STUDSES OF YHYROIE) FUNCTSDN EN THE CHWKEN Thesis 5m ihe Degree cf Ph. D. MiCHIGAN STATE UMVERSWY Aii‘? Singh 1966 mum: This is to certify that the thesis entitled Studies of Thyroid Function in the Chicken presented by Ajit Singh has been accepted towards fulfillment of the requirements for Ph'D° _ degree in__Ph_ISi°108Y (k/ .10 :7?" 2,14 :-j/ '5- ' ‘. ‘4"! Major professor Date November 28L 1966 0-169 LIP R 4‘9 Univcmty Dalia . slim S {are i v3 A a H ABSTRACT STUDIES OF THYROID FUNCTION IN THE CHICKEN by Ajit Singh The thyroid secretion rate (TSR) in chickens of different ages was determined by four methods: (I) goiter prevention, (II) thyroid hormone substitution, (III) direct output and (IV) thyroxine degradation. In method I, the daily dose of thyroxine, which gave thyroid weight of tapazole-treated birds equal to the un- treated controls, was taken as the TSR. In method II, TSR was measured by determining for each group the dose of thy- roxine (T4) or triiodothyronine (T3) required daily to maximally block 1131 release in normal or tapazole-treated chickens. In method III, thyroidal I131 output rate was de- termined by daily counts, the thyroids were removed, ana- lysed for total iodine and TSR was estimated as the product of daily 1131 output rate (K4) x thyroidal iodine x 1.529 (I4 iodine equivalent» In method IV, half life (tl/Z) of T4 was determined from successive plasma counts at 3-hour intervals after 113l-T4 administration. Thyroxine distribution space (TDS) was calculated. Protein-bound iodine (PBI) was Ajit Singh analysed and extrathyroidal thyroxine (ETT) estimated as PBI x 1.529 x TDS. TSR was estimated as the product of ETT x K. Method IV was also applied to determine parameters of tri— iodothyronine degradation. TSR of bobwhite quail and coturnix was estimated by method IV. The TSR values obtained in different experiments within each method were sufficiently close to indicate good repeatability. The representative TSR of chickens estimated by I, II, III and IV methods, respectively, were 2.28, 2.00, 1.10 and 2.03 pg/lOO g/day. TSR of adult chicks, goitrogen— treated chicks, bobwhite quail and coturnix as estimated by method IV averaged 1.59, 1.02, 2.49 and 2.78 pg/lOO g/day, respectively. TSR of goitrogen—treated chickens by method II was higher. There was no age difference in TSR measured by method III in growing chicks in the range of 1-9 weeks of age. An increased plasma radioactivity was noticed in the 12-hour samples taken in the degradation experiments. This phenomenon seems to result from discharge of unchanged hormone from the liver. Methods III and IV yielded lower TSR, TDS, ETT and thyroidal iodine content, but higher K4, zero time percent uptake (U), and almost no difference in tl/2 and FBI of (iodine deficient chickens. Method II revealed no TSR Ajit Singh difference between chickens fed adequate or deficient iodine diets. Iodine deficiency retarded growth rate. T3 and T4 were found to be equally potent in chickens by method II. The representative tl/2 of T4 in blood of chickens, bobwhite quail and coturnix were 3.23, 4.60 and 5.55 hours, respectively. The t1/2 of T4 was identi- cal to that of T3 in all birds. T4 had a significantly greater t1/2 in adult chickens. The representative TDS of chickens, bobwhite quail and coturnix, respectively, were 29.39, 28.08 and 55.29 ml/100 g b.w. Adult chickens had lower TDS/unit b.w. T3 distribution spaces of all birds were higher than of T4. The FBI and thyroidal iodine were analysed by using dilute ceric ammonium sulphate following alkaline ashing. The representative PBI of chickens, bobwhite quail and coturnix were 1.12, 1.76 and 1.26’ug % respectively. Tapazole retarded growth rate, feather and comb growth of chickens. Two—three ug thyroxine /100 g/day counteracted these effects. Larger doses of T depressed 4 growth of normal and tapazole-treated birds. Thyroxine in small doses improved growth of normal chicks. Metabolic rate (M.R.) of chickens was determined in a closed circuit type manometric system. T3 and T4 produced a small and transitory rise in M.R. Ajit Singh The following indices of thyroid function were found to be significantly related: Age and K age and thyroidal 4, iodine, K4 and thyroidal iodine, K'4 and U, thyroidal iodine and body weight, T3 distribution space and body weight. These investigations support the conclusion that the thyroid function in chickens differs from that of mammals in lower PBI, shorter tl/Z of thyroid hormones, their equal physiological potency and insignificant effect on M.R. The author believes that the direct output and the T4 degradation methods should measure true TSR in chickens provided all their known and unknown factors are properly accounted for. Consideration of the known factors strongly suggests that as presently applied, the direct output method most nearly represents the true TSR in chickens. STUDIES OF THYROID FUNCTION IN THE CHICKEN BY Ajit Singh A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1966 Ei!.,.'EII.IHJ! ! LE ACKNOWLEDGMENT S The author wishes to express his deep gratitude to Dr. E. P. Reineke for his wise council and guidance in planning and conducting the investigations and in the prepar- ation of this manuscript. Besides, his gentle behavior and encouragement at all times were great impetus in accomplish- ment of this work. The writer considers himself privileged to have had the opportunity to associate with Dr. R. K. Ringer, who took a constant interest in this project and at times helped in actual conducting of the work. Special thanks are also due to Dr. W. E. Cooper for his guidance in statistical analysis of the data. A sincere appreciation is expressed to Mrs. Judianne Anderson, who with untiring efforts made iodine determin- ations and helped in some experiments. Thanks are also due to Mr. Larry Paulik and Mrs. Linda Allison for analysing.some samples; to Miss Barbara J. Brace and Miss Jill A. Hart for doing artwork; to Mrs. Beverly Wandel, Mr. Kenneth Gallagher ander. Stephen Unger for taking care of the birds. Financial aid in the form of a fellowship, provided by the Rockefeller Foundation is greatly appreciated. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . 4 Goiter Prevention Method 4 Thyroxine Substitution Method 5 Direct Output Method 10 Thyroid Gland Uptake and Release of 1131 11 Thyroxine Degradation Method 13 Thyroid Function in Chickens l6 Metabolism and Excretion of Thyroid Hormones in Chickens l9 Protein-Bound Iodine 20 Effect on Growth and Metabolic Rate 21 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 24 Birds and their Feed 24 Chemicals and Drugs 25 Counting Apparatus and Procedure 27 Goiter Prevention Method 28 Thyroid Hormone Substitution Method 29 Direct Output Method 30 Thyroid Hormone Degradation Method 33 Uptake of Thyroid Hormones by Red Cells 35 Determination of Metabolic Rate (M.R.) 36 Statistical Analyses 37 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 38 Goiter Prevention Method 38 Thyroid Hormone Substitution Method 40 Direct Output Method ‘ 44 Thyroid Hormone Degradation Method 54 Biological Half Lives (tg) 54 Thyroid Hormone Distribution Space (TDS) 56 Increased Plasma Radioactivity Under Stress 61 Protein-Bound Iodine (PBI) 63 Effect of Iodine Intake on Thyroid Function 65 iii Page Effects of Thyroxine on Growth, Feather and Comb Development 70 Metabolic Rate (M.R.) 74 Comparison of the TSR Methods 78 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 84 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 88 APPENDICES . . . . . . . . . . . . . . . . . . . . . 97 iv Table LIST OF TABLES Thyroid secretion rate in chickens by thy- roxine and triiodothyronine substitution methods Direct output data in chickens Statistical analysis of turnover data of thyroxine and triiodothyronine in chickens Statistical analysis of turnover data of thyroxine and triiodothyronine in quail Percent increase of radioactivity in blood as calculated in the 12—hour sample taken under stress Protein-bound iodine (pg per 100 m1 plasma) in chickens and quail . . Effect of dietary content of iodine on body weight, thyroid weight and thyroidal io- dine of 7- week-old chickens . Effect of dietary iodine content on various parameters of thyroid secretion rate de— termined by different methods in chickens Results of one way analyses of variance on M.R. in chickens Page 41 46 57 58 62 64 66 67 75 Figure 10. 11. LIST OF FIGURES Goiter prevention assay Substitution of thyroxine or triiodothyro- nine in normal or tapazole-treated chicks on diets adequate or deficient in iodine Typical TSR determination by direct output method . . . . . . . . . . . . . . . . . Relationships between age, K4, thyroidal iodine and TSR in chickens . . . Relationship between K' and zero time per- cent uptake (U) in thyroids of chicks from one to nine weeks of age Relationship between thyroidal iodine and body weight in chicks from one to thirteen weeks of age Relationship between thyroidal iodine and thyroid weight in adult birds Typical TSR determination by T4 degradation Effects of thyroxine and tapazole on growth of chickens . . . . . . . . Influence of thyroxine and/or tapazole on feather and comb growth . . . . Change of metabolic rate in chickens after administration of thyroid hormones vi Page 39 42 45 48 50 52 53 55 71 73 76 LIST OF APPENDICES Appendix A 1'11 0 '11 M q Data on TSR of individual subjects by direct output method . . Thyroxine turnover in individual subjects Triiodothyronine turnover in individual subjects Additional data on thyroid iodine and FBI of chickens and quail . . . Thyroid iodine analysis PBI analysis 63-S chick starter diet Rat feed mixture SR—l Diet fed to 56—week old chickens Diet fed to quail Effect of a single dose of (4. 0,ug/100 g. b. w. ) thyroid . Effect of daily administration of (6.0_ug/ 100 g b.w.) thyroid hormones on M.R. in chickens . . . . . . . . . . . . . vii Page 97 100 103 105 109 110 111 112 113 114 115 116 I NTRODUCTION The thyroid gland is a major regulator of metabolic and productive processes. Study on chick thyroids is not only important from the standpoint of comparative physiology, but also to exploit economic traits of this species. Avian thyroid hormones are intimately related to the physiological processes associated with the energy metabolism, growth rate, feather and comb growth, molting, egg production, etc. etc. With recognition of meat and eggs as sources of high quality human nutrition and with increased development of poultry farming all over the world, it has become more pertinent to explore further in the fowl thyroid. This may help to step up greater and more efficient food production for the ever increasing human population. It has been recognized that the thyroid status is best understood by quantitative measurement in terms of thy— roid secretion rate (TSR). Other indices such as estimation of protein bound iodine (FBI), metabolic rate (M.R.), I131 uptake and release rates, and the triiodothyronine (T3) test have been shown to give pnly qualitative information about thyroid gland function. Also, there are some marked differ- ences of these indices between mammals and birds. Their usefulness in measuring thyroid gland activity in chickens is thus even more questionable. Previously only the goiter prevention and the T4 substitution methods have been employed for measuring thy— roid secretion rate in chickens. Their results indicated a large TSR difference. Furthermore, only meagre information is available about the other indices of thyroid function such as protein bound iodine (PBI), biological half life (tg), thyroid hormone distribution space (TDS), thyroidal iodine uptake, release rate, thyroidal iodine content and the effects of the thyroid hormones on M.R., growth rate, feather and comb growth. Quail breeding has recently been gaining favour among the poultry farmers. However, practically no work has been reported on the thyroid function of this species. In View of the above, it was proposed to study the thyroid function of chickens and quail by measuring their thyroid secretion rates. Investigations were also under- taken on related aspects, affording information on the thy— roid hormone functions in these birds. In the present sutdy, the thyroid secretion rate of chickens was measured by four methods, viz. goiter prevention, the thyroid hormone substi— tution, the direct output and the thyroid hormone degra- dation (turnover) methods. The results of these methods were compared. Effects of dietary iodine content on various parameters of TSR and upon body and thyroid weights were determined. Information was also sought on relative poten— cies of triiodothyronine (T3) and thyroxine (T4) in chicks. The effects of T4 on growth rate, feather and comb growth, and that of T3 and T4 on M.R. were determined. Further tg, TDS, extrathyroidal thyroxine and triiodothyronine (ETT), FBI and thyroidal iodine content were investigated in both chickens and quail. There are some, previously unknown, but important relationships between age, 1131 daily output (K4), and thyroidal iodine, between fractional output (K'4) and zero time percent uptake (U), between thyroidal iodine, body weight and thyroid weight, and between body weight and TDS. These relationships have also been determined. REVIEW OF LITERATURE Variations within and between species, and the scientists' endeavor to ever improve upon methodology have led to the development of several methods for measuring thyroid secretion rate. This has brought about an enormous amount of literature, out of which, only the references pertinent to this project have been cited in the following review. Goiter Prevention Method The discovery of goitrogenic compounds enabled Dempsey and Astwood (1943) to develop a technique in rats for determining average thyroid secretion rate. Mixner, Reineke and Turner (1944) described a similar type of assay using one-day-old chicks. The method is based on the action of goitrogens inhibiting endogenous formation of the thyroid hormones in the test subject. This permits an increased out- put of TSH, which causes a compensatory enlargement of the thyroid gland. If, however, thyroxine is given exogenously, the TSH is held in check, whereby thyroid enlargement is pre- vented to an extent in proportion to the amount of thyroxine given. The daily dose of thyroxine needed to give thyroid Weight of goitrogen-treated birds equal to the untreated controls is taken as the adequate daily requirement of the hormone to maintain normal thyroid-pituitary balance and is considered as the secretion rate. The goiter prevention method has been most extensively employed for determining thyroid secretion rate in many species of small animals. Average levels of secretion equivalent to 2.70 and 0.75_pg dl-thyroxine daily were reported in two—week-old chicks. Maximum of this range was observed in fall and minimum in the summer season (Reineke and Turner, 1945). Thyroxine Substitution Method A large step forward in the development of methods for measuring thyroid secretion rate was made with the ad- vent of radioactive iodine. Among the methods involving use of 1131 , the thyroxine substitution method has been most widely used in all animals. This method is based upon the ability of the thyroid to rapidly accumulate a great pro- portion of ingested iodine, as also the fact, that adminis— tration of exogenous thyroxine will reduce both thyroidal 1131 uptake and output. This suggested to Perry (1951), the possibility of an indirect thyroid assay by determining the amount of thyroxine required to block release of 1131 from the thyroid. Perry used this procedure in rats and by vary- ing dosage between different groups, he demonstrated a re- lationship between the amount of thyroxine given and the de— gree of inhibition of hormone release. Henneman, Griffin and Reineke (1952) developed this method for measuring thyroid secretion rate in individual sheep. Furthermore, Reineke and Singh (1955) have devised a procedure based on thyroxine substitution whereby thyroid secretion rate could advantageously be measured in individual and smaller groups of animals than required earlier. The procedure involves, the taking of external thyroid counts 2-3 days after 1131 injections. Daily thyroxine dose is increased progressively at 2-day intervals, a thyroid count being taken before each increment in dosage. The end point then is the amount of 131 output from exogenous l - thyroxine needed to block the I the thyroid. The latter measurement is made by expressing each thyroid count as percentage of previous count until the 100 percent point is reached. In chickens, the thyroxine substitution method was first adapted by Pipes, Premachandra and Turner (1958). They reported thyroid secretion rate (TSR) values ranging from 2.0 to 5.0‘pg 1-thyroxine/ 100 gm/ day. Mellen and wentworth (1959) adapted a modified procedure. They reasoned that the effect of a single injection of thyroxine in chickens may not persist for 24 hours and so they gave thyroxine in one, two or four equally spaced injections. From the average regression of thyroid radioactivity as per- cent of previous count on thyroxine dose, they estimated group secretion rates as 3.02 to 4.29’pg/ 100 g/ day. These results do not show any consistent difference in estimated TSR due to injection schedule. In the same publication, Mellen and wentworth (1959) reported results of another ex- periment where thyroxine was administered once daily and the dosage increased progressively at 48—hour intervals. The successive counts formed the basis of the individual re- gression of percent of previous count on thyroxine dose. By this procedure, they estimated TSR for individual chicks ranging from 3.25 to 5.17/ug/ 100 g/ day. Mellen and wentworth (1960) again reported a comparison of chicks TSR by radioiodine and goiter prevention methods. The former method gave values from 3.5 to 4.5, while from the latter method, TSR was estimated as 1.40’pg/ 100 9/ day. The workers using thyroxine substitution methods in chickens invariably mix a goitrogen in the feed to minimize reutilization of 1131 from metabolized hormone and to ac- celerate release of the isotope from the thyroid. Reineke and Singh (1955) pointed out that when goitrogen is used in the substitution method, the estimated TSR is higher than that without goitrogen and that thyroxine fails to com- pletely block 1131 output from the thyroids of thiouracil— treated rats. On the other hand Turner-gg_g1. (1959) and Mellen (1961) believe that TSR is not affected by goitrogens. Himeno §E_§l- (1961) compared thyroid secretion rate, measured with or without thiouracil in 4Aweek—old.cockerels. They grouped the birds as that on (1) normal diet, (2) thi- ouracil mixed diet started 24 hours before substitution, (3) thiouracil mixed diet started 48 hours before substi- tution, (4) thiouracil mixed diet started 120 hours before substitution. Average thyroid secretion rates inlug/100 g/day were obtained as (1) 4.61 (2) 7.00 (3) 7.97 (4) 11.67. These results are confusing, but nevertheless demonstrate that TSR in thiouracil-fed chickens increase significantly over those on normal diet. Also, the TSR increases further with length of thiouracil feeding. These authors believe that use of thiouracil is not adequate for precise determi- nation of TSR in chickens. Reineke (1965) reported another influence of thiouracil on release rate. He noted that treatment with thyroxine prior to and during thiouracil ad- ministration blocked 1131 output from rat thyroids for 3-4 days and then output was resumed at the same level as when thiouracil was given prior to thyroxine. There has been little agreement among different workers on the end point reached, with regard to total or partial suppression of TSH in the substitution method. In the work with normal rats (Reineke and Singh, 1955) and sheep (Henneman, Griffin and Reineke, 1952) the daily thy- roxine dose which maintained 100 percent previous count in the thyroid was taken as end point. In thiouracil-treated rats (Reineke and Singh, 1955) 92 percent previous count was taken as the end point. More recent data (Reineke and Lorschieder, unpublished) show that in rats on a 48-hour counting sequence thyroxine injections will only hold thyroidal 1131 at 97.5 percent of the preceeding count. Himeno and Turner (1961) took 95 percent previous count as their end point. They also recorded higher TSR in tapazole- treated birds than in normal birds. Tanabe and Kbmiyama (1962) reported a modified thy- roxine replacement method, based on partial inhibition of thiouracil—induced acceleration of 1131 release from the chick thyroid. Daily TSR is measured as the amount of thy- roxine which inhibits goitrogen induced acceleration of 1131 release from the thyroid gland and returns the retention of 131 thyroidal I to the rate before the start of thiouracil administration. A regression equation is solved for thy— roxine dose vs. 1131 retention rate. By this method, they estimated thyroid secretion invpg l-thyroxine/lOO g/day as 1.50 to 1.80 for 6 to 7—week-old cockerels and 0.58 for 12— month-old hens. They noted that comparatively these values are 60 percent of those obtained by complete inhibition techniques; 85 percent of those obtained by the same pro— cedure with non thiouracil-treated chickens, and close to the value derived from the goiter prevention assay. Tanabe §£_§1. (1965) compared effects of thiouracil, propylthiouracil and methimazole on thyroids and thyroxine metabolism in chicks. They reported that methimazole is the most potent and thiouracil the least with regard to 1131 up- take and release. All these goitrogens at 0.1 percent level 'in the diet had little effect on thyroid secretion rate in 10 6 to 7—week—old cockerels. Further, thiouracil and propyl- thiouracil decreased (by 10 percent) deiodination of radio- thyroxine and increased (by 20-30 percent) faecal excretion. On the other hand, neither methimazole nor KClO4 had such effects. Tanabe (1964) also observed a rough correlation be- tween thyroid secretion rate and the levels of alkaline phosphatase in chicken serum. Wagai_gt_a1. (1965) de- termined thyroxine secretion rate in chicks by a microhisto— metric assay. Direct Output Method The direct output method for estimation of thyroid secretion rate in the rat was first worked out by Reineke (cited by Bhatnagar, 1963). This method involves measuring of thyroidal 1131 turnover and the thyroidal iodine content. The product of these two parameters multiplied by a factor to account for the different activities of thyroxine and tri- iodothyronine gives an estimate of thyroid secretion rate in terms of T4 released daily. No thyroxine or goitrogen is administered in this method. Sorensen (1958) described a direct output method to determine thyroid secretion rate in cattle and pigs. Radio- activity of the thyroid gland was measured for a certain period after injection of 1131. In the same duration RBI and FBI131 were determined from the blood samples. The rate 11 131 turnover is calculated from the constant for thyroidal I declining radioactivity of the thyroid. The secretion rate of labelled hormone is found by multiplying the rate con- stant by the amount of thyroid 1131. Assuming that the spe- cific radioactivity of the circulating thyroid hormones and that of the hormone just secreted is identical, thyroid se— cretion rate can be calculated as: Serum PBI Secreted hormone iodine ;ug/hr. =;Eg/100ml Secreted hormone - 1131 percent dose/hr. Serum PBI131 %dose/100ml Reineke (1964) compared the effect of iodine intake on thyroid secretion rate in rats as determined by thyroxine substitution and direct output methods. The results differed. In the substitution method, no significant differ— ence was noted between groups given varying levels of iodine, while in the direct output method TSR values increased pro— gressively with iodine intake. No literature seems to be available involving compu— tation of TSR in chickens by the direct output method. Thyroid Glandngptake and '7 Release of I131 Thyroid gland uptake and release of 1131 have been measured to estimate thyroid functions. Their usefulness as indices of true thyroid secretion is uncertain. However, their values in qualitative and clinical thyroid conditions 12 are. recognised. Goyings, Reineke and Schirmer (1962) sug- gested a method of diagnosis of hypothyroidism in dogs by measuring 24 hour thyroidal 1131 uptake. They reported average ratios of thyroid to thigh counts as 11:1 and above in normal dogs and from 1:1 to 11:1 in the hypothyroid animals. Recently Greenberg (1966) suggested the possibility of differentiating between primary and secondary hypothyroid- ism in humans by measuring the release rate of I131. A rapid release suggests primary thyroid pathology whereas a slow release rate supports pituitary pathology as the cause of the thyroid insufficiency. Turner et a1. (1959) reported that the uptake of I131 by the thyroid is affected by many factors such as the size, weight and colloid content of the gland, variations in iodine content of the ration, kidney functions, state of pregnancy and lactation. Lodge, Lewis, Reineke and McGillard (1958) found no correlation between 1131 uptake and esti- mated TSR in calves. Flamboe and Reineke (1959) did not ob- serve a relationship either between TSR and percent uptake or 1131 or between TSR and 1131 output rate in the goat. However, in the sheep, Hoersch, Henderson, Reineke and Henneman (1961) observed a low negative correlation (r = -.255) between TSR and zero time percent uptake. They pointed out that the 1131 uptake in itself is not a reliable quantitative estimate of thyroid hormone production. 13 Goitrogens decrease thyroidal retention of 1131, and if administered after the normal uptake, these drugs enhance release rate. Tanabe §E_§1. (1965) reported a comparison of these effects of different goitrogens in chickens. March §£_§1. (1964) observed that the thyroid gland and the thy- roidal uptake of 1181 were greater in chicks fed higher levels of protein. Supplementation of lysine in the diet, however, aggravated the amino acid imbalance and signifi- 131 cantly depressed the thyroidal uptake of I per chick. Thyroxine Degradation Method The plasma thyroxine turnover or thyroxine degra- dation technique has been largely applied in man both in health and disease of the thyroid (Sterling §E_a1. 1954, 1956; Ingbar and Freinkel, 1955; Gregerman, 1962). Thel technique comprises intravenous injection of a tracer amount of thyroxine. Serum or plasma radioactivity is measured in samples taken at different intervals. Biological half life, fractional turnover rate and thyroxine distribution space are calculated from the decline in radioactivity. Thyroxine degradation is then worked out from these parameters and the chemical PBI of the blood samples. The validity of this method depends upon the assumptions that for a steady state, hormone degradation is equivalent to hormone production. 131 __ Furthermore, the administered I tagged 1 - thyroxine be- haves in vivo in precisely the same fashion as the natural 14 hormone secreted by the thyroid gland. However, in contrast with other methods, the turnover technique measures only thyroxine degradation and not the thyroxine equivalent of the biological effectiveness of secreted T4 and T3. With regard to studies in man, Ingbar and Freinkel (1955) reported that in myxedematous patients, the volume of thyroxine distribution space (TDS) was significantly di- minished and the fractional rate of thyroxine turnover was slightly reduced. Sterling and Chodes (1956) also made similar observations. They reported that extra thyroidal organic iodine pools, fractional turnover rate and the thy- roxine degradation rate were diminished in myxedema and in- creased in thyrotoxicosis. Gregerman_§§_§1. (1962) employed the degradation technique in 73 euthyroid men of different ages. According to them, TDS decreased with age, but ap- parently only after decade 6. The fractional turnover rate also decreased with age but only till decade 7. The thy— roxine degradation decreased by about 50 percent over the age span measured, roughly 20 to 80 years. The authors suggest that these age dependent variations may result from the de- crease of metabolic rate with age. Recently Oddie, Meade and Fisher (1966) made a statistical analysis of human data published in 30 papers plus additional information communi- cated through numerous authors. They observed that sex and pubertal state showed no significant effect on thyroxine ‘turnover. The thyroxine distribution space (TDS) increased 15 with increase in weight, but decreased in such clinical states as hepatitis and obesity. There is no effect of height or age on TDS. The fractional degradation is lowered as age advances. It is independent of weight and height. It decreases in hypothyroidism and hypometabolism (non thy- roid) but increases in hyperthyroidism, hypermetabolism (non thyroid) and continuing antithyroid drug therapy. A signifi- cant increase in PBI occurs in hepatitis, in hyperthyroidism under treatment with antithyroid drugs, in hypothyroidism, nephrosis and diabetes. The thyroxine degradation technique has been employed to estimate TSR in certain animals. Freinkel and Lewis (1957) first used it in sheep. Post and Mixner (1961) de- termined TSR in dairy cattle using two thyroxine turnover methods. They compared within animals, the results of (1) an isotope dilution procedure based on decline in specific activity of PBI following injection of 1131 - labelled thy- roxine, and (2) normal thyroxine turnover method based on the decline in plasma PBI after injection of non—radioactive thyroxine. The latter method was proposed earlier by Mixner and Lennon (1959). Daily thyroid secretion rates (per 100 lb body weight) estimated by (1) and (2) methods, re- spectively, averaged 0.40 and 0.39 mg in young calves and 0.14 and 0.13 mg in non lactating cows. These values com— pared favourably with those of the thyroxine replacement 'method. The authors stated that of the two turnover methods 16 the isotope dilution method is the most accurate. They also presented evidence that recycling of 1131 through the thy— roid during the first 96 hours does not influence signifi— cantly the turnover rate of 1131 labelled thyroxine. In rats, TSR obtained by the radiothyroxine turnover method (Gregerman, 1963) was generally in agreement with those gotten by other methods. The fractional turnover rate and thyroxine degradation were higher in female and in male and female rats exposed to cold. Thyroxine distribution space per unit body weight was found to be greater on ex- posure to cold and, unexpectedly, in old senescent rats. Since, changes in TDS reflect thyroxine degradation, the latter was also found to be increased in senescent rats. This finding is in contrast with the results obtained in man (Gregerman et a1., 1962) and in rats reported by other workers employing other methods. The degradation method has not previously been used for estimation of TSR in chickens. Thyroid Function in Chickens The function of the chick thyroid remains relatively unexplored. Although thyroid functions in birds and mammals are believed to be generally alike, nevertheless, the avail- able literature points out important differences, which merit some discussion. 17 Both thyroxine (T4) and triiodothyronine (T3) have been isolated radiochromatographically from chicken plasma (Mellen and wentworth, 1959a), and thyroid extracts (Shella- bargar and Pitt-Rivers, 1958; Mellen and Wentworth, 1959a). Recently, however, Rosenberg §£_§1. (1964) could not find T3 in the hydrolysates of cockerel thyroids. They did not ascribe any reasons for this. Wentworth and Mellen (1961b) reported that the two thyroid hormones are found in blood of chickens, turkeys and ducks at the ratio of 60 percent thy- roxine to 40 percent triiodothyronine. The two thyroid hormones are reported to be bound to serum albumin. Unlike in mammals, the chicken and duck serums show no alpha-globulin-like thyroxine-binding protein (Ringer, 1965). Farer, Robbins, Blumberg and Rall (1962) found thyroxine-binding prealbumins and albumins, but no thyroxine—binding globulins in the blood of chickens, turkeys and pigeons. Tata and Shellabarger (1959) reported an equal binding affinity of albumin for T3 and T4 in chickens, but more recently, Heninger (1962) has shown that the two thyroid hormones are unequally bound to chicken plasma proteins. He 131 observed that one hour following the injection of I labelled hormones, 50.4 percent of T and 24.8 percent of T 4 were bound to the albumin fraction of the plasma proteins. 3 Ig_vitro, T3 was taken up by erythrocytes at a faster rate and to a greater extent than was T thus showing that T 4’ 3 has a markedly lower affinity for plasma proteins than T4. 18 A comparison of the protein binding of thyroid hormones in rat, chicken and human serum was drawn in the report of Dubowitz, Myant, and Osorio (1962). While noting a difference in binding of T3 and T4 by chicken serum, they also observed that chicken serum and rat serum resemble each other in their binding of T3 and T4 more closely than either of them resembles human serum, although relative potency of T3 and T4 in the rat is similar to that in man but differs in the chicken. Owing to the protein binding differences, T3 is ex- pected to be more potent than T4. However, the opinions differ. T4 has been reported to possess less potency than T in blocking TSH release (Gilliland and Strudwick, 1953). 3 At variance with this, and to the situation in mammals, it was found that T is equally potent as T by the chick thy- 4 3 roid goiter prevention test (Shellabarger, 1955) and in stimulating heart rate (Newcomer, 1957). Furthermore, evi- dence suggests that T4 is more potent than T3 by such com- parisons as (1) in reducing goiter (Newcomer, 1957; Mellen and Wentworth, 1959b), (2) radioiodine assay (Mellen and Wentworth, 1959b) and (3) in promoting oxygen uptake of chick myocardium (Newcomer and Barret, 1960). Protein bind- ing is thus not an adequate explanation for differences in and T potencies of T in avian species (Heninger, 1962). 4 3 19 Metabolism and_Excretion of Thyroid Hormones in Chickens There is not much information available concerning metabolism and excretion of thyroid hormones in chicks. Recently, however, Hutchins and Newcomer (1966) reported that (l) the principal route of excretion of radioactive T4 and T3 was via bile instead of urine during a 4-hour col- lection period, (2) T3 was excreted at a more rapid rate than T4 via both bile and urine, (3) the principal metabo- lites of labelled T4 and T3 were conjugated and deiodinated thyronines present in the bile and 113l-iodide in the urine of chickens, (4) the percent of radioactive T3 in chicken plasma decreased at a greater rate than did T4, indicating that T3 was metabolised peripherally and excreted at a greater rate than T4. Heninger and Newcomer (1964) reported that half lives of T4 and T3 in chicken plasma were almost identical, although in cardiac tissue, they noted that T had a mean 3 half life of 3.9 hours, while that of T was 4.9 hours. In 4 case of Japanese quail, McFarland, Yousaf and Wilson (1964) observed that the fractional turnover rate (k) of thyroxine was higher (4.02 percent loss/hour) in birds kept at 70°F than that (2.56 percent loss/hour) in those kept at 90°F. Hypothalamic lesions in quail also decreased k values. In contrast to the short half lives of thyroid hormones in (birds, it may be interesting to note that T4 and T3 20 respectively have half lives in man, 6.7 and 2.7 days (Sterling, 1955), in guinea pig 31.3 and 30.2 hours, (Ray and Premachandra, 1964); in rat for T4 19 hours (Feldman, 1957) and in dairy cattle 46 to 48 hours (Post and Mixner, 1961). Protein-Bound Iodine Protein—bound iodine (PBI) is an estimation of the concentration of thyroid hormones in the blood. Using 4 week-old New Hampshire chicks, Bumgardner and Shaffner (1957) reported a mean value of 1.12‘pg percent. They found no significant difference in PBI values between the controls and birds treated with thiouracil and thiouracil plus up to 8‘pg T4 per day. They also pointed out that repeatability of determinations of chick PBI was not good. Mellen and Hardy (1957) made comparisons between PBI levels of some birds and mammals. They reported values ranging from 1.13 to 1.22 pg percent in 8 and 20 months old chickens, and al- most similar values in Pekin ducks. These values are far be- low those for rat, cow or man. Rosenberg_§£_§1- (1964) 7 and 1131) in studied thyroidal metabolism of iodine (I12 chickens and rats by equilibration of injected iodine with existing thyroidal iodine. They reported an average PBI of 0.51 mg percent in 70-day-old cockerels maintained on low iodine diet. Supplementation of low iodine diet produced an 21 iJisignificant increase in PBI of chickens. (0.63’ug percent) In rats, however a high iodine diet produced a significant rise in PBI. Iodide supplementation also produced large in- creases in total and free iodide in both rats and chickens. Thyroidal content of iodide 1127 in all animals was in— creased 3 to 4 fold by iodide supplemented diet. Rate of trapping of iodide was essentially the same in thyroids of rats on two kinds of diet. In thyroids of chickens on iodide supplemented diet, the rate was 5 - fold higher than in rats. Effect on Growth and Metabolic Rate That thyroidectomized chicks grow less than controls is well known, but levels of replacement therapy, which can bring back normal or near normal growth is not too clear. Winchester and Davis (1952) claimed to have stimulated growth of thyroidectomized chicks to 91 - 99 percent of con— trol body weight by daily injections of 2 or 4’ug of d1- thyroxine/100 g.b.w. Almost similar results were obtained by Clegg, Ericson and Hein (1959). Ringer (1965) made a comprehensive review on effects of thyroidectomy and hypothyroidism on growth of chickens. He stated that goitrogens have been used to increase growth or improve carcass quality through increased deposition of .fat. The rationale of using goitrogens is to depress the 22 thYICXid.activity. This is then reflected in a reduced meta- bolic rate, which in turn could produce a gain in weight. Results contrary to the above have been obtained by some workers. Chickens fed a ration with thiouracil at 0.2 percent level (Herbert and Brunson, 1957) and methimazole at levels above .001 percent (Wilson and MacLaury, 1961) showed a de- crease in weight gains. Combining diethylstilbestrol with thiouracil, however, improved growth in chickens (Andrews and Bohren, 1947). Such combinations of stilbestrol with methimazole improved both growth and carcass quality in turkey broilers (miner gt_gl., 1959). Effect of thyroprotein on the growth of normal fowl is also not clear. White Plymouth Rocks fed less than 0.1 percent thyroprotein showed enhanced growth rate up to 6 weeks but not at 12 weeks (Irwin, Reineke and Turner, 1943). At other occasions, feeding of therprotein did not produce any growth gain (Boone, Davidson and Reineke, 1950), or the growth rate was even depressed (Turner, Irwin and Reineke, 1944; Oloufa, 1955). Recently, Snedecor and Camyre (1966) have shown an interaction of androgen and thyroid involving comb growth, but could not find any such clear effect on body weight of cockerels. Increased liver glycogen and liver weight following hypothyroidism was noted by Snedecor §£_él° (1964, 1965, 1966). 23 A single injection of thyroxine in mammals stimulates metabolic rate (M.R.) and the action is prolonged over a period of several days. Unlike this, Mellen (1958) reported that thyroprotein-fed birds show increased metabolic rate only for a short time during the first few hours after fast- ing. After 12—14 hours of fasting, M.R. was consistently low except at 22—24 hours. A depression in M.R. following thyroidectomy occurs in many avian species (Lee and Lee, 1937; Winchester, 1939; Marwin and Smith, 1943; Mellen and Wentworth, 1962). The stimulating effect of therprotein on M.R. in chickens lasts for as long as supplementation is maintained (McCartney and Shaffner, 1950). Increase in metabolic response proportional to graded doses of thyro— protein (at levels greater than 5 gm/cwt) was noted by Singh and Shaffner, (1950). They also reported that increasing the caloric value of the ration increased metabolic response to thyroprotein. Strite and Yacowitz (1956) while working out a modified method for measuring 02 consumption of young chicks noted a slight rise in M.R. with therprotein. MATERIALS AND METHODS Birds and their Feed A total of 399 white leghorn chicks and 46 quail were used in this study. The chicks were obtained as day— old cockerels from a single hatchery. They were placed in brooders adjusted to 350C and kept on 14 hours per day lighting period. All the birds had free access to fresh drinking water. According to design of experiments, the chicks were fed ad libitum with the following rations: 1. Michigan State University 63-S chick starter krumbles. This is manufactured by King Milling Company, Lowell, Michigan. The formula is given in Appendix G. This was adequate with iodine. 2. Rat ration mixture SRr2, with iodine supplemented to provide 1.3,hg/gm of diet. 3. Rat feed mixture SR—2. This was deficient in iodine. The formula of this diet is given in Appendix H. Bobwhite (Colinus virginianus) and Japanese quail (Coturnix coturnix japanica) were procured as adult male birds, 56 to 68-week and 10-week old, respectively, from the Poultry Science Department, Michigan State University. Twelve white leghorn roosters 56-week old, used in degradation 24 25 Stxuiies, were also obtained from the Poultry Science De- partment. The diets of those birds were standard quail and poultry rations containing adequate iodine (Appendices J and I). Chemicals and Drugs Methimazole (l-Methyl-2-Mercaptoimidazole, or tapa- zole) was obtained from Eli Lilly and Company. It was used as a goitrogen at the levels of()JH5percent in the ration or 0.025 percent in drinking water. Thyroxine and triiodothyronine, stock solutions of lOng/ml of Sodium-L-thyroxine (merck) and triiodo—L—thyro- nine (Smith, Kline and French) were prepared and stored in the refrigerator for a short period of time. For equimolar quantities of the two hormones, 1.0’ug of T is needed for 4 each 0.84 pg of T In preparing solutions, the crystalline 3. hormones were first dissolved in a small amount of 0.1 N NaOH. Enough 0.1 N HCl was then added to make the solution slightly cloudy which signifies the point where the mono- sodium salt is formed. This suspension was diluted with an appropriate amount of normal saline solution to make up the desired concentration. The solutions for injection were made up as needed from the stock solution. In one experiment on metabolism, T and T3 solutions were mixed in the ratio of 4 60:40 for treating a group of chicks. 26 In tracer experiments, ten microcuries of carrier- free 1131 as NaI, made up to a volume of 0.25 cc in normal saline solution, was injected subcutaneously; to each bird. A drop of chicken plasma was added to the diluted 1131 solu- tion to minimize adsorption to glass. Standards containing 1/10 of the injected dose were kept in small glass planchettes. These were stabilized by addition of a drop of casein suspension containing an excess of K1 and NaHSO3. The radioactivity contained in the standards was measured every time a determination was made on the thyroid and at similar geometry. Percent of the injected dose was then de- termined by comparison with the standards. Radioactive thyroxine and triiodothyronine (triomet) labelled with 1131 were obtained from the Abbot laboratories as 50 percent propylene glycol solutions. They had specific activities of 38.4 and 28.2 mc per mg, respectively. They were diluted with normal saline solutions containing a drop of chicken plasma and were administered intravenously in doses of 10 pc per chick and bobwhite quail and 5‘pc per Japanese quail. The standards containing 1/100 to 1/50 of the dose in the same volume of fluid were kept in plastic vials similar to those used for the plasma samples. The standards were counted at the same geometry each time plasma samples were counted. After making correction for the back- ground count, percent of the injected dose in plasma was calculated. 27 Standards were based on the amount of protein- precipitable radioactive material in the labelled hormones. Gregerman, §£_§1. (1962) reported that thyroxine content of commercial solutions was approximately 90 percent. In this study also, it was observed that the recovery of counts after TCA precipitation of the plasma containing labelled hormone in 8 trials was 89.8, 90.9, 93.4, 92.4, 93.9, 91.0, 93.3 and 91.3 percent of the initial radioactivity. It was thus thought that a correction of the standards by an average factor of 0.919 is necessary, as otherwise, the distribution space measurements would be over estimated, since the impure component would probably be eliminated much more rapidly than the hormone. Counting Apparatus and Procedure The gamma radiation from the thyroid region of birds was counted on a scintillation counter (Nuclear Measurements Corporation) with a 2" crystal connected to a radiation analyzer and a laboratory scaler. A count rate meter was connected in the circuit to help in determining the position of the chick for the highest counts. The unanesthetized bird was placed in a specially improvised plastic cone. This cone served both as an immobilization apparatus and a device for location of the thyroid geometry for the maximum counts. Body background counts were taken over the thigh 28 region. Thyroid counts and the counts in the standards were corrected, respectively, for body and room backgrounds. Plasma samples and the thyroids when removed from the body were counted in a well type scintillation counter connected to a radiation analyzer and laboratory scaler. Goiter Prevention Method The goiter prevention assay described by Mixner, Reineke and Turner (1944) was employed on week-old cockerels. In one experiment 47 chicks were divided into 6 groups. One group served as a normal control while all birds in the other five groups were given 0.025 percent tapazole in drink- ing water. Out of these five, a group receiving no thyroxine served as a hypothyroid control. The remaining four groups received, respectively, 0.5, 1.0, 2.0 and 3.0 pg L—thyroxine per 100 gm b.w. daily for the duration of the experiment. On the 20th day, the birds were killed with ether and the thyroids dissected out cleanly and weighed on a Roller—Smith torsion balance. Thyroid weights in mg per 100 gm b.w. were plotted against thyroxine dosage (Figure l). The thyroid secretion rate was determined by the dose of thyroxine at which the thyroid weight curve of the injected birds inter- cepted the normal thyroid weight line. In this study, the original goiter prevention tech- nique was modified such that all birds were weighed at 3—day 29 intervals to determine the effects of various treatments on growth rate. The results of the first experiment prompted the author to undertake a combined study on body growth, feather and comb development with the goiter prevention assay in the second experiment. One hundred l-week old chicks were divided into 10 groups. Group 1 served as the normal control. Groups 2, 3, 4, and 5 received, respectively, 0.5, 2.0, 3.0 and 4.0‘pg L—thyroxine per 100 gm b.w. daily. Group 6 was put on tapa- zole water and served as hypothyroid control. Groups 7, 8, 9 and 10 received both tapazole water and daily injections of 1.0, 2.0, 3.0 and 4.0‘pg L-T4/100 gm b.w., respectively. The weights of all birds were regularly recorded. Growth of feathers was estimated by dying wing and tail feathers with picramic acid at the beginning and measuring the newly grown parts on the 12th, 20th and 32nd day. The comb height was measured only on the 32nd day when chicks were killed for TSR determination from their thyroid weights. Thyroid Hormone Substitution Method In general the T4 substitution method of Reineke and Singh (1955) for determination of thyroid secretion rate in rats was adapted to chicks. Sixty male chicks aged 2 — 6 weeks were used in 6 experiments. The experiments were de— signed to test results of the thyroid hormone substitution method as follows: 30 1. Comparison with other methods of TSR determination. 2. Influence of dietary iodine intake in estimation of TSR. 3. Influence of goitrogens in estimation of TSR. 4. Determination of the comparative potency of T3 and T4 on normal and tapazole-treated chicks. Forty-eight to seventy-two hours were allowed for maximum thyroidal uptake of 1131 after administration of the isotOpe. The initial counts were taken and the injections of T4 or T3 were then started. Where effects of the goitrogens on the apparent TSR were to be determined, the birds were put on tapazole in feed or water on the day when hormone in— jections were started. The injections were given daily but counts were made on alternate days and after each count, the dose was increased gradually till the maximum percent pre— vious counts were obtained. This dosage level of the thy- roid hormones per 100 gm b.w. was taken as the daily TSR. Goitrogens enhance release rate of 1131 from the thyroid and thus to avoid risk of losing considerable radioactivity from the thyroid, the increment in hormonol dose in certain cases was given daily after taking thyroid counts. This modifi- cation apparently had no effect in the chicken. Direct Output Method The technique of Reineke (1963, 1964) for rats was applied to birds. Sixty 1 to 9—week old cockerels were used 31 11165 different experiments, with the idea of comparing: 1. results of TSR computation with that by other methods. 2. effect of age and iodine content in feed intake, thyroidal iodine content, thyroidal iodine turnover (re— lease rate). 3. correlation, if any, between these different parameters. Forty-eight to seventy-two hours after administration of I131, radioactivity of the thyroid was determined daily consecutively for 5 to 6 days. Percent of the injected dose in the thyroid was plotted on the ordinate of semi-logarithmic paper against time in days on the abscissa. The output curve was fitted by inspection and extrapolated to time zero. The maximum uptake and biological radio iodine half—life were read from the curve. The birds were killed for re— covery of thyroids, which were subjected to total iodine analysis. The following calculations are made for esti- mation of TSR. I131 .693 output rate constant: k =-—E:— or 2 k = 2 302 (log At - log Ao) ' t Fractional output daily (fractional turnover): = k'4 = 1 - e“kt (t = 1 day) 32 l I 31' daily output corrected for recirculating iodine: k = k'4 U = Percent maxi— mum uptake Maximum uptake at zero time 100 Daily iodine output = k4 x total thyroidal iodine (From iodine analysis of thyroid) Daily thyroxine output = Daily iodine output x 1.529 (thyroxine equivalent of iodine) Daily TSR per 100 gm b.w. = Daily thyroxine output X 103 (gm) Rosenberg et al. (1964) did not find T3 in the hydro— lysates of chick thyroid. Mellen and Wentworth (1959a) ob— served 86 parts of T and 14 parts of T in thyroid digests 3 I131 4 at 96 hours after injection. Shellabarger and Pitt- Rivers (1958) also reported that both T3 and T4 131 are present in the thyroids of chicks 24 hours after I injection, but that T3 radioactivity does not exceed 5 percent of T4 activity. In view of such disagreements, as also of the al— most identical potencies of the two hormones as observed in our work on chickens and that reported by other workers, it was decided that the factor 1.53 which is applied for ad— justment of T4 equivalent in proportion to the potency of the mixture of T4 and T assumed to be released in rats, 3) may not be applied in case of chickens. 33 Thyroid Hormone Degradation Method In general the methods used in man (Ingbar and Frienkel, 1955; Sterling and Chados, 1954; Gregerman §£;§1., 1962) were applied in birds. Fifty-four, six, seven and fifty-six-week-old chicks, twenty-six bobwhite and twenty Japanese quail, all males, were used in thyroxine and tri- iodothyronine degradation experiments. Six-week—old chicks were fed on diet deficient in iodine while all other birds were on diet adequate in iodine. The blood samples were taken from the wing vein opposite to the one used for ad— ministration of labelled hormones. Sufficient blood was drawn every 3rd, 6th, 9th and 12th hour to yield 0.5 cc and 0.1 cc plasma in case of chicks and quail, respectively. In some preliminary experiments on chickens, blood samples were collected 24 hours after administration of the tagged hor- mones, but no appreciable counts could be detected at that time. Thus sample collection at 3-hour intervals was re- sorted to. The last or 12—hour blood samples in many cases were collected by heart puncture. These samples were large enough for use both for counting radioactivity and for PBI determination. The percent of the injected dose in the plasma as calculated from each sample was plotted against time on semi—logarithmic graph paper (Figure 8). The curve was fitted by inspection and extrapolated to time zero. The maximum radioactivity at zero time, thyroxine distribution 34 space (TDS), biological half life (tk), fractional turnover (k) were calculated. In certain birds, especially the quail it was noticed that the samples obtained by heart puncture contained more radioactivity than the earlier samples Obtained from the wing vein. A similar type of overdischarge was particularly well marked when the last samples were taken by decapitation of quail in certain earlier experiments not included for collection of this data. In view of this, the last samples in T3 degradation experiments with quail, were taken both by venous and heart punctures and the percent radioactivity com- pared in the two. From this, percent discharge was calcu— lated. In case of the subjects where fourth samples were collected only by heart puncture, percent increase in radio— activity was calculated from the difference between this point and that of the curve extrapolated from the earlier samples. The thyroxine turnover was calculated as below. Thyroxine distribution space: _ 100 (in mls) (TDS) - % dose at 0 time in 1 m1. plasma 131 _ .693 T4 - I turnover rate constant. k —-—E;- (hours) Fractional turnover: k = l - e-X Daily thyroxine degradation or TSR = ETT x k x 24 (Multiply by 1.529 to convert thyroxine iodine into its equivalent as thyroxine. Multiply by 24 to convert hourly degra- dation to degradation per day.) Daily TSR in’hg per 100 gm b.w. = Daily TSR x 100 .w. (gm) 35 _Uptake of Thyroid Hormones by Red Cells In order to study uptake of T4 or T3 by avian erythro— 131 cytes, a drOp each of I labelled T4 and T3 was added into two equal chicken blood samples. Whole blood was counted and then incubated at the chicken body temperature (41.60C) for 2 hours. Plasma was separated from the red cells after centrifuging. The cells were washed thrice with normal saline solution, centrifuged and the wash mixed everytime with the plasma. The cells and the diluted plasma were then counted and necessary corrections made for geometry and de- cay. Percent of the hormones bound to R.B.C.'s was determined. Determination of Metabolic Rate (M.R.) Reineke's multiunit metabolic apparatus, a modifi- cation of the Maclagan and Sheahan procedure was used for determining metabolic rate in chickens. This multiunit sys- tem consists of 12 desiccators connected by three—way stop cocks to mercury manometer, vacuum and oxygen lines. The animal is weighed and placed in a tightly sealed desiccator of known volume. The desiccator is charged with oxygen and contains soda lime for the absorption of C02. As 02 is used, the pressure in the chamber decreases proportionately. O 2 consumption is computed directly from the decline in pressure 36 and the net volume of the chamber minus volume occupied by the animals. Forty-eight.3'U35-week old chicks were used in this study. In the first series of experiments, 24 birds were equally divided into 4 groups comprising controls, T4, T 3 and a combination of T4 + T3—treated birds. Each treated bird received 6.0‘pg/100 gm b.w. daily of either single or combined hormones. The birds were fasted for 12 hours be- fore determination of M.R. Of the 4 determinations made in this series, two were done after 24 hours and one each 2 and 3 hours after the last hormone administration. The first determination was made 7 days after initiation of injection. In the second series, two experimental groups of 8 birds each were injected subcutaneously with 4.0.pg/100 gm b.w. of T4 and T3. The third group.containing the same number of birds was injected similarly with normal saline solution and served as a control. M.R. was determined 1, 2 and 7 hours after single injections of the hormones. Calculations: . P 273 60 02 consumption (mls./Hour) — (V - Va) x 76 x 273+t x T Where V = net vol. of unit in ml. Va Volume of animal P = Pressure difference in mm. Hg. t = temperature in desiccator T = Time of determination in minutes 37 Statistical Analyses The following methods were used for statistical an- alyses of the data: One- and two-way analysis of variance and linear re- gression for finding correlation coefficients and estimation of confidence intervals, means and standard errors were done by the methods given in Li (1964). The Mann-Whitney U test for comparison of two independent samples and the Spear- man rank correlation coefficient for determining relation- ships between T3 distribution space (TDS) and body weight of chicks, were done by the methods given in Siegal (1956). RESULTS AND DISCUSSION Goiter Prevention Method TSR values (2.32 and 2.25 pg T4/lOOg/day) from the two experiments on the goiter prevention method (Figure l) are in close agreement. The slight difference is probably due to experimental variation. In the climate of Michigan, it is expected that there would be no seasonal variation during the span of time covered by the two experiments. Also, there were only 12 days age difference between the groups of birds used in the two experiments. It may be noted in Figure 1 that the thyroid weights were greater in the group receiving tapazole plus daily in- jections of 0.5 Pg T4 than those in the group receiving tapa- zole only (39.40 and 38.95 mg per 100 g.b.w. respectively). This phenomenon is explained by the fact that in a critical dose range, T4 potentiates goitrogenesis by a goitrogenic drug. Sellers and Schonbaum (1965) believed that this action of T4 is mediated via an effect on the adenohypophy- sis or higher centres. 38 39 .mmmmm coaucm>mnm kuaoo .H muomam . a as ooH\m1\omon oConumnB I A O.¢ m.m O.m m.N O.N m.H O.H m.O O 1 d ._ 4 q u, A I# J OH 1 ON #2. mamo mm How con wmmmm 9|.I.IL . / a OOH mhmo.om Mom com hmmmm cllllJ jTI ll 1:: oaa 'M'q m5 cor/'5m nufirem pIOJqu 4O Thyroid Hormone Substitution Method Table l and Figure 2 show that the values of TSR ob- tained by substitution of T4 and T3 in normal birds (2.00 and 2.17 respectively) are significantly lower than those in the tapazole-treated birds (4.14 and 3.86 respectively). Differences of TSR by the T substitution method be- 4 tween normal and goitrogen-treated subjects were also ob- served in rats (Reineke and Singh, 1955) and in chickens (Himeno, Tanabe and Komiyama, 1961; Himeno and Turner, 1961). Turner_gt_al. (1959) in rats and Mellen (1961) in chickens could not find TSR differences between normal and goitrogen- treated animals. Table 1 also shows that for both T4 and T3, points of percent previous counts in the normal birds the end averaged higher than that in the goitrogen—treated birds. This again is supported by the work of Reineke and Singh (1955) and Reineke and Lorscheider (unpublished) in rats. Tanabe and Komiyama (1962) suggested a different end point in a modified substitution method in chickens. TSR in that procedure was estimated as that amount of T4 which inhibits thiouracil-induced acceleration of 1131 release from the thyroid gland and returns the retention of thyroidal 1131 to the rate before start of the goitrogen. Ringer (1965) is of the view that the physiological basis for computation of TSR in the method of Tanabe et a1. is not clear, since such 41 Table l. Thyroid secretion rate in chickens by thyroxine and triiodothyronine substitution methods. Thyroidal 1131 Retention at Estimated No. of Age TSR Estimation, TSR‘pg/loo 9 Group Birds (weeks) Percent b.w./day Previous Count T normal 9 . 5.5 96.5 2.00 4 i .67 .167 |+ T4,.tapazole treated 7 2 90.5 4.14 .i 5.93 .i .34 T3, normal 9 5.5 94.0 2.17 i 2.11 .i .2762 T3, tapazole treated 7 2 85.4 3.86 .i 6.0 .1 .34 Mean i standard error. Estimated TSR values are significantly greater in tapazole—treated than in normal groups. (For T4 U = l, P< 00020 For T3 U: 6’ P: 0020) TSR differences between the two normal groups and be- tween the two tapazole-treated groups are non significant (U = 37.5,“? > .05 and U = 20, P = .620 respectively) by Mann-Whitney U test. PERCENT PREVIOUS COUNT I00 I ’r"\ . 9° ’ 5M 4M 80 '5' 70 / 5 60 I / I 50 4 6 i 40 30.1...L_L_I._L_l_.l__l_.l_.l_l__|_.l_ O. 5 Lo I.5 2.0 2.5 3.0 3.5 4.0 4.5 50 5.5 6.0 T4 OR T3 DOSE IN 7/l00 GM. aw. SUBSTITUTION OF: (I) T4 - NORMAL CHICKS (2)T4- TAPAZOLE TREATED CHICKS (3) T3 - NORMAL CHICKS (4IT3-TAPAZOLE TREATED CHICKS (5) T4- NORMAL CHICKS LOW IODINE OIE‘r (6)T4- TAPAZOLE TREATED CHICKS, LOW IODINE DIET (AVERAGE CURVES) Figure 2. Substitution of thyroxine or triiodothyranine in the normal or tapas le-treated chicks on diets adequate or deficient in iodine. 43 birds are still releasing labelled products from the thyroid at a rate equivalent to the normal one. In the present study (Table 1, Figure 2) no signifi— cant difference was found in the thyroid suppressing effect of T3 and T4 as indicated by TSR values of the two normal or the two tapazole-treated groups. Per unit weight of the hormones, the potencies of T and T4 are not significantly 3 different. On the iodine basis, however, T3 is slightly more active. The end point for T4 was 2.0‘pg, equivalent to 1.31 ug of iodine. For T3 it was 2.17 pg equivalent to 1.27‘pg iodine. In view of the experimental variation to be ex- pected in this type of comparison and the smallness of the difference in activity, whether on a weight or iodine basis, the entire thyroidal iodine output has been calculated as T4 in subsequent experiments by the direct output method. The results of different workers differ with regard to relative potencies of the two hormones in the chicken (Gilliland_g§_§l., Shallabarger, Newcomer, Newcomer and Barret, Mellen and Wentworth—-a1ready cited). Some workers report that T3 is more potent than T Others report the 4. opposite and still others think both hormones are equally potent. In mammals T3 is generally considered 4-6 times more potent than T Recently, Bauman, Pipes and Turner (1965) 4. observed that T3 is 2.6 times more potent than T4 by the substitution method in rats. Rat substitution assay in this laboratory shows T to be 4 times as potent as T4 per unit 3 44 weight and 4.6 times as potent as T4 per unit of iodine (Reineke and Lorscheider unpublished). 4 or T3 dose representing estimated TSR, blocks maximally the 1131 output Figure 2 also shows that when a given T from the thyroid, a further increment in the hormone dose was not able to hold that blocking level. Apparently some leakage of the iodine does occur, despite administration of sufficient hormone which should supposedly suppress com- pletely the pituitary TSH secretion. Similar observations have been made in rats (Reineke and Singh, 1955) and in chickens fed thiouracil (Mellen and Wentworth, 1958). Direct Output Method Figure 3 shows typical TSR determination by the direct output method. There is a close agreement in the TSR of different groups of chickens as estimated by this method (Table 2). The statistical analysis showed no significant difference in TSR among birds of different ages. A trend for higher TSR is, however, noticed in week-old chickens. The highest TSR was observed in 4.5-week—old chickens, which could possibly be due to their higher thyroidal iodine con— tent. This group was fed with the chick starter diet (Ap- pendix G). All other groups were given either SR-l or SRr2, supplemented with iodine (Appendix H). A study of the ef— fect of three diets on body weight, thyroid weight and Percent I131 dose in thyroid o C) c> CDI‘ .04 CD iii A or Figure 3. — _ J Days 45 t . $5 4 8 days K'4 (slope) .13438/day K4 TSR 1.23’pg/100 gm/day .l376/day Typical TSR determination by direct output method. The output curve was fitted by in- spection and t% was read from 0.693 ts x 1.529 2 body wt. in 100's of 19m“ I I I I 6 7 8 9 10 the curve; Then K'4 = 46 .Cmoe on» ca omooHocH mcoaum>ummno mo HOAESZRR . .amem. H.me.m mamoom uanOS moon How omumonOm no: .mooum wasp mo Mme Hmuou haamo mmmnm>ms .Amo. A.m .mm.H u om.mmv mocmaum> mo mammamcm >n3 moo an ucmoamacmam so: mum mcwxowsu mo mmooum ucoummmwo mGOEm .3.n Em ooa\dma mo woodenmmwfia .HOHHO oumocwum + mosam> Cmmz om. H. HH.H~ H. mmoo.u. mmao.H. am.mm H. 0A a emo.a om.eam mmeo. mama. o.emeH I I. 1«Aoflw Reloaw. _I mmH. + ~m.¢ + Ho.+ whoo.+ 6H.HH + m m.e om-a oo.em NHHH. oeoo. ma.eme mm.H H. emao.M. meoo.u. 10H *4 mm.ma News. mmeo. mHH. H. eae.a H. omo.u. moao.u. ae.oa H. OH m mam. mm.m memfl. memo. oo.oo~ I I I «Lemmy I I mom. + on. + Rmo.+ mmmo.+ mam.m + OH H moom.a ao.m momm. memo. om.am e a SCU\.3.Q CCHOOH & exhumo unmoumm AEmv momxuaao AmeOSV em ooa\xme Hmeaousaa Hmuoa mafia omen Assam: seom Co .02 was .mcoxoflno ca mumo pomuoo DOOHHQ .N magma 47 thyroidal iodine revealed no significant difference between thyroidal iodine of chickens fed chick starter diet and SR~2 supplemented with iodine. The relationship between daily 1131 output from the gland (K4) and age of chickens is presented in Figure 4. K4 decreases significantly in older birds. There is a strong correlation coefficient (r) = —.8351, P < .005 in chickens from 1-9 weeks of age. Figure 4 also shows a relationship between thyroidal iodine per unit body weight and age of chickens. This again has a strong correlation coefficient of -.8869, P < .005. It thus seems that as the birds grow older, decrease in output rate is balanced by an increase in thyroidal iodine content such that the thyroid secretion rate per unit body weight is little affected. This fact is further indicated by an inverse relationship between the K4 and the thyroidal iodine per 100 gm body weight. This re- lationship is significant, with a correlation coefficient of -.8285, P < .005. The thyroid secretion rate in the direct output method is finally estimated (Figure 3) through a series of calculations involving the above mentioned parameters. The relationship between TSR and thyroidal 1131 release rate or uptake are biased because these parameters are related. Nevertheless, the K'4 and the U (Zero time percent uptake) being independent observations, their relationship was 48 .30..I .28w- -—28.0 g .6 .244- -24 0 (En /-> IIIe-\ k; 8 .204 -20.0 Q m c -H .16- «16.0 '8 «4'0 c H m .12— —12.0 .g -H o 08 ’9. 8.0 33 m .04-1I - 4.0 0 - m IV; L , =1. F 1 4 T A. I H 1: 1 5 7 9 ll 13 56 I Age (weeks) Figure 4. Relationship between (I) K4 and age of chicks. (II) Thyroidal Iodine/100 gm b.w. and age of chicks. (III) K4 and thyroidal iodine/100 gm b.w. of chicks. This graph shows mean values i standard errors. Regression and correlation coefficients for each relationship are given below. Curve (IV) shows TSR/100 gm b.w./day by the direct output method. II III K4 age Thyroidal Iodine K4 vs. thyroidal 1 g YXf= log a /100 gm b.w. (up iodine/100 gm b.w. + b log x to 13 wks.) vs. (up to 9 wks.) b = —.7691 age lOg'Yx = log a a = -.5620 ‘Yx = a + bx + b 109 x _ P b = 1.8522 b a — 8384 r = - 8351 a = -.2873 a = -.O438 P (.005 P (.005 r = .8869 r = —.8285 ‘pg of daily TSR/100 gm b.w. 49 determined in the growing chicks up to 9 weeks of age. They have a correlation coefficient of -.6719 and P < .005 (Figure 5). The direct output method does not seem to have been previously employed in chickens. This method has also been little used in other animals and thus information about its various parameters is lacking. The knowledge available about the thyroidal 1131 uptake has usually been obtained for a particular time after I131 administration. Under these situations it is largely agreed that in normal animals no simple relationship exists between the TSR and the I131 uptake or release rate (Turner et a1., Lodge §t_al., Flamboe and Reineke, already cited). In sheep however, a low nega- tive correlation between the TSR and the zero time percent uptake was found by Hoersch, Henderson, Reineke and Henneman (1961). The thyroidal I131 uptake and release rates are af- fected by a wide variety of factors such as size, weight, colloidal content, dietary iodine content, kidney function, pregnancy and lactation, etc., etc. It is thus not sur- prising that many workers could not observe relationships be- tween other parameters and the TSR. In this study on chickens, daily 1131 output rate has been seen to be significantly higher in the younger birds and the thyroidal iodine per unit body weight signifi- cantly higher in older birds. It therefore appears that as .6 CO 50 log Yx =loga+bx 0 b = -3.8008 a = -.6922 r = -.6719 p <.005 O Dotted lines are 95% confidence 0 intervals for predicted average. E. +—- o L Figure 5. Relationship between K' and zero time per- cent uptake (U) in _ thyroids of chicks - from one to nine weeks of age. lll«llllllll4 .02 .04 .06 .08 .10 .12 .14 .16 .18 .20 .22 2 4 Zero time percent uptake (U) 51 the bird grows, its thyroid weight increases, the gland ac- cumulates increased 1127 which is associated with a decrease in thyroidal iodine release rate. It is for this reason that the researchers working with mature or nearly mature chickens find an almost flat output curve. For this reason some workers have used a goitrogen to accelerate the release rate. It is interesting to notice a strong relationship between ug of total thyroidal iodine and gram body weight of growing chicks from 1 to 13 weeks of age (Figure 6). On a log-log plot the regression of thyroidal iodine upon body weight is nearly a perfect fit with a correlation coefficient of .97845, P < .005. This relationship does not hold for mature chickens. In adult birds, including chickens, bob— white quail and coturnix, the total thyroidal iodine per unit body weight has been correlated with the thyroid weight per unit body weight (Figure 7). These two measures are again highly related with r = .9416, P < .005. Both these relationships are close enough to allow a good estimation of the iodine content in thyroids from body weights of growing chicks and from thyroid weights of adult birds, provided the dietary content of iodine is adequately controlled. In grow— ing chicks it may also be possible to estimate their thyroid secretion rate by the direct output method without killing them for recovery of their thyroids and iodine determination. 2. Log total thyroidal iodine (pg) N O H '1—0 m 0 52 o /' .' 0 . log Yx = log a + b log x ‘b = 1.76864 . a = -3.12194 ‘ P (.005 ' ' r = .97845 ./ . ///o Dotted lines are 95% confidence interval for predicted average. thyroidal iodine and body weight in chicks. Figure 6. Relationship between 0 o from one to thirteen . / weeks of age. a Q’// //3. o I l E I 2.0 2.5 3.0 3.5 Log body wt. (gms) Thyroidal iodine‘pg/lOO gm b.w. 53 D Bobwhite quail o Coturnix A Chicks —120 / / . —100 / YX = a + bX / b = 6.65 ‘—90 a = -22.59 P <.005 r .9416 F80 Dotted lines are 95% confidence intervals for .70 predicted average. //// f O I _30 K] ZS A / u 0 Figure 7. Relationship between -20 d9 n thyroidal iodine and '.s thyroid weight in a A0 D adult birds. _ uV ‘10 (D [)9h3 1. 5 10 15 20 0 9V T I I l L Thyroid wt. mg/lOO gm b.w. 54 Thyroid Hormone Degradation Method Thyroid secretion rates of 7-week normal chicks, 7- week goitrogen—treated chicks, 56-week normal chicks, 56 to 58-week bobwhite quail and 10—week coturnix, as determined by the thyroxine degradation method averaged 2.03, 1.59, 1.02, 2.49 and 2.78lug/100 g/day, respectively. It shall be seen from Figure 8 that the TSR in this method is calculated from three parameters: the biological half life (t%), the thyroxine distribution space (TDS) and protein-bound iodine (PBI). The values of these parameters influence the esti- mated TSR. The t%, TDS and PBI were also determined for all birds in the T3 degradation experiment but the TSR was not calculated as the exact information with regard to plasma-protein binding of T3 and its contribution to the PBI of chickens is not available in the literature. The thyroid hormone degradation method has not been previously used for estimation of TSR in birds. There is, thus, little infor- mation with which to compare the results obtained in this study. Biological Half Lives (tg) The biological half lives of T4 and T3 are not sig- nificantly different in bobwhite and Japanese quail (Table 4). The apparent differences are due to an interaction Percent I131 dose in 0.5 m1 plasma % dose at 0 time (U) 55 2 TDS/100 gm b.w. 20.49 ml t% = 3.2 hours K = .19467 PBI = .858‘pg % TSR = 1.25 ug/lOO gm/day 1.- .09— 008(- .07“ 06’ 05- .04— .03— .03- Figure 8. Typical TSR determination by T4 degradation. The degradation curve was fitted by inspection, and t% was read.from the curve. Then K =-0'693; TDS = 100 § % dose at 0 time., ET = TDS x PBI; TSR = ETT x K x 24 x 1.529. .0 l l l 41 3 6 9 12 Hours 56 between the two hormones and the two breeds of quail. In the case of chickens, only the t% of T in 56—week normal 3 chickens was significantly greater than in all other groups of chickens treated by both T and T4 (Table 3). The inter- 3 action between the three groups of chickens and the two hormones is significant. It is interesting to note that the biological half and T lives of T are relatively much shorter in birds than 4 3 in man or other mammals (see literature review). Heninger and Newcomer (1964) reported mean half lives of 4.9 and 3.9 hours of T4 and T3 respectively in the cardiac tissue of 4 3in the chicken plasma observed in this study. McFarland, Yousaf chickens. These values are close to the t% of T and T and Wilson (1964) reported fractional turnover rates of T4 in coturnix, which when expressed as t% ranged approximately from 17 to 27 hours at 700 to 90°F. These values are much higher. A further check of their report revealed that they had taken cardiac blood samples, which have been observed in the present study, to cause a previously unnoticed reaction resulting in an increased radioactivity in the blood and an overestimation of t;5 values. Thyroid Hormone Distribution ,Space (TDS) The distribution spaces of T3 were significantly higher than of T in both chickens and quail (Tables 3 and 4). 4 .mouan o tweamucoo mooum numm mmooum Hmaaaam .ucmoamacmaw co: omuoom Hopes .Hm>oa moo.o um uomoamacmamsee .H0>0H mo.o um unmeamacmams .m can m .n weapon mum ucmsahmmxm :Oamemummo ma Eonm .O can m .m mm omaump MHO>Huoomme mum quEflummxm coaumOmumoo as Eoum mcoxoaso mo mmsoum Hmsuoc .mx3 om pow ooumouu comonuaom .mx3 h .HmEHo: .mx3 n mom.a «66.- mom.u me o o 0 ¢ mom HI sow mom a HmEHoz owumwua cmmonuaoo Hmeuoz mOCOEuom .mx3 mm .mx3 5 .mx3 n 57 .mOCOEHOE oaoumnu 03p on» cam mcoxoflno mo mmooum COHEN on» coo3p Ion coauomuouofl mafi3oom mooam> mu How mamme topmonom mamas om>ummno mo magma O o mMOm o O ¢MOmN O I o mammz m m n . O m m U m a n m m Ramos Omcmm OHQHDHDZ 302 hem.a om oa.os om Honum Ree¢¢.oH mem.mm N mm.m .m©.mom N coauomHODCH «Ramm.am 64.64 A Itemm.mo~ mm.smeva a ma 666 ea ssena.om m¢.o¢ m NH.N so.m¢H N mcoxoano m mm some .m.o m mm CmoE .m.o wousom Amusomv my =.3.A .Em ooa Hem .HE woman coaquflHuMHn CH moacoumnuoooflauu ocm mcflxoumnu mo mumo Hw>ocnsu mo mammamcm Hmoaumaumum .mmumoflamon m nuw3 mocmwum> mo mwmhamcm mm3 O39 .mcwxowno. .m magma 58 Table 4. Statistical analysis of turnover data of thyroxine and triiodothyronine in quail. Two way analysis of variance with 9 replicates. Distribution space ml. per 100 gm. b.w. tg (hours) Source d.f. mean ss F d.f. mean ss F Quail 1 .4482 64.49*** 1 .04656 3.033 T4 and T3 1 .7606 109.43*** 1 .02083 1.357 Interaction l .0501 7.21** 1 .14350 9.348*** Error 32 .00695 32 .01535 New Multiple Range Test* (Distribution Space) D C B A Log. means 1.96276 1.81428 1.74673 1.44891 Table of observed minus adjusted means showing interaction between two groups of quail and the two thyroid hormones. Distribution Space tg Hormones Bobwhite Coturnix Bobwhite Coturnix T4 -.0373 .0373 -.0631 .0631 T3 .0373 -.0373 .0631 -.0631 Variance homogeneity was checked by Bartlett's test. HO being rejected, log transformation was done on the data and variance homogeneity checked again. Two Bob- white samples from T4 and one from T3 groups, whose PBI values were off the normal range were eliminated to make equal numbers. ' ' ’ Groups A and B are Bobwhite and Coturnix in T4 degradation experiment. Groups C and D are Bobwhite and Coturnix in T3 degradation experiment respectively. *Significant at 0.05 level. **Significant at 0.025 level. ***Significant at 0.005 level. 59 Among the three groups of chickens treated by T3, there were no significant differences in their distribution spaces, but in the case of T4, the distribution spaces in 7-week normal chicks were significantly higher than in 56-week normal chicks. The TDS of both T4 and T3 in coturnix were signifi— cantly higher than those in bobwhite quail. A significant interaction existed between the quail and the thyroid hormones. The decrease of TDS of T4 in older chicks could be an age effect. Gregerman §t_§1. (1962) reported an age de- crease of TDS in man after decade 6 and he suggested that the decrease of metabolic mass with age could influence the distribution space. Contrarily, Oddie, Meade and Fisher (1966) observed that the distribution space is independent of age and height in man. The above mentioned workers also reported a positive correlation between the distribution space and the body weight in man. In the case of 6- to 7- week old chickens regression of the thyroxine distribution space on body weight was not found to be significant (F l, 13 = 3.28, P > .05). The relationship between the triiodothyronine distribution space and body weight of 7-week normal and goitrogen-treated and 56-week normal chickens was tested by the Spearman rank correlation coefficient. This showed a significant positive correlation with values of rs = .9023 and P < .02 (two tailed). 60 Following administration of the labelled hormones, the plasma samples taken after 3 to 12 hours contained half or nearly half the percent radioactivity for 1131—T 3 1131-T4. This indicates that T3 disappears from the plasma as for at a faster rate than does T4. Hutchins and Newcomer (1966) also observed that T3 was metabolised and excreted at a greater rate than T4 in chickens. Further, Flock, Owen and Paris (1966) reported that T3 in rats was deiodinated and conjugated at a faster rate than T4. In an in vitro experiment on the uptake of T4 and T3 avian erythrocytes, it was observed that the red blood corpuscles took up 1.11 percent of 113l-T as compared to 4 4.71 percent of IlBl-T after 2 hours incubation of the 3 chicken blood mixed with 1131 labelled hormones. Qualita— tively similar results regarding the differential uptake of the two hormones by the avian RBCs were reported by Heninger (1962), although percent uptake of the two hormones varied in his experiments. Rapid deiodination, metabolism and relatively larger uptake of T3 by RBCs may account for higher TDS for T3 than for T4. This view is further supported by the observation that the triiodothyronine distribution spaces in young sexually immature and old sexually mature chickens are not significantly different (Table 3). Sexually mature males have a higher number of erythrocytes and the androgens, are 61 believed to be responsible for the difference (Sturkie, 131 1965). Thus, more RBCs in older birds take up more I —T3, which may result in a decreased radioactivity in the plasma and an increased calculated TDS. The coturnix have higher T4 and T3 distribution spaces than chickens and bobwhite quail. This could possibly be due to a relatively smaller size, or a species characteristic of coturnix. The t% in T4 and T3 turnover of this quail was, however, not signifi— cantly different from that of other birds. Increased Plasma Radioactivity Under Stress During the course of plasma sampling it was noticed that in some birds their 12-hour samples (in some cases earlier samples) contained higher radioactivity than some of their earlier samples (Table 5). This kind of increased I radioactivity was seen (1) more in 1131—T4 than Il3l-T3 turnover experiments, (2) more in coturnix than in bobwhite quail, (3) more in goitrogen-treated and normal older birds than in the normal younger birds, (4) the discharge was especially well marked in the samples taken by heart punCture, (5) the discharge also seemed to be related to the amount of the blood drawn from the bird. Presumably, the in- creased radioactivity resulted from a stress to the birds at this stage. The discharge did not seem to have any relation- ship to the blood volume, as the heavier chickens showed more than the lighter ones. 62 .me03 CH woman «0 modes .osonm ca woman mo HOQEUZR .Houum onmocmum + cme om.oH H. mo.m H. Hm.m H. oe.~ H. oR.HH. m mm.eo mo.v~ km.e mm.o mm.m a- H Hma om.ma H. aa.a H. em.oHH. om.o H. om.eu. a eo.ema m~.mm mm.m~ mo.mm me.m EIHmHH stxoac so «Ixoouomo RAH Isaomo to stake so sixes so omummuu xacuouou mufl£3nom HmEHoz Cmmouuaow HmEHoz Hamoo 666x0Aa0 .mmoupm Amoco coxmu OHmEmm HSOEINH may do ooumaoono mm OOOHQ CH >ua>apum0aomn mo Ommwuoca unmouwm .m Canoe 63 In studies on rats, dogs and men, Taurog et al. (1951, 1952) and Albert and Keating (1949) observed that the thyroxine concentrates in the liver even when it is ad— ministered intravenously. They thought that T probably 4 undergoes an enterohepatic circulation. Recently, Gorman _§E_al. (1966) isolated and perfused livers of rats at l to 20-hour intervals following administration of the thyroid hormones. They observed that the unchanged T4 was released to the perfusing blood until an equilibrium was reached. They also reported that the livers of rats given labelled T3 released only a small amount of the hormone to the per— fusing blood. These differences between T3 and T4 together with a lower affinity of T3 for the plasma proteins and a faster metabolism of T may account for higher discharge 3 seen in the 12-hour samples in the T4 than in the T3 degra- dation experiment. Protein-Bound Iodine (PBI) The results of PBI analyses are given in Table 6. There was little overall difference in PBI levels of differ- ent groups of chickens and coturnix. The bobwhite quail, however, showed higher PBI, both in analyses of individual samples, as well as in the pooled plasma. The author is not aware of any report already published about PBI of quail. In the chicken, the results obtained in this study are in agree- ment with those reported by Bumgardner and Shaffner (1957) 64 Table 6. Protein bound iodine (pg per 100 m1. plasma) in chickens and quail. ‘ Number Iodine of Age Content Birds Birds (wks.) of Diet PBI Chickens 43 5-7 Adequate .1226 i.-0367 Chickens 18 5—6 Deficient 1.0135 .i .0483 Chickens ll 56 Adequate .2840 i.-0975 - - 18 .7572 Bobwhite quail 10* 56—68 Adequate .i .0637 .4770* . 17 Japanese quail 10* 10 Adequate .2603 1,-1320 .9344* Mean i standard error. *PBI of pooled plasma from 10 other quail. 65 and Mellen and Hardy (1957). The values reported by Rosenberg_§£_§1. (1964) were relatively lower. The chickens on the diet deficient in iodine had slightly lower levels of PBI than those on adequate iodine diet. Similar non— significant differences were noted by Rosenberg gtyal. (1964). Lower levels of PBI in birds are in marked contrast to the situation in mammals whose average PBIs range from 3-6,ug percent. The absence of a Specific thyroxine—binding alpha—globulin in avian blood may contribute to this differ- ence. Further, short biological half lives of the thyroid hormones in birds as observed in the turnover studies and an insignificant increase of M.R. following T4 injections in birds, may be associated with lower levels of PBI, as the latter is an estimation of the thyroid hormones in the blood. Effect of Iodine Intake on Thyroid Function The effects of dietary content of iodine on body weight, thyroid weight, thyroidal iodine and the estimated TSR by different methods are given in Tables 7 and 8. A significant decrease of body weight in the iodine-deficient group shows how a low iodine intake can impair thyroid function which in turn depresses growth gains in chickens. Excessive amounts of this mineral produce iodine toxicity. This is associated with cessation of egg production and 66 Table 7. Effect of dietary content of iodine on body weight, thyroid weight (mg/100 gm b.w.) and thyroidal iodine (pg/100 gm b.w.) of 7 week-old chickens. One way analysis of variance Body weight Thyroid weight Source d.f. mean 33 F d.f. mean ss F Treatment 2 19515.03 2 66.86 Error 27 3699.85 5.274* 27 6.14 10.89*** New Multiple Range Test* A B C C A B Means 21.40 702.80 637.30 11.32 7.23 6.53 One way analysis of variance Thyroidal Iodine Source d.f. mean 53 F Treatment 2 385.26 *** Error 26 19.32 19°94 New Multiple Range Test* A B C 12.68 8.56 0.47 Treatments A, B and C are respectively the diets chick starter, SR2 with 1.3.pg/gm feed, and SR2 deficient in iodine. *Significant at .05 level. ***Significant at .005 level. non significant. Underscored 10 chicks in all the groups, 9 in thyroidal iodine With B treatment group. 67 N00.V H m0.A ha m0.A hm N00.V 0 N00.V 0 mo.v OH m0.v m.0N Am.mvm mvHo. + NO0NH.0 Amwmvm 00. + NH.H Am.mvm mm. + mm.m Am.mvw 000.H + mh.0H Amwavoa moo. + ¢R.H Amwavoa oHo. + Hooa.o Amhioa 0000. + 0N00.0 I a; hmo. + 0000m.0 Ame 6 M00. + 00.0 AIS; 0N. + vv.m Is; m50.m + mm.mm Amwevo so. + om.e Am.eooa H0. + NHH.0 Am.oooa 0h00. + 0000.0 RH m1 gem a ma.Hmm Amuoomv a mammam CH 4» .3.n Em ooa\asmoe .3.Q Em 00H H00 01. mcaooH Hmoaonsas ex wamumb Eosflxmz x wEHu OMONV D m D umwa D hmcuHEBIcomz an mocmoamacmwm Hmoaumaumum mo Hw>mq OCHUOH CH DCOHOHMOQ uwfln OCHOOH ca mumsomom umao kuwEmHmm .mcmxoano Ca moooume ucmummmao hp UOCHEHODOO oumu coaumuowm oaoumzu mo mumumEmHmm wooaum> co ucmucoo mafiooa mumwwao mo uowwmm .m OHQmB 68 fl .va 01.0uca uuo>ooo ou Ammm.H0 uomam>flowm wcfloofl B £ua3 >a0aua52s .smo\.3.n m ooH\ma.ca ma mme .mcoxoaoo mo Aeneas 0cm Amxmm3 CH0 mom on» >H0>auoommmu mum mammoucmumo DOOEDHB 0cm casua3 mosam> mop 3oHoQ monomam .HOHHO unannouw + mooam> cow: Am.mvm Ame coaumowumoo moo.v o poo. + m6.o ms. + mo.m as as may Am.ewoa Am.w0o moo.v o Hmo. + oe.o mma. + om.a 660066 nuanao an mos mouan omummuu Awym AMVo maowmmmu co coausu mo.A oH AHA. + om.o em. + eH.e Iaumnom as sn_mme mouse Aewba Am.m0m HmEHOC 0 EOHusu mo.A em moo. + oo.H boa. H.oo.~ -aumnsm a so mes 69 decreased fertility (Perdomo, 1966). It is therefore recom— mended that poultry rations should be supplemented by an adequate amount of iodine. The thyroidal content is sig- nificantly increased in the chicken fed adequate iodine diet. This indicates the capacity for iodine retention in the chick thyroid. Rosenberg §E_a1. (1964) reported that in thyroids of chickens on iodide supplemented diet, the rate of trapping was fivefold higher than in rats. The differ— ences in iodine content of the thyroid due to differences in dietary iodine intake also place limitations on the re- lationships between the thyroidal iodine and body weight in young chicks and thyroidal iodine and the thyroid weight of adult birds. These relationships hold good under controlled dietary iodine contents. The zero time uptake of 1131 and K4 are significantly increased by inadequate intake of io- dine. This is possible because iodine deficiency is associ- ated with increased TSH secretion, which in turn stimulates the thyroid gland whereby the U uptake and the daily output rate are both increased. The thyroxine distribution space is significantly decreased with deficient iodine intake. The TDS reflects the calculations of ETT which is also ac- cordingly affected. Iodine content of the diet does not seem to alter PBI and t% of thyroxine in the plasma. The thyroid secretion rates determined by direct out- degradation methods are significantly increased put and T4 70 when the diet is adequate in iodine, but there was no sig- nificant difference due to dietary iodine intake in TSR estimated by the T4 substitution method in normal and tapazole-treated birds. The results of the direct output and substitution methods in chickens are in keeping with those in rats reported by Reineke (1965). Effects of Thyroxine on Growth, Feather and Comb Development Effects of thyroxine and tapazole on growth of chickens is shown in Figure 9. Groups 2, 3, 4, and 5 re— ceiving respectively 0.5, 2.0, 3.0 and 4.0’ug of T4 per 100 g b.w./day gained more weight than the normal control group. Growth gains in T4—treated groups were in the order of the doses given. The growth of birds in Group 6, treated with tapazole only (hypothyroid control, Tx) was most retarded. Groups 7, 8, 9, and 10 receiving both tapazole and replace- ment therapy at the rates of 1.0, 2.0, 3.0, and 4.0,ug T4/100 g b.w./day, respectively, were heavier than the normal and the hypothyroid controls. Groups 8 and 9 receiving 2.0 and 3.0’pg T4, however, gained more weight than group 10 re- ceiving 4.0‘pg T4. In another series of experiments, not included in Figure 9, the tapazole-fed birds given daily injections of 3.0 and 5.0‘pg of T4/100 g b.w. showed relatively less growth than those receiving 2.0 pg T4/100 g b.w. Moreover, PERCENT GAIN PERCENT GAIN 540 SIG 480 450 420 390 360 330 300 270 240 2l0 I80 I50 I20 90 so 30 540' SIG 480 450 420 390 380 330 300 270 240 2IO I80 I50 I20 90 60 30 0 DAYS 0 Figure 9. — GROUP I .06... sump 2 --GROUP 3 --CR0IIP 4 ---GROUP 5 --—GROUP 6 ....... enoup 7 -—-OROUP a ---- GROUP 9 ----GROUP I0 3 6 IO 71 2 I CONTROL .5, T4 27 T4 31 74 ‘7 T4 HYPOTHYROIO CONTROL (Tn) T,+ l7 T4 ,- T,* 2; T4 .I' T,.+ 37 T4 ,-’ TI: " 41 T4 ’1. 20 25 32 Effects of thyroxine and tapazole on growth of chickens. 72 in experiments on determination of M.R. growth of the birds receiving a daily dose of 6.0‘ng T4 or T3 per 100 g b.w. for 26 days was depressed as compared to that of controls. These observations indicate that thyroxine in small doses improves growth of chickens, but when administered be— yond physiological doses, depresses growth rate. T4 in toxic doses accelerates catabolic processes, and then the body weight is reduced. The observations described above also indicate that higher doses of T administered to the 4 tapazole-treated birds retarded growth rate more than when given to the normal birds. Although no explanation is readi- ly available for such action of T4, some unknown interaction may be possible between higher doses of the hormone and the goitrogen on a line similar to the one demonstrated by Seller and Schonbaum (1965) involving a goitrOgen potenti- ating effect of small doses of T and propylthiouracil. 4 The effect of thyroxine and tapazole on growth of wing and tail feathers and comb growth is shown in Figure 10. The hypothyroid group showed retarded growth of feather and comb. The feathers of the birds from this group looked fringed and lacked barbules. In all other groups to whom T4 was given, either additionally or as a replacement therapy, the comb and feather measurements were normal or nearly normal. The size and shape of feathers is influenced by the gonadal and thyroidal hormones (Sturkie, 1965). Snedecor 73 .fisonm 0.800 one .8450.“ so oaoudmdp .Hohocm ofiUnoEo. 00 006.09% 0200 0002.50... 4.5. D 0mm:...<0 N0 ...< 00 In 0><0 N0 Ib2~20030<~2 0800 0><0 N0 I 20.0.20 00 0 0><0 ON I 20.0.20 02 N 0><0 N. I 20.0.).0 F0 . 0.. 000.0 0w... \\N /h \ . IIIIIIII 1.1! \i\. I\ / /. HHH HH J J, own 3- o a I 0 9 U “4- O m 3 u 5 8 O T: w om ma 3 O m .d. D. I 8 om p 1 O D O U 1- I 2. m. 77 hours. The maximum effect was produced by T4, two hours after its administration. Only at this time, the rise in M.R. by T4 approached a significance level of 0.05. At no other time did any treatment given produce a significant rise in M.R. The M.R. was depressed 24 hours after injection. The depressing effects of T3 and T3 + T4 at this time were statistically significant. No prior information is available regarding the ef- fects of administration of T T + T or T on M.R. 3’ 3 4 4 McCartney and Shaffner, (1950) and Mellen (1958), however, measured M.R. in thyroprotein-fed birds. The stimulating ef- fect lasted for as long as supplementation was maintained or for a short time during the first few hours after fasting. Mellen (1958) also reported that M.R. was lower than in con- trols after 12—14 hours of fasting. The effect of T3 or T3 in combination with T4 is less than of T4 alone. This is expected in view of the faster metabolism of T3 than T4. A short time action of the thyroid hormones on M.R. in chickens may be related to short biological half lives of these hormones as noted in the turn— over experiments. The lower PBI levels in the chicken may be another factor worthy of consideration. Furthermore, differences of thyroxine-binding protein between birds and mammals may also be responsible for variation in thyroxine effect on M.R. The specific thyroxine-binding alpha—globulins 78 are not present in birds. There is no increase of M.R. in Rainbow trout following thyroxine administration (Fromm and Reineke, 1956). Comparison of the TSR Methods Only the goiter prevention and the thyroxine substi- tution method have been previously employed for estimation of TSR in chickens. In the present study, the direct output and the thyroxine degradation methods were also used, and four methods were compared. The values of TSR obtained in different experiments within each method were sufficiently close to indicate good repeatability. The average esti- mated TSR by the goiter prevention, the T4 substitution, the direct output and the T4 degradation methods, respectively, measure as 2.28, 2.00, 1.10 and 2.03 )19 of T4/ 100 g/day. Thyroid secretion rates measured by the substitution of T4 on tapazole-treated birds and T3 on both normal and tapazole— treated birds and the results of T degradation are excluded 3 from this comparison for the reasons discussed separately under each method. Mellen and Wentworth (1960) compared goiter preven- tion and radio-iodine assay for determination of TSR in chickens. Mean values obtained by the former method were 65 percent of those derived from the latter method. They reasoned that the lower values from the goiter prevention assay resulted because the method was applied at higher 79 temperature and that because T4 is administered on a per bird basis, but the final estimation of TSR is made by con- verting data on body weight basis. Tanabe §£_al. (1965) obtained equivalent values of TSR by employing goiter pre- vention and radioiodine assay methods. The radioiodine assay method adapted by Tanabe §£_al. is somewhat different and the values obtained by them are relatively lower than those generally reported by other workers. Further, a care- ful examination of their reports reveals that their experi- mental birds were fed on rations containing approximately 0.5 ’pg iodine per gram feed, which is rather low in View of the minimum iodine requirement of about 1.0,ng/gm in growing chickens (Wilgus, Gassner, Patton and Harshfield, 1953). The use of the goiter prevention assay is based on the assumption that the estimated TSR is the dose of T4 re- quired to suppress the output of TSH in goitrogen-treated animals to that of the control. The variability of the goiter prevention assay was questioned from time to time (Escobar del Ray §t_al., 1962; Jagiello and McKenzie, 1960; Van Middlesworth §t_§l., 1959). A serious objection has been in the use of goitrogen like thiouracil which decreases deiodination of thyroxine. This extrathyroidal action of the thiouracil is thought to give faulty TSR values. In View of this, methimazole, which has the least extrathyroidal effects, was used as the goitrogen in the present study. 80 Some other workers have, however, used both thiouracil and methimazole in the goiter prevention method and got identi- cal TSR values in rats and chickens (Wiberg §E_gl., 1964; Tanabe et al., 1965). In case of the substitution method, an assumption is made that the TSR is the quantity of exogenous T4 necessary to suppress thyroidal iodide release, or in other words, the T4 dose which completely blocks TSH, is the TSR in this method. The values obtained by the substitution method in goitrogen-treated animals are undoubtedly higher as compared to any other method. Both the T4 substitution and the goiter prevention assay, in which thyroxine is injected once daily, can be questioned on the ground that daily administration of thyroxine neglects important consideration of its short half life. In rats the plasma thyroxine concentration de- clines by about 75 percent in 24 hours after injections (Gregerman, 1963). For chickens, the position will be still worse be- cause the tk of thyroxine is much shorter than in rats. It may also be noted in the present study (Table 8) that the use of the T4 substitution method in both normal and goitrogen-treated birds on diet deficient in iodine gave similar TSR values as in the birds on diet adequate in io— dine. However, significantly lower values were obtained in chickens with iodine deficiency by use of the direct output 81 and the T4 degradation methods. Similar results were ob— served in rats in comparison between the direct output and the T4 substitution methods (Reineke, 1964). It, therefore, seems that the direct output and the T4 degradation methods should give more accurate estimations of TSR. Presently, however, these methods do not yield absolute values. In the calculations for the direct output method, an assumption was made that the entire daily iodine output from the thyroid of the chicken is in the form of T A 4. factor of 1.529 for T4 equivalent of iodine was used to con— vert the rate of iodine released daily into TSR. In fact, iodine may be released from the thyroid as T T and iodide 4’ 3 with possibly some M.I.T. and D.I.T. The exact proportion of T3 in the chick thyroid is not too well known. However, the results from the present study and the data from the other workers show that T4 and T3 in chickens. The released iodotyrosines may be negligible. are almost equally potent It is also believed by many workers that not more than 10 percent of iodine is released as iodide. In view of this, no other unknown factor, including T3 was taken into account and the factor 1.529 was used. Presently, TSR values ob- tained by the direct output method are about half as large as observed in the other methods. Nevertheless, any error in this method that would arise if a portion of the iodine 82 released were in non—hormonal form (Iodide, M.I.T., D.I.T.) would result in overestimation rather than underestimation of the TSR. As pointed out earlier, there is too little and T in chickens to 3 4 account for a significant error in the calculations. Also, difference between the potencies of T consideration of the known factors in this method indicate rather strongly that as presently applied, the direct output method most nearly represents the true TSR of the chicken. The T4 degradation method is delicate and there are, several factors involved which influence the TSR values. I. The thyroxine distribution space may generally be overestimated as follows: (1) 1131 released from the deiodination of the labelled hormone is partly taken up by the thy- roid and is partly excreted. (2) Impurities in the commercially available 1131-T4 and the possibility of the non-thyroxine com- ponent being more rapidly eliminated may affect the TDS. (3) Increased radioactivity in the plasma samples may result due to efflux of unchanged hormone stored in the liver. Numbers 2 and 3 were noted in the present study. Also, the thyroidal uptake of released I131 was checked for comparison by the administration of tapazole in a group of chickens. In this experiment, the estimated TSR approached 83 the values obtained by direct output (l.59,ug vs 1.10 by the direct output method). II. The ratio of T4 : T3 as circulating thyroid hormones, the intensity of the plasma protein binding of the two hormones and the exact contribution of T3 in the PBI are yet not fully understood in chickens. The extent to which these unknown factors have in- fluenced the estimated TSR in the present study is difficult to determine. It is, however, thought that when all the known and unknown factors have been properly accounted for, the values obtained by the T4 degradation method should be close to those in the direct output method. SUMMARY AND CONCLUSIONS The thyroid secretion rate (TSR) in chickens of different ages has been determined by four methods: (1) goiter prevention, (2) thyroid hormone substitution, (3) direct output and (4) thyroxine (T4) degradation. The results of these methods were compared. The values of TSR obtained in different experiments within each method were sufficiently close to indicate good repeatability. The representative estimated TSR by l, 2, 3 and 4 methods re- spectively measure 2.28, 2.00, 1.10 and 2.03‘ng/100 g/day. TSR of 7-week goitrogen-treated chicks, 56-week normal chicks, bobwhite quail and coturnix as estimated by the T4 degradation method,respectively averaged 1.59, 1.02, 2.49, and 2.78dpg/100 g/day. TSR estimated by the thyroxine (T4) and triiodothyronine (T3) substitution methods on goitrogen— treated chickens was much higher. There was no significant difference in TSR measured by the direct output method in growing chicks in the range of l to 9 weeks of age. An increased plasma radioactivity was noticed in the lZ-hour samples taken in the degradation experiments. It is presumed that such excessive radioactivity results due to discharge of unchanged hormone from the liver under a stress at that stage. 84 85 Effects of dietary iodine content on various para- meters of TSR as determined by different methods and upon body and thyroid weights and thyroid iodine were determined. With diet adequate in iodine, TSR by the direct output method and the degradation method, thyroxine distribution space (TDS), extrathyroidal thyroxine (ETT), thyroidal iodine con- tent and growth rate of chicks were significantly higher, but K4, U uptake and thyroid weight were significantly lower. There was no difference of t%, PBI and the TSR by the substi— tution method between the chickens on the diets containing adequate and inadequate iodine levels. T3 and T4 were found to be almost equally potent by the method of substituting hormones to maximally block 1131 release. The representative biological half lives of T4 in blood of chickens, bobwhite quail and coturnix were 3.23, 4.60 and 5.55 hours, respectively. The half life of T3 in each kind of bird was not significantly greater. The representative thyroxine distribution space in ml/100 g b.w. of chickens, bobwhite quail and coturnix, re— spectively, measured 29.39, 28.08 and 55.29. TDS/unit body weight of 56-week old chickens is significantly lower than in 7-week chickens. T3 distribution spaces in all birds are higher than of T4. The representative protein—bound iodine of chickens, bobwhite quail and coturnix were measured as 1.12, 1.76 and 86 1.26 Jag percent, respectively. PBI of bobwhite quail is higher than of chickens and coturnix. Tapazole (a goitrogen) retarded growth rate, feather and comb growth of chickens. Thyroxine in the doses of 2-3 )ug/lOO g/day counteracted completely the growth inhibiting effects of tapazole. Thyroxine in small doses (0.5 to 4.0 ,ng/lOO g/day) improved growth of normal chickens. But, 6’ng of the hormone depressed growth of normal and 4,ug depressed growth of goitrogen-treated birds. T T3 or a combination of T4 + T3 produced a small 4, and transitory rise in M.R. of chickens. Only at 2 hours after T4 administration did the rise in M.R. approach the 0.05 significance level. The depressing effect of T3 and T3 + T4 at 24 hours after their injection was found to be significant. The following indices of thyroid function were found to be significantly related: Log K.4 Thyroidal iodine and age. r = .8869, P < .005. and log age. r = -.8351, P < .005. Log K4 and log thyroidal iodine. r = -08285, P< 0005. Log K' and U uptake. r = —.67l9, P < .005. 4 Log thyroidal iodine and log body weight in growing chicks. r = .9784, P < .005. Thyroidal iodine and thyroid weight in adult chickens and quail. r = .9416, P < .005. T distribution space and body weight of chickens (Spearman rank correlation coefficient) rs = .9023, P < .02. 87 The data in the present studies apart from other observations support the conclusion that the thyroid function in chickens differs in general from that of mammals. Chickens have lower PBI values, shorter half-lives of T3 and T4 in the plasma, almost equal physiological potency of T3 and T4 and show an insignificant rise of M.R. following ad- ministration of the thyroid hormones. The merits and demerits of the four TSR methods used in this project have been discussed. It is thought that the direct output and the degradation methods should yield values closer to TSR, provided that all their known and unknown factors are properly accounted for in the calculations. How— ever, consideration of the known factors involved in these methods indicates rather strongly that as presently applied, the direct output method most nearly represents the true TSR of the chicken. BIBLIOGRAPHY Albert, A.3 and F. R. Keating 1949 Metabolic studies with I1 l labelled thyroid compounds. J. Clin. Endocrinol. 9:1406. Andrews, F. N., and B. B. Bohren 1947 Influence of thiouracil and stilbestrol on growth, fattening and feed efficiency in broilers. Poultry Sci. 26:382. 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MacLaury 1961 The effect of tapazole on growth of hybrid cockerels. Poultry Sci. 40:890. Winchester, C. F. 1939 Influence of thyroid on egg pro- duction. Endocrinol. 24:697. 96 Winchester, C. F., and G. K. Davis 1952 Influence of thyroxine on growth of chicks. Poultry Sci. 31: 31. APPENDIX A DATA ON TSR OF INDIVIDUAL CHICKS BY DIRECT OUT-PUT METHOD U Total _ _ Percent K'4 K4 Thyroidal TSR/ Ch1ck We1ght Max1mum Iodine 100 gm b.w. No. gms. Uptake ‘Pg /day 1—week—old 401 88 .014 .1627 .1650 1.84 .5268 402 90 .034 .1555 .1610 2.71 .7387 403 90 .024 .2264 .2320 2.48 .9761 404 85 .041 .2379 .2480 3.65 1.6274 405 100 .5214 3.10 2.4705 406 97 .2469 5.46 2.1260 407 103 .084 .2125 .2319 1.87 0.6446 408 78 .036 .3604 .3738 1.26 0.9246 409 87 .021 .1975 .2017 3.19 1.1590 410 95 .046 .3347 .4272 1.18 0.8112 3—week—old 189 184 .106 .0819 .09166 13.63 1.040 190 240 .110 .0861 .09670 10.28 0.630 191 230 .150 .0703 .08271 12.82 0.700 192 242 .069 .1521 .16338 17.43 1.800 195 190 .028 .2063 .21221 3.41 0.580 196 218 .055 .1344 .14220 6.42 0.640 197 140 .072 .0930 .10020 6.21 0.670 198 210 .062 .2601 .27734 4.57 0.920 199 170 .076 .0810 .08769 8.17 0.640 200 182 .074 .1026 .11075 6.61 0.610 11 .055 .1307 .1383 15.68 12 .056 .1131 .1198 11.55 13 .049 .1131 .1189 7.39 14 .062 .0957 .1020 24.81 97 98 U Total ' . Percent K'4 K4 Thyroidal TSR/ Ch1ck We1ght Max1mum Iodine 100 gm b.w. No. gms. Uptake Pg /day 15 .093 .1220 .1345 11.72 16 .062 .1393 .1485 13.37 17 .048 .1307 .1372 14.96 18 .052 .1220 .1287 16.32 19 .088 .1131 .1240 15.66 20 .063 .1220 .1302 8.36 4w5-weekeold 184 404 .082 .0810 .0883 31.25 1.043 122 462 .093 .0718 .0792 44.54 1.166 199 450 .051 .1270 .1338 51.90 2.359 187 452 .024 .1344 .1376 26.55 1 235 185 395 .037 .1707 .1773 21.67 1.487 193 360 .069 .0955 .1026 19.35 0.843 182 420 .098 .0683 .0756 36.40 1.002 110 450 .082 .0810 .0883 52.57 1.576 181 430 .054 .1204 .1273 22.20 1.005 108 363 .070 .0951 .10223 4-5-week—old (Diet deficient in iodine) 27 568 .085 .2420 .2646 6.81 .4853 28 515 .090 .2473 .2717 7.16 .5774 29 550 .096 .1086 .1201 10.84 .3619 30 414 .090 .1324 .1454 4.63 .2481 31 396 .062 .0979 .1043 9.37 .3772 32 371 .110 .1918 .2156 6.16 .5473 33 523 .082 .1263 .1375 10.12 .4069 34 294 .072 .1537 .1656 6.16 .5302 35 427 .098 .2181 .2417 9.38 .8117 36 455 .140 .1157 .1346 6.87 .3108 99 U Total . . Percent K24 K4 Thyroidal TSR/ Ch1ck We1ght Max1mum Iodine 100 gm b.w. No. gms. Uptake yg /day 9-weekuold 101 1475 .210 .0310 .03921 270.00 1.09 102 1475 .110 .0325 .0365 291.25 1.10 103 1475 .160 .0703 .0837 131.25 1.13 104 1425 .091 .0297 .0326 108.12 0.38 105 1440 .125 .0273 .0312 216.87 0.72 106 1720 .110 .0208 .0233 228.12 0.47 107 1350 .097 .0703 .0778 144.68 1.27 108 1350 .190 .0267 .0330 231.87 0.86 109 1450 .280 .0741 .1029 248.75 2.69 110 1400 .145 .0284 .0332 294.68 1.06 APPENDIX B THYROXINE TURNOVER IN INDIVIDUAL BIRDS r i j ._—1 ETT .P9;* Thyroxine TDS (TDS/100 gm degradation Bird Weight t % ml/lOO gm PBI b.w. x PBI,Dg/100 gm No. gms Hours b.w. ,ng % ‘pg/ml.) b.w./day Chickens: 5-6-week-old (Diet deficient in iodine) 24 580 4.1 8.61 1.0538 .09073 .5178 25 430 3.0 8.98 .7762 .06970 .5273 33 485 5.1 10.89 1.6315 .17767 .8271 40 360 3.0 12.39 1.3003 .16111 1.2190 42 260 4.1 15.70 .9475 .14876 .8488 37 355 3.0 5.75 1.0836 .06231 .4715 30 375 4.0 12.63 1.0584 .13367 .7798 38 395 4.6 11.34 1.0886 .12345 .6334 Chickens: 6—7—week_old 6 605 3.0 34.52 .6854 .23660 1.79 20 530 .5 34.67 .9937 .34451 2.27 45 705 3.0 23.28 .8717 .20293 1.54 46 660 3.0 24.86 1.1245 .27955 2.11 48 675 4.5 45.33 1.0800 .48956 2.56 53 732 3.5 36.92 .8820 .32563 2.15 58 710 1.0314 5 59 765 4.25 50.05 .9759 .48843 2.69 60 660 3.0 25.78 .6860 .17685 1.34 95 840 3.25 29.56 .8034 .23748 1.80 339 744 3.3 26.84 1.1440 .30704 2.13 330 564 3.7 28.58 .8360 .23893 1.49 338 572 3.0 36.51 1.0120 .36949 2.79 344 692 3.4 28.86 .9900 .28576 1.93 337 568 2.9 32.35 .8250 .26695 2.08 328 732 3.1 23.24 1.0450 .24287 1.78 100 101 ETT 2993!" Thyroxine TDS (TDS/100 gm degradation Bird Weight t % m1/100 gm PBI b.w. x PBI’pg/loo gm No. gms Hours b.w. .ng % ,ng/ml.) b.w./day Chickens: 7—week—o1d, goitrogen treated 332 560 2.4 17.64 1.0890 .19215 1.76 333 590 3.3 25.95 1.0450 .27121 1.88 346 732 2.5 19.61 1.0560 .20709 1.84 345 610 3.1 _l8.83 .8580 .16156 1.15 336 752 2.8 20.36 .8690 .17697 1.42 348 552 3.0 20.81 .9680 .20140 1.52 Chickens: 56-week—old 7095 2120 3.6 20.64 .8140 .16801 1.07 7096 1935 4.7 10.79 .8690 .09376 .47 7097 2237 4.2 9.78 1.2650 .12369 .69 7093 2285 3.7 10.58 1.110 .11751 .73 7080 2110 3.7 27.56 1.2430 .34257 2.14 7092 2267 3.3 7.79 1.8920 .14744 1.03 Bob white quail: — 68-week-old 1535 166.6 3.8 17.79 6053 177.3 3.8 19.19 1.2210 .23429 1.41 3771 183.7 4.7 40.66 2.5810 .04950 5.31 295 262 6.0 21.92 1.3690 .30005 1.21 281 193 4.4 25.06 1.9140 .47966 2.58 3600 188 5.6 37.07 290 173 4.7 21.25 2.8610 .60798 3.28 6612 211.5 3.8 28.03 296 211 4.4 31.10 1.2210 .37971 2.04 1219 180.4 3.5 30.87 1.440 .44451 2.95 6477 178.5 5.3 25.71 .9680 .24890 1.12 12174 176 3.5 18.65 ' 12165 177 5.41 7.31 102 — h *- J ETT ’ng* Thyroxine TDS (TDS/100 gm degradation Bird Weight t.% m1/100 gm PBI b.w. x PBI‘pg/loo gm No. gms Hours b.w. ypg % ‘pg/ml.) b.w./day 12033 190 3.75 12.72 12018 162 5.0 29.85 10581 203 5.33 18.10 Japanese quail: 10—week—old 293 114 4.5 67.17 2.3410 297 97.5 3.9 66.37 .8000 .53096 3.17 284 97.7 3.8 72.35 .9460 .68445 4.18 291 118.8 4.0 57.29 1.2000 .68748 4.01 299 109.5 8.0 52.45 1.1650 .61100 1.86 280 107.5 4.1 55.14 .9860 .53375 3.04 287 109.2 4.4 43.15 1.0560 .45572 2.43 276 115.5 8.0 49.73 1.4410 .71656 2.18 288 95.5 8.2 45.82 1.0120 .46371 1.38 300 92.7 1.0000 *Multiply with T4 iodine equivalent (1.529) to con- vert into‘pg T4. APPENDIX C TRIIODOTHYRONINE TURNOVER IN INDIVIDUAL SUBJECTS ETT ~ TDS ng Bird Weight t lg m1/100 gm (TDS/100 gm b.w. PBI No. gms Hours b.w. x PBI pg/ml) pg % Chickens: 7-week-old 331 554 4.0 60.16 .71470 1.1880 421 660 3.7 68.87 .06059 1.5400 425 612 3.7 65.36 .84118 1.2870 347 682 4.0 50.56 .80643 1.5950 342 582 4.0 58.04 .72782 1.2540 329 680 3.2 59.78 .72333 1.2100 Chickens: 7—week~o1d goitrogen treated 326 752 3.8 53.19 .70795 1.3310 343 664 3.6 65.47 .87140 1.3310 327 824 4.2 69.74 .31808 1.8900 335 912 4.3 57.71 .66655 1.1550 341 526 3.8 52.22 .55718 1.0670 340 406 3.5 84.93 .07436 1.2650 Chickens: 56—week—old 7090 2315 10.4 79.99 .07346 1.3420 7100 2415 9.8 53.08 .56636 1.0670 7085 2057 14.4 67.52 .95811 1.4190 7098 2117 .3 51.34 .72286 1.4080 7088 1775 6.4 65.51 4.0150 7091 2295 6.8 50.66 .85818 1.6940 Bobwhite quail: 56 - 68-week-old 3857 185.3 5.5 59.96 .79807 1.3310 4753 175.6 10.0 79.08 .88569 1.1200 3020 194.0 3.8 55.42 .48160 .8690 103 104 ETT TDS ng* Bird Weight t % ml/lOO gm (TDS/100 gm b.w. PBI No. gms Hours b.w. x FBI.pg/ml) 4pg % 4950 169.5 11.4 59.59 .83247 1.3970 12088 164.0 9.5 64.18 .7148 4.2300 5135 189.6 6.7 67.61 .77346 1.1440 13555 186.8 8.8 68.63 1.72878 2.5190 3409 200.0 9.3 64.93 1.08562 1.6720 3139 182.1 3.5 61.01 .87244 1.4300 3683 187.6 6.8 74.03 .7340 2.3430 Japanese quail: 10—week—old 283 94.0 4.1 99.41 .36198 2.3760 286 94.6 4.5 62.17 .77961 1.2540 294 107.2 4.2 79.04 .92161 1.1660 279 89.1 4.1 102.02 .52622 1.4960 277 94.1 4.2 96.60 .03072 1.0670 278 100.6 4.4 124.25 .61276 1.2980 450 85.2 4.5 73.35 .95208 1.2980 285 113.5 4.4 102.45 .19456 1.1660 139 82.4 4.3 102.84 .76576 1.7176 N.B. 92.0 1.2980 *Multiply with T3 iodine into Jug T3 . equivalent (1.71) to convert APPENDIX D ADDITIONAL DATA ON THYROID IODINE AND PBI OF CHICKENS AND QUAIL Bird No. Weight gms. 7—week-old chickens (chick starter diet) 21 22 23 24 25 26 27 28 29 30 695 730 750 640 765 775 720 685 660 795 Total Thyroid Thyroidal Weight Iodine PBI mg - )19 )lg % 34.0 74.81 47.0 59.38 37.0 49.81 72.0 163.69 62.0 105.31 47.0 80.88 61.5 143.19 65.0 103.13 39.5 35.38 52.5 89.44 7—week—old chickens (Diet SR~2 added I, 1.3‘ug/gm feed) 1 \omqmmbww [-4 O 718 760 620 770 780 770 695 575 700 642 30.0 1.0670 52.0 33.69 1.2760 36.0 33.00 1.0010 37.5 59.12 1.3420 57.0 92.12 1.2870 54.0 49.02 1.3420 57.0 113.44 1.1550 47.0 81.40 1.4300 40.5 42.35 1.2100 46.0 31.56 1.2100 7—week—old chickens (Diet SR—2 with no I added) 11 12 665 600 48.5 3.91 .9900 50.0 2.61 .9350 105 106 Total Body Thyroid Thyroidal Bird Weight Weight Iodine PBI No . gms . mg jig pg % 13 520 55.0 0.73 .9130 14 650 114.0 1.03 .9900 15 582 64.0 1.45 .6930 16 680 72.0 5.69 .9570 17 645 95.0 5.98 .1220 18 723 47.0 1.76 .8690 19 623 75.0 3.13 .9130 20 685 100.0 4.37 .9240 11 week-old-chickens (Adequate iodine diet) 284. 387. 158. 338. 383. 344. 283. 181. 191. 168. l omqmmbwm H O 13 weekeoldechickens (Adequate iodine diet) 111. 99. 156. 131. 130. 93. 191. 180. 1 mummbwm 1415 1400 1100 1827 1352 1434 1203 1270 1365 1404 1450 2035 1372 1668 1332 1635 1851 1531 97. 117. 81. 194. 115. 150. 78. 76. 100. 73. 0 OOOOOOU1U10 5 NOOOUTOLH 342. 544. 484. 455. .25 387 281. 427. .52 492 46 75 73 25 46 52 80 50 73 96 37 50 60 07 32 02 107 l4-month-old chickens (Adequate iodine diet) 165. 179. 122. 183. 83. 179. 166. 105. 167. 127. 7093 7096 7095 7097 7088 7091 7090 7098 7100 7080 2285 1935 2120 2237 1775 2295 2315 2117 2415 2110 0 U'IOOOOOOOO 14-17—month—old Bobwhite 3857 4753 3020 4950 12088 13555 5135 3409 3139 3681 281 1219 296 295 3771 185. 175. 194. 169. 164. 186. 189. 200. 182. 187. 193. 180. 211. 262. 183. 3 \noopomwommomom 9. 12. 9. 12. 9. 8. 44. 26. 14. 29. 13. 12. 11. 13. 9. WOU‘OUIOUIUIUTWOOOOUI 613 575 479. 511. 244. 560. 490. 362 406. 535. quail (A 17 19 19. 33 21. 332 118. 21. 115 51. 27 41 46 18. Total Body Thyroid Thyroidal Bird Weight Weight Iodine No. gms mg. ’pg 1554 92.0 220.27 10 1556 102.3 375.87 .80 .47 16 17 20 34 77 .12 34 7O dequate iodine diet) .55 .95 12 .75 07 .40 .65 65 02 .13 35 .97 .40 .87 82 108 Total Body Thyroid Thyroidal Bird Weight Weight Iodine No. gms. mg. P9 6612 211.5 49.0 237.87 290 173.0 16.0 61.17 6477 198.5 18.0 97.50 3600 188.0 18.0 88.87 1535 166.6 14.5 62.50 6053 177.3 12.5 57.10 10-week—old Japanese quail (Adequate iodine diet) 283 94.0 7.0 23.20 286 94.6 .0 21.87 294 107.2 8.0 26.25 279 89.1 5.5 16.36 277 94.1 4.5 7.07 278 100.6 5.0 7.49 450 :85.2 4.5 13.69 285 113.5 5.0 16.25 139 82.4 8.5 33.75 N.B. 93.0 11.5 58.20 276 115.5 5.5 18.24 284 97.7 6.5 15.49 287 109.2 4.0 28.62 300 92.7 6.5 13.01 280 107.5 7.5 37.42 291 118.8 3.0 6.80 293 114.0 6.0 16.95 297 97.5 4.0 1.87 288 95.5 4.0 2.67 299 109.5 3.5 3.35 APPENDIX E THYROID IODINE ANALYSIS Method of Barker and Humphrey (1950) for analysis of protein-bound iodine has been modified for determining total thyroidal iodine (Reineke, unpublished). Iodine Determination Place 1 cc of 4 N Na CO into a pyrex test tube con- taining the thyroid. Dry overnight at 90-95°C. Dried residue is then incinerated in muffle furnace for 2% hours at 600-6250C. Dissolving iodine from ash Add 2 cc 2N HC1 2 cc 7 N H 80 and *21 cc glass distilled water. Mix and stir until no more effervescence appears. Centrifuge for 20 minutes. Colorimetry Treat each sample and the reagent blank in duplicate. Take *1 cc of the digest into colorimeter tube. Add *4 cc glass distilled water and 0.5 cc arsenious acid reagent (Hycel). Place in water bath at 27°C. Allow tube a few minutes to reach that temperature. Add 0.5 cc dilute** ceric ammonium sulphate reagent (Hycel) at 30—second intervals to each tube. Let stand in bath exactly 15 minutes after addition of ceric ammonium sulphate. ’Then add 0.5 cc of 1% brucine solution at 30—second intervals. Read samples in Coleman spectrOphotometer at a wave length of 480 milli—microns. Subtract regent blank value and read final value in‘pg of iodine from the Standard curve*** prepared earlier at the same temperature and duration of reaction. *May vary according to the expected iodine content in the aliquot. **2 CC Hycel reagent diluted to 5 CC with glass dis- tilled water. ***Curve for high iodine value at 27°C for 15 minutes ranges from 0 to 0:2,ug of iodine. 109 APPENDIX F PBI ANALYSIS The method is similar to the thyroid iodine analysis. Plasma Protein Precipitation 1 cc plasma + 7 cc glass distilled water + 1 cc of 10% ZnSO4-+1 cc 0.5 N NaOH. Add NaOH slowly with constant stirring to aid in the formation of the precipitate. Wait for 5 minutes. Centrifuge, dis- card supernatent liquid and wash protein thrice with 10 cc glass distilled water. Centrifuge and discard supernatent each time. In order to eliminate any free iodine that may affect PBI analysis, dual precipitation was done as follows: 1 cc plasma + 10 cc glass distilled water + 4 cc 11.2% trichloroacetic acid. Wash precipitate once with 3% TCA, dissolve in 7 cc glass distilled water. Add 1 cc ZnSO4 solution and 1 cc NaOH to reprecipi- tate proteins. Centrifuge and wash the precipitate. Drying and Ashing Dissolve precipitate in 1 cc 4 N N82CO3- Dry over— night at 90°C. Incinerate at 600-625°C for 2% hours. Dissolving iodide from ash Add 2 cc 2 N HC1 2 cc 7 N H2804 and *7 cc glass distilled water. Stir and centrifuge. Colorimetry Take *5 cc aliquot in duplicate. Add 0.5 cc arsenious acid reagent (Hycel). Place in water bath at 50°C. Allow tubes a few minutes to reach that temperature. Add 0.5 cc dilute** ceric ammonium sulphate (Hycel) at 30-second intervals to each tube. Let stand in bath exactly 20 minutes after addition of ceric ammonium sulphate. Stop reaction with 0.5 cc 1% brucine solution. Read in colorimeter. Sub- tract blank value and read final value in,ng of iodine from the standard curve*** prepared at the same temperature and duration of reaction. *May vary according to the expected iodine content in the aliquot. **2 cc Hycel reagent diluted to 5 cc with glass dis— tilled water. ***Curve for low iodine value at 50°C for 20 minutes ranges from 0 to 0.05 ’pg‘iodine. 110 APPENDIX G MICHIGAN STATE UNIVERSITY 63-S Chick Starter KRUMBLES Fine Ground Yellow Corn Ground Oats (38-40#/bushe1) Wheat Middlings 17% Dehydrated Alfalfa Meal 50% Meat/Bone Scraps 45% Protein Soybean Meal 55% Fish Meal Vitaproil Dried Whey Ground Limestone Dicalcium Phosphate (24% Calcium 18.5% Phosphate) Salt, Iodized Vitamin Trace Mineral Premix NFZ added Manufactured by KING MILLING COMPANY LOWELL, MICHIGAN 111 1060 100 100 80 50 500 40 40 10 10 5-10 2001 + RAT FEED MIXTURE APPENDIX H SR—l Add 0.5 lbs.of special mineral salt premix SR—l per each 100 lbs. of basic feed mix and mix thoroughly. A. Basic Feed Mix: Amount/100 lbs. 112 Ingredient of Mix Shelled Yellow Corn ground through 1/8" screen 68.9 lbs. Soya-bean oil meal (50% protein) 28.0 lbs. Dicalcium phosphate 1.8 lbs. Lime stone 0.6 lbs. Dawes & Forbes Vitamin B supplement 0.1 lbs. Dawes & Forbes Vitamin B12 supplement 0.2 lbs. Standard Brands 9F yeast 9000 I.V. (Vit. D2/g) 5.0 gm Pfizers Vitamin A supplement (10,000 I.V. Vit. A/g) 15.0 gm Mineral Salt Premix SR-l: Element % Element Compound gm/100 lbs. *Iodine 0.010 KI-10% Ca Stearate 0.653 (Pfizer) Zinc 0.800 ZnSO4.7H20 (Baker) 160.574 Manganese 0.542 MnS04.H20 (Baker) 73.12 Iron. 0.270 FeS04.7H'20 (Baker) 60.782 Copper 0-054 CuSQZ. (anhydrous 6.169 Baker) Sodium Chloride (plain) (Morton's) 4234.72 *This feed mixture minus iodine is termed SR-2. APPENDIX I DIET FED TO 56-WEEK OLD CHICKENS Corn - free choice plus Ground Yellow Corn 1320 lbs Soybean Meal, dehulled, 50% protein 310 Alfalfa meal, dehyd. 17% protein 60 Meat & Bone Scraps 50% protein 50 Fish Meal 55% protein 60 Dried Whey 40 GrOund Limestone 100 Dicalcium phosphate 20 Salt, iodized 6 Vitamin trace mineral premix 10 (contains iodine) Choline chloride 25% 2 Zinc oxide (80% zinc) 0.25 Animal Fat 25 2003.25 lbs 113 APPENDIX J DIET FED TO QUAIL Protein 25% Ground Yellow Corn Soymeal dehulled 50% prot. 17% Alfalfa Meal Dried Whey Meat/Bone Scraps Fishmeal (Mehhaden),60% Ground Limestone(CaC03) Dicalcium phosphate Salt Iodized Vit. Premix l Nopcosol”M44‘(containsTiodine) Fat 114 412. 370. 50. 25. 25. 25. 50. 15. 20. OOOOOOOOU‘I OU'I 1000 lbs. APPENDIX K EFFECT OF A SINGLE DOSE OF (4.0’pg 100 gm b.w.) THYROID HORMONES 0N M.R. IN CHICKENS. 0 Consumption 2 ml/hour/kg. b.w. Hours after Injection Chick Treatment Number 1 2 7 326 1086 1237 1474 327 1182 1552 861* 328 1271 1468 1446 329 1313 1495 C°ntr°l 330 1547 1516 1595 331 1795 1483 1380 332 1553 2180 1540 333 1163 2270 1619 334 1203 2383 1627 335 1150 1659 1138 336 1505 1944 1402 T 337 1370 1917 1325 4 338 1584 2220 1538 339 1220 2160 1512 340 2042 1770 341 1442 2068 1399 342 1611 1697 1679 343 1754 2055 1649 344 1386 2174 1376 T 345 1209 1503 3 346 1487 1854 ’ ‘ 347 1479 1653 1623 348 1573 2010 898* 349 1970 1878 1921 *Eliminated from statistical analysis by Chauvent, criterion. p. 47. Documenta Geigy. Scientific tables, (Body weight of chickens 250—350 gm.) 115 5th Edition, APPENDIX L EFFECT OF DAILY ADMINISTRATION OF (6.0 ,ng/lOO gm b.w.) THYROID HORMONES ON M.R. IN CHICKENS. 02 Consumption ml/hour/kg. b.w. Days of Administration Chick 15 14 7 11 Treatment Number Hours after injection 2 3 24 24 151 1746 2006 2256 1828 152 1812 1922 1963 1692 153 1567 1697 1469 1905 contr°1 154 1971 1940 155 1620 1663 1811 1810 156 1666 1528 1569 2117 157 1755 1302 1614 2074 158 1826 1902 1921 2169 T 159 2236 1844 1598 2099 4 160 1537 2032 161 1495 1720 1754 1979 162 1779 2149 2066 1645 163 1887 1709 1370 1681 164 1859 1456 T 165 1894 2026 1518 1661 3 166 1577 1163* 1568 167 1646 1723 1448 1571 168 1688 1824 1811 1624 170 1753 1901 1120 2066 171 1347 1944 1029 1636 T + 172 1909 1622 1695 1489 3 173 ' 2400* 1543 1760 174 1903 1949 1608 1899 175 1512 1825 1599 1110 *Eliminated from statistical analysis by Chauvent criterion. Documenta Geigy. Scientific tables, 5th Edition, p. 47. (Body weight of chickens 120-225 gm.) 116