Am» u mmv Am.m a m.v em. H mm.m mo.m mdm w; m H a Am.m a mm.v Ace n mHv Am.m a e.v mm. H 3; and an? on; mm a 0 SJ - we; 3m . my 35 .. m; 2 mm. H mad sad «.3 mm; R s m 5 $6 .. om; Rm .. o: 3.: .. m; cm. a 2.4 mm.m QR. SA 8 m 4 .m.m .>¢ Amwnmmv .>¢ Aowmmmv .>d Aowmmmv .>« .omqmnmwwawmoam wwwMHMMM macaw oHapoE .oo wwwwww mpmow adoaw adage: Hapoa noapmapnoonoo mo onooaom oasao> .oz .02 E .mpmow womaBOpomoaoamns hampoaaaoo use madmdpamm use pompaH ad mamasomnm pom macaw oHapoz Haves one .noaumapmooaoo macaw .anoam ofldpoz unooaom .masHo> soaom mo mamaazm .m wands 53 per cubic centimeter and total number of motile sperm per ejaculate in billions. No statistically significant differ- ences in concentration existed among any of the groups of goats although the lowest average concentration occurred in group-E cretinoid goats which received belated thyroxine treatment. A "Students" 3 test for significance revealed that the .89 billion sperm per ejaculate for group B and the .59 billion sperm for group F were significantly lower (P.01) Goats within groups 17 51.73 3.04 1.72 (P E'.05) Ejaculates within goats 141 250005 1 077 ——_ m. J Group D is not included in this analysis since only one goat in this group produced semen and no group variance occurred. The "F" ratio of 3.46 among all groups of goats lies between the one and five percent level of probability according to Snedecor's (1954) tables of "F” ratios. That considerable 54 variation in total sperm per ejaculate existed, is evidenced by the 1.72 "F" value for goats within groups. This value was significant at the five percent level of probability. 3. Abnormal spermatozoa were counted for each ejacu- late collegted. The summary of the percent of abnormal sperm cells is presented in Table 7. A significantly greater average number of morphologically abnormal sperm cells were found in groups C, E, and F goats than in group-A goats. TABLE 7. Summary of Percent Abnormal Spermatozoa Produced by Control and Thyroidectomized Goata e ibnormal * Group Goats Ejaculates Spermatozoa S.E. A 7 60 16.0 31.2 B A 30 19.1 12.7 C 4 27 23.9* 13.0 D 1 6 21.9 15.6 E 3 23 - 22.S* ‘i2.8 F 3 18 22.6* {12.9 a? ; .0657 S.E. = Standard Error A protoplasmic extrusion frequently projected out from the galea capitus of the sperm in smears examined from both the control and thyroidectomized goats. This did not aPpear to be associated with immaturity of the sperm cells Since the remaining portion of the head, midpiece, and tail usually showed no abnormality when the droplet was present. 55 Ejaculates from the completely thyroidectomized goats showed damaged midpieces, with the head separated from the tail, more frequently than did ejaculates from the controls. Bent and tightly coiled tails were the other frequently seen types of abnormality. Only a small percentage of ejaculates showed protoplasmic droplets along the midpiece or tail section. The latter abnormalities were about equally distributed among the control and thyroidectomized groups. C. Libido'Measurements Sexual desire varied considerably among the control and partially thyroidectomized goats. The results of the libido trials are shown in Table 8. Two of the control goats (174 and 689) failed to mount an estrous doe on repeated opportunities. They seemed to fear the presence of the doe in the pen. On the other hand, placing these bucks back in the pen with other bucks often resulted in immediate attempts to mount them. Relatively normal libido was exhibited by group-B males and by two males in group C. The latter showed sexual interest even after exhibiting early myxedema and rough hair coat. The two bucks to which thyroprotein was given soon after thyroidectomy failed to show active sexual interest in the estrous does throughout the eXperiment. They did, however, attempt to mount other bucks penned with them regularly and frequently were noted to be masturbating. 56 TABLE 8. Summary of Libido Trials Among Control and Thyroidectomized Goats. ==a========================================================= Group Goats Trials Failure to Mount A 7 41 2? f B 3 22 6 C 3 17 5 D 2 15 15 E 3 l2 9 F 3 21 2O H , ..-. -. um ‘.".“—‘51 One of the three goats (173), thyroidectomized, then allowed to develop pronounced symptoms of hypothyroidism, and then treated with therprotein, showed normal sexual interest. The other two (169 and 144) would not mount an estrous doe but were noted to obtain erection in her pres- ence. None of these bucks showed any sexual interest prior to the thyroid replacement therapy. None of the thyroidectomized bucks showed sexual interest during the libido trials except on one occasion when goat 168 mounted but did not obtain erection. It appears that partial thyroidectomy does not essentially affect the ability of male goats to exhibit normal sexual desire. On the other hand, replacement therapy With therprotein in completely thyroidectomized goats did not effect resumption of normal libido. Either the level 0f therapy was not Optimum for these goats or variations in response to the stimulus of an estrous doe did not result 57 in exhibition of normal sexual desire. The latter seems possible since two of the control goats showed similar be- havior. No measurable differences in libido occurred among the various times of the year when trials were made. D. Postmortem Findings 1. Gross pathology a. Reproductive opgans. Immediately following death and examination for external pathological changes the testes were removed from the scrotum. Each was checked for the pres- ence of possible adhesions which might have resulted from earlier testicular biopsy. As shown in Table 9, 10 of 24 goats which had been biOpsied showed minor adhesions be- tween the parietal and visceral surfaces of the tunica vagi- nalis surrounding the testis. In three of the goats, (99. 166, 756) enlarged epididymides were observed. Upon incision, a caseous core was found in the center of each enlargement. Goat 756 had not produced semen during the experiment. Although the semen passage from the testis might have been impaired by the epididymal lesion noted in goat 756, no anatomical ab- normality of the other testis which should have impaired 8perm movement could be found on gross examination. The point of entry of the biopsy needle was identi- fied in 11 of 25 goats by calcified streaks along the path 58 TABLE 9. Tabulation of Occurrence of Testicular Adhesions, Testis Calcification, Acces- sory Thyroid Tissue, and Joint Lesions in Control and Thyroidectomized Goats on Gross Post-mortem Examination. ———‘—-» fl Testicular Calcifica- Accessory Tibio- Goat Adhesions tion in Thyroid Tarsal Testis Tissue Joint Erosions 99 + + + - 886 + + - - 135 + - - - 163 - - - - 166 + +(2) - - 174 -(1) -(1) + - 689 - - - - 778 - — +1.53m. - 137 - - + .7sm. +(3) 164 - + +2.0gm. - 165 + - +1.45m. - 158 - - - +(4) 899 - + - +(4) 161 - - +.1gm. +(3) 167 - - - - 149 + + +.lgm. +(3) 756 - +(2) - - 144 + + - +(6) 169 - + - +(3) 173 + + - - 59 TABLE 9 (Continued) —— i Testicular Calcifica- Accessory Tibhr- Group Goat AdhesionS‘ tion in Thyroid Tarsal Testis Tissue Joint Erosions F 132* + - - +(4) 168 - - - + 613 + + +.3gm.(5) +(3) 681* ~<1) -<1) - +<4) 159* -(1) -(1) - +(4) 758* -(1) -(1) r + *Died before end of experiment. (l)No biopsy (2 Dranuloma present (3)Slight (4)Marked (5)Non-functional (6)New cartilage 60 of the biopsy. These streaks extended into the testicular parenchyma for a distance of one or two cm. Small flecks of calcium were noted occasionally scattered through the testis. Grossly no changes in the tissue adjacent to the calcified areas was evident. Calcification did not occur more fre- quently in one eXperimental group than another, with the ex- ception that all group-D goats showed calcified deposits. Testicular weights for the various groups of goats are presented in Table 10. The accessory glands were not weighed in all goats completing the eXperiment in 1958. When it was discovered that some bulbourethral glands exhibited cystic changes it was decided to weigh all the accessory gland structures in the remainder of the goats. TABLE 10. Mean Weights of Reproductive Organs in Control, Partially Thyroidectomized and Thyroidectomized Goats. W . Bulbo- . Mean Eipidi- urethral Seminal Goats Body Testis ymis Glands Vesicles Wt. (1) (1) (1) Group Ngg, Kg. ‘gm. gm. _gp£_f 5p, A 7 61.7 265.7 44.6 3.75 15.98 B 1+ 55.0 264.6 44.7 3.35 8.00 C 3 39.7 190.3 41.1 3.55 6.80 D 2 50.1 178.7 48.6 2.45 8.30 E 3 32.2 153.9 32.2 2.65 9.45 F 3 26.7 112.8 31.1 2.55 8.10 (1) 'Weights represent four goats in ”A" group, two goats in each of the remaining groups. 61 Although the mean testicular weight for group-A goats (265.7 gm.) was much greater than in the athyroid group-F goats (112.8 gm.), the significance of the difference dis- appears when these weights are correlated with body weight. Table 11 contains the mean ratios of tissue weights to body weight for the various experimental and control groups. The ratio of epididymis, bulbourethral gland, and seminal vesicle weight to body weight was non-significantly higher for goats in group-F than those in group A. Cystic bulbourethral glands were seen in three thyroid- ectomized and two control goats. The center of the gland in each case was filled with gelatinous fluid. In one control goat (163) concretions approximately two centimeters in diameter were seen within the fluid. TABLE 11. Ratio of Mean Reproductive Organ Weight to Body Weight in Control and Thyroidectomized Goats. Epidid- u;:££3;1 Seminal Goats Test:s y?ijh Glap34 V68(§3:8 Group N94; _X10 :1 X10 :1 X19 :1— V§10 {1* A 7 43.1 6.7 .57 2.41 B 4 48.1 7.2 .56 1.29 C 3 47.9 9.3 .81 1.55 D 2 35.6 10.6 .69 1.66 E 3 47.8 10.8 .88 2.68 F 3 42.2 10.6 .87 3.23 ‘ —_‘ T (1) Weights represent four goats in group-A, two goats in each of the remaining groups. 62 No gross pathological changes were seen in the seminal vesicles, ampullae or vas deferentia of the control or thy- roidectomized goats. b. .Skin and subcutaneous tissue. In the com- pletely thyroidectomized goats which were unsupplemented with iodinated protein the skin about the face and neck was con- sistently wrinkled, dry, scaly, and showed marked alopecia. Peeling of the outer keratinized layers was observed around the scrotum and inner surface of the thighs. The skin did not appear greatly thickened nor crusted as is characteristic of hyperkeratosis and parakeratosis. A comparison of the gross appearance from a control and thyroidectomized goat is seen in Figure 5. Group-C goats in which thyroidectomy was completed between six and eight months of age did not show the marked a10pecia of groups E and F. Group-E goats which had been fed iodinated protein after cretinism de- veloped showed little scaling and scurfing but did have marked alopecia. In groups B and D, partially thyroidecto- nnsed and thyroprotein supplemented, respectively, the skin appeared, grossly, identical to that of the controls. In the subcutaneous tissue of thyroidectomized goats in groups C and F varying amounts of a mucoid type of edema were seen. This myxedematous condition was most pronounced in the tissue beneath the skin of the neck and in the axil- lary and inguinal regions. c. Accessopythyppid tissues. Careful examina. tion for tissue containing thyroid follicles was made at 63 autopsy. In Table 12 there is a summary of the findings as to the relative amount of accessory thyroid tissue in con- trol and incompletely thyroidectomized goats. From 7.1 to 33 percent of the injected dose was detected by counting 131 present in the thyroid region of control goats. the I Surgically thyroidectomized goats had from 0 to 16 percent of the injected dose present in the thyroid or neck region. All goats showing .5 percent or less uptake of injected 1131 proceeded to show cretinoid symptoms without further treat- ment. On the other hand when as little as 1.7 percent of the injected I131 was detected in the thyroid region the goats gained normally and required treatment with heavy doses of 1131 or repeat surgery to destroy or remove the accessory tissue. Ten of nineteen goats showed accessory tissue caus- ing .5 percent or more uptake of the injected I131. Results following repeat surgery or a thyroid destroying dose of 1131 are presented in Table 13. Although some tissue remained after repeat thyroidectomy it was insufficient to prevent development of cretinism or myxedema. Similarly, the thyroid destroying dose of 1131 caused regression of any warrining thyroid tissue. Accessory tissue was found at autopsy in one control goat, four partially thyroidectomized and three supposedly completely thyroidectomized goats. One-tenth-gram amounts of tissue were present in group-C goat 161 and.group-D goat 149. Even though these goats underwent repeat surgical 64 Showing Comparison of 1131 Counts per TABLE 12. - Minute in Control, Partially Thyroid- ectomized and Completely Thyroidec- tomized Goats, Goat Group Net Percent of Percent Location No. .C.P.M.. Injected Dose Control Count 847 A 11,320 7.1 - Sub-Laryngeal 886 A 12,690 8.4 - " " 135 .A 57.970 37.6 - " " 163 A 23,240 18.3 - " " 166 A 17,180 13.5 - " " 174 A 21,080 16.6 - " " 689 A 43,380 33.3 - " " 778 B - 400*= 0 Thoracic Inlet 137 B 6,200 4.9 23.0 Lower cervical 164 B 20,280 16.0 77.0 " " 165 B 3,580 3.0 14.4 Laryngeal 158 0 3,300 ..1 12.1 " 899 0 2,920 1.9 10.7 " 161 C 13,400 10.7 51.2 Thoracic Inlet 167 C 2,200 1.7 8.8 Laryngeal 149 D 4,640 3.7 18.0 Mid-cervical 756 D 300 .2 1.1 Lower cervical 144 E - 54o * O O - 169 E -1,180*‘ 0 O - 173 E 10,180 8.1 38.8 Laryngeal and lower cervical 65 TABLE 12 (Continued) ~———-:-_ _ W —..— M— Goat Group Net Percent of Percent Location No. C.P.M. Injected Dose Control Count 132 F 890 .5 3.3 Intermandibular 168 F 500 .4 1.9 Laryngeal 613 F 7,260 5.8 27.8 Laryngeal and lower cervical 159 F - 200* 0 o - 681 F -2,080* 0 0 - *Indicates thyroid count less than body background count. 66 thyroidectomy for removal of accessory tissue, small frag- ments still remained. One group-F goat (613) had a piece of thyroid tissue still remaining in the ventral neck region after radioactive iodine destruction. The cellular appear- ance of this will be discussed under hist0pathological re- sults. Figure 6 depicts the location of an accessory thy- roid gland found on aut0psy. TABLE 13. Radio-active Iodine (I131) in Thyroid Region Following Surgery or 1131 Thyroid Gland Destruction. ‘— J. —-‘_-— Goat no. 3:: 31:32: InfigzgggtDose Outcome 158 360 I; —F Cretinoid 899 no count Cretinoid 167 2250 .3 Myxedema 161 4650 .8 Myxedema 149 90 - Treated 173 790 . .l Cretinoid 132 no count Cretinoid 613 1550 .3 Cretinoid d. 2225... Some atelectasis was observed in the thyroidectomized goats. This was notably present in the ventral portions of the diaphragmatic lobes. Several of the. lungs observed in the 1958 group of goats had small nodules palpable on the dorso-lateral surface of the diaphragmatic lobes. No gross changes were evident in the trachea and 57 larynx which might have suggested the cause of the marked ‘stertorous breathing noted in the thyroidectomized goats. One of the goats dying before the end of the experiment (goat 159. group F) had extensive pneumonia and pleural ad- hesions. e. Urogenital system. Three of the thyroidec- tomized goats had calculi in the renal pelvis and urinary bladder. Two of these (681 and 758) died as a result of calculi lodging in the urethra. The hilus of the kidney in these two goats was moderately distended. The calculi removed from the kidney hilus of goat 173 in group E were soluble in dilute hydrochloric acid but insoluble in acetone. f. Rumen and abomasum. Since bloating had been pronounced in group-F thyroidectomized goats the rumen was examined carefully for possible differences from the con- trols. Although the papillae of the rumen wall were smaller and the wall thinner than in the controls no gross pathologi- cal changes were seen. Small grayish white nodules were present on the sur- face of the abomasal wall in several of the group-F goats. These may have been the site of entry of parasitic larvae although none were seen in the abomasal contents. g. Locomptor system. Joint lesions, manifested as shallow erosions of the articular cartilages, were found in the athyroid untreated goats but not in the control nor partially thyroidectomized goats. A tabulation of their 68 occurrence is included in Table nine. In one goat (159) hemorrhage was present in the joint cavity. Erosion of the medial oondyle of the femur in the stifle joint was noted in one goat (899). Lesions were seen in the joints of the anterior limb in only one thyroidectomized goat. The erosions were prone to occur at points where the carti- laginous surfaces suffered the most contact. In goats which were made cretinoid and then treated with iodinated protein some healing of the eroded areas was noted. A thin layer of cartilage appeared to overlie the erosions. Grayish white streaks were found in the muscles of the posterior limb of athyroid but treated goat 169. Changes were most pronounced in the gastrocnemius muscle. Muscular changes of this nature were not detected in any other goats. h. Qipoulatory changes. Two thyroidectomized goats showed marked roughening of the aortal walls just dor- sal to the heart. These roughened areas did not appear to be calcified on gross examination. In goat 169 from athy- roid group E the heart muscle was extremely pale. i. Endocrinegglppgs. Weights of the adrenal, thyroid, and pituitary glands are presented in Table 14 along with the ratios of average glandular weights to the average body weights for each experimental group. The average weight of the pituitary glands of goats in group F was .31 gm. or less than half that of the controls (.69 gm.). The ratio 69 of gland weight to body weight was not significantly differ- ent, however. Differences at the five percent level of significance existed between the ratios in control group A and groups B, C, and D. TABLE 14. Mean weights of Endocrine Glands and Ratio of Endocrine Gland Weight to Body Weight in Control and Thyroid- ectomized Goats. —.'r—_fi_,v_ 'Pitui- Pitui- tary Adrenal . Goats . Body tary Adrenal Thyroid Gizsogy Gl:Bo y 1 Group No. Kgp 4152; gm4l1 gm. x;0' .1 X10'1:1 61.7 .69(1) 3.08 4.2 .llo‘l) A 7 .501 B 4 55.0 .97 2.68 1.4 .170* .498 C 3 39.7 .83 1.70 .03 .208* .430 D 2 50.1 .85 1.6 -- .171* .522 E 3 32.2 .38 2.1 -- .120 .644 F 3 26.7 .31 2.1 -- .129 .927 (1) Average of six goats. ' * Significant at 5% level. Adrenal gland weight to body weight ratios were not signifi- cantly different among groups. No gross abnormalities in either the adrenal or pitui- tary gland were seen. The pituitary fossa appeared slightly compressed vertically in the thyroidectomized goats in groups B and F. 7O 2. Histopathology a. Rc-agrodugéle sxstem. (1). .Testis. The principal changes detected in the reproductive system occurred in the testis of thyroidectomized goats. Cellular changes occurred in.both the intra- and inter- tubular tissues. To some extent the cellular picture was affected by the fixative used to preserve the testis. Figures 8, 9 and 10 show the differences encountered. Formalin fixing pre- served the interstitial structures better than Zenker's fluid or Bouin's fluid but did not preserve the cells within seminiferous tubules as well. Bouin's fluid caused more separation of tubules from the intertubular reticulum but preserved the cells within the seminiferous tubules well. Zenker's fluid gave results intermediate between the other two and permitted more intense staining of nuclei with hematoxylin. In hematoxylin and eosin stained sections the number of seminiferous tubules showing active spermatogenesis with developing Spermatozoa was smaller in athyroid than in the control goats. Most of the control goats showed 60 percent or more of the tubules to contain spermatozoa. In the athyroid untreated goats less than 50 percent of the tubules contained developing apermatozoa. A pronounced hyalinizing of the secondary spermato- cYtes and Spermatids occurred in the thyroidectomized goats, 71 Compared to the controls the athyroid goats had a greater number of spermatogenial cells and primary spermatocytes along the basement membrane which contained vacuolated cyto- plasm and pyknotic nuclei.. See Figures 11 and 12. When formalin-fixed, testicular tissue was stained with Sudan IV there was a much greater concentration of sudanophilic material in the seminiferous tubules of the athyroid group. Large sudan0philic droplets were found along the basement membrane as well as in the lumen. The sudano- philic substance present in the tubules of control goats appeared as finely diapersed droplets while in the thyroid- ectomized goats it appeared more coarsely globular. J, Sudan IV-stained sections of testes from a control and thyroidectomized goat are shown in Figures 13 and 14. Multinucleate cells were more numerous in the lumina of the seminiferous tubules of athyroid goats than in the control. (Figure 15). Goats in group E, treated with iodin- ated protein after cretinism had developed, showed testicu- lar changes similar to the completely athyroid goats. Histologically, necrosis and calcification occurred along the path of the biOpsy needle in testes which had been biopsied. Adjacent to the area of calcification there was complete absence of spermatocytes, apermatids and de- veloping spermatozoa. The epithelium of the seminiferous tubules had become cuboidal. The width of the area varied considerably, ranging from approximately 100 microns to three millimeterS. 72 The Leydig cells in control goats contained large oval nuclei surrounded by abundant cytoplasm which contained finely dispersed granules. In thyroidectomized goats the Leydig cells were scarcer and frequently had pyknotic nuclei and vacuolated, scanty“ cytoplasm (Figures 16 and 17). Heidenhain's modification of Mallory‘s aniline blue stain of testicular tissue showed a slight increase in inter- stitial connective tissue in the thyroidectomized goats. This stain also served well in identifying the Leydig cells in the interstitial tissue. Sections stained with aniline blue are shown in Figures 18 and 19. Sudan IV staining of the inter- stitial tissue revealed a faint reddish orange tinge in the cytoplasm of the Leydig cells which was absent in most of these cells in the thyroidectomized goats. Sertoli cell changes, if any, could not be adequately assessed. Developing spermatozoa in athyroid goats migrated toward the Sertoli cells much as they did in the controls. The increased number of sudanophilic cells noted in the athyroid goats suggests either some change in activity of the cells of Sertoli type or that fatty changes were occur- ring in the spermatogonia. .Those goats in groups B and D had testicular histology similar to that found in the control goats. The degenerative changes in group-C goats were not as pronounced as those in groups E and F. (2). Testicular biopsies. Testicular biopsies were 73 made at approximately three, six, and twelve months of age in goats from all groups. Not more than two biOpsies per goat were made. Sufficient tissue for histologic examina- tion was obtained in 21 of 26 biopsy attempts. At three months of age neither the athyroid nor the control goats showed spermatogenesis. A few of the spermatogonial cells had begun to divide in both groups. There were greater numbers of Leydig cells showing prominent cytoplasmic granules (Figure 20) in the control than in the athyroid goats, (Figure 21). At six months of age testicular biopsy specimens from goats in the control group-A, showed large numbers of primary and secondary spermatocytes and spermatids which had migrated to the Sertoli cells (Figure 22). Testicular tissue was not obtained in two biOpsy specimens from athyroid goats at six months of age. However, a biOpsy specimen obtained from a Group—F goat at nine months showed only limited spermatogenesis. Vacuolation of spermatocytes and absence of granulation in the cyt0p1asm of the Leydig cells was also noted (Figure 23). Leydig cell activity and spermatogenesis continued to be more prominent in bi0psy specimens taken at twelve months of age from the control goats. ‘ (3). Epididymis. There was decreased epithelial height, more hyalinization, fewer secretory granules, fewer stereo-cilia and fewer sperm in the proximal portions 74 of the epididymis in the thyroidectomized goats than in the controls. Goats in groups A, B, and D showed tall columnar epithelium with numerous secretory granules at the tips of the cells in the head of the epididymis. The tail portion of the epididymis was distended with spermatozoa. Treatment of cretinoid goats with iodinated protein did not improve this condition. Sections of epididymides in control and thyroidectomized goats are depicted in Figures 2A and 25. A sperm granuloma resulting from biopsy trauma of the epididymis is seen in Figure 26. (A). Accessory glands. The seminal vesicles did not show marked histologic variation between the control and thyroidectomized goats. There was less secretory fluid in the lumina 0f the seminal vesicular glands of athyroid goats. Bulbourethral glands did not show consistent histo- logic variations between the control and experimental groups. What appeared to be cystic glands on gross examination was merely distention of the central ducts with secretion. b. Endocrine glands other than gonads (l). Thyroid gland. The thyroid glands removed from the young experimental goats showed follicles of moderate size which contained colloid. Two thyroid accessory glands removed from goats previously partially thyroid- ectomized showed marked hypertrophy of the acinar epithelium 75 and absence of colloid (Figure 27). At autOpsy the goats having accessory thyroid glands showed acinar epithelial hypertrophy roughly in- versely pr0portiona1 to the amount of tissue present. The epithelial cells were irregularly tall columnar cells vary- ing from 15 to 30 microns in height. The epithelium pro- jected to the center of many of the acini where little or no colloid existed. Very little hyperplasia was noted in the accessory thyroid tissues. Goat 613 from group F which had been radio- thyroidectomized had degenerative thyroid tissue inter- spersed in a lymph nodule. The epithelial cells were atrophic and most of the follicles were filled with erythrocytes. No colloid material was seen within the acini. (2) Parathyroid glapgp. Although careful exami- nation was made for the parathyroid glands they were re- covered in only two athyroid goats at aut0psy. No hist0pathologic changes were detected in these tissues. (3). Pancreas. Islet cell nuclei showed more early pkynosis in the thyroidectomized than in the control goats. This observation is of doubtful significance in relation to other more profound changes occurring in the athyroid goats. (4). Pituitary. The anatomical structure of the -76- goat pituitary is similar to that described for the bovine (Jubb and McEntee, 1955). BaSOphilic cells comprise most of the cells seen in the medullary portion of the anterior pituitary gland. In hematoxylin—eosin stained sections the basophilic cytoplasm of cells in the central area take on a faintly bluish hue. AcidOphilic cells are found largely at the periphery of the pars distalis. Occasional acidophils occupy the central zone. The beta basophilic cells lie chiefly adjacent to the relatively large sinusoids in the .central pars distalis. However, they are also scattered among the acidophils in the peripheral portion. Some delta basophilic cells are found in the central portion of the pars distalis but most are found close to the vascular net among the acidophils. Both the acidOphils and basophils tended to be arranged in clumps which are closely invested with capillaries and small vessels. Sections made from Bouin-fixed portions of the glands preserved the architecture well but tended to give poorer differentiation of the acidophils from the basophils with hematoxylin and eosin stain. Benin-fixed sections did not take the PAS stain sufficiently well to differentiate cell types. Periodic acid-Schiff-positive material stained a bright rose purple in the formalin-fixed sections. Maximum numbers of PAS-stained cells occurred in the control goats. Large polyhedral cells which correlate with 77 beta baSOphilic cells described by Jubb and McEntee (1955) occupied the anterior portion of the pars distalis nearest the pituitary stalk. In the completely thyroidectomized goats these cells are degranulated and largely vacuolated, leaving only a spider web-like cytoplasm and sometimes a poorly stained nucleus. Occasional beta cells contained some PAS-positive material which was only faintly granulated. In the control goats, PAS-positive beta and delta cells showed granules ranging from faint through marked granule formation to intensabrstained cytoplasm in which the granules were obliterated. Table 15 shows, subjectively scored, the number of vacuolated beta basophils, delta baSOphils and acidOpils This scoring was made on PAS-stained sections. Not all of the pituitary glands were scored because formalin-fixed sec- tions of the median portion of the gland were not available. Examination of the table reveals that vacuolation and degranulation is much more pronounced in the thyroidectomized than in the control or partially thyroidectomized goats. More vacuolation and degranulation was apparent in the delta basophilic cells in the thyroid-deficient goats than in the controls. In the thyroidectomized goats which were treated with iodinated protein, granulation and vacuola- tion were variable. Figures 28 and 29 compare the histologic Structure of the pituitary in a control and thyroidectomized goat. 78 TABLE 15. Summary of Vacuolation, Cell Degran- ulation, and Type of Granulation Pre- sent in Pituitary Glands in Various Thyroid States. V— Approx. no. 1 Cells Type of 3 Group Goat Vacuolationl Degranulatedz Granulation B D" B D ,A B D A 886 0 174 l 163 2 689 o 166 1 B 778 o 137 2 C 158 3 167 3 D 756 3 681 2 E 144 2 169 3 F 758 3 3 A o o 1 1 o o 1 1 1 1 o 1 o 1 613 2 __A___ 1 0 4 3 3 1 1 3 3 3 2 1 2 2 2 l 1 3 2 3 1 3 3 2 2 l O 4 3 3 3 1 2 3 ' 4 4 1 O l 3 3 l l 3 3 3 1 1 2 3 3 2 2 2 2 2 2 2 2 3 3 l l 3 3 3 2 1 1 2 3 2 l 1 2 UWHUNMMWHOMNMHO O O l l O O 1 2 2 2 l l O 2 2 -¥ lVacuolation: 3 - marked; 2 - moderate; l — slight; O - None. gProportion of cells degranulated: 4 - 100%; 3 - 67.100% 2 — 34-66%; 1 - 0~33%. . 3Type of granulation: 4:1ntense stain, granules not visible; 3:Pronounced stain, granules prominent; 2:moderate stain, granules faint; l:faint stain, no granules B=Beta BasOphils; DzDelta BasOphils; A=Acidophils 79 (5). 'Agggagl_sl§gggp Histologic examination of the adrenal glands revealed significant variations between the euthyroid and completely thyroidectomized goats. Hema- toxylin and eosin stained sections revealed a marked differ- ence in the relative thickness of the cortical zone between athyroid and control goats. The average cortex depth measured 2.4 mm. in three group-A goats and 1.4 mm. in four goats from groups E and F. The medullary diameter in three group-A goats averaged 1.9 mm. while in goats from groups E and F it was 2.8 mm. These differences in width were not suspected until microscopic examination was made. Figures 30 and 31 compare differences in depth of cortex and diameter of the medulla. \ The hematoxylin and eosin sections revealed that there is little vacuolation of the cytoplasm of the glomerulosa. The glomerulosal layer also extended deeper into the cortex in the control goats than in the athyroid group. Sudan IV staining of the adrenal gland revealed much more lipid material in the zona glomerulosa cells in the thyroidectomized than in control goats. The zone fascicu- lata also showed greater sudanOphilia. Partially thyroid- ectomized group-B and iodinated protein supplemented group- D athyroid goats did not show the greater sudanophilia in the adrenal cortex. Treatment of athyroid goats after cretinism developed did not effect a return to the histologi- cal state shown in the control goats (Figures 32 and 33). 80 o. thra-endocrinevligéugé (l). §gig. The skin of the athyroid goats consist- ently showed hyperkeratinization. The epidermis was thrown up in folds which accounted for the rough appearance of the skin grossly. The Malpighian layer of cells was thinner in the thyroid deficient than in the control goats. In one goat (613) only one or two layers of the stratum spinosum cells remained. In the hair follicles excessive keratinization of the Malpighian layer of the external root sheath and the cuticle of the hair shaft occurred in the thyroidectomized goats. In many of the follicles almost complete degeneration of the hair follicle by hyperkeratinization was noted (Figures 34 and 35). The sebaceous glands in some of the deficient goats had pyknotic cell nuclei. Increased vacuolation of the cyto- plasm in these cells was observed. In some areas keratinized epithelium appeared to completely close the excretory ducts leading from the sebaceous glands into the hair follicle. The papillae of the dermal layer had largely dis- appeared in the athyroid goats. Edema was prevalent in the connective tissue of the derma and hypoderma. The bluish cast elicited by this edema in hematoxylin-eosin-stained sections suggested that it was mucin. Smith and Jones (1957) describe a similar appearance for the mucoid degeneration seen in myxedematous tissue. —-——-—-—-—_:—l‘—..' ' .T'_ - ‘ _ ___ 81 The cytoplasm of the smooth muscle cells in the erector pili muscles was much more vacuolated in the athy- roid than in the control goats. These cells were differ- entiated from connective tissues and possible nerve trunks by applying Heidenhain's aniline blue stain and Neil's myelin stain to unplicate sections. (2) Rumen and abomasum. The keratinized epithelium was not as thick in the thyroidectomized goats as in the controls. In some of the deficient goats ballooning degenera- tion of the Malpighian layer was noted. The papillae were less well develOped in the athyroid than in the control goats. The smooth muscle cells in many areas of the rumen wall were vacuolated and their nuclei pyknotic in the thy- roid-deprived goats. (3) Kidney. The kidneys of the thyroidectomized un- supplemented goats showed pyknosis and coagulation necrosis of many of the epithelial cells in the proximal and distal convoluted segments of the nephrons. Hyaline casts were also present in the tubules. Some early interstitial fibrous tissue was observed to be formed about the glomerulus. Figure 36 shows the kidney from an athyroid goat which was typical of the changes seen. (4). gusculo-skeletal_tissues. One of the thyroid- ectomized. goats (169) showed marked degeneration of the skeletal muscles of the posterior limb, particularly the gastrocnemius muscle. Microscopically the muscle fibers 82 appeared first to become hyalinized exhibiting a bright homo- geneous red color in eosin and hematoxylin stained sections. Where the cross striations were absent, the sarcolemma ap-’ peared to have disintegrated leaving bundles of hyaline fibrils. Some of the nuclei were pyknotic while others re- mained vesicular. The nuclei tended to accumulate along the junction between degenerating and healthy tissue. No in- ' flammatory cells were present and fat cells had extensively replaced the necrosed muscle tissue. In the heart the changes were somewhat different. The hyalinization was not as pronounced. Following necrosis the cells appeared to have fragmented into long fibrils. Lacuna- like spaces formed about the nuclei which gave the muscle the appearance of hyaline cartilage. There was little replace- ment of necrosed muscle with fat. No calcification was evi- dent in either the heart or skeletal muscle. Figures 37 and 38 show skeletal and heart muscle changes. Fig. 2. Appearance of goats at conclusion of experi- ment. From left to right goats are from control group A, partially thyroidectomized group 3, late thyroidectomized group C, early thyroidectomized grouer. Fig. 3. Goat on left is from group A. Two goats in Center were thyroidectomized early (group D) but received .04 gm. iodinated protein per ten pounds body weight. Goat on extreme right is from group F. Fig. 4. Goat from group-E showing arrested horn growth. Extreme cretinoid symptoms were present before iodinated protein therapy was instituted. T‘we-e‘ blasts} . \\ . ’k ‘51.. ' ‘ ,‘ - - :‘;:;-E"f:5’«-e-Léfév}é 7:91,?“ 1’ - I/ ., Fig. 5. Strips of back skin removed from control and athyroid goats at necropsy. Top: control goat--note absence of alopecia. Lower: skin from athyroid goat. Much loss of hair and hyperkeratosis. J ,. amt. f , /.A Fig. 6. Accessory thyroid tissue in group-B goat located near anterior and of the sternum on the ventral sur- face of the trachea. “'Illl.‘llu‘ ' ‘ 7 ~ .HH!IIIIIHHIHHIHHIHH IHIIIIH HHIHH N '4 ‘10 l 1 1/2 113 14 --_._‘_> Fig. 7. Condylar surfaces of the bones of the tibia- tarsal Joint. Group-A goat on the left and group-F goat on the right. Arrows indicate eroded areas of cartilaginous surface. ._.v I" r' n»..-_ . :3}.I‘ .5 1‘3”! -¢¢_ 13.? '3‘6: ', ' 1*} \ , “i' i ii . C v ~ ' .g‘s‘o.‘ 1‘ 3 ‘ .. I; 4:,- % \ l I 4' 1 . c . , - A :1 1 1 '1‘».°" ' "" .n '- ‘\ ‘3 wig.“ . $491555“- vi-sf’t $31.13.": A a? Fig. 8. Testis section showing seminiferous tubules from control goat. Tissue was formalin fixed. Note good retention of interstitial tissue but loss of cellular ele- ments fro ' . ". _.'.;t:.)f3: ‘: ‘Qj. k - ~1u ‘. . ' ’ I... dh‘. .‘ I. “121% ri‘-:§ ' . ,7 ., Fig. 9. Testis section from same goat as in fig. 8 Zenker-fixed. Some separation of tubules but cell nuclei stained more intensely. H & E x 110. 87 Fig. 10. Testis section from same goat as in fig. 8. Bouin-fixed seminiferous tubules are widely separated, but there is good retention of intratubular cells. HE x 110. .1 . .' . J. A ‘ 1 Fig. 11. Testis from control goat. Note absence of vacuoles among primary and secondary spermatocytes (a & b) and spermatids (c). Maturing sperm cells regularly lined d up at tips of Sertoli cells ). Zenker-fixed. H & E x 575. 1.2.1" 1 1M. Fig. 12. Testis from group-F goat. Spermatogenic cells vacuolated (A). There is disruption of the spermatogenetic wave in the epithelium with pyknosis (B) and karyorrhexis of nuclei (C) in many spermatocytes. Zenker-fixed. H & E x 600. \ ‘ ‘Vb ‘ 9 I .—- (a! fi'v-l a.- a \ | Sudan IV stained , 'JT . a Fig. 13. Testis fr section counterstained with Harris' hematoxylin. spermato- Only genic cell nuclei stain intensely with counter stain. cells and cell debris near lumen (A) are Sudanophilic. x 150. ' \- -‘.‘V\ 1‘ . ~ '1‘ .-..(-. ‘._--..‘,~ '.-'\’I 625‘ .,-"' \. '>,.T.. . \ I '. l . . ' ' ' ..' I n I‘ . ‘ .. . t b .. s ‘ . . _. , ‘ 0 ‘ " ‘ l ‘ l' . I ' ' ' '. " \.‘ . _ ' ‘ . ‘ . ._ _ . ‘ in n . - . . ' V. ‘_ 1 — ‘ '\* f} It -. _, in-” ”'7, ,7,_ 11, _‘*- (.1‘ . . .{31 ‘. .. - , 5“ ‘ _ . __ " . Fig. 14. Testis from athyroid goat. Sudan IV stained Note rela- Sudan- section counterstained with Harris' hematoxylin. tive absence of nuclear stain, compared to fig. 13. ODhilic cells along basement membrane (A) and in lumen of tubule (B). x 150. 90 Fig. 15. Seminiferous tubule from athyroid goat showing atrophy of germinal epithelium (A) de enerating spermatids (D) and multinucleate cells in lumen M). H & E x 590. 1 ° 1' I ’ ' ' Q t; ”o «O f H . ‘ ‘ ‘ II \ " , ‘ ‘5 . . t" No 9’" i‘fi’oe' . a ‘ \‘ ( 81“!" ‘t‘. " . g 9' P f. a , 5‘.) ‘ ‘.r, r ' ‘g \‘13 4: is '4‘ ' o I 3’ I . ' Q9. , s~ :irc. Du .‘ : 5 . g «9 ., .' _‘ \ b m: * ‘ «I o , . ..-- . ' 0 EA, - ' . ’_ v ‘0‘} - . ) _ .0 1‘.."' "i _ , 1 , 11,4, 7 ,, _ _;.’§A,1,' Fig. 16. Section showing Leydig cells in testicular interstitial tissue from group-A goat. Nuclei are rounded and abundant cytoplasm shows slight granulation (L). Zenker- fixed. H & E x 590. 1mi' ' I. .. L , ,,,,A I ‘ .7 ,‘ ,1 Fig. 17. Section showing Leydig cells in testicular interstitial tissue from group-F goat. Pyknotic nuclei are evident (P), vacuolation is present in cytoplasm (V), granulation of the cytoplasm is faint. Zenker-fixed. H & E stain x 600. '0 ‘11"? Fig. 18. Heidenhain' s aniline blue stain of testis from control group-A goat for collagenous fibers (C) in base- ment membrane and interstitial tissue. Also note prominent granules in cytoplasm of Leydig cells (G). x 625. --.,., \N“ ‘5‘ Fig. 19. Heidenhain' s aniline blue stain of testis from athyroid group-F goat. Collagen increased in basement mem- brane of seminiferous tubules (C). Leydig cells (L) are small and have little cytoplasm. x 625. Fig. 20. Testicular biopsy section from control goat at three months of age. Leydig cells prominent Spermatogonia inactive except for occasional primary sperma- tocyte. H & E x 625. .,~ _r::3s f!”". 2.5 A , A-1_ ‘Lu- . - his. _. ‘3 ._17A cular biopsy from athyroid group-F goat at three months of age. Interstitial cell nuclei are small and cytoplasm is scanty (L). Occasional Spermatogonia have divided and moved toward lumen of seminiferous tubules. H & E x 675. Fig. 21. Testi Fig. 22. Testicular biopsy section from group-A goat at six months. Active spermatocytogenesis present, with developing spermatids lined up (S) at tips of Sertoli cells. H a: E X 6750 _‘ W V ‘ ' ., ’t _" I! \*"“‘.‘“3§¥§‘N' \\ .. ix _., f“ \:I;;(3x""h\§' ‘. 333:1)“ ._‘" ~.' _ '1 K 3 y ' ‘-\ r A -“' R‘QE'? ; a. In ‘ “I A l 1 ‘ \o ~\ ,4.” ‘ %~"»»3u;"3y:§1’ip -. 3%; J: 8:1. ’ p_~s 11 Fig. 23. Testicular biopsy section from group-F goat at age nine months. Limited Spermatogenesis is evident. There is much vacuolation of primary and secondary Spermat- ocytes and degeneration of spermatids (S). H a E x 160. F13. 24. Section from head of epididymis of control 3 prominent stereocilia (S), secretion droplets, relat ve absence of vacuoles in epithelium and many sperato- lzoa in the lumen. H & E x 110. a. o" \ .' -._M Fir. 25. Section from head of epididymis ZrOUP-F goat- 4 1 “x 1 Circular body at A), epithelium much vacuolated n). Few tereocilis, s armatozoa, and secretion granules in the ‘ H a E x 160. a lumina of the epididymal ducts. . ~ '. n I . t 5- t. \v \~ ' :4 .. . I. , ._ hk‘iisfiawt‘ .‘ «ma ;. ?6. Grenulomatous reaction in testis following bioySy with Vim—Silverflan biOpsy needle. Calcification of spermatozoa has occurred at (A), giant cell formation is shown at (B). Seminiferous tubules immediately edjccent show degeneration and atrophy. H & E x 175. \ . ‘ Fir 1 rm i 2 k . x)~ u‘ ( ‘ _ . a \ ‘x ;- t, .- . u .. t a ‘.- ' .1 , o ‘- Q ‘ > . ~ r , x» '_, Fig. 97. Accessory thyroid tissue removed+at six months from group-C goat originally thyroidectomized at one month of age. Note hypertroyhy of acinar epithelium at (A) and hyperplasia at (B). H & E X 375- Q ' Q. , . . i 5451.“? Fig. 28. Anterior pituitary gland from group-A goat. Central area consisting primarily'of basophilic cells to the left of (B). Acidoph‘ilic cells in outer zone at (A). PAS - Orange G stain x 230. c ‘ 4",“ "." t Q‘ K ' . . / ' ' o . . Q! ' I ‘ o‘\ ‘ A l - ‘ z t . 7 .4 Fig. 28 a. ‘Higher power magnification of fig. 28. Beta basophilic cell showing intense PAS positive mucoprotein (3). Acidophilic cell (A). Smaller delta basophilic cells showing moderate granule fromation (D). PAS - Orange G X 650. ' - £1:- .- VJ; ~,_r~ ‘5: ' ‘5;-.-~ ‘ ‘O i ,‘4 Fig. 29. Anterior pituitary gland from group-F goat. Acidophilic zone (A), vacuolated beta cell at (B), delta cell containing PAS positive cell at (D). Other delta cells to left of (D) are vacuolated. x 230. V .5 Fig. 30. Adrenal gland from group-A goat. (G) Zona glomerulosa, (F) Zona fasciculata, (M) Edge of medullary zone. H & E x 45. ‘7, _ I“. “M.- ‘- . a. . ‘_ z . .- 1.2.. ,. Fig. 31. Adrenal gland from group-F goat. Compare fig. 30. Cortical zones (G) and (F) much shallower than in control goats. H & E x 45. '.I ‘ 4 . '1‘." .‘ . QPezfQ‘ - g . U I I ‘ A ‘.‘ -‘ ‘r “.1 "*' A_ 1" 31;} '; l-Jh- :fl TrfiN“..—¥mfi- .‘u'i‘n I Fig. 32. Adrenal gland from group-A goat stained with Sudan IV. Sudancphilic substance equally distributed in zone glomerulosa (G) and zone fasciculata (F). Sudan IV x 150. ' \V“~'i\‘ .S ' "33’, \ \ u .\.\‘s 153 N \‘ . u. dffi*‘\ . 1’ r‘ ' w‘ ‘ ‘ V.\ I ' ‘ J. ! _ . 2' . .-*:;=‘"-':~iWse eat -. ,- 0..., i Fig. 33. Adrenal gland from group-F goat stained with Sudan IV. Intense sudanOphilic staining in zone glomerulosa (G). Decreased stain intensity in fascicular zone (F). x 150. L!".e’g".}u,: . .3g—1 Fig. 34. Skin fro bac of group-A control goat. Ex- cessive keratinization of epidermis is absent (0). Smooth muscle cells (M) show little vacuolation. H & E x 130. \x‘ " 4" » "vw ? ' Fig. 35. Skin from back of group-F athyroid goat. Showing hyperkeratosis of stratum corneum (C), degeneration of hair shaft (S), and vacuolation of smooth muscle (M . H & E x 115. 102 Section from kidney of group-E goat which had Fibrous H & E x (0). Cast within tubule at (F) present around glomerulus. 61713. P Fig. 36. calculi in renal tissue proliferation 115. Fig. 37. Gastrocnemius muscle from group-E goat show- i hyalinization (H), muscle nuclei (S) and fatty tissue (g? which has replaced destroyed muscle fibers. H a E x 160. . ~. ': .\" ' o .x _ .<.,§\\ . | - __\ _ > 1’“... , Fig. 38. Mycardium showing lacunae like spaces around Burkinde fiber nuclei (D) and fragmentation of cardiac muscle fibers. H & E x 160. V. DISCUSSION A. Relation of Clinical Symptoms to Thyroid Deficiency The onset of symptoms about two weeks following com- plete thyroidectomy correlates closely with the observations of Reineke g; éi° (1941) and jimpson (1974a?.. The latter author reported that rate of gain began to decline on the nineteenth day after thyroid removal. The failure of many thyroidectomized sheep to show cretinism, was reported by Marston and Pierce (1932). At autOpsy varying amounts of accessory thyroid tissue were found. The data from the eXperiment reported here indicate that accessory thyroid tissue is frequently present in goats. This may be found in varying locations extending from the larynx to the thoracic cavity. The thyroid uptake data suggest that a large part of the normal thyroid gland can be removed without clinical im- 2 pairment of body functions. Radioactive 11"1 uptake measure- ment provides a convenient method of determining the presence of thyroid tissue soon after surgical ablation. Although a reasonable attempt was made to standardize the counting proce- dure for each goat some variations existed which could account for differences in Percent uptake of the injected dose. Nevertheless goats showing less than .5 percent or the in- jected dose present in the thyroid region went on to become 104 105 cretins. Those showing more than 1.7 percent of the injected dose showed no cretinoid symptoms. Low counts in the thyroid region may be obtained in the complete absence of thyroid tissue. Bustad gt_gl. (1957), Fletcher gt 3;. (1956), Flexner gt gl. (l9h2), Logothetopoulos and Myant (1956), Taurog §t_gl. (1959) have shown that salivary tissue can concentrate iodine. 131 treated goats The symptoms of athyroidism in I were slower in developing than when all tissue was removed by surgery. Bustad §£.§ly (1957) report that destruction of the iodine concentrating power required one month and nine 1131 per day months in ewes fed 1800 and 240 microcuries respectively. It appears that eight millicuries of 1131 given subcutaneously was a reasonable dose to destroy the remaining accessory thyroid tissue. The lethargy, stiffness, bloating, alopecia, skin dryness, myxedema and reduced horn growth noted in the athy- roid goats in this experiment agree with similar observa- tions made in goats by Simpson (1927 a and b), and Liddel and Simpson (1926). In sheep similar symptoms were reported after thyroidectomy by Marston and Pierce (1932) and after radiothyroidectomy by Bustad gt 3;. (1957). Replacement therapy with iodinated casein when symptoms of cretinism first appeared, at a level suggested by the work of Flamboe and Reineke (1959), gave essentially the same growth rates and other clinical features shown by goats with intact thyroids. Delaying therapy until cretinism was 106 well established resulted in only partially alleviating the symptoms. The cessation of libido in thyroidectomized goats was similar to that reported by Maqsood (l951h) in rams dur- ing the summer season. Libido was restored in thyroidecto- ndaxigoats when thyroxine was replaced as soon as symptoms of cretinism appeared. However, replacement therapy failed when cretinism was prolonged. This might explain why some aged animals with a history of prolonged infertility fail to reapond to thyroid therapy. B. Semen Production and Sperm Morphology Several factors enter into the semen data which bear discussing. First, no attempt was made to limit feed intake of controls to that of the therprival goats. It was felt that to do so would not be fair to the control goats whose energy requirements would be considerably greater than that of the athyroid goats. Secondly, the necessity for using electroejaculation to obtain semen creates quite different results than those obtained by collection in an artificial vagina. Thirdly, the collection frequency was less than would have been Optimum. This may have resulted in a differ- ence in Sperm concentration, motility, and volume between the thyroidectomized and control goats, because of the lack of masturbation in the former and its frequency in the latter. Fourthly, the output of semen could have been affected by 107 the testicular biOpsies performed. However it was felt that, since testicular biOpsy was performed in both control and thyroidectomized goats, similar relative differences should occur between groups. So far as could be determined this is the first eXperi- ment in which semen was collected by electroejaculation from young completely thyroidectomized goats. The data indicate that, though spermatozoan production is not completely in- hibited, significantly fewer motile spermatozoa per ejacu- late were Obtained from them than from the control goats. The decreased apermatogenesis encountered in athyroid goats confirms the findings of Maqsood (1950b, 1951a) that thyroid inhibition by thiouracil exerts similar effects in young rabbits and rams. The average semen volume for the control goats was higher (l.4cc.vs. .65 cod, the concentration lower (2.63 vs. 2.72 billion per ch and the total spermatozoa per ejaculate higher (3.180 vs. 1.658 billion) than values for goats re- ported by Eaton and Simmons (1952). That no detectable Seasonal variation occurred in semen production or in numbers of abnormal spermatozoa was attributed to the fact that the goats were kept inside a building which greatly modified extremes of outside environ- mental temperatures during the summer and winter months. The percent of abnormal Spermatozoa seen in the con- trol goats was considerably higher than that reported in 108 goats bylhuonandSimnrs Q9321216.0% vs. 8.45%). It is speculated that this difference might be eXplained by the effect which testicular biopsy had on spermatids which were in the process of deve10pment at the time of tissue removal. The proto- plasmic extrusion seen on heads of spermatozoa from both the control and athyroid goats constituted approximately one half of the total abnormalities seen. It is possible that this may be a normal structure which would not be seen by ordinary light microscopy. C. Pathological Changes The general pathological changes noted in the com- pletely thyroidectomized goats agree closely with those noted by Goldberg (1927). Hematology and blood chemistry were also similar ex- cept that Goldberg (1927) noted a larger drop in blood sugar in the athyroid goats than was obtained in this experiment. Reference to the erythrocytes with pointed processes in young goats was not found in the literature. These abnormal erythrocytes persisted longer in young athyroid goats than in the controls. Goldberg and Simpson (1925) noted that thyroidectomized sheep and goats showed greater than normal numbers of megakaryocytes, formative erythroblasts and granu- lar leucocytes. Whether those two observations are correlated remains to be determined. Bustad,gtwgl. (1957) report that serum calcium in lambs born to ewes fed 240/kc 1131 per day was lower and the serum 109 inorganic phosphate higher than in the control lambs. In the present eXperiment the athyroid goats had lower serum cal- cium and higher serum phosphorus than the controls. Whether parathyroid gland functionwas impaired is a matter for some conjecture. Marston and Pierce (1932) stated that sheep have two pairs of parathyroids, one embedded in thymic tissue dorsal to the thyroid gland and the other intimately bound to the thyroid gland. Simpson (1912) reported that acute fatal tetany followed the removal of the thyroid and parathy- roid gland in lambs five to seven weeks old. In the experi- ment reported here no tetany was seen among the athyroid goats. It was assumed that parathyroid tissue remained in these goats even though it was found in only two of those which were completely thyroidectomized. Urinary calculi were not mentioned by Goldberg (1927) as one of the findings in thyroidectomized sheep and goats, though renal pathology was noted. The acid solubility of the calculi found in the present eXperiment indicates they were not steroid in nature. In view of the increase in inorganic phosphorus in the serum it is speculated that increased urinary phosphorus may have followed, by itself or in combina- tion with other factors causing conditions favorable to the formation of phosphatic calculi. Further eXperimental work will be necessary to elucidate the role of hypothyroidism in zrinary calculus formation. Joint erosions, particularly in the tibio—tarsal joint have not been reported before in thyroidectomized goats in 110 so far as could be ascertained. Silberberg and Silberberg (1955) reported that aging mice of certain hypothyroid strains develop lesions of the articular cartilage similar to those seen in the thyroidectomized goats in this eXperiment. Ulcer- athniof the articular cartilage in mice however was followed by ossification of the underlying bone, marginal outgrowth of cartilare and bone in the joint cavity, and fibrosis and ingrowth of the synovial membrane into the epiphyseal marrow. The latter changes were not characteristic of the arthritic ulceration seen in goats although the lesions may not have persisted long enough to permit development of the chronic form. Lung changes were difficult to correlate with the hypothyroid condition. Although the atelectasis was-more extensive in the athyroid group it also occurred in some of the controls. The marked stertorous breathing noted during life, however, was confined to the athyroid groups of goats. Bustad gt El- (1957) reported that ewes whose thyroids had 131 experienced increased sensitivityto been destroyed by I cold and were subject to reapiratory infection. The adrenal gland changes in goats confirm the ob- servations of Deaneand Greep (19$?) concerning the increased Sudan IV~staining and shrinkage in the cortex. Decrease in Size of the adrenal gland in proportion to body weight in the thyroidectomized goats was not noted, however. No men- tion was made in the literature reviewed about the relative 111 increase in diameter of the medulla accompanying the decrease in depth of the cortex. Whether this medullary increase is some form of compensatory enlargement remains a matter for conjecture and further investigation. The pituitary gland changes in thyroidectomized goats agree closely with those observed by Jubb and McEntee (1955b) in the thyroidectomized bull and with those of Halmi (1950), and Purves and Griesbach (1951b) in hypothyroid rats. The large beta basophilic cells in the central part of the pars distalis which stain PAS pesitive become degranulated and vacuolated following thyroidectomy. A subjective decrease in the small delta cells suggested but did not establish that the gonadotroph cells described by Jubb and McEntee (1955b) in bulls were decreased in the group-F goats subjected to prolonged athyroidism. Although the testes and accessory glands were smaller in thyroidectomized goats from groups B and F than in the controls, the size in relation to body weight was the same as for the controls. This is somewhat contradictory to the evidence presented by Maqsood and Reineke (1950) in mice, Scow and Simpson (1945) in rats, Maqsood (1950b) in rabbits, and Greenwood and Chu (1939) in cookerels that thyroidectomy or thiouracil caused reduction in testicular size. The failure of thyroidectomy to cause decrease in relative testicular size in goats many have resulted from removal too late to prevent testicular development. p.) H "O The histological changes in the testis (atrophic and degenerative changes in the seminiferous tubules and the interstitial tissue) are similar in thyroidectomized goats to those reported in thyroid-inhibited mice by Maqsood and é"? \s. U) 4 ), in 1 . ‘1’ 3 5 RD Reineke (1970), in rats by Scow and Slaps A rabbits and rams by MaQSood {lu505). The observation that the degenerative changes occur principally in secondary spermatocytes and epermatids agrees with the findings of Knudsen (1954). He observed that in bulls, whose testes were subjected to thermal insulation for short or extended periods, primary spermatocytes failed to show normal mitotic activity. Sudan IV-staining of testicular sections revealed increased sudanophilic substances in cells along the basement membrane and in cells in lumina of the seminiferous tubules. Whether this increase in sudanophilic substance was due to accumulated neutral fat or a possible increase in ketosteroid materials was not determined. Deans and Greep (1947) by special techniques showed that increased sudanOphilia in the glomerulosa of the adrenal gland was due to an increase in ketosteroids. Since the Sertoli cells are believed to pro- duce estrogenic subspances in certain pathologic states such as Sertoli-cell tumors (Smith and cones 1957} it seems that \ :3 C CD 1 the increased sudanophilia wight be due to ketos+ .oiis. Interstitial tissue changes approximated those reported by several authors studying the effect of thiouracil 113 feeding on Spermatogenesis in rams, rabbits, and mice (Maqsood and Reineke, 1950; Maqsood 1951a, 1951b). Thyroid- ectomy of rams resulted in atrophy of testicular interstitial tissues in studies reported by Berliner and Warbritton (1937). In the present study there were fewer Leydig cells in testes from thyroidectomized goats and a greater preportion showed nuclear pyknosis, and cytoplasmic atrophy. The aniline blue stain (Heidenhainb modification) differentiated the Leydig cells well from the collagenous interstitial fibers. In the control goats the majority of the Leydig cells stood out as large ovoid cells with large round nuclei and a distinct granular cyt0p1asm. Sudan IV stained the cytoplasm of these cells very faintly. In the athyroid goats the cytoplasmic granules were much diminished or absent. Pronounced changes in the epithelium of the epididymis suggest diminished function in this portion of the reproduc- tive organs. Few changes in the epididymis have been re- ported following thyroid inhibition. Maqsood and Reineke (1950) reported a decrease in seminal vesicular weight in 'young mice fed thiouracil. From these studies it is concluded that total thyroid- ectomy is necessary to produce the typical effects of de- creased reproductive fUnction. 'Thyroidectomy apparently has its most profound effect on libido. However, since libido is dependent on neurological as well as endocrine factors the extent to which other conditions such as arthritis and 114 its accompanying pain affected this function could not be accurately determined. D. Testicular BiOpsy as a Tool for Measuring Testicular Function Testicular biOpsy was successful in the majority of the goats in which it was tried. It is doubtful that indis- criminate repeated biopsy with the Vim-Silverman needle should be performed since some permanent damage to testicular tissue occurs. Barker (1949) reported that, of three methods of testicular biopsy, the Vim-Silverman biOpsy needle caused the least damage. The technique of Barker (1949) using the dorsa- lateral portion of the testis of goats proved effective in obtaining specimens with the Vim-Silverman needle. It was determined that the onset of spermatogenesis was delayed in the thyroidectomized goats. The results of the biOpsies indicate that care must be exercised in young goats not to puncture the head of the epididymis since escape of spermato- zoa will result in the formation of a Sperm granuloma. VI. SUMMARY AND CONCLUSIONS The clinical and pathological effects of complete and partial thyroidectomy on reproductive function in young male goats were studied. Because of the complete lack of libido in athyroid goats electroejaculation was employed to collect semen from all goats. It was further observed that compared to the controls the athyroid goats: 1. 2. 3. 7. Produced significantly fewer motile spermatozoa per ejaculate (.59 billion vs. 1.71 billion.) Produced significantly more abnormal spermatozoa. Had lower absolute mean testicular weights, but equivalent ratio of testicular weight to body weight. Showed more cytoplasmic degranulation and atrOphy and more pyknosis of Leydig cells. Had more vacuolation and hyaline-like change in secondary spermatocytes and spermatids. Exhibited greater sudanophilia among cells in the seminiferous tubules, particularly in the lumen and along the basement membrane. Showed more vacuolation, fewer stereocilia and the formation of laminated bodies resembling corpora amylacea in the epithelium of the head of the epididymis. 115 116 8. Had more pronounced degranulation and vacuolation of the large beta basophils in the anterior lobe of the pituitary gland. 9. Had a shallower adrenal cortex and a greater medullary diameter in the adrenal gland. lO. Showed hyperkeratosis of the epidermis and hair follicles and vacuolar degeneration of smooth muscle in the dermis. 11. Had consistently appearing shallow erosions of articular cartilages of the tibiotarsal Joints while the controls had none. It was concluded that athyroidism more profoundly af- fected libido than it did Sperm production even though the latter was significantly reduced. Reproductive and other functions were not impaired when male goats were only partially thyroidectomized. Like- wise administering iodinated protein soon after complete thyroidectomy permitted normal growth and semen production. Replacement therapy after pronounced cretinoid symptoms develOped did not restore normal growth nor reproductive function but did alleviate other symptoms of cretinism. This may explain in part the failure of bulls and other male animals eXperiencing chronically reduced reproductive func- tion to respond to thyroid or other therapy. When complete thyroidectomy was delayed till about six months of age there was a non-significant decrease in sperm production and some 117 1089 in libido. Had this latter group of goats been kept longer it is probable that reproductive function would have steadily declined. It was concluded that surgical thyroidectomy must be followed by 1131 uptake studies to determine whether acces- sory thyroid tissue remains in goats used for athyroid eXperi- mentation. Approximately eight millicuries of 1131 destroyed the remaining tissue without causing other undue local lesions in incompletely thyroidectomized goats. Testicular biOpsy provided an adequate means of assess- ing testicular histology but resulted in some local necrosis and calcification along the path of needle entry. It was found that extreme care should be taken to prevent puncture of the epididymis, or when it is punctured, the escaping sperm invariably cause formation of a granuloma. LITERATURE CITED Armstrong, D. T. and Hansel, H. 1956. The Effect of Age and Plane of Nutrition on Growth Hormone and Thyrotrophic Hormone Contents of Pituitary Glands of Holstein Heifers. J. Animal Sci. 15:640. Asplund, R. 0., McLaren, G. A., Henderson, H. 0., and Porter- field, I. D. 1959. Unusual Magnitude and Variation of Plasma Protein Bound Values of Dairy Cattle. J. Dairy Sci. Barker, 0. A. V. 1949. Testicular BiOpsy TechniQues in Bulls. Report of . the Ontario Vet. College, 130. Bauman, E. J. and Marine, D. 1945. Involution of the Adrenal Cortex in Rats Fed With Thiouracil. Endocrinology 36:400.- 405. BeaCh, F. A. 1947. A Review of Physiological and Psychological Studies of Sexual Behavior in Mammals. Physiol. Reviews. 27:240 ~ 307. Berliner, V. and Warbritton, V. 1937. The Pituitary and Thyroid in Relation to Sperm Production in Rams. Am. Soc. Animal Prod. Proc. Black, W. G., Schick, A. L., Pope, A. L. and Casida, L. E. 1950. Some Effects of Testosterone Proprionate and Thyro- protein in Lambs. J. Animal Sci. 9:186. Blaxter, K. L., Reineke, E. P., Crampton, E. W., and Petersen, .. , W. E. 19kg. The Role of Thyroidal Materials and of Synthetic Goitrogens in Animal Production and an Appraisal of Their Practical Use. J. Animal Sci. 8:307 - 352. Blivaiss, B. B. ~ 1947: Interrelation of Thyroid and Gonad in the Develop- ment of Plumage and Other Sex Characters in Brown Leghorn Roosters. Physiol. Zoology 20:67 - 107. 118 Bogart, R. and Mayer, D. T. 1946. Environmental Temperature and Thyroid Gland Involve- ment in Lowered Fertility of Rams. Mo. Agr. Res. Bulletin. 402. Brenton, 0., Griffith, W. 8., Patrick, T. E., Johnston, J. E., " and D'Arensbourg 1955. Seasonal Trends inG Plasma Protein-Bound Iodine (PBI) Levels, Semen Production and Fertility of Bulls. J. Dairy Sci. 38:602. Bratton, R. W., Musgrave, S. D., Dunn, H. 0., and Foote, R. H. 1959. Causes and Prevention of Reproductive Failures in Dairy Cattle. II Influences of Underfeeding and Overfeeding from Birth to 80 Weeks of Age on Growth, Sexual Development, and Semen Production of Holstein Bulls. Cornell Univ. Bulletin. 940. Brody, S. 1945. Bioenergetics and Growth. The Reinhold Publishing Co. New York. Bustad, L. K., George, L. A.. Jr., Marks, 8., Warner, D. E., Barnes, C. M., Horde, K133. and Kornberg. H. A 1957. Biological Effects of I Continuously dministered to Sheep. Radiation Res. 6: 380 - 413. Deane, H. w. and Greep, R. O. 1947. A Cytochemical Study of the Adrenal Cortex in Hypo— and Hyperthyroidism. Endocrinology 41: 243 — 257. Dutt, R. H. and Bush, L. F. 1955. The Effect of Low Environmental Temperature on Initiation of the Breeding Season and Fertility in Sheep. J. Animal Sci. 14: 885 - 896. Dutt, R. H. and Simpson, E. C. 1957. Environmental Temperature and Fertility of South Down Rams in the Breeding Season. J. Animal Sci. 16:136 - 143. Dyrendahl, Sven. 1949. Some Effects of Feeding Iodinated Casein for a Long Time to Cattle, Swine and White Rats. Royal Veterinary College, Stockholm. 116p. Dziuk, P. J., Graham, E. P., Donker, J. D., Marion, G. B., and Petersen; W. E. 1954. Some Observations in Collection of Semen from Bulls, Goats, Boars and Rams by Electrical Stimula— tion. Vet. Med. 49:455 - 458. Eaton, 0. N., Schott, R. 1., Simmons, V. L. and Frank, A. R. 1948. The Effects of Feeding. hyr orcceic.or Seaen Charac- teristics of Rams. J. of Animal Sci. 71449-454. E3t0n. 0. N. and Simmons, V. L. 1959. A Semen Study of Goats. Am. J. Vet. Res. 13:32? 544. Fiaccavento, W. 1952. Histological Changes in the Testicle Following Treatment with Thiouracil. Policlin. Sez. Chir. 59:183 - 192. Cited in Excerpts Med. Sec. 6.. 6 28 1953. K}: , e, C. H. and Subbarow, Y. 925. The Colorimetric Determination of Phosphorus. J. Biol. Chem. 66:375 - 400. Ht}: Flamboe, E. E. and Reineke, E. P. 1957. Thyroxine Secretion Rates of Lactating and hon- 1actating Dairy Goats. J. Anima Sci. 16:1061 abstr. - Flamboe, E. E. and Reineke, E. P. 1959. Estimation of Thyroid Secretion Rates in Dairy Goats and Keasurement of 115* Uptake and Relaese w th Regard to Age, Pregnancy Lactation e d eason of the Year. J. Animal Sci. 18:1135 - 1148. Fletcher, K., Honour, A. J., and Rowlands E. N. 1956. Studies on the Concentration of Radio-Iodide and Thiocyanate by Slices of the Salivary Gland. Biochemical J. 63:194 - 199. Flexner, J., Brug rer, M., and F.emoer S. 194?. The Exc retio of Iodine by the Salivary and 3astric Glands. Fed. Proc. 1:109. Flipse, R. J., Snyder, J. W., Thai ker, D. L. and Almquist, J. O. 1953. Effect of Tot a1 Digest ible I.utrient Intake on Grewth and Reproductive DevelOpment in Dairy Bulls. Penn. State Univ. Frog. Rept. 104. L, A,, Jr.,Barnes, C. N., i d Bu st1d, L. K. . A Procedure for Thyro Hides omy in Sheep. J. A. V. Q m 0 +41 \0 '\ U] (D "‘1. 3. er Changes in Thyroidectomiz;d Goldberg, S. A. and Sizes .1 c. EXp. 3101. and Red. 23:13? - 133. 1995. Osseous and Ana 3 q 4. O C O L. 1 ha k-J (D O ’ #13 U1 (7’ ' Rennenan, Henneman, I?e&-o13»n’ H. u) KDm it i" h 3 PJ F: ‘—’. A... n n -vt~ ,u D ~‘ chance: in Cr ans oi invro N .. A‘ . V‘.r n goats. Soar. J. Eat : s : A Few Stain for Elastic Tissue. ~. J. Clin. Path. 20: FF5 _ 666, "R A. V. And Chu, J. r. On one Relation Between Thyroid ani Sen Gland Functionin: in LTOWH Leghorn Fowl. lugr. J. Etp. Fh35101. 2;:111 119. W. E. Relationshin of the Bosonhil Cells and Thyrotronhic Formone Production. G::. You. fortns. L:€Al - 2L7. itcd in Etcernt? Veiica, Sec. 3. 6:”29, 195?. W. E. And Purves, V. D. Tue TignifiCWnce of t?e Rasorhil hanges in the “i‘wi+1r" Accomrenyin: Verious F‘rrs of Thyroxine (eficiency. Eri . J. of Ex:. Fear. 25:15 - 17. S. Tro Type: of Bancrhils in the Anterior Pituitary Cf the Rat and The‘r Resrective Cvtoohyciological Significance. Eniocrinolo"y A7:-: — 299. 1 1. The Effects of Graied Doses of Thyroxin on the Anterior Tituitarv of Ryrotr yroii Aloi:c Rats. Anat. Record. 112:17 — 16. i n the of Sheen as Affected 3 Y1 o S 3 ed, fre3ntncy ani La -ctati:n. A Tr s‘e for u. . Deeree, Kich. State Univ. . P. and Griffin, S. A. we Thyroid Section Rate of Sneen as Affected ‘ I‘Rreed, Pre~nancy and Lactation. 14:43.9 - 434. A. and Madden, F. W. The Artific al Insemination f Dairy Cattle. A Ear ioooa and Labor tory Manwal. Lucas Brothers, Columoia. Ao. . S , Delfs, E. and Foot, E. C. r ffect of Thioura il Hypothyroidism on Repro- von in the Rat. Endocrinology 38:33? - 344. {‘me 45.8 h \I 11 O T“ H 4.4 I A t 122 Jubb, K. V. and McEntee, K. 1955a. Observations on the Bovine Pituitary Gland. I. Egvie: of Literature on the General Problem of eno ypOp yseal Functional Cytology. Cornell Vet. 453576 ' 5920 Jubb, K. V. and McEntee, K. 1955b. Observations on the Bovine Pituitary Gland. II. Architecture and Cytology with Special Reference to Basophil Function. Cornell Vet. 45:593 - 641. Kirkpatrick, Charles M. 1955. The Testis of the Fox Squirrel in Relation to Age and Seasons. Am. J. of Anat. 9:229 - 243. KnUdS en, 00 1954. Cytomorphological Investigations into the Spermio- cytogenesis of Bulls with Norman Fertility and Bulls with Acquired Disturbances in Spermiogenesis. Acts Pathologica et Microbiologica Scandinavica Supple- mentum 101. Lennon, H. D., Jr. and Mixner, J. P. 1958. Relationships Between Plasma Protein Bound Iodine and Certain Measures of Reproductive and Lactational Performance in Dairy Cattle. J. Dairy Sci. 41:740. Levin, M. E. and Daughaday, W. H. 1955. The Influence of Thyroid on Adrenocortical Function. J. Clin. Endocrinology. 15:1.499 - 1511. Lewis, R. C. 1952. Protein Bound Iodine Levels in Dairy Cattle Plasma Milk, and Colostrum. Thesis for Ph. D. Degree. Mich. State Univ. Lewis, R. C., Lodge, J. R., and Reineke, E. P. 1957. The Thyroxine Secretion Rate of Dairy Calves Estimated by the Extrapolation Technique. J. Animal Sci. 16:1063. Lewis, R. C. and Ralston, N. P. 1953. Protein Bound Iodine Levels in Dairy Cattle Plasma. J. Dairy Sci. 36:33 - 38. Liddel, H. S. 1925. The Growth of the Head in Thyroidectomized Sheep. Anat. Record. 30:32? - 332. Liddell, H. S. and Simpson, E. D. 1926. The Onset of the Pot-Belly in the Thyroidectomized Sheep and Goat. Am. Journ. Physiol. 76:195 - 196. Lodge, J. R. 1957. A Study of Thyroid Activity in Dairy Calves Using Radioactive Iodine as an Indicator. A Thesis for Ph. D. Degree. Mich. State Univ. 123 Lodge, J. R., Lewis, R. C., and Reineke, E. P. 1957. Estimating the Thyroid Activity of Dairy Heifers. J. Dairy Sci. 40:209 - 215. Logothetcpoulos, J. H., and Myant, N. B. 1956. Concentration of Radio-Iodide and S - Thiocyanste by the Salivary Glands. J. Physiol. 134:189 - 194. Maqsood, M. 1950a. The Effect of Varying Levels of Thyroidal Stimula- tion on Adrenals under Different Environmental Temperatures. J. of Endocrinology 7:82 - 85. Maqsood, M. 1950b. The Role of the Thyroid in Sexual Development in the Male. Nature, London, 166:692. Maqsood, M. 19500. Thyroid Secretion in the Rabbit Determined by the Thiouracil-Thyroxin Equilibrium Method. Nature, London. 166:735 - 736. Maqsood, M. l951e. Influence of Thyroid on Spermatogenesis. Science Naqsood, M. 1951b. Some Aspects of Thyroid Physiology in the Male. Ph. D. Thesis, Cambridge Univ. Cited by Maqsood, M. in Biol. Reviews 27:281 - 319, 1952. Maqsood, M. l951c. Seasonal Variations in the Testis Histology of the Ram. Vet. Rec. 63:597. Maqsood, M. l951d. Thyroxine Therapy in Male Subfertility. Nature, London. 168:466 - A67. Maqsood, M. l951e. An Abnormality of Mammalian Spermatozoa. EXper- ientia. 7:304. Maqsood, M. 1952. Thyroid Functions in Relation to Reproduction of Mammals and Birds. Biol. Reviews, Cambridge Philosoph. SOC. 27:28]. "' 3190 Maqsood, M. 1954. Effects of the Thyroid State, Season and Castration on the Adrenal Glands in Male Rabbits. J. of Endocrinology 11:10} - 109. F4 D) i.» Naqsood, M. and Reineke, E. P. 1950. Influence of Environmental Temperatures and Thy“oid Status on Sexual DevelOpment in the Male Mouse. Am. J. of Pr ysiol. 162$ - 30. Parks, 8., Dockum, N. L., and Bustad, L. K. 1957. HistOpathology of the Thyroid Gland of Sheep in Prolonged Administration of 1131.1m. J. Pttt. 33: 219 — 250. Marston, H. and Pierce, A. W. 1932. The Effects Following Thyroidectomy in Merino Sheep. Austral. J. Exp. Biol. and Ked. Sci. 10:20} - 218. McKenzie, F. F. and Berliner, V. 1937. The Reproductive Capacity of Rams. Mo. Agr. Exp. Sta. Res. Bulletin. 338. Meites, J. and Chandrashaker, B. 1948. Effects of the Thyroid Status on Response of the Gonads to Pregnant Marc's Serum in Two Different Species. J. Animal Sci. 7:542. Millikan, C. H. and Haines, S. F. 1953. The Thyroid Gland in Relation to Neuromuscular Disease. Archiv. of Int. Med. 92:5 - 39. Mixner, J. P. and Upp, C. w. 1947. Increased Rate of Thyroxine Secretion by Hybrid Chicks as a Factor in Heterosis. Poult. Sci. 26:389 - 395. Hockel, H. 1937. Zur Physiologie des Zeigenbock Sperms (In Hinblick auf die kunstliche Besamung). Vet. Med. Dias. Leipzig. Abstr. in Zucktungs Kunee, 13:366 (1937). Kossman, H. W., Hoffman, R. A., and Kirkpatrick, C. M. 1955. The Accessory Genital Glands of Male Gray and Fox Squirrels Correlated with Age and Reproductive CycleS. Am. J. Anat. 97:25? - 302. Oloufa, N. If., Bogart, R., and ”svenz*s, F. F. 1951. Effect of Environmental Temperature and the Thyroid Gland on Fertility in the Male Rabbit. Oregon Aw State College Sta. Tech. Bulletin, 2;. 1944. Anterior Pituitarym - Thyroid Relationships in the Fowl. Anat. Rec. -- :33? - 33)C-. Petersen, 1941. Phillips, 1943. Pipes, G. 1957. 125 J. Technique of Artificial Insemination in Goats. Better Goatkeeping, 2:291 - 292. F. A Method for Measuring Thyroid Hormone Secretion in the Rat with its Application to the Bioassay of Thyroid Extracts. Endocrinology.48:463 - 650. W. E., Spielman, A., Pomeroy, B. S. and Boyd, W. L. Effect of Thyroidectomy on Sexual Behaviour of the Male Bovine. Proc. Soc. EXp. Biol. and Med. 46: 1 " 170 R. W., Schott, R. G., Eaton, O. N. and Simmons, V. L. Seasonal Variations in the Semen of Sheep and Goats. Cornell Vet. 333227 - 235. N., Premachandra, N., and Turner, C. W. Technique for In Vivo Measurement of Thyroidal I in Cattle. J. Dairy Sci. 40:341 - 350. 131 Premachandra, B. N., Pipes, G. W., and Turner, C. w. 1957. A Study of Thyroxine Secretion in Cattle. J. Animal Sci. 16:1063. Puntriano, G. and Meites, J. 1951. Purves, H. 1951a. Purves, H 1951b. The Effects of Continuous Light or Darkness on Thyroid Function in Mice. Endocrinology 48:21? - 224. ' D. and Griesbach, W. E. The Site of Thyrotrophin and Gonadotrophin Produc- tion in the Rat Pituitary Studied by McManus- Hotchkiss Staining for Glycoprotein. Endocrinology 49:244 - 264. . D. and Griesbach, w. E. Specific Staining of the Thyrotrophic Cells of the Rat Pituitary by the Gomori Stain. Endocrinol- ogy 49:42? - 428. Reineke, E. P. 1946. Reineke, 1941. Reineke, 1945. The Effect of Snythetic Thyroprotein on Sterility in Bulls. The Problem of Fertility. Edited by E. T. Engle, Princeton Univ. Press, Princeton, N. J. E. P., Bergman, A. J., and Turner, C. W. Effect of Thyroidectomy of Young Goats upon Certain A. P. Hormones. Endocrinology. 29: 306 - 312. E. P. and Turner, C. W. Seasonal Rhythm in the Thyroid Hormone Secretion of the Chick. Poult. Sci. 24:499 - 504. .xiCKE‘Y, 4.4. 1925. The Thyroid Influence on the Behaviour of the White Rat. COT‘To Pr53"03'701. Hono3r. Cited by Madsood, H. 195?. Biol. Reviews 27:29 - 319. 1952. :00“, Robert O. and 3impson. "iriam E. .f" ‘TI '35“ , f‘ “4' "T *9LD. T-:‘\'rfiiLl(4n+Orfi" in ti-p - LO"-~"J1"¢A 3:11;). {In-w. v. F‘?CO:‘.L AA/ 91:1,? 9 - ‘_(;f). .1 9- 1? ‘.CIA '3’ D3. :3 ’l" . n3. Turner, C. erminotion of the Rate of Thyroxine Secre tic n in Do: e.stic Animals. Ho. Agr. EXp. Sta. 1c tin, Columbia, 392. C 1 Synthetic Therprotein on . J. D531?" ,‘T‘n 55343.13; .. 7.: w. J hi" 0 é./ O 1’ r/‘. h T we , -n : 1- .. -iloerberg, A. and 511 Graem" . T :4 3““ I" -. V .- :- v . 39/“: “°~"P019 Joint Lseaou in r1Cc .1 various Ages. PO A] .arCij-r.1\. Cf PEltll. :n_ :vaf? - 235. Silberber3, K. and 311 berle R. 1955. Role of Thyroid Horn one in the Pathog-ne:is of Joint Disease in files. J. Bone and Joint Surg. 37A=537 - 543. ‘3 lberberg, P., 0.33. Silberberg, M. 19542. Skeletal Effects of Rciio-iodine Inducgd Thyroid Dsficiency in Mice as Influenced by Sey, Age, and Strain. Am. J. of Anat. 95:26} - 290. Silberberg, z. and Silberberg, E. 19541. Joint Disease ingfiice Th yroidector.ized with Radio-iodine 113*. Proc. Soc. EXp. Biol. N. Y. 27°44? - 45?. Simpson, Ethyl D. 1927a. The E fec t of Thyroidec ctomy on Skeletal EMU; cle ir. Srieep. Q1131“. J0 E3