1: 11! um; {film}!!! 1»! M w manna"! "3 ll LIBRARY1 Michigan State ‘ University ‘; . ‘E‘V’Ig”, u t . ,. ,nr 2‘ vhxx _:f('I'/\“' ..V t. ‘A. : OVERDUE FINES: 25¢ per day per item RETUMIMS LIBRARY MATERIALS: ___________—-——- Place in book return to remve charge from circulation records EFFECT OF DIETARY IODIDE ON PITUITARY AND THYROID HORMONE SECRETIONS IN HOLSTEIN HEIFERS by Kwan-yee Leung A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree Master of Science Department of Dairy Science 1979 ABSTRACT EFFECT OF DIETARY IODIDE ON PITUITARY AND THYROID HORMONE SECRETIONS IN HOLSTEIN HEIFERS BY Kwan-yee Leung The effect of various quantities of dietary iodide on development of the pituitary-thyroid axis was studied in dairy heifers. Heifers ranged in age from 11 to 15 weeks at the start of experiment and were randomly assigned to one of four treatment groups to be given 0, 50, 250 or 1250 mg supplemental iodide in the form of ethylenediamine dihydriodide for 25 weeks. Supplemental iodide at a dose of 1250 mg per day decreased both basal tri-iodothyronine (T3) and thyroxine (T 4) concentrations relative to comparable values for controls. Basal concentrations of thyrotropin (TSH) in serum of these heifers was greater than that of controls on day 78 of treatment. Thyrotropin releasing hormone (TRH) was given on day -5, at 28 day intervals during treatment and on day 10 post-treatment. During iodide feeding period, TSH release by TRH was greater in heifers being fed 1250 mg iodide than in controls. However the increase in T3 and T 4 in serum of these heifers in response to the TRH-induced increase of TSH were not different among groups. Daily body weight gains of groups fed either 250 or 1250 mg iodide were less than that of controls during the first 21 and 77 days respectively, of the treatment. Prolonged decrease in daily weight gains of heifers fed 1250 mg supplemental iodide daily resulted in lower body weights for these heifers at 10 months of age. Heifers fed 1250 mg dietary iodide had increased thyroid weights when expressed on a per 100 kg body weight basis. Iodide feeding did not affect either adrenocortical function or time of onset of puberty. I conclude that the concentrations of iodide normally fed to dairy heifers in Michigan does not affect the development of their pituitary- thyroid axis. ACKNOWLEGEM ENT I wish to express my gratitude to Dr. H.D. Hafs, chairman of the Dairy Science Department, for providing funds and facility for my graduate training. I would also like to thank Dr. S.H. Wittwer, Director of Agricultural Experiment Station, for funding this project. My appreciation is also extended to the members of my graduate committee, Drs. H.A. Tucker, J .W. Thomas, and R.K. Ringer for their advice and assistance in my master's program. To my major professor, Dr. E.M. Convey, I am deeply indebted for his constant encouragement and unending patience throughout my graduate studies and in the preparation of this thesis. I owe specific thanks to Dr. D. Hillman for his assistance in preparation of this thesis when Dr. J .W. Thomas was absent. I wish to acknowledge Drs. G.H. Conner, D. Haggard and J. Main for their veterinary assistance in animal health. I am also grateful to Larry Chapin, Barbara Irion and Chuck Wallace for their invaluable technical advice and assistance during the experiment. Appreciation is also expressed to Drs. J .L. Gill and R.R. Neitzel for their statistical advice and computer programming. Finally, to my fellow graduate students and lab technicians, I express my sincerest appreciation and thanks for their advice and assistance in all aspects of this study. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF APPENDICES INTRODUCTION REVIEW OF LITERATURE A. Embryology I. Hypothalamus and Anterior Pituitary II. Thyroid a. General b. Follicles Anatomy 1. Gross a. Hypothalamus and Anterior Pituitary b. Thyroid II. Microscopic a. Hypothalamus and Anterior Pituitary b. Thyroid Synthesis and Secretion of Thyroid Hormones and Thyrotropin l. Iodide Uptake II. Thyroid Hormones III Thyrotropin Control of TSH Secretion I. General H. ThyrotrOpin Releasing Hormone III. Neural Regulation of TRH Secretion IV. Short L00p Feedback V. Feedback from Thyroid Hormones Mechanism of Thyrotropin Action Effect of Excess Iodide vi vii 0101 A“ ca 09 “N MN code: 05 co MATERIALS AND METHODS I. Experimental Animals II. Assays III. Statistical Analysis RESULTS 1. Body and Tissue Weights II. Serum Hormonal Response to TRH Injection A. Thyrotropin (TSH) (1) Prior to iodide feeding (2) During iodide feeding (3) Following iodide feeding Thyroxine (T 4) (1) Prior to iodide feeding (2) During iodide feeding (3) Following iodide feeding Tri-iodothyronine (T3) (1) Prior to iodide feeding (2) During iodide feeding (3) Following iodide feeding 15 17 18 19 19 24 24 24 24 31 31 31 34 34 34 34 39 III. Effect of exogenous TRH on basal concentrations of thyrotropin, thyroxine and tri-iodothyronine IV. Total glucocorticoids V. Age at puberty VI. Normal endocrine pattern of pituitary - thyroid axis in heifers DISCUSSION SUMMARY and CONCLUSIONS APPENDICES I. Radioimmunoassay for Serum Thyrotropin II. Radioimmunoassay for Serum Thyroxine III. Radioimmunoassay for Serum Tri-iodothyronine BIBLIOGRAPHY iv 39 43 43 45 54 56 58 60 Table 2 Table 2 Table 3 Table 4 LIST OF TABLES Doses of iodide (mg/kg body weight/day) during supplemental iodide feeding. Effect of supplemental dietary iodide on body, ‘ thyroid, adrenal glands and pituitary weight. Serum total glucocorticoid concentrations (ng/ml) of heifers during treatment. Basal and thyrotropin releasing hormone-induced increase in thyrotropin, tri-iodothyronine and thyroxine. 16 23 42 44 LIST OF FIGURES Daily weight gain of heifers during and after feeding supplemental iodide. Basal thyrotropin (TSH) concentration in serum of heifers 5 days before (PRE), during and 10 days after (POST) supplemental iodide feeding. Thyrotropin (TSH) response to thyrotropin releasing hormone (TRH) five days prior to beginning iodide feeding. Thyrotropin (TSH) response to thyrotropin releasing hormone. Repprted are areas under the TSH response curves (ng ml min) 5 days before (PRE), during and 10 days after (POST) supplemental iodide feeding. Thyroxine (T ) concentration in serum of heifers after thyrotropin rileasing hormone (TRH) five days prior to beginning iodide feeding. Basal thyroxine (T ) concentration in serum of heifers . 5 days before (PR , during and 10 days after (POST) supplemental iodide feeding. Tri-iodothyronine (T ) concentration in serum of heifers after thyrotropin rel asing hormone (TRH) five days prior to beginning iodide feeding. Serum tri-iodothyronine (T ) concentration in serum of heifers 5 days before (PRE , during and 10 days after (POST) supplemental iodide feeding. vi 21 26 28 30 33 36 38 I. H. III. Appendices Radioimmunoassay for Serum Thyrotropin Radioimmunoassay for Serum Thyroxine Radioimmunoassay for Serum Tri-iodothyronine 56 58 59 INTRODUCTION The endocrine system regulates growth, reproduction and lactation of domesticated animals. Understanding the mechanism by which these events are controlled may allow manipulation of the endocrine system to achieve maximum expression of economically important traits in animals including dairy cattle. Hillman in 1975 received a substantial number of complaints from Michigan dairy farmers concerning poor health and reduced milk production among cows in their herds and also reduced growth rate of their calves. Preliminary investigations indicated that 13 of 17 problem herds were fed iodide in excess of National Research Council requirements (unpublished observations). In some cases, daily iodide intake was 80 times the recommended requirement. Iodide is often added to protein and mineral supplements, concentrates, trace mineralized salt and vitamin-mineral pre-mixes. Diets for cattle usually consist of combinations of these feedstuffs. In addition, other iodide compounds such as ethylenediamine dihydriodide are usually prescribed by veterinarians to treat or prevent footrot, fungal infections, and respiratory diseases. Thus, iodide intake could greatly exceed the recommended daily quantity. In addition, McCauley _e_t a_1 (1972) and Wallace (1975) described toxic reactions that could follow routine use of iodide compounds. The purpose of this thesis was to investigate the effects of dietary iodide on development of the pituitary-thyroid axis of dairy heifers. REVIEW OF LITERATURE A. Embryology I. Hypothalamus and Anterior Pituitary The hypothalamus develops from the walls and floor of the diencephalon. A medial depression in the floor of the diencephalon forms the infundibular stalk. The anterior pituitary originates as an outgrowth from the roof of the embryonic pharynx. This diverticulum migrates upward toward the infundibular stalk. It retains its connection with the pharynx via a slender duct until the formation of the skull. With the disappearance of this duct, the diverticulum becomes an enclosed vesicle. The caudal portion of this vesicle contacts the inf undibular stalk and develops into the pars intermedia. The rostal portion proliferates and becomes the pars distalis. The residue of the duct, at least in humans, forms the pharyngeal hypophysis which may contain adrenocorticotropin, prolactin and thyrotropin (Allen and Mahesh, 1976). II. Thfloid a. General The thyroid is the earliest diverticulum arising midline on the pharynx between the first and second pairs of pharyngeal pouches. Thyroid primordial cells lying in the wall of this bilobed evagination penetrate the underlying mesenchyme and migrate caudad. During migration, the thyroid remains connected to the floor of the foregut via the thyroglossal duct. This duct later solidifies and disappears. At the point of origin of the thyroid gland, there remains a depression on the pharynx named the foramen caecum. 3 The thyroid moves caudad under the hyoid bone and laryngeal, cartilage, finally reaching its permanent position at the ventral and anterior surface of the trachea. As the thyroid proliferates laterally, the postbranchial bodies which arise from the fourth pharyngeal pouches become incorporated into the gland. Whether or not true thyroid tissue is formed by proliferation of these bodies is still unanswered (Patten, 1968, Langman, 1969). By this time, the thyroid has acquired its final shape, i.e. two lateral lobes linked by a medial isthmus. b. Follicles The initial primordial thyroid consists of a solid columnar cell mass arranging radially about a small lumen. As cells divide and get smaller, vascular mesenchyme which will develop into highly vascularized connective tissue facilitating the transport of thyroid hormones, seperate them into cords of cells. These epithelial cords then break up into nests of cells and form central lumens. Later in embryonic development, colloid, a major product of thyroid epithelial cells, starts to accumulate in the lumens (Valdes- Dapena, 1957). B. Anatomy 1% a. Hypothalamus and Anterior Pituitary The hypothalamus is located at the ventral medial portion of the brain. It forms the floor and lateral walls of the third ventricle and is delineated anteriorly by the optic chiasma and posteriorly by the mammillary bodies. It is attached to pituitary by the infundibular stalk. The anterior pituitary is located in a deep depression in the sphenoid bone called the sella turcica. It is separated from the intracranial cavity by the diaphrama sella which is penetrated by the infundibular stalk. The anterior pituitary is generally divided into three regions, i.e. pars intermedia which is attached to the posterior pituitary; pars tuberalis which surrounds the anterior and lateral surfaces of the infundibular stalk; and pars distalis which forms the major mass of the pituitary body. The superior hypophyseal arteries which arise from the internal carotid arteries form the capillary network in the median eminence of the hypothalamus and infundibular stalk. From here, blood is collected and conducted via portal vessels to the sinusoids of the anterior pituitary. The venous return empties into the hypophyseal veins leading to the cavernous sinus. There is no evidence that sympathetic or parasympathetic innervation of the anterior pituitary play any part in regulating activity of this gland (Harris, 1955). b. Thyroid The thyroid gland is composed of spherical follicles which are lined with a single layer of epithelial cells and contain colloid in their central lumen. Thyroglobulin is the major component of this colloid. An extensive network of capillaries surrounds each follicle and between these capillaries are lymphatic vessels which drain into the cervical lymph node. Blood is mainly supplied by the thyroid branch of the thyrolaryngeal artery. The caudal thyroid artery, if present, enters the caudal end of the lobe. The thyroid veins run parallel to the arteries and empty into the external jugular vein (Getty, 1975). Both sympathetic and parasympathetic fibers innervate the thyroid gland. These nerve fibers run along and terminate mainly on blood vessels. Although in some instances, they appear to make direct contact with follicular epithelial cells. Experiments wherein the thyroid was transplanted or denervated have shown that neural control has no effect on thyroid secretion (Turner and Bagnara, 1976). II. Microscopic a. Hypothalamus and Anterior Pituitary Neurons in the hypothalamus have been categorized into two groups, i.e. magnocellular and parvicellular neurosecretory cells (Knigge and Silverman, 1974). The former group has large cell bodies and is located primarily in the supraoptic and paraventricular nuclei. These neurons are responsible for the synthesis of oxytocin and vasopressin. Parvicellular neurons which are relatively small and diffusely located are believed to synthesize and secrete releasing hormones into the hypophysial portal vessels in the infundibular stalk to influence anterior pituitary functions. The anterior pituitary is composed of irregular cords of glandular cells which are intimately related to an extensive network of sinusoids. Recent deveIOpment of immunohistochemical stains enables one to establish the cellular origin of various protein hormones produced in the anterior pituitary. Thyrotropin (TSH) is produced in thyrotrophs which are of polygonal shaped and have granules that are among the smallest in the pituitary (Bloom and Fawcett, 197 5). The concentration of thyrotrophs is much greater in the medulla than in the cortex of the anterior pituitary gland (Jubb and McEntee, 1955). b. Thyroid Follicular epithelial cells vary in height according to the functional activity of the gland. In general, these cells tend to be cuboidal or low columnar in shape. An extensive endoplasmic reticulum, prominent Golgi apparatus, numerous mitochondria and colloid dr0plets are the major features of this cell. There are also microvilli extending from the apical end of the cells into the lumen. The number of these microvilli increases following thyrotropin treatment (Turner and Bagnara, 1976). N In the interfollicular spaces and between follicular cells, there are lightly stained cells called parafollicular cells, or C-cells. These cells secrete calcitonin which decreases serum calcium concentrations. C. SYNTHESIS AND SECRETION OF THYROID HORMONES AND THYROTROPIN Thyroid hormone synthesis and secretion have been extensively reviewed by DeGroot and Stanbury (197 5). A brief description follows. I. Iodide Uptake Dietary iodine is reduced to iodide before it is absorbed from the gastrointestinal tract into blood. Concentrations of inorganic iodide in plasma of cows is about 180 ng/ml (Convey _e_t _a_l_. 1978). The epithelial cells of the thyroid have the greatest ability to concentrate iodide among all tissues of the body. The concentration gradient of iodide between thyroid and plasma can reach 20:1. Iodide transport activity of the thyroid gland is augmented by TSH and decreased when TSH secretion is suppressed. Although the salivary glands and gastric mucosa also actively transport iodide and establish a concentration gradient between cytoplasm and plasma, TSH does not alter iodide uptake by these glands. Iodide is excreted from the body primarily by the kidney but also via the mammary gland during lactation. H. Thyroid Hormones Thyroglobulin, the precursor of the thyroid hormones, is the major glycoprotein in colloid. The protein portion of thyrogloban is formed at membrane bound polyribosomes on the endoplasmic reticulum and sugars are added both at the endoplasmic reticulum and Golgi apparatus. Newly formed molecules are then transported into colloid and iodination occurs just outside apical ends of the follicular cells. The iodination process requires peroxidase, H202 and tyrosine from thyrogloban as an iodide acceptor. Radiographs of thyroid sections demonstrate protein bound iodine 15 to 20 seconds after intravenous administration of 1311. Of iodide formed in the thyroid, 90 to 9596 is bound to tyrosyl residues in thyroglobulin. Iodination of tyrosine yields mono-iodotyrosine (MIT), which is further iodinated to form di—iodotyrosine (DIT). It has been suggested that the linear form of thyrogloban is iodinated. After iodination, thyrogloban develops a coiled secondary conformation and places some of the iodinated tyrosines close to one another. The coupling of two DIT yields thyroxine (3,5,3',5'-tetra-iodothyronine or T 4) and coupling of MIT and DIT yields tri-iodothyronine (3,5,3'—tri-iodothyronine or T3). These couplings proceed at a much slower rate than does iodination of tyrosine. The ratio of DIT/ MIT (or T 4/T3) in the thyroid is a function of availability of iodide. When the supply of iodide is plentiful, much thyroxine or DIT are found. With a low iodide diet, there is a greater probability of coupling of MIT and DIT, thus decreasing the ratio T 4 to T3. Under TSH stimulation, thyroglobulin reenters the follicular cells by endocytosis and intracellular droplets are formed. These droplets fuse with lysosomes to form phagosomes. Thyroid hormones are cleaved from thyroglobulin by proteolytic enzymes and are released into the blood. In blood, more than 99% of serum thyroxine is bound to thyroxine binding globulin, albumin and prealbumin, whereas tri-iodothyronine is 8 weakly associated with binding globulin and albumin (Ingbar, 1963). This weak association with serum protein may be the reason for the high turnover rate of tri-iodothyronine. The biological half-11f e of tri-iodothyronine is about 1 to 2 days and that of thyroxine 6 to 7 days in man. Sterling (1970) suggested that one third or more of tri-iodothyronine in blood may arise from peripheral conversion of thyroxine in organs such as liver and kidney. Reverse tri-iodothyronine (3,3',5'-tri-iodothyronine) which is also found in blood is synthesized by the thyroid and also produced by peripheral deiodination of thyroxine. However, no physiological function of reverse tri-iodothyronine has been demonstrated. III ThyrotrOpin Thyrotropin is composed of 2 peptides, an a subunit which is common to TSH and the pituitary gonadotropins and a B subunit in which hormonal specificity resides. Only non-covalent forces are involved in subunit-subunit interaction (Pierce e_t g. 1976). Immunohistochemical studies showed that both subunits are synthesized in the same cells in the pituitary (Baker e_t _a_1., 1972). In normal pituitaries there is a large pool of free a subunit relative to 6 subunit, in addition to thyrotropin. D. CONTROL OF TSH SECRETION I. General Grafe and Grunthal (1929) reported that dogs with massive diencephalic damage had a low metabolic rate. Houssay and coworkers (1935) demonstrated that hypothalamic lesions in the infundibulotuberal regions in toads led to reduced cell height of the thyroid epithelium. These studies provided early specific evidence that the anterior pituitary is at least partially controlled by the central nervous system. Green and Harris (1947) described the vascular portal system between the hypothalamus and pituitary. Scharrer and Scharrer in 1954 discovered that the hypothalamus was capable of secreting hormones. These observations formed the basis for the portal vessel chemotransmitter hypothesis. This hypothesis proposed that the hypophysiotropic hormones were synthesized by neurons in the hypothalamus, transported to and stored in the neural endings in the stalk median eminence, released into interstitial spaces around the portal capillary plexus and distributed throughout the anterior pituitary via the portal vessels. II ThjrotrOpin Releasing Hormone The earliest documented studies of thyrotropin releasing hormone (TRH) are those by Shibusawa and coworker (1956). Schreiber _e_t g. (1961) demonstrated that incubation of a purified extract of bovine hypothalamus with rat pituitaries led to an increased release of thyrotropin into the medium. Five years later, Schally gt 31. (1966) reported that TRH is consisted of an equal molar ratio of 3 amino acids i.e. glutamate, histidine and proline. By the end of the decade, laboratories of Guilleman and Schally independently reported the amino acid sequence of TRH isolated from ovine and porcine hypothalami (Burgus e_t a_l., 1969 and Bowers e_t g” 1970). Subsequently, specific radioimmunoassays for TRH were developed and total hypothalamic TRH measured in hypothalami of various species. Demonstration of TRH in the hypophysial portal blood of rats (Wilber and Porter, 1970 and Oliver _e_t g, 1973) and increased thyrotropin concentration in rats after infusion of TRH into the hypOphysial portal vessel (Porter _e_t _a_1., 1971) provided further support to the portal vessel chemotransmitter hypothesis. 10 TRH has also been found in: l) extrahypothalamic brain tissue of rats; 2) spinal cord of rats; 3) bovine, ovine and porcine pineal glands and; 4) cerebrospinal fluid of rats and humans (reviewed by Reichlin _e_t_ _a_1., 1976). In fact 80% of total brain TRH was found in extrahypothalamic areas. TRH has also been measured in serum and urine, but these findings may be artifact. For example,ca1culations based on TRH secretion rate, metabolite clearance rate and urinary excretion, estimate that the amount of TRH present in blood should be less than 5 pg/ ml, which is below the level of sensitivity of TRH assays. Vagenakis gt 9.1. (1975) also suggested that measures of TRH in urine are probably not correct. ‘ Mitnick and Reichlin (1972) reported that TRH formation continued in the presence of inhibitors of protein synthesis such as cycloheximide and puromycin, and that TRH synthesis required an ATP dependent soluble enzyme system. These observations led to a proposal that TRH was synthesized by a nonribosomal system mediated by an enzyme "TRH synthetase". The half-life of TRH in serum is very short, of the order of 3 to 7 minutes (reviewed by Sterling and Lazarus, 197 7). III. Neural Reggation of TRH Secretion The neurosecretory neurons that synthesize and release TRH are believed to be capable of transforming a neural message into hormone output. They serve as the link between the central nervous system and anterior pituitary. Experiments in which various neurotransmitters were added to incubated hypothalamic fragments showed that both dopamine and norepinephrine increased and serotonin decreased release of TRH into culture medium (Grimm and Reichlin, 1973). Disulfiram, which inhibits the action of dopamine B hydroxylase to convert dopamine to norepinephrine, blocked the TRH response to dopamine, but not norepinephrine. Clonidine, a noradrenergic receptor agonist, caused a significant increase of thyrotropin in serum (Annunziato gt a_l., 197 7). These studies support the idea of positive control of TRH secretion by noradrenergic neurons and negative control by serotoninergic neurons. IV. Short Loop Feedback In, 1958, Halasz and Szentagothai first hypothesized the existence of a short-loop feedback in which anterior pituitary hormones antagonized their own synthesis and release. Motta e_t a_l. (1969) reported that thyrotropin given to thyroidectomized rats for 16 days lowered hypothalamic TRH concentration. The presence of thyrotropin in the stalk median eminence (Bakke and Lawrence, 1967) and subependymal network that drains blood from the anterior pituitary toward the hypothalamus (Torok, 1964) is consistent with the possibility of a short loop feedback of thyrotropin. ‘ V. Feedback from Thyroid Hormones The pituitary-thyroid axis is a classical example of a negative feedback system. The anterior pituitary secretes thyrotropin which stimulates the secretory activity of the thyroid. In turn, thyroid hormones regulate thyrotropin secretion through a direct action on the anterior pituitary (Reichlin, I966). Inhibitors of protein synthesis such as puromycin, cycloheximide and actinomycin D prevent inhibition of thyrotropin release by thyroid hormones (Bowers fl a_l., 1968). In addition, exogenous tri-iodothyronine decreased the thyrotropin response to TRH only if serum T3 concentration remained elevated for at least 48 hours (Wartofsky _e_t 91., I976). Collectively, these results suggest that the negative feedback effect of thyroid hormones on thyrotropin secretion requires a period during which protein synthesis takes place (Sterling and Lazarus, 1977). 12 E. MECHANISM OF THYROTROPIN ACTION The mechanism of thyrotropin action on the thyroid function has been reviewed by Tong (1974). Thyrotropin binds to a receptor on the epithelial membrane of thyroid cells. Following binding, adenyl cyclase activity increases as does the intracellular concentration of cyclic adenosine monophosphate (cAMP). cAMP is believed to serve as a second messenger stimulating colloid endocytosis, release of thyroxine and tri-iodothyronine, glucose oxidation and synthesis of thyroidal RNA, phospholipid and protein. The question remains as to how so many responses of such diverse character can be triggered so rapidly in virtually simultaneous fashion. F. EFFECT OF EXCESS IODIDE The acute effect of excess iodide on thyroid function was first observed by Wolff and Chaikoff in 1948. Increasing doses of iodide caused a decrease in macromolecular organification. Excess iodide also causes a decrease in thyrotropin induced iodide concentrating activity (DeGroot and Stanbury, 1975). Formation of hypoiodous acid, tri-iodide and I2 (Nagataki, 1974) and protein-iodide complexes (VanSande e_t Q. 197 5) were suggested as active agents that caused this acute inhibitory action of iodide. Prolonged thyroid exposure to serum iodide concentrationa 100 times greater than normal could not maintain the acute effect of iodide for longer than 26 hours (Wolff _e_t- _a_1., 1949). Nagataki e_t _a_l. (1966) demonstrated that in rats given high doses of iodide (45 to 405 ,ug/day) for at least 10 days, l3 formation of thyroidal organic iodide was several times greater than that in controls fed 5 jig/day. However, concentration of serum iodothyronine and the disappearance rate of labeled thyroxine were not altered by excess iodide. Since the release rate of thyroid hormones did not increase, the thyroid must either increase storage of the increased amounts of organic iodide or secrete it in a nonhormonal form. In support of this view, Ohtaki e_t g. (1967) demonstrated increased release of nonhormonal iodide from the thyroid of patients on a high iodide diet. Braverman and Ingbar in 1963, showed that thyroid glands which have adapted to high iodide actually have a lower intrathyroidal iodide concentration than in controls. Nagataki _e_t al. (1970) suggested that iodide treated rats decreased the reutilization of intra-thyroidal iodide derived from the deiodination of iodothyronine freed from thyroglobulin. Of human patients exposed to excess iodide, few show evidence of goiters (Nagataki, 1974). Newton gt 21. (1974) also reported that Holstein bull calves fed up to 200 ppm iodide had normal thyroid weight, but had decreased weight gains and feed intake. However, Wallace in 1975 observed thyroid hypertrophy and adrenocortical hyperplasia in Georgia dairy herds fed an average of 107 mg iodide daily. McCauley gt _a_l. (197 4) demonstrated that beef and dairy herds in Minnesota, which were fed ethylenediamine dihydriodide (EDDI) for prevention of footrot, showed labored breathing, coughing, excessive nasal and lacrimal secretion, sluggishness, inappetence, salivation and fever. These symptoms gradually disappeared after the termination of EDDI treatment. Swanson (1972) reported that milk production decreased in Holstein cows fed 100 mg iodide as KI beginning about 8 weeks prepartum. Daily milk yield of cows receiving supplemental dietary iodide averaged 27.8 14 kg/day compared with 31.6 kg/day for the controls. Increased iodide concentration in milk from cows fed 100 mg iodide was also observed. However, concentrations of plasma thyroxine, thyroxine disappearance rate and thyroxine secretion rate were not different between treatment groups. MATERIALS AND METHODS 1. Experimental Animals Forty Holstein heifers were purchased in Indiana. Heifers ranged in age from 11 to 15 weeks at the start of experiment. They were randomly assigned to one of four treatment groups to be given 0, 50, 250 or 1250 mg supplemental iodide daily for 25 weeks. Iodide was given in the form of ethylenediamine dihydriodide (EDDI) orally via drench. The day iodide treatment began was designed day zero. Analyses of iodide content in the pelleted concentrate, alfalfa hay and drinking water revealed that all heifers were fed approximately 2.8 mg (0.51 pm) iodide daily at the onset of experiment. This quantity is about twice that (.25 ppm) recommended for growing heifers by the National Research Council (Jacobson e_t g. 1978). Heifers in the other three treatment groups were fed 32, 160 and 800 times the daily recommended requirement of iodide. The amount of iodide fed each group was constant during the treatment period. Therefore, iodide doses calculated on a body weight basis had decreased by approximately one half by the end of iodide feeding relative to that fed at the start of treatment (Table 1). Five heifers in each group were randomly selected to be given thyrotropin releasing hormone (TRH, 15 ug/100 kg body weight) via jugular cannula at 5 days before the start of iodide treatment, 28 day intervals during treatment and 10 days post-treatment. The same five heifers from each group were used throughout. Those heifers given TRH were taken off iodide 1: 16 mm H came on monam> m Hm. H m.m we. H w.n ow. H o.m sq. H o.o~ we. H o.HH omma we. H o.H co. H m.~ co. H m.~ no. H n.~ co. H m.H omm Ho. H N. Ho. H m. Ho. H m. Ho. H m. No. H v. om .. In I. u- mmoo. H mo. o ooH VOH on wv om mxmw\wav opHpoH psoEHmoHP mo mama IWMMMMM mcHnooa ouHcoH HmpcoEonmsm wcHHSQ flxmu\H:wHoz xwon mx\wav ovHuoH mo momoo .H ofiaae 17 feeding after 172 days whereas the other five heifers from each group were fed iodide until the day of autopsy (=190 days). This experiment took place between September 1976 and March 1977. Animals were housed in groups of 3 to 5 with controlled lighting (18L:6D) and temperature (lo-15°C). Heifers were fed 1.8 kg pelleted concentrate daily and alfalfa hay E1 Mm. All heifers were weighed 1 or 2 days prior to each TRH injection. Jugular blood was collected via veni-puncture at weekly intervals from all heifers. Selected samples (see Results) from these bleedings were assayed for thyrotropin, thyroxine, tri—iodothyronine, progesterone and total corticoids. In addition, blood was obtained from jugular cannula at -30, 0, 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180 and 240 min relative to TRH injection. Serum thyrotropin, thyroxine and tri-iodothyronine concentration of these samples were determined by radioimmunoassay. Autopsy of these heifers was performed during a two week period beginning 12 days post-treatment. Thyroid, adrenal and pituitary glands were removed, trimmed and weighed. Since final body weights were not obtained from these animals at autopsy, body weights obtained on day 9 post-treatment were used to adjust thyroid, adrenal and pituitary weights for body weight differences. II. Assays Serum thyrotropin was assayed by double antibody radioimmunoassay (Appendix 1). Various dilutions of two pools of bovine sera containing low or high concentration of thyrotropin were assayed in each assay to ensure parallelism between standard sera and standard curves. Thyroxine and tri-iodothyronine concentrations in serum were assayed using commercial radioimmunoassay reagents (Appendix 2 and 3, respectively). 18 Dilution curves of pooled bovine serum were parallel to both thyroxine and tri-iodothyronine standard curves. Known quantities of thyroxine or tri-iodothyronine added to bovine serum were quantitatively recovered (111.7% for thyroxine assay and 99.6% for tri-iodothyronine). Six to eight serum samples from a pooled bovine serum were assayed in each assay. Within and among assay coefficient of variation determined from 12 thyroxine assays were 5.5 and 4.596. Comparable values for 7 tri-iodothyronine assays were 5.396 and 10.496. Selected serum samples were assayed for progesterone and total glucocorticoids by radioimmunoassay previously described by Louis _t a_l. (197 3) and Smith _e_t g. (197 2), respectively. III. Statistical Analysis Basal serum hormone concentrations prior to TRH, body weight gains, body weight and tissue weights were analyzed by one way analysis of variance. Changes in serum hormone concentration following TRH and basal hormone concentration over time were analyzed by split-plot analysis of variance for repeat measurements (Gill and Hafs, 1971). Significance of differences among treatment were determined by Dunnett's t test (Kirk, 1968). Onset of puberty data were analyzed using a Chi-square test of contingency table. RESULTS Two heifers from the group fed 1250 mg of iodide as EDDI died of bronchopneumonia during the treatment period. Data from these animals are excluded from results discussed below. Those heifers that received TRH every four weeks were not fed iodide after day 162 (see Materials and Methods), whereas those not given TRH were fed iodide until autopsy (=190 days). A within treatment comparison between heifers given TRH and those not given TRH revealed that differences in body weight, body weight gain, and endocrine gland weight adjusted for body weight were not different (P >.10). Therefore, values presented in Figure 1 and Table 2 represent means for all animals within each treatment group. 1. Body and Tissue Weights Body weights of these heifers averaged 121 kg at 8 days prior to the onset of iodide feeding and differences among treatment groups were not significant (P>.10). After 21 days of feeding supplemental iodide, daily weight gains for heifers fed 250 and 1250 mg of iodide were (0.28 and 0.09 kg/day respectively) lower (R.0005) than that of controls (0.50 kg/day) (Figure 1). This lower rate of gain persisted in heifers fed 1250 mg of iodide up to day 77. Between day 77 and 161 of treatment, heifers in all groups gained body weight at similar rates. During 10 days following cessation of treatment (day 162 to 172), heifers in the group fed 1250 mg iodide gained less rapidly than those in the other three groups. However, one way analysis of variance revealed no difference among groups (P=.18). l9 20 Fig. 1. Daily weight gain of heifers during and after feeding supplemental iodide. 21 Scam Nb 2 .2 2 no.2 t. 2 me 2 _m 2 .2 So 838 R :5 mice _m .80 @480 .. / .un / . f. / J / fl / ”H ”H” . / W”. H. ”a / ”H. w” .u .H n.” "m u. .H” .I a a W H H. m. . .H. ./ . .H Omm_ mm. Cam I. H 2., z o D 30325 362 _oEmEmEqam (flop/6») U109 Mme/w l 22 This prolonged decrease in daily weight gains of heifers fed 1250 mg supplemental iodide daily resulted in lowered (P<.05) body weights for those heifers on day 21, 49, 77, 105, 161 during treatment (data not shown) and on day 9 post-treatment (final body weight; Table 2) as compared to that of controls. Thyroid, adrenal and pituitary weights of heifers at slaughter are reported in Table 2. One-way analysis of variance revealed no significant effect of dietary iodide on thyroid (P=0.20) and adrenal (P=0.20) weights unadjusted for body weight. Averaged over all groups, thyroids and adrenals weighed 21.7 g and 12.3 g, respectively. . Pituitaries from heifers fed 250 or 1250 mg of iodide daily averaged 1.1 g and 1.0 g, respectively, which was less (P<.05 ) than that of controls (1.4 g). In addition, there was a tendency for 50 mg of supplemental iodide to reduce pituitary weight relative to controls, however this difference was not statistically significant. In view of the fact that heifers fed 1250 mg of supplemental iodide had lower final body weights than those in the control group. Thyroid, adrenal and pituitary weights were adjusted on a per 100 kg body weight basis (Table 2). Adjusted pituitary and adrenal weights were not different among treatment groups. However, analysis of variance revealed a difference (P=.05) between adjusted thyroid weights of control heifers (7.4 g/100 kg bw) and those heifers fed the highest dose of supplemental iodide (10.0 g/100 kg bW). The enlargement of the thyroid in our heifers was mainly due to accumulation of large amount of colloid (Sleight and Mangkoewidjojo, personal communication). 23 mo. n a ”machucoo ofinmpwmsoo 509m acouommHo as mo.vm mmfionucoo oanmhmmeoo scam “conommHn a ucmHoz mem a mm.H cams ops mosfim> m mo. + ow. q.o + H.m ..H.H + o.o~ .vo.o + o.fi m.o + N.HH w.~ + V.H~ s.NH + mNN omma S.Hms. fiouoé maufim .Noéu H; Youofi Smuwém ~.w Hoom , SN S.Hmv. Scum: Baum.“ Baum; Hanoi mSHHéL M: MEN 8 8. H3. nonoé Yon s.“ 85H 2.; You. 5.2 m.oH v.8 Rm HAHN o .5 my. 8:» lllll m I?! QSBE meHHsuHm namcohp< pHonxzh meszuHm camcohu< pHouxgp H;MHoz ochoH xwom Hmch Hancos uoammsm 332.. 8532 £33: 333. apcmHoz meHHsuHm wan mvcafio Hmcoau< .wHouxzb .xwom co owHuoH meuoHa Lmucosoammsm mo Huommm .N mam.10) among the four groups prior to onset of iodide feeding and when averaged over all groups was 3.1 ng/ml (Figure 2). In addition, basal TSH was not affected by iodide feeding except on day 78 of treatment when serum TSH of heifers fed 1250 mg of iodide was greater (P=.05) than that of controls. Following TRH (Figure 3) , TSH concentration increased (P<.001) in blood of heifers in all groups reaching highest concentration of 14 to 17 ng/ ml within 15 minutes. By 240 min, TSH returned to values equal to basal concentrations. No difference in responses to TRH was detected among the four groups of heifers during this control period. The TSH secretory pattern induced by TRH was qualitatively similar after each TRH challenge. Therefore, each response during and following iodide feeding will not be graphically presented here. Rather, data will be presented as area under the TRH response curve (ng mll min) as in Figure 4. (2) During iodide feeding: After 22 days of supplemental dietary iodide, amount of TSH released (ng mil min) by TRI-l was greater (P<.005) in heifers fed 1250 mg of iodide as compared to that of controls (1713 vs 671 , Figure 4). Even though heifers fed 250 mg of iodde daily tended to have an increased response to TRH on day 22 (857 ng ml-1 min), this increase was not significant. Magnitude of TRH induced TSH release continued to be greatest Fig. 2. Basal thyrotropin (TSH) concentration in serum of heifers 5 days before (PRE), during and 10 days after (POST) supplemental iodide feeding. —-—-—— —_—— ____— ____— ——_—— —-——— -——— VF —..—_.—— —-————— w——-— *— W—A - _d—f s. -3 53° 8 a s g3".@ «02 b h 10.0 '- 26 UVVtVVTVIIUVfVVVVVCVVVV OOOOOOOOOOOOOOOOOOOOOOO. POST vvv'jVVVVVVVVVVV1VVVVV OOOOOOOOOOOOOOOOOOOOOOO 162 34 3‘. 0 Warsaw 3 WI/I/I/I/I/I/I ‘3 ‘5 cu E ............ V l I ‘5 O O 0.0.0.0...0.0.0.0.0.0...0.0.0.0.0.0.0.0.0 2 2 I- * “5 vvvvvvvvvvvvvvvvv .: OOOOOOOOOOOOOO0.00.0. a 8 5 .1 ( [Ill/DU ) ugdoquul 27 Fig. 3. Thyrotropin (TSH) response to thyrotropin releasing hormone (TRH) five days prior to beginning iodide feeding. 28 O¢N CON. 8 Dow D 4 On 0 0 32:2... £3. 3206235 E25 25... ON. om CO 9 ([tll/DU) ugdoquul L ON 29 Fig. 4. Thyrotropin (TSH) response to thyrotropin releasing hormone. Reported are areas under the TSH response curves (ng ml-1 min) 5 days before (PRE), during and 10 days after (POST) supplemental iodide feeding. 30 ST . O - P 'V‘VVVvav W ”II/[IA ”III 7 62 134 IOG V vvw vvv Viv‘i‘fvv OOOOOOOOOOOOOOOOO. 78 v'fvvvv'vvw i ——v- 'vvvvf' 0.00.0.0...OOOOOOOOOOOOOOOO. O O O. so Vvv"wvva .00....OOOCCCOOO0.000.000... O 50 Cl IS! I 250 [3] l250 Iodide (mg/day) Supplemental *22 PRE _ l l __l O ID 0 (20' x 11111.1le 611) sung esuodsaa HSJ. Japun 081V (day) Length of Treatment 31 in heifers fed 1250 mg iodide throughout the entire treatment period. Interestingly, the TSH response to TRH gradually decreased with time in all four treatment groups (Figure4 ). In fact, there was a significant negative correlation between duration of treatment in days and area under TSH reSponse curve (ng ml"1 min) within all groups except those fed 50 mg of iodide. Correlation coefficients were -.48 (P<.01), -.25 (P = .18), -.37 (P<.05) and -.65 (P<.001) for heifers fed 0, 50, 250 and 1250 mg of iodide, respectively. (3) Following iodide feeding: On day 10 after cessation of iodide feeding (Figure 2), basal concen- tration of TSH averaged over treatment was 4.4 ng/ml, and differences among groups were not significant. Although heifers which had been fed 50, 250 and 1250 mg of iodide daily for 162 days had a greater TSH response to TRH compared to controls, no statistical difference was detected (P z .17; Figure 4). B. Thyroxine (T 4) (1) Prior to iodide feeding: Changes in serum T 4 concentration after TRH are shown in Figure 5. Although basal serum T 4 concentrations (time 0) for heifers assigned to be fed 250 mg of iodide daily were greater and values for those assigned to be fed 50 and 1250 mg of iodide were less than that of controls, analysis of variance revealed no difference among groups (P 2 0.17). After TRH, T 4 increased linearly with time and had not reached a plateau at 240 minutes (Figure 5). Analysis of variance revealed a significant difference (R0.05) in the T responses to TRH among the four groups. Assuming that there 4 should not be any difference in serum T concentration prior to onset of 4 iodide supplementation, all T data collected during and after treatment 4 32 Fig. 5. Thyroxine (T 4) concentration in serum of heifers after thyrotropin releasing hormone (TRH) five days prior to beginning iodide feeding. 33 3,5 2:. 03.. ON. cm 0 0 0mm. x 0mm 0 0m 4 ON 0 0 >25on 2: o. A _EcocwoaLWG 0? 7 om IE. Cm 3162 35:20 :E 255623;. OO. (Iw/DU) 171 34 were adjusted by covariance for pretreatment differences. Actual data are presented in Figure 5 and 6. 1 The secretory pattern of T 4 after TRH shown in Figure 5 was qualitatively similar at each subsequent TRH challenge. In addition, there was no significant time by treatment interaction in T 4 response to TRH. For these reasons, only basal T concentrations relative to duration of supplemental iodide 4 feeding are presented in Figure 6. (2) During iodide feeding: After 22 days of feeding supplemental iodide, basal concentration of T 4 was not altered (P 2 .11) when adjusted to pre-treatment values (Figure 6). However, on day 50, T in serum of heifers fed 1250 mg iodide daily 4 (32.1 ng/ml) was less (P<.005) than the comparable value for controls (49.7 ng/ml). Lower concentrations of T in serum of heifers fed 1250 mg of 4 supplemental iodide persisted throughout the entire feeding period. (3) Following iodide feeding: At 10 days after cessation of treatment (Figure 6), serum of heifers that had been fed the highest dose of supplemental iodide still had lower (P<.01) basal concentrations (ng/ml) of T 4 than did controls (45.9 vs 61.4). C. Tri-iodothyronine (T3) (1) Prior to iodide feeding: Average concentration (time 0) were not different among groups 5 days prior to onset of treatment (Figure 7). After TRH, T3 increased linearly and reached peak values at 120 minutes. Analysis of variance revealed no difference in T3 response to TRH among the four groups prior to start of treatment. (2) During iodide feeding: The pattern of change in T3 concentration after TRH described in Fig. 6. Basal thyroxine (T 4) concentration in serum of heifers 5 days before (PRE), during and 10 days after (POST) supplemental iodide feeding. 36 W N 'l'l/I/I/I/I/I/I/I/I/I/Il. 9 vvvvvrjvf VVVVVVV OOOOOOOOOOOOOOOOC 134 V‘v‘fi'fivvvvv ' 106 Length of Treatment (day) ’78 vvvvvvvvvfivvvv ’50 W Vl/I/I/I/I/I/I/I/I I“ 3‘. _O 23 -rfifi--n-fi------fiv :008 CO eeeeeeeeeeeeoeeoeeeeeeo “E '0 ‘0 Lu 5" °‘ 53 gs -” 1 aaljllEl :‘U m2 L 4 1 1 1 1 a 1 1 1 O O O O 8 co .. N (Int/5L1) augxoMul 37 Fig. 7. Tri-iodothyronine (T3) concentration in serum of heifers after thyrotropin releasing hormone (TRH) five days prior to beginning iodide feeding. 38 EN 222.: Ow. 00 0mm. x 0mm. a On 4 o o 3835. 2.62 6.58235 $162 85:20 :E. .5288me Wu QM (um/511ml 39 Figure 7 is qualitatively representative of changes during subsequent challenges. Additionally, no difference in magnitude of T3 response to TRH among treatment groups was detected by any TRH challenge. Therefore, only basal T3 concentrations relative to duration of treatment are presented in Figure 8. After 22 days of supplemental iodide feeding, heifers fed 1250 mg iodide had a lower (P<.01) basal T3 concentration than controls. This pattern continued throughout the treatment period. On day 78, heifer concentrations in all treatment groups showed lower concentrations of serum T . The 3 cause of this observation was unknown. (3) Following iodide feeding: Groups of heifers that had been fed 1250 mg iodide had less (P<.01) T3 in serum than did controls at 10 days post-treatment (Figure 8). III. Effect of exqgenous TRH on basal concentrations of thyrotropin, thyroxine and tri-iodothyronine. Selected serum samples collected by venipuncture at various stages of this experiment were assayed for TSH, T3 and T4 to determine the effect of periodic injections of TRH on basal hormone concentrations. Hormone values revealed no evidence of either a direct TRH or TRH by supplemental iodide feeding effect on the pituitary-thyroid axis (data not shown). IV. Total glucocorticoids At 5 days prior to onset of treatment, total glucocorticoids concentration in serum averaged overall groups was 3.9 ng/ml (data not shown). Differences between groups were not significant (P: .48). Total corticoids in serum of heifers after various duration of iodide feeding (Table 3) provided no evidence of altered adrenocortical function due to treatments (P>.50) although 40 Fig. 8. Serum tri-iodothyronine (T3) concentration in serum of heifers 5 days before (PRE), during and 10 days after (POST) supplemental iodide feeding. viivvvvvvvvvvv'vvvvvvv Posr ‘V'v‘fiffivvav‘vvvvva OOOOOOOOOOOOOOOOOOOOOO 162 134 106 H A 78 Length of Treatment (day) 'I/I/I/I/I/I/I/I/I/ W‘ IIll/I/I/I/I/I/I/I/I/I/I/IIIII/III[a 'I/I/I/I/I/I/I/I/I/I/I/IIII/II. 50 3: - 3 2 \ C O " E o o O e'evefevove'e'eve'e'e'o'efeveve'o'o'e'eve'e'e'e'e'e'eve e E, V '0 3 3 ' uJ — 0 a, -— I: ° '3 I ‘°' 6?) 2 A Q 0 0. 0. IO "" o (nu/gu) augquu10po! -11; 42 TABLE 3. Serum Total glucocorticoid Concentrations (ng/ml) of Heifers During Treatment. Supplemental Iodide (mg/day) Duration of Treatment (Days) 8 16 37 58 79 100 121 0 3.2 5.4 5.9 2 5 6.2 3.0 5.4 50 3.4 3.1 4.6 2.9 8.8 4.2 6.2 250 3.3 1.8 3.2 3.1 5.3 4.1 5.0 1250 2.3 2.7 5.0 2.6 5.6 3.7 2.7 AVG 3.1 3.3 4.7 2.8 6.5 3.8 4.8 AVG 4.5 4.7 3.7 3.5 4.1 Standard Error Due to Treatment: .58 |+ Standard Error Due to Time: :_.45 Standard Error Due to Each Cell: i.°89 43 differences between day of sampling were significant (P<.005). V. Age at puberty Selected serum samples from all heifers were assayed for progesterone to determine time of onset of puberty. Animals were considered to have reached puberty if serum progesterone values were equal to or above 1.0 ng/ml i.e. had functional corpora lutea. Two heifers fed 50 mg of supplemental iodide were later discovered to be free-martins at autopsy and were therefore dropped from this aspect of the experiment. By day 142, one heifer from each treatment group had deve10ped a functional corpus luteum. At the end of the experiment, 7 of 8, 3 of 10 and l of 8 heifers from groups fed 50, 250 and 1250 mg of iodide as EDDI respectively, had exhibited behavioral estrus. These data were not different from those of controls (5 out of 10 heifers, P>.05). VI. Normal endocrine pattern of pituitary-thyroid axis in heifers Hormone data (Table 4) from heifers (n=5) which served as the controls in this experiment provided us an opportunity to evaluate TSH and thyroid hormone changes in normal growing heifers between 3 and 9 months of age under conditions of controlled photoperiod and temperature. In these heifers, basal TSH concentration remained constant (P>.05) during this period averaging 3.1 ng/ ml, while serum T3 and T 4 concentrations fluctuated greatly (P<.0005) over time. We did note that the TSH response to TRH decreased with age. However, the increase in serum T3 and T 4 concentrations that occurred between 0 and 120 min after TRH was similar at each TRH challenge. 44 .mOHSCHE ONH OH O EOHM GOHHwHHCQOQOU QQOEHOS Cw OWE—MSG u < e .ou::Ha HuHE m: :H one o>Hsu uncommou :mH Hows: on Hammmn .mm.H memos mam mosfim>m S.Humfi m.~.....m.em 35H 35 Edna; mSHNmN 31¢.ng m o.N.H 3.2 flaunts axons; 85.ng omHHmem Rowe...“ w fimnod Yanmé. coaufid 8.o.Ho.N S Hamm m.on.N H mAHmd eAHmé. 35....25 oodnmg SHHHS N.oHo.m o maufifl Saunas modHEd BSHTN 02H Sm Sousa m mgued «Sufi? Bangs moduw; SSH HS noHNH a. SAME: fimHoém Edufld 2.on4 2. Heoo TOMB... m < Human < Human o>qu Hows: Human e a e a 8.2 a U ocHxOszb ocflconxzuovoHnHHe :Hmopuonxgb anacosv om< woconuxzk wcm ocH:OHx:uoonuHHH .cHaonuouxze :H ammoHocH woo:p:H ocosho: wchonom :HQOHHOHxah can Hemmm .v m4m