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Some aspects of thyroid metabolism in the chicken Poon, Raymond Wai-Man 1977

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SOME ASPECTS OF THYROID METABOLISM IN THE CHICKEN by RAYMOND WAI-MAN POON B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF POULTRY SCIENCE We accept t h i s t h e s i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA October , 1977 © Raymond Wai-Man Poon, 1977 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements fo r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e fo r reference and study. I f u r t h e r agree tha t permiss ion for e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copy ing or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department of POULTRY SCIENCE The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date October 1977 ABSTRACT The study of t h y r o i d metabol ism u s u a l l y invo l ves the employment of a n t i t h y r o i d compounds and exogenous t h y r o i d hormones in order to e l u c i d a t e the t h y r o i d f u n c t i o n through the understanding of the e f f e c t s of these compounds. In t h i s s tudy , the e f f e c t s of d i e t h i o u r a c i l and rapeseed meal go i t rogens were examined in c h i c k s . The g o i t r o g e n i c e f f e c t s of the t h i o u r a c i l were mani fested r a p i d l y by increased t h y r o i d we ight , t h y r o i d e p i t h e l i u m and uptake of r a d i o i o d i n e . The g o i t r o g e n i c e f f e c t s of rapeseed meal were s i m i l a r to the e f f e c t of t h i o u r a c i l except that a t the l e v e l of meal f e d , the e f f e c t s were less pronounced and ev ident on ly a f t e r prolonged f e e d i n g . When an iod ine d e f i c i e n t d i e t was fed to growing c h i c k s , the g o i t r o g e n i c e f f e c t was less than when t h i o u r a c i l or rapeseed meal was f e d . H i s t o l o g i c a l s t u d i e s of the t h y r o i d glands o f a d u l t b i r d s fed an iod ine d e f i c i e n t d i e t showed s l i g h t h y p e r p l a s i a wi thout t h y r o i d a l enlargement and w i t h p e r s i s t e n c e of c o l l o i d , w h e r e a s growing c h i c k s fed an iod ine d e f i c i e n t d i e t d i s p l a y e d severe h y p e r p l a s i a and l o s s of c o l l o i d . By c o n t r a s t , the e f f e c t s of t h i o u r a c i l were independent of the age of c h i c k s . A combinat ion of t h i o u r a c i l and rapeseed meal produced g o i t r o g e n i c e f f e c t s g rea te r than those o c c u r r i n g when t h i o u r a c i l or rapeseed meal was fed s i n g l y . - i i -T h i o u r a c i l fed in an iod ine d e f i c i e n t d i e t , on the other hand, showed no g o i t r o g e n i c i t y f o r c h i c k s . High doses (4.0 ug) of t h y r o x i n e admin is te red d a i l y to t h i o u r a c i 1 - t r e a t e d c h i c k s a l l e v i a t e d the a n t i t h y r o i d e f f e c t s of t h i o u r a c i l as i n d i c a t e d by the decrease in t h y r o i d w e i g h t , t h y r o i d e p i t h e l i u m and r a d i o i o d i n e uptake . Low doses (1.0 ug or l e s s ) of thy rox ine a d m i n i s t r a t i o n , however, produced the o p p o s i t e e f f e c t s . N e v e r t h e l e s s , h igh doses of t h y r o x i n e d i d not r e l i e v e the growth depress ing e f f e c t s of t h i o u r a c i l c o m p l e t e l y . A u t o r a d i o g r a p h i c s t u d i e s on t h y r o i d s of t h i o u r a c i 1 - t r e a t e d or rapeseed m e a l - f e d c h i c k s showed a uni form d i s t r i b u t i o n of r a d i o i o d i n e in the c o l l o i d w i t h i n f i v e hours of a d m i n i s t r a t i o n of t r a c e r . D i f f e r e n c e s in a c t i v i t y were observed among the i n d i v i d u a l f o l l i c l e s in each g l a n d . The s m a l l e r f o l l i c l e s concent rated more iod ine than the l a r g e r ones. Furthermore, very l i t t l e i od ine was observed in the e p i t h e l i a l c e l l s . These f i n d i n g s , together w i t h prev ious repor ts that i no rgan ic i od ine was present in the t h y r o i d a f t e r i t s a d m i n i s t r a t i o n , suggested that i o d i n a t i o n d i d not occur in the a p i c a l membrane of the f o l l i c u l a r c e l l s border ing the c o l l o i d . D i f f e r e n t rapeseed c u l t i v a r s c o n t a i n d i f f e r e n t amounts of g l u c o s i n o l a t e s which are r e s p o n s i b l e fo r the g o i t r o g e n i c e f f e c t of the rapeseed meal . G o i t r o g e n i c i t y of three d i f f e r e n t k inds of rapeseed meals was compared in growing c h i c k s and the r e s u l t s i n d i c a t e d that Candle meal had the l e a s t g o i t r o g e n i c a c t i v i t y than the Tower and Span meal . - i i i TABLE OF CONTENTS f a 9 £ ABSTRACT i TABLE OF CONTENTS . . . i i i LIST OF TABLES v i LIST OF FIGURES ix LIST OF TABLES IN APPENDIX x i i LIST OF ABBREVIATIONS xv ACKNOWLEDGEMENTS xv i INTRODUCTION .1 LITERATURE REVIEW 3 BIOSYNTHESIS AND RELEASE OF THYROID HORMONES 3 1. Uptake of iod ide in to the t h y r o i d . 4 2. Generat ion of an o x i d i z i n g agent 6 3 . Synthes is of t h y r o g l o b u l i n 6 k. Ox ida t ion of iod ide to a r e a c t i v e higher va lence s t a t e and the b ind ing of iod ine to t y r o s i n e 7 5. Formation of iodothyron ines in thyrog1obu1 in 9 6. Storage and p r o t e o l y s i s of t h y r o g l o b u l i n 10 EFFECTS OF TSH ON THE THYROID GLAND 11 EFFECTS OF IODIDE ON THE THYROID GLAND 13 ANTITHYROID COMPOUNDS AS A SOURCE OF G0ITR0GENS AND THEIR MECHANISM OF ACTIONS 15 1. Monovalent anions other than iod ide 17 2. Thionamides 18 - iv -3 . Aminoheterocycl i c compounds 21 4 . Iodide 22 5. M i s c e l l a n e o u s i n h i b i t o r s 24 6 . N a t u r a l l y o c c u r r i n g got t rogens 24 GOITROGENIC SUBSTANCES IN RAPESEED MEAL 24 1. In f luence of L ~ 5 - v i n y 1 - 2 - o x a z o l i d i n e t h i o n e on t h y r o i d gland of ch ickens 27 EXPERIMENTS . 31 Exper i ment 1: E f f e c t s o f i od ine d e f i c i e n c y and t h i o u r a c i l on the t h y r o i d h i s t o l o g y of White Leghorn hens 31 Experiment 2 : The e f f e c t s of rapeseed meal and t h i o u r a c i l s i n g l y and in combinat ion on the t h y r o i d glands of growing c h i c k s . . . . 41 Experiment 3 : Factors a f f e c t i n g the g o i t r o g e n i c i t y of t h i o u r a c i l in the c h i c k . . . . 5 4 Experiment 4: E f f e c t s of a d m i n i s t r a t i o n of d i f f e r e n t amounts of t h y r o x i n e on the t h y r o i d weights of c h i c k s in response to t h i o u r a c i l 71 Experiment 5= E f f e c t s of graded doses of D , L - t h y r o x i n e on the t h y r o i d glands of the growing c h i c k s t r e a t e d w i t h t h i o u r a c i l . . .. .81 - V Experiment 6: Au to rad iog raph ic s t u d i e s of the uptake and r e l e a s e of f-125 in t h y r o i d glands of c h i c k s t rea ted w i t h t h i o u r a c i l or fed w i t h rapeseed meal 98 Experiment 7'• E f f e c t s of rapeseed meals w i t h high or low c o n c e n t r a t i o n of g l u c o s i n o l a t e s on the t h y r o i d a l uptake and r e t e n t i o n of i od ide 119 SUMMARY AND CONCLUSIONS 129 BIBLIOGRAPHY 1 3h APPENDIX 152 - v F -LIST OF TABLES Tab 1e Page I Composit ion of d i e t s fed in Experiment 1 . . . . . . 36 II Average t h y r o i d a l weights at t e r m i n a t i o n of Experiment 1 37 III Average percentage of e p i t h e l i a l t i s s u e in the t h y r o i d glands of b i r d s in Experiment 1. . . . . . . 38 IV Composit ion of d i e t s fed in Experiment 2 . . . . . . ^9 V Average t h y r o i d weights of c h i c k s a t 1 week of age in Experiment 2 . 5 0 VI Average weight of e p i t h e l i a l t i s s u e of c h i c k s a t 1 week of age in Experiment 2 51 VII Thy ro ida l uptake of r a d i o i o d i n e - 1 3 1 by c h i c k s at 2 weeks of age in Experiment 2 52 V I M Treatments employed in Experiment 3 ^ IX Composit ion of d i e t s fed in Experiment 3 ^3 X Average body weights of c h i c k s of a l l t reatments in Experiment 3 ^ XI Average t h y r o i d weights of c h i c k s of a l l t reatments at d i f f e r e n t t ime i n t e r v a l s in Experiment 3 ^5 XII Chick t h y r o i d e p i t h e l i a l t i s s u e s at the age of 20 days in Experiment 3 ^6 XIII Composit ion of d i e t s as fed in Experiment h 76 " v i i XIV Experimental t reatments imposed in Experiment 4^ ^ 77 XV Growth rates of c h i c k s on a l l t reatments in Experiment 4 78 XVI Thyro id weights of c h i c k s in Experiment 'i 79 XVII Composit ion of d i e t s fed in Experiment 5. 91 XVIII D i f f e r e n t t reatments imposed in Experiment 5 . . . . 92 XIX Averaged body weight of c h i c k s at the age of 7> 13 and 19 days in Experiment 5 93 XX Thyro id weights of c h i c k s in d i f f e r e n t t reatments at day 8 , 14 and 20 in Experiment 5 .94 XXI Percentage e p i t h e l i a l t i s s u e of t o t a l t h y r o i d f o r d i f f e r e n t t reatments at d i f f e r e n t days in Experiment 5 95 XXII Thy ro ida l uptake of 1—131 f o r d i f f e r e n t t reatments a t d i f f e r e n t t imes a f t e r i n j e c t i o n in Experiment 5 96 XXIII Composit ion of d i e t s as fed in Experiment 6 . . . . 109 XXIV Average t h y r o i d weights of c h i c k s at the age of 8 weeks ( l e f t lobe o n l y ) , in Experiment 6 . . . . 1 1 0 XXV Uptake of 1-125 by the l e f t lobe of the t h y r o i d glands of c h i c k s at the age of 8 weeks in Experiment 6 I l l XXVI Percentage compos i t ion of d i e t s fed in Exper iment 7 124 - v i i i -.7 XXVI I Average body weights of c h i c k s at 5 weeks of age in Experiment 7 125 XXVIII Thy ro ida l uptake of 1-131 by c h i c k s at 12 days of age in Experiment 7 126 XXIX Thyro ida l uptake of 1-131 by c h i c k s at 5 weeks of age in Experiment 7 127 i X -LIST OF FIGURES F i g u r e Page 1 P h o t o m i c r o g r a p h o f t h y r o i d g l a n d o f i o d i n e d e f i c i e n t b i r d (400x) . . . . . . 39 2 P h o t o m i c r o g r a p h o f t h y r o i d g l a n d o f c o n t r o l b i r d (400x) 39 3 P h o t o m i c r o g r a p h o f t h y r o i d g l a n d o f t h i o u r a c i 1 -t r e a t e d b i r d (400x) kO h T h y r o i d a l 1 —131 u p t a k e o f c h i c k s a t two weeks o f age, k, 8, 16 and 32 hours a f t e r 1-131 a d m i n i s t r a t i o n 53 5 I n c r e a s e i n t h y r o i d w e i g h t i n re s p o n s e t o d i f f e r e n t t r e a t m e n t s 67 6 P h o t o m i c r o g r a p h o f t h e t h y r o i d g l a n d o f i o d i n e d e f i c i e n t ( R - l ) b i r d s (400x) 68 7 P h o t o m i c r o g r a p h o f the t h y r o i d g l a n d o f c o n t r o l (R-2) b i r d s (400x) 68 8 P h o t o m i c r o g r a p h o f t h e t h y r o i d g l a n d o f t h i o u r a c i 1 - t r e a t e d and i o d i n e d e f i c i e n t (R~3) b i r d s (400x) 69 9 P h o t o m i c r o g r a p h o f t h e t h y r o i d g l a n d o f t h e t h i o u r a c i 1 - t r e a t e d (R-4) b i r d s (400x) 69 10 P h o t o m i c r o g r a p h o f t h e t h y r o i d g l a n d o f t h e t h i o u r a c i 1 - t r e a t e d and T^-supplemented (R~5) b i r d s (400x) 70 11 Photomicrograph of the t h y r o i d gland of the TSH- t reated (R-6) b i r d s (400x) . . . . . . 70 12 Average t h y r o i d weight as a percentage o f body weight in t h i o u r a c i 1 - t r e a t e d c h i c k s w i t h respect to i n c r e a s i n g dosage of D.L-T^ 80 13 Thy ro ida l I —.13-1" uptake of c h i c k s a t the age of 3 weeks in Experiment 5 97 14 Average percentage 1-125 uptake by t h y r o i d of c h i c k s at the age of 8 weeks 112 15 Autoradiograph of t h y r o i d gland of c o n t r o l b i r d 5 hours a f t e r 1-125 i n j e c t i o n (400x) 113 16 Autoradiograph of t h y r o i d gland of t h i o u r a c i l -t r e a t e d b i r d 5 hours a f t e r 1-125 i n j e c t i o n (400x) . .113 17 Autoradiograph of t h y r o i d gland of rapeseed mea l - fed b i r d 5 hours a f t e r 1-125 i n j e c t i o n (400x). . 114 18 Autoradiograph of t h y r o i d gland of c o n t r o l b i r d 10 hours a f t e r 1-125 i n j e c t i o n (400x) . . . . . 114 19 Autoradiograph of t h y r o i d gland of t h i o u r a c i l -t r e a t e d b i r d 10 hours a f t e r 1-125 i n j e c t i o n (400x). .115 20 Autoradiograph of t h y r o i d gland of rapeseed m e a l -fed b i r d 10 hours a f t e r 1-125 i n j e c t i o n (400x) . . . 115 21 Autoradiograph of t h y r o i d gland of c o n t r o l b i r d 15 hours a f t e r 1-125 i n j e c t i o n (400x) 116 22 Autoradiograph of t h y r o i d gland of t h i o u r a c i l -t reated b i r d 15 hours a f t e r 1-125 i n j e c t i o n ( 4 0 0 x ) . . . ] 16 23 Autoradiograph of t h y r o i d gland of rapeseed m e a l -fed b i r d 15 hours a f t e r 1-125 i n j e c t i o n ( 4 0 0 x ) . . . .117 2k Autoradiograph of t h y r o i d gland of c o n t r o l b i r d 20 hours a f t e r 1-125 i n j e c t i o n (kOOx) 117 25 Autoradiograph of t h y r o i d g land of t h i o u r a c i 1 -t r e a t e d b i r d 20 hours a f t e r 1-125 i n j e c t i o n (kOOx). . 118 26 Autoradiograph of t h y r o i d gland of rapeseed m e a l - f e d b i r d 20 hours a f t e r 1-125 i n j e c t i o n (kOOx). , . 1 1 8 27 Thy ro ida l uptake of 1-131 at 12 and 35 days of a g e . .128 - x i i -LIST OF TABLES IN APPENDIX Table Page 1(A) Experiment 1: S t a t i s t i c a 1 a n a l y s i s comparing the average weight of t h y r o i d glands in White Leghorn hens at t e r m i n a t i o n of Experiment 1 153 11(A) Experiment 1 : . S t a t i s t i c a l a n a l y s i s comparing the average percentages of e p i t h e l i a l t i s s u e in the t h y r o i d glands of b i r d s 154 I I I(A) Experiment 2: S t a t i s t i c a l a n a l y s i s comparing the average weights of t h y r o i d glands of c h i c k s at 1 week of age 155 IV(A) Experiment 2: S t a t i s t i c a l a n a l y s i s comparing the average weights of e p i t h e l i a l t i s s u e of c h i c k s a t 1 week of age 156 V(A) Experiment 2: S t a t i s t i c a l a n a l y s i s comparing t h y r o i d uptake of r a d i o i o d i n e - 1 3 1 by c h i c k s at 4 , 8 , 16 and 32 hours a f t e r i n j e c t i o n 157 VI(A) Experiment 3- S t a t i s t i c a l a n a l y s i s comparing the average body weights of c h i c k s in d i f f e r e n t treatment at d i f f e r e n t ages 159 VI I(A) Experiment 3- S t a t i s t i c a l a n a l y s i s comparing the percentages t h y r o i d body weight of c h i c k s at day 9 , 13 and 20. . . 161 VI I I(A) Experiment 3= S t a t i s t i c a l a n a l y s i s comparing the percentages of e p i t h e l i u m of t o t a l t h y r o i d gland of c h i c k s a t 20 days of age 163 IX(A) Experiment 3' S t a t i s t i c a l a n a l y s i s comparing the est imated weights of t h y r o i d a l e p i t h e l i a l t i s s u e s of c h i c k s at the age of 20 days . .164 X(A) Experiment 3- S t a t i s t i c a l a n a l y s i s comparing the est imated weights of t h y r o i d e p i t h e l i u m per 100 g body weight of c h i c k s at 20 days of age 165 XI(A) Experiment 4: S t a t i s t i c a l a n a l y s i s f o r comparing the average body weights of c h i c k s at 3~ l/2 weeks of age 166 x i i i -XI I(A) • Experiment 4: S t a t i s t i c a l a n a l y s i s fo r comparing the t h y r o i d weights of c h i c k s in R-1 to R-8 a t . 3 - l : / 2 weeks of age 167 XI I I(A) Experiment 4: S t a t i s t i c a l a n a l y s i s f o r comparing the t h y r o i d weights of c h i c k s in R - l , RT$ and R-10 at 3 - 1 / 2 weeks of age 168 XIV(A) Experiment 5 : S t a t i s t i c a l a n a l y s i s f o r comparing the average body weights of c h i c k s a t the age of 19 days . . . . ' 169 XV(A) Experiment 5: S t a t i s t i c a l a n a l y s i s f o r comparing the average t h y r o i d weights of c h i c k s a t day 8, 14 and 20 170 XVI(A) Experiment 5: S t a t i s t i c a l a n a l y s i s f o r comparing the percentage e p i t h e l i a l t i s s u e of t o t a l g land at day 8 , 14 and 20 . . 172 XVI I(A) Experiment 5- S t a t i s t i c a l a n a l y s i s f o r comparing the percentages of i n j e c t e d dose in c h i c k s at va r ious times a f t e r a d m i n i s t r a t i o n at 1-131 174 XVI I I (A)Experiment 6: S t a t i s t i c a l a n a l y s i s f o r comparing the average weights of t h y r o i d l e f t lobe of c h i c k s a t the age of 8 weeks 178 XIX(A) Experiment 6: S t a t i s t i c a l a n a l y s i s f o r comparing the average weights of t h y r o i d l e f t lobe of c o n t r o l and rapeseed m e a l - f e d c h i c k s at the age of 8 weeks 179 XX(A) Experiment 6: S t a t i s t i c a l a n a l y s i s fo r comparing the percentage uptake of 1-125 in the t h y r o i d l e f t lobes of c h i c k s a t 8 weeks of age 180 XXI(A) Experiment 7- S t a t i s t i c a l a n a l y s i s fo r comparing the average body weight of c h i c k s at 5 weeks of age 182 XXI I(A) Experiment 7- S t a t i s t i c a l a n a l y s i s fo r comparing the percentages of t h y r o i d a l 1—131 uptake by c h i c k s at the age of 12 days 183 XXI I I (A)Experiment 7- S t a t i s t i c a l a n a l y s i s of comparing the percentages of t h y r o i d a l 1—131 uptake by c h i c k s a t 35 days of age 184 - x i v -XXIV(A) C a l c u l a t e d crude p r o t e i n c o n c e n t r a t i o n f o r a l1 r a t i o n s in d i f f e r e n t experiments 186 - X V -LIST OF ABBREVIATIONS B.W. cAMP DIHPPA DIT H2°2 125 1-131 .+ ,U. . C U . urn P9 mg MIT NADH NADPH PBI PBI-131 PTU RSM SBM V D,L -T 4 TSH body weight c y c l i c adenosine 5 1 -monophosphate d i iodohydroxyphenopyruv ic a c i d d i i o d o t y r o s i ne hydrogen perox ide r a d i o a c t i v e iod ine 125 r a d i o a c t i v e iod ine 131 i od i n i urn i n ternat ional u n i t i n t e r n a t i o n a l c h i c k u n i t micron microgram m i l l igram mono i odotyros i ne n i c o t i n a m i d e adenine d i n u c l e o t i d e (reduced form) n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate (reduced form) p ro te in -bound iod ine p ro te in -bound r a d i o a c t i v e iod ine 131 p r o p y l t h i o u r a c i1 rapeseed meal soybean meal t r i i odothyron i ne D , L - t h y r o x i n e thy ro t rop i n TU t h i o u r a c i1 _ xv i ACKNOWLEDGEMENTS The author wishes to express h i s g r a t i t u d e to P r o f e s s o r B.E. March of the Department of P o u l t r y Sc ience who o r i g i n a l l y suggested the study and superv ised the research reported h e r e i n . Her c o n t r i b u t i o n to my academic development and h e l p f u l adv ice are deeply a p p r e c i a t e d . A f u l l measure of a p p r e c i a t i o n is a l s o extended to the o ther members of my Thes is Committee: Dr. D.B. Bragg Chairman, Department of P o u l t r y Sc ience Dr. C.R. Kr ishnamurt i Department of Animal Sc ience Dr. R.C. Fitzsimmons Department of P o u l t r y Sc ience Dr. A . F . Burton Department of B iochemis t ry S p e c i a l a p p r e c i a t i o n i s g iven to Dr. A . F . Burton f o r h i s a d v i c e and help on l i q u i d s c i n t i l l a t i o n count ing and to Dr. R.E. Ba r ton , Department of B i o c h e m i s t r y , who k i n d l y permits me to use the U n i l e x II l i q u i d s c i n t i l l a t i o n counte r . In p a r t i c u l a r , Dr. C .E . S l o n e c k e r , Department of Anatomy, must r e c e i v e s p e c i a l mention in accord w i t h h i s a d v i c e and help in performing the a u t o r a d i o g r a p h i c s t u d i e s . The author a l s o wishes to express h i s a p p r e c i a t i o n to Mr. R. Soong and Miss A. Wong, P o u l t r y N u t r i t i o n Labora to ry , f o r t h e i r f requent a s s i s t a n c e when r e q u i r e d . - 1 -INTRODUCTION The t h y r o i d gland was one of the f i r s t endocr ine glands s t u d i e d . Al though much i n f o r m a t i o n concern ing the t h y r o i d gland has been a c q u i r e d , many aspects of i t s metabol ism remain u n c l e a r . The exact mechanism of t h y r o i d hormone s y n t h e s i s and c o n t r o l under normal p h y s i o l o g i c a l c o n d i t i o n s i s s t i l l u n c e r t a i n . When normal t h y r o i d f u n c t i o n i s i n h i b i t e d by an a n t i -t h y r o i d substance such as t h i o u r a c i l or the go i t rogens in rapeseed meal , the system becomes more compl icated s i n c e t h y r o i d f u n c t i o n and hormone s y n t h e s i s a re e i t h e r a f f e c t e d or b l o c k e d , depending on the mode of a c t i o n of the a n t i t h y r o i d compounds i n v o l v e d . Whi le numerous s t u d i e s have concentrated on the e f f e c t s of go i t rogens i n -t h y r o i d metabol ism, e s p e c i a l l y on those r e l a t e d to t h y r o i d hormone s y n t h e s i s , few i n v e s t i g a t i o n s have examined the e f f e c t s of s y n e r g i s t i c a c t i o n s of the d i f f e r e n t go i t rogens and t h e i r e f f e c t s on both a d u l t and young a n i m a l s . Ne i ther have many s t u d i e s been made on the e f f e c t s of t h y r o x i n e supplementat ion d u r i n g go i t rogen a d m i n i s t r a t i o n . Furthermore, the g o i t r o g e n i c i t y of rapeseed meal has been s tud ied e x t e n s i v e l y s i n c e the meal can be used as a feed f o r animals or as a source of p r o t e i n f o r human f o o d s . N e v e r t h e l e s s , there i s s t i l l a need fo r f u r t h e r research because new c u l t i v a r s of rape have been developed which c o n t a i n d i f f e r e n t p ropor t ions of v a r i o u s p r o g o i t r o g e n i c substances . The present study i s d i v i d e d in to severa l s e c t i o n s , in order to examine the e f f e c t s of a n t i t h y r o i d compounds. The f i r s t - 2 -s e c t i o n examined the e f f e c t s of feed ing g o i t r o g e n i c d i e t s to a d u l t b i r d s and growing c h i c k s . The second s e c t i o n of the study examined the s y n e r g i s t i c a c t i o n s of t h i o u r a c i l and the go i t rogens present in the rapeseed meal on iod ine metabol ism in the t h y r o i d gland and h i s t o l o g i c a l changes w i t h i n the g l a n d . Par t three of the i n v e s t i g a t i o n was concerned w i t h the e f f e c t s of exogenous t h y r o i d hormone on the t h y r o i d f u n c t i o n dur ing the feed ing of the a n t i t h y r o i d compound, t h i o u r a c i l . In the f o u r t h s e c t i o n , the d i s t r i b u t i o n of i od ine in the t h y r o i d glands of ch icken fed rapeseed meal and t h i o u r a c i l was s t u d i e d to determine whether there a re d i f f e r e n c e s in the mode of a c t i o n of the d i f f e r e n t go i t rogens . , It has been i n d i c a t e d j n the l i t e r a t u r e that the mode of a c t i o n of rapeseed meal go i t rogens i s d i f f e r e n t from that of o ther a n t i t h y r o i d compounds. The f i n a l par t of the study invo lved the comparison of the g o i t r o g e n i c i t y of rapeseed meals manufactured from d i f f e r e n t c u l t i v a r s of rapeseed. S tud ies of the a c t i o n s of rapeseed meal go i t rogens on the t h y r o i d gland are important s i n c e the presence of these go i t rogens may l i m i t the use of rapeseed meal as p r o t e i n supplement f o r animal f e e d s . More g e n e r a l l y , i t was hoped that the study would prov ide in fo rmat ion which would f u r t h e r our understanding of t h y r o i d metabol ism. - 3 -LITERATURE REVIEW BIOSYNTHESIS AND RELEASE OF THYROID .HORMONES A cont inuous supply of iod ine i s necessary f o r t h y r o i d hormone s y n t h e s i s s i n c e t h i s element i s an e s s e n t i a l c o n s t i t u e n t of the t h y r o i d hormones. I ts r o l e in t h i s c a p a c i t y represents the on ly recognized ins tance of a p h y s i o l o g i c a l l y use fu l f u n c t i o n f o r i od ine (Tong, 1971)• Absorp t ion of iod ine from the g a s t r o -i n t e s t i n a l t r a c t i s v i r t u a l l y complete i r r e s p e c t i v e of the endogenous iod ine l e v e l . The absorbed iod ine in the c i r c u l a t i o n i s main ly concentrated by the t h y r o i d g l a n d . The t h y r o i d gland inco rpora tes the ino rgan ic i od ide i n t o t h y r o i d hormones and re leases these hormones i n t o the c i r c u l a t i o n f o r t r a n s p o r t to the p e r i p h e r a l t i s s u e s . P h y s i o l o g i c a l and pharmacolog ica l a n t i t h y r o i d agents , as we l l as an inadequate supply of iod ine can h inder the r e g u l a r b i o s y n t h e s i s of t h y r o i d hormone, hence, i n t e r f e r i n g w i t h normal t h y r o i d metabol ism. In the present d i s c u s s i o n , t h y r o i d hormone s y n t h e s i s and i t s r e l a t i o n s h i p to iod ine w i l l be rev iewed. D i f f e r e n t types of g o i t r o g e n s , t h e i r mechanism of a c t i o n , and t h e i r e f f e c t s on the phys io logy of t h y r o i d g land w i l l be a l.so cons i d e r e d . It is g e n e r a l l y accepted that the processes of t h y r o i d hormone fo rmat ion inc lude the f o l l o w i n g in termediary steps - h -(DeGroot and N i e p o m n i s z c z e , 1977): 1) t r a n s p o r t o f i o d i d e i n t o the t h y r o i d ; 2) g e n e r a t i o n o f an o x i d i z i n g agent (presumably H^O^); 3) s y n t h e s i s o f the r e c e p t o r p r o t e i n , t h y r o g l o b u l i n ; 4) o x i d a t i o n o f i o d i d e t o a r e a c t i v e h i g h e r v a l e n c e s t a t e ; 5) b i n d i n g o f i o d i n e t o t y r o s i n e p r e s e n t i n t h y r o g 1 o b u 1 i n ; 6) f o r m a t i o n o f i o d o t h y r o n i n e s i n t h y r o g l o b u l i n by c o u p l i n g o f i o d o t y r o s y l r e s i d u e s ; 7) s t o r a g e and p r o t e o l y s i s o f t h y r o g l o b u l i n i n t h e f o l 1 i c l e ; 8) d e i o d i n a t i o n o f f r e e i o d o t y r o s i n e s ; and 9) r e l e a s e o f t h y r o x i n e and t r i i o d o t h y r o n i n e t o the b l o o d . UPTAKE OF IODIDE INTO THE THYROID The k i n e t i c s o f i o d i n e m e t a b o l i s m have been s t u d i e d e x t e n s i v e l y and i t i s c o n v e n i e n t t o c o n s i d e r i o d i n e k i n e t i c s i n terms o f a t h r e e compartment system (Rapoport and DeGroot, 1971), a l t h o u g h t h i s i s p r o b a b l y an o v e r - s i m p l i f i c a t i o n . There a r e t h r e e major i o d i d e p o o l s , e x t r a t h y r o i d a l i n o r g a n i c i o d i d e , t h y r o i d a l i o d i d e and e x t r a t h y r o i d a l o r g a n i c (hormonal) i o d i d e . However, P i v i e r e e t aj_. (1965) s u g g e s t e d t h a t t h e r e a r e f o u r compartments. These a r e t h e i o d i d e p o o l s a l r e a d y mentioned t o g e t h e r w i t h a n o t h e r i o d i d e compartment r e p r e s e n t i n g both t h e t r a p p e d i o d i d e and t h e r a p i d renewable i n t r a t h y r o i d a 1 o r g a n i c i o d i d e p o o l . The e x t r a -t h y r o i d a l i n o r g a n i c i o d i d e pool a c c u m u l a t e s i o d i d e d i r e c t l y from t h r e e s o u r c e s . These a r e v i z . the d i e t , t h e e x t r a t h y r o i d a l o r g a n i c i o d i d e pool as a r e s u l t o f p e r i p h e r a l d e i o d i n a t i o n o f t h y r o i d hormones and a s m a l l q u a n t i t y . w h i c h i s l e a k e d d i r e c t l y from the - 5 -t h y r o i d (DeGroot, 1 9 6 6 ) . . .The' iod ide balance is mainta ined by the e x c r e t i o n of ino rgan ic i od ide in the u r i n e and conjugated t h y r o i d hormone in the b i l e . However, a sma11 q u a n t i t y of t h y r o i d hormone i s a l s o excreted d i r e c t l y from the e x t r a t h y r o i d a l o r g a n i c i od ide pool v i a u r i n e and f e c e s . The t h y r o i d can accumulate i od ide by d i f f u s i o n and a c t i v e t r a n s p o r t (Evered, 1976) . However, there i s o n l y . n e g l i g i b l e uptake of i od ide by d i f f u s i o n a t p h y s i o l o g i c a l plasma c o n c e n t r a t i o n . The iod ide which enters through t h i s route i s a l s o a v a i l a b l e f o r t h y r o i d hormone b iosynthes is ( P i t t - R i v e r s e t a l . 1958) . The major par t of the i o d i d e e n t e r i n g the t h y r o i d does so by an a c t i v e t r a n s p o r t process s i n c e i t i s c l e a r that iod ide i s t ranspor ted i n t o the t h y r o i d a g a i n s t an e l e c t r o c h e m i c a l g r a d i e n t (Woodbury and Woodbury, 1963) . The f a c t that t h i s iod ide t r a n s p o r t mechanism i s a b o l i s h e d by agents that i n h i b i t o r uncouple o x i d a t i v e p h o s p h o r y l a t i o n - ( F r e i n k e l and Ingbar, 1955; W o l f f , 1964; Wyngaarden et a 1. 1953) i n d i c a t e s that the t r a p p i n g mechanism i s probably an a c t i v e process r e q u i r i n g energy r e l a t e d to the N a + , K + -ATPase -dependent t r a n s p o r t system (Turk ing ton , 1962; Rapoport and DeGroot, 1971) . Much a t t e n t i o n has been focused on the s i t e of t h i s " i o d i d e pump" mechanism, which i s probably a membrane receptor s i t e loca ted both a t the basal c e l l s u r f a c e to t r a n s p o r t i od ide from the e x t r a c e l l u l a r f l u i d i n t o the c e l l and at the a p i c a l ee l 1 s u r f a c e to t r a n s p o r t iod ide from the c e l l i n t o the f o l l i c u l a r lumen (Halmi , 1961 ; Tong et_'aj_. 1962; Tong, 1971) . - 6 -GENERATION OF AN OXIDIZING AGENT A l l systems d e s c r i b e d so f a r as forming i o d o t y r o s i n e s and iodothyron ines in v i t r o h a v e involved perox idases and are c a t a l a s e s e n s i t i v e (Coval and Taurog, 1967; Taurog, 1970; Nagasaka et a l . , 1971; Danner and M o r r i s o n , 1971) . Thus, i t i s w ide ly accepted that i h v ivo iod i nat i ons r e q u i r e genera t ion of hydrogen perox ide (H^O^)• Four d i f f e r e n t mechanisms have been proposed as p o s s i b l e sources of ^2^2 ' n t ' i e P r e s e n c e °f s u i t a b l e s u b s t r a t e s and c o f a c t o r s . The proposed enzymes a re NADPH-cytochrome c reductase ( S u z u k i , 1966 ; Nagasaka et a l . , 1971; Yamamoto and DeGroot, 1975) , NADH-cytochrome b^ reductase (Ohtaki et a j_ . , 1973) , monoamine ox idase mechanism (F isher et a l . , I968) and xanth ine ox idase (F i scher and Lee, 1973)- However, Nagasaka e_taj_. (1970 commented that NADPH-cytochrome c reductase i s most l i k e l y to be the enzyme i n v o l v e d . SYNTHESIS OF THYROGLOBULIN T h y r o g l o b u l i n i s syn thes i zed in the polyr ibosomes of the endoplasmic r e t i c u l u m of the t h y r o i d c e l l s (Nad 1 er et_ aj_. , 1964; Thomson and Goldberg , 1968; P i t t - R i v e r s , 1967). The p r o t e i n formed i s passed c o n t i n u o u s l y in to the c i s t e r n a e , making i t s way from there to the Go lg i zone where the carbohydrate moiety i s . a d d e d . From the Golg i zone, the r e s u l t i n g g l y c o p r o t e i n moves in to the a p i c a l v e s i c l e s -- 7 -which reach the a p i c a l eel 1 s u r f a c e , fuse w i th the ee l 1 membrane and break up to r e l e a s e t h e i r contents i n t o t h e : c o l l o i d (Doniach, I967). The s i t e of the i o d i n a t i o n has been the t o p i c of much, e x t e n s i v e s tudy . By employing techniques such as l i g h t m i c r o -scop ic autoradiography and e l e c t r o n m i c r o s c o p i c auto rad iography , i t has been found that : the major s i t e of i o d i n a t i o n of t h y r o g l o b u l i n is the c o l l o i d present on ly in the lumen of the t h y r o i d f o l l i c l e s i n c e l i t t l e r a d i o a c t i v i t y i s d e t e c t a b l e in the cytoplasm or c o l l o i d d r o p l e t s . I ns tead , a uni form d i s t r i b u t i o n o f s i l v e r g r a i n s i s observed in the lumen (Nadler and Leb lond , 1954; N a d l e r , 1965; Simon and Droz, 1965; Lupulescu and P e t r o v i c i , 1965; Doniach, I967). However, i t i s apparent that the r a d i o i o d i n e f i r s t appears in r i n g s around the per iphery of the luminal space and l a t e r d i f f u s e s to g i ve an uni form b l a c k i n g of c o l l o i d . C ro f t and P i t t - R i v e r s (1970 observed that there are more g r a i n s over the c e l l s than over the c o l l o i d s h o r t l y a f t e r r a d i o i o d i n e a d m i n i s t r a t i o n and no i o d i n a t i n g a c t i v i t y i s found w i t h i n the c o l l o i d e x t r a c t s (Gross et a l . , 1965). I o d i n a t i o n probably o c c u r s , t h e r e f o r e , at the membrane of the f o l l i c u l a r c e l l s border ing the c o l l o i d ( P i t t - R i v e r s , 1967; L i s s i t z k y e_t a_1_., 1969). OXIDATION OF IODIDE TO A REACTIVE HIGHER VALENCE STATE AND THE BINDING OF IODINE TO TYROSINE An enzyme capable of u t i l i z i n g H ^ f o r p e r o x i d a t i o n of iod ide to i o d i n e , and c a t a l y z i n g the two -s tep r e a c t i o n of i od ide - 8 -p e r o x i d a t i o n f o l l o w e d by b i n d i n g t o t y r o s i n e has been i s o l a t e d by s e v e r a l i n v e s t i g a t o r s (Hosoya and M o r r i s o n , 1967; Taurog e t a l . 1970; DeGroot et_a_l_. j 1972)'. I t has a l s o been i s o l a t e d i n membranes o f the a p i c a l b o r d e r s o f t h e t h y r o i d e e l 1 (Strum and K a r n o v s k y , 1970). The p e r o x i d a s e i s a haeme p r o t e i n w i t h a p r o s t h e t i c group s i m i l a r t o p r o t o p o r p h y r i n IX. A number o f r e a c t i o n mechanism have been s u g g e s t e d f o r t h e mechanism o f o x i d a t i o n w h i c h l i n k s i o d i d e t o t y r o s i n e . These p o s s i b l e mechanisms have been review e d by DeGroot e t a 1 . (1972); and DeGroot and N i e p o m n i s z c z e (1977); who c o n c l u d e t h a t t h e most l i k e l y mechanism i n v o l v e s a f r e e r a d i c a l . I o d i d e i s o x i d i z e d w i t h a one e l e c t r o n l o s s t o the f r e e r a d i c a l , w h i c h i n t u r n combines w i t h a f r e e r a d i c a l formed by a one e l e c t r o n o x i d a t i o n o f t h e p h e n o l a t e i o n t o form i o d o t y r o s i n e . T h i s proposed sequence o f e v e n t s assumes t h a t .the t h y r o i d p e r o x i d a s e i s o x i d i z e d by H^O^ w h i c h then r e a c t s w i t h i o d i d e t o g e n e r a t e a f r e e i o d i n e r a d i c a l . The p e r o x i d a s e can then r e a c t e i t h e r w i t h a n o t h e r f r e e i o d i n e r a d i c a l o r w i t h t y r o s i n e t o form a f r e e t y r o s y l r a d i c a l . Then th e i o d i d e and t y r o s y l r a d i c a l can combine t o form i o d o t y r o s i n e w h i l e the enzyme r e t u r n s t o i t s o r i g i n a l u n o x i d i z e d s t a t e . The p r o c e s s as d i s c u s s e d above, p r o b a b l y o c c u r s a t t h e m i c r o v i l l o u s i n t e r f a c e between c e l l and c o l l o i d . I t seems l i k e l y t h a t an e x c e s s o f i o d i d e would i n t e r f e r e w i t h t h e b i o s y n t h e s i s o f t h y r o i d hormone here because t h e r e would be an e x c e s s o f f r e e i o d i n e r a d i c a l s which would condense t o ' f o r m i o d i n e r e n d e r i n g them u n a v a i l a b l e f o r i o d i n a t i o n . - 9 -FORMATION OF IODOTHYRONINES IN THYROGLOBULIN A f t e r i o d o t y r o s i n e i s generated in t h y r o g l o b u l i n ? a seconda ry c o u p l i ng react ion o c c u r s , . 1 e a d i ng to the format ion of i n t r a t h y r o g l o b u l i n t h y r o x i n e and t r i i o d o t h y r o n i n e (DeGroot et a l , , 1972), T h e o r e t i c a l 1 y , t h i s cou ld proceed through i o d i n a t i o n of i n t r a t h y r o g l o b u l i n t h y r o n i n e , c o u p l i n g of two i n t r a t h y r o g l o b u l i n i o d o t y r o s y l groups or c o u p l i n g of f r e e i o d o t y r o s i n e w i th p e p t i d e -l i n k e d iodotyrosy 1. However, thyron ine i t s e l f does not e x i s t in n a t u r e , except perhaps as a degradat ion product of t h y r o i d hormone. T h e r e f o r e , fo rmat ion of t h y r o x i n e and t r i i o d o t h y r o n i n e must invo l ve the c o u p l i n g o f two i o d o t y r o s y l molecules (DeGroot and Niepomniszcze , 1977)-There are two mechanisms whereby iodothyron ines can be formed in the thy rog lobu l i n (DeGroot e_t_ aj_. , 1972). An i n t r a m o l e c u l a r rearrangement i s one route that can lead to the fo rmat ion of thy rox ine from d i i o d o t y r o s i n e (DIT). The mechanism f o r the r e a c t i o n is an o x i d a t i o n lead ing to the format ion of an i o d o t y r o s y l f ree r a d i c a l o r p o s i t i v e l y charged i o n , which can be t r a n s f e r r e d w i t h i n the t h y r o g l o b u l i n to another i o d o t y r o s y l group to form the i o d o t h y r o n i n e . An a l t e r n a t i v e c o u p l i n g mechanism cou ld be the p a i r i n g of a f r e e DIT molecule or a d e r i v a t i v e and a DIT group in t h y r o g l o b u l i n . This concept was supported by r e c o g n i t i o n of di iodohydroxyphenopyruvi c a c i d (.DIHPPA) in the t h y r o i d ( S u r k s e t a l . , 1968), s i n c e DIHPPA can be formed by o x i d a t i v e deamination of the DIT mo lecu le , and i t e x i s t s in water in e q u i l i b r i u m w i t h i t s enol - 10 -tautomer (Toi et_ aJL , 1963; 1965)- P e r o x i d a t i o n o f t h e enol form o f DIHPPA l e a d s t o the'. f o r m a t ion o f D l R P P A - p e r o x i d e , w h i c h can then be c o u p l e d w i t h a n o t h e r DIT m o l e c u l e i n t h y r o g l o b u l i n t o . f o r m t h y r o x i n e . A l t h o u g h t h e r e q u i r e m e n t s have been met f o r t h e f o r m a t i o n o f i o d o t h y r o n i n e s t h r o u g h t h e c o u p l i n g o f f r e e DIHPPA w i t h p e p t i d e - 1 i i o d o t y r o s i n e ( i g o e t a 1 1968; Bl as i e t a l . , 1969 a, b) , the e x t e n t o f t h i s pathway i n t h e t h y r o i d i s unknown. S i n c e an a c t i v e t y r o s i n e d e i o d i n a s e i s p r e s e n t i n t h e t h y r o i d and would r a p i d l y d e h a l o g e n a t e i o d o t y r o s i n e , i t i s p o s s i b l e t h a t t h e r e may be l i t t l e s u b s t r a t e f o r d i r e c t g e n e r a t i o n o f DIHPPA. Thus t h i s mechanism f o r the c o u p l i n g r e a c t i o n i s c o n s i d e r e d u n l i k e l y a t t h e p r e s e n t time (DeGroot and N i epomn i s z c z e , 1977)• STORAGE AND PROTEOLYSIS OF THYROGLOBULIN T h y r o i d hormones a r e s t o r e d i n the f o l l i c u l a r c o l l o i d where they a r e c o n s t i t u e n t s o f t h e t h y r o g 1 o b u l i n p o l y p e p t i d e . In a d d i t i o n t o c o l l o i d d r o p l e t s , two f u n c t i o n a l l y d i f f e r e n t types o f v e s i c l e s a r e o b s e r v e d i n t h e a p i c a l r e g i o n o f t h y r o i d f o l l i c l e c e l l s ( . S e l j e l i d e t a 1 . , 1970). These a r e the e x o c y t o t i c and e n d o c y t o t i c v e s i c l e s . E x o c y t o t i c v e s i c l e s a r e i n v o l v e d i n t h e t r a n s p o r t o f newly s y n t h e s i z e d p r o t e i n to t h e f o l l i c l e lumen f o r s t o r a g e (Ekholm et_ a_l_. , 1975; Bjorkman e t _ . a l . , 1976).. . The e n d o c y t o t i c v e s i c l e s may be r e s p o n s i b l e f o r . t h e . r e e n t r y o f t h y r o g l o b u l i n i n t o the c e l l f o r r e l e a s e t o t h e c i r c u l a t i o n ( . S e l j e l i d , 1968). - 11 -Autorad iograph ic s t u d i e s show that newly syn thes i zed hormone is i n i t i a 1 l y ' s t o r e d . a d j a c e n t • t o the e p i t h e l i a l border . Cont inu ing s y n t h e s i s may produce c o n c e n t r i c lamel lae of c o l l o i d , w i t h o l d e r t h y r o g l o b u l i n i n . t h e center (Rapoport and DeGroot, 1971)-Schneider (.1964) showed that t h y r o i d a l i od ide s to red l a s t , presumably near the e p i t h e l i a l m i c r o v i 1 l o u s border , i s sec re ted f i r s t on s t i m u l a t i o n . He termed t h i s the " l a s t come, f i r s t s e r v e d " p r i n c i p l e of iod ine turnover in the t h y r o i d iod ine p o o l . Th is is supported by the s t r u c t u r a l and f u n c t i o n a l heterogene i ty among the f o l l i c l e s (Nad 1 er et_ a K :, ] 954> Studer et aj_. , 1972). Once r e - e n t e r e d i n t o the t h y r o i d c e l l , r e lease of the t h y r o i d hormones from the thyrog lobu1 in is achieved by p r o t e o l y s i s , by a c i d proteases and pept idases conta ined in the lysosomes (Wol lman e_t£ a l . , 1964; Se 1 j e 1 id , 1968). The lysosomes migrate to the a p i c a l part of the c e l l on s t i m u l a t i o n to meet the incoming c o l l o i d d r o p l e t , and the iod ina ted o r g a n i c compounds are s p l i t o f f from the t h y r o g l o b u l i n (Evered, 1976). The t r i i o d o t h y r o n i n e and thy rox ine are then a v a i l a b l e f o r s e c r e t i o n and the uncoupled MIT and DIT are d e i o d i n a t e d w i t h i n the t h y r o i d c e l l , r e l e a s i n g iod ide f o r c o u p l i n g to t y r o s y l res idues at the a p i c a l s u r f a c e of the c e l l once more (Johnson, 1974). EFFECTS OF TSH ON THE THYROID GLAND Almost every in termediary step in thyroid :hormone b i o -s y n t h e s i s and re lease i s regu la ted in one way or another by t h y r o t r o p i n (TSH) from the p i t u i t a r y g l a n d . - 12 -W i t h i n a s h o r t t i m e a f t e r TSH s t i m u l a t i o n , i n c r e a s e d t h y r o i d a l m e t a b o l i c a c t i v i t y , such as i n c r e a s e d p h o s p h o l i p i d t u r n o v e r i s d e t e c t a b l e ( S c o t t et_ aj_. , 1968) . F o r m a t i o n o f pseudopods a t t h e a p i c a l e e l 1 b o r d e r i s o b s e r v e d , f o l l o w e d by i n c r e a s e d numbers o f i n t r a c e l 1 u l a r c o l 1oid d r o p l e t s ( K e t e l b a n t -B a l a r s e et_ aJL , 1976), a p i c a l m i g r a t i o n o f lysosomes, and s e c r e t i o n o f t h y r o i d hormone i n t o . t h e c i r c u l a t i o n . I t has a l s o been o b s e r v e d t h a t TSH s t i m u l a t i o n causes an i n c r e a s e i n t h y r o i d w e i g h t , mean a c i n a r c e l l h e i g h t , t h y r o i d h y p e r t r o p h y and a l o s s o f c o l l o i d and p r e v i o u s l y s t o r e d r a d i o i o d i n e from the t h y r o i d g l a n d ( K e a t i n g et_ aj_. , 19^5; Sasson and Rosenberg, 1963). F u r t h e r m o r e , TSH can combine w i t h a r e c e p t o r s i t e on the t h y r o i d c e l l membrane and s t i m u l a t e s t h y r o i d hormone p r o d u c t i o n a t a number o f p o i n t s i n t h e s y n t h e t i c pathway. TSH i n c r e a s e s i o d i d e t r a p p i n g a f t e r a l a t e n t p e r i o d by i n c r e a s i n g t h e i o d i d e pump a c t i v i t y (Sherwin and Tong, 1974; W i l l i a m s and Malayan, 1975). There i s an i n c r e a s e o f r i b o s o m a l p r o t e i n s y n t h e s i s i n response t o TSH l e a d i n g t o i n c r e a s e d f o r m a t i o n o f t h y r o g l o b u 1 i n and the enzymes i n v o l v e d i n hormonogenesis. Moreover, TSH stimu1 a t e s i o d i n a t i o n o f t y r o s i n e and t h e p r o t e o l y s i s o f t h y r o g l o b u l i n l e a d i n g t o r e l e a s e o f i o d o t h y r o n i n e s i n t o t h e c i r c u l a t i o n ( E v e r e d , 1976). These a c t i o n s a r e mediated by a c t i v a t i o n o f adenyl c y c l a s e and can be s t i m u l a t e d d i r e c t l y by c y c l i c AMP (P a s t a n and K a t z e n , 1967; F i e l d , 1975; E v e r e d , 1976). E x c e l l e n t reviews o f the a c t i o n o f TSH on t h y r o i d m e t a b o l i s m a re a v a i l a b l e (Dumont, 1971; S t e r l i n g and L a z a r u s , 1977)-- 13 -In a d d i t i o n to the e x t r a t h y r o i d a l c l a s s i c a l negat ive feedback c o n t r o l by t h y r o i d hormones and the h y p o t h a l a m i c : c o n t r o l by t h y r o t r o p i n r e l e a s i n g hormone v i a the hypotha1amohypophysea1 p o r t a l system CScha 11 y et_ aj_., 1969; Burgus and Gui11emin, 1970; Brenenman and Rathkamp, 1973), there.seems to be an i n t r a t h y r o i d a l autonomous mechanism whereby . thy ro ida 1 i od ine metabol ism i s regu lated (_Barakat and Ingbar, 1965; Studer and Greer , 1968; Socolow et a 1., 1968; S t e r l i n g and Lazarus , 1977)• It a l s o appears to a l t e r t h y r o i d hormone s e c r e t i o n , independently of TSH supply (.Green and Ingbar, 1962; Onaya and. Halm'*, 1967; Gaf n i et_ aj_. , 1977) -Solomon and Dowling (.i960) hypothes ized tha t au to regu la t ion of t h y r o i d a l i o d i d e t r a n s p o r t a l lows an i o d i d e - d e f i c i e n t animal to main ta in i t s i n t r a t h y r o i d a l hormone s t o r e s . The accumulated supply may then be re leased by the e x t r a TSH secreted in response to a lower iodothyron ine l e v e l in the b l o o d . EFFECTS OF IODIDE ON THE THYROID GLAND Iod ide , as we l l as TSH, can a f f e c t every step in the s y n t h e s i s and s e c r e t i o n of t h y r o i d hormones ( i ngbar , 1972). Hence, an excess or d e f i c i e n c y in iod ine in take can a f f e c t normal t h y r o i d metabol ism. When the in take o f iod ine d e c l i n e s , h y p e r p l a s t i c f o l l i c u l a r c e l l s are observed (Feldman, 1961). Furthermore, in iod ine d e f i c i e n t r a t s , the r a t i o o f T^/T^ in the t h y r o i d decreases . This may compensate f o r the iod ine d e f i c i e n c y s i n c e the oxygen - 14 -consumption and h e a r t r a t e o f t h e a nimal remains normal (Emrieh e t a 1 . , 1971). R i e s c o et_ aj_. (I977) o b s e r v e d i n . r a t s . c h r o n i c a l l y d e f i c i e n t i n i o d i n e t h a t T ^ . l e v e l i n b l o o d was u n d e t e c t a b l e w h i l e t h e l e v e l was low but s t e a d y . However, TSH l e v e l r o s e t o a ve r y h i g h l e v e l whereas t h y r o i d i o d i n e c o n t e n t reached i t s l o w est v a l u e a t f i f t e e n weeks and showed no f u r t h e r d e c r e a s e . On t h e . o t h e r hand, t h y r o i d w e i g h t appeared t o i n c r e a s e p r o g r e s s i v e l y . : DeGroot and Niep o m n i s z c z e . (1977) commented t h a t the main e f f e c t o f i o d i n e d e f i c i e n c y on the f o r m a t i o n o f t h y r o i d hormone i s a f e e b l e i o d i n a t i o n o f thyrog1obu1in w i t h an i n c r e a s e d MIT/DIT r a t i o and i n c r e a s e d T^/T^ r a t i o . The i n c r e a s e d p r o d u c t i o n o f T^ i s a r e s u l t o f poor t h y r o g l o b u l i n i o d i n a t i o n and.the r e l a t i v e l y i n c r e a s e d MIT/DIT r a t i o . Augmented TSH may a l s o p l a y a r o l e s i n c e T^ f o r m a t i o n i s s t i m u l a t e d by and dependent upon TSH. As the l e v e l o f t h y r o g l o b u l i n i o d i n a t i o n f a l l s below 0.1%, i o d o t h y r o n i n e format i o n drops p e r c i p i t u o u s 1 y . Presumably, t h i s r e p r e s e n t s a s t a t e o f decompensation due t o i o d i d e d e f i c i e n c y and may l e a d t o h y p o t h y r o i d i s m . D i m i n i s h e d T^ and T^ s y n t h e s i s leads t o low plasma hormone 1 eve 1s, e l e v a t e d TSH and t h e t y p i c a l g o i t r o g e n i c response t o i o d i d e d e f i c i e n c y . Excess i o d i d e had been c l a s s i f i e d as a c l a s s o f a n t i t h y r o i d a g ents by Greer et_ a_l_. (1964). Hence, the e f f e c t s o f ex c e s s i o d i d e w i l l be reviewed i n the s e c t i o n on a n t i t h y r o i d compounds. A n a l y s i s o f d i s t r i b u t i o n o f iodoamino a c i d s i n . t h y r o i d t i s s u e s has shown l i t t l e d i f f e r e n c e between t h e a v i a n and the mammalian g l a n d s (Shel 1 aba r g e r and P i t t - ^ R i v e r s , 1953- Mel 1 en and Wentworth, 1959). Hence a d i v e r s i t y o f mechanisms has not been r e c o g n i z e d f o r t h y r o i d hormone s y n t h e s i s i n d i f f e r e n t s p e c i e s . - 15 -U l t r a s t r u c t u r a l s t u d i e s of t h y r o i d gland of fowl a re a 1 so . found to be s i m i l a r to those of mammals except in the subce l1u la r d i s t r i b u t i o n of dark bodies and the s i z e ' o f 1ysosomes (French and Hodges, 1977)• However, i t should be noted that t r i i o d o t h y r o n i n e has a h igher potency than an equimolar q u a n t i t y of thy rox ine in other animals whi1e the iodothyronines have.no d i f f e r e n t r e l a t i v e potenc ies in b i r d s .(Hutch ins and Newman, 1966;' S h e l l a b a r g e r , 1955; Newcomer, 1957; Robbins and Rau, 1957)- Furthermore, Tata and S h e l l a b a r g e r (1959) found that t h y r o x i n e - b i n d i n g g l o b u l i n is absent in ch icken blood which probably e x p l a i n s > t h e . f a i 1 u r e . o f p ro te in -bound iod ine (PBI) to serve as a c r i t e r i o n of measurement of t h y r o i d hormone metabol ism i n the b i r d . ANT ITHYROI DAL COMPOUNDS AND THE IR MECHANI SMS OF ACTION The e x i s t e n c e of substances which can n e u t r a l i z e e x c e s s i v e amounts of t h y r o i d hormone and thus exer t a n t i t h y r o i d e f f e c t s has been e n t e r t a i n e d s i n c e i t f i r s t became c l e a r that the t h y r o i d gland possessed endocr ine f u n c t i o n . Var ious unsubstant ia ted c la ims f o r the e x i s t e n c e of such a substance were made. The s t u d i e s of Barker (1936); R i c h t e r and C l i s b y (1941); and Mackenzie et_aj_. (1941) s t i m u l a t e d a great deal of s c i e n t i f i c endeavour in t h i s a r e a . Astwood et a 1. (1943) and the Mackenz ie ' s (1943) pub l i shed s t u d i e s on the nature of the a n t i t h y r o i d e f f e c t s of t h i o u r e a d e r i v a t i v e s and :sulphonamides which c l e a r l y i n d i c a t e d that these m a t e r i a l s acted by i n h i b i t i n g format ion of t h y r o i d hormone. - 16 -Astwood (1955) commented that when f i r s t encountered; the compounds which i n h i b i t e d t h y r o i d hormone s y n t h e s i s seemed to f a l l i n to two c a t e g o r i e s , t h i o u r e a and r e l a t e d substances and d e r i v a t i v e s of a n i l i n e . However, when more compounds were s t u d i e d , many except ions to t h i s g e n e r a l i z a t i o n were encountered. Many of t h e s e . a n t i t h y r o i d agents can produce i n h i b i t i o n of t h y r o i d funct ion in v i v o , a t dosage l e v e l s which do not have major adverse e f f e c t s on o ther organ systems. The i r a d m i n i s t r a t i o n to i n t a c t man or animals can lead to decreased l e v e l s of c i r c u l a t i n g t h y r o i d hormone, a c t i v a t i o n of t h y r o i d s t i m u l a t i n g hormone (TSH) s e c r e t i o n , and u l t i m a t e l y to t h y r o i d enlargement or g o i t e r (Green, 1971). Greer et_ a_K (1964) d i v i d e d the a n t i t h y r o i d compounds in to f i v e s p e c i f i c c l a s s e s . These are the th ionamides , a n i l i n e and a m i n o h e t e r o c y c l i c compounds, monovalent anions other than i o d i d e , iod ide and o ther misce l laneous i n h i b i t o r s i n c l u d i n g s u b s t i t u t e d pheno ls . These i n h i b i t o r s can be f u r t h e r d i v i d e d in to two c a t e g o r i e s : agents that i n h i b i t t h y r o i d i od ide t r a n s p o r t and those that i n h i b i t the complex of r e a c t i o n s invo lved in o r g a n i c b i n d i n g and c o u p l i n g p rocesses . Monovalent anions belong to the former category w h i l e th ionamide , a m i n o h e t e r o c y c l i c compounds, s u b s t i t u t e d phenols and iod ide belong to the l a t t e r group (Green, 1971; Ingbar and Woeber, 1974). - 17 -MONOVALENT ANIONS OTHER THAN IODIDE The a c t i v e t r a n s p o r t mechanism r e s p o n s i b l e f o r c o n c e n t r a t i n g ino rgan ic i od ide in the t h y r o i d is a l s o a c t i v e toward s e v e r a l other monovalent anions whose molecu la r s i z e is s i m i l a r to that of i od ide (Wol f f , 1964; Greer , Kenda11 and Smi t h , 1964). Thus, p e r c h l o r a t e (CIO^ ) , pe r technetate (TcO^ ) , perrhenate (ReO^ ) and t e t r a f l u o r o -borate (BF^ ) , can be concent rated by the t h y r o i d , w i thout undergoing s i g n i f i c a n t metabo l i c t r a n s f o r m a t i o n (Green, 1971)- These anions can c o m p e t i t i v e l y i n h i b i t the a c t i v e t r a n s p o r t of i o d i d e . Thus, when iod ine in take i s low, these anions can l i m i t iod ine accumulat ion so markedly that hypothyro id ism and g o i t e r r e s u l t . Other anions are a l s o found to i n h i b i t i od ine c o n c e n t r a t i o n or promote d ischarge of iod ide from the t h y r o i d glands of ra ts c h r o n i c a l l y t r e a t e d w i t h p r o p y l t h i o u r a c i l . They are c h l o r a t e , h y p o c h l o r i t e , p e r i o d a t e , i o d a t e , b i i o d a t e and n i t r a t e (Wyngaarden et a 1. , 1952). However, p e r c h l o r a t e was found to be the most potent among these anions w h i l e n i t r a t e was the l e a s t e f f e c t i v e . Thiocyanate (SCN ) is a l s o a potent i n h i b i t o r of i o d i d e t ranspor t and has a molecu la r s i z e s i m i l a r to o ther t r a n s p o r t i n h i b i t o r s (Wol f f , 1964). But i t d i f f e r e d in severa l aspects from the anions d iscussed p r e v i o u s l y . F i r s t l y , i t i s not concentrated by the t h y r o i d t i s s u e (Maloof and Soodak, 1959)• Secondly , i t is a c t i v e l y metabol i zed by the t h y r o i d in V ivo and in c e l l - f r e e p repara t ions (Maloof and Soodak, 1959; 1966), F i n a l l y , th iocyanate - 18 -i s an i n h i b i t o r of o r g a n i c i o d i n a t i o n at c o n c e n t r a t i o n s s l i g h t l y g reate r than those which i n h i b i t i o d i d e t r a n s p o r t (Greer et a l . , 1066). It has been proposed that t h i o c y a n a t e , in a d d i t i o n to i n h i b i t i n g t h y r o i d a l i od ide t r a n s p o r t , is a l s o a c o m p e t i t i v e s u b s t r a t e of the t h y r o i d a l i o d i d e perox idase (Green, 1971)• There fo re , t h i s can e x p l a i n : i t s b i o t r a n s f o r m a t i o n and i t s a b i l i t y to i n h i b i t i o d i n a t i o n u . Both t h i o c y a n a t e a n d - p e r c h l o r a t e have been used c l i n i c a l l y to t r e a t h y p e r t h y r o i d i s m . However, due to t h e i r t o x i c i t y , n e i t h e r of them is any longer w ide l y used c1 in ica11y , a l though they are u s u a l l y e f f e c t i v e agents in t h i s respect ( ingbar and Woeber, 1974). THIONAMIDES As a c l a s s , the thionamide compounds, which inc lude the c l a s s i c a n t i t h y r o i d agents such as t h i o u r e a , t h i o u r a c i 1 , p r o p y 1 t h i o u r a c i 1 , 1 -methy l -2 -mercapto imidazo le (methimazole) and c a r b i m a z o l e , are the most potent i n h i b i t o r s of t h y r o i d hormone fo rmat ion known. They do not i n h i b i t i od ide t r a n s p o r t , and thus t h e i r a d m i n i s t r a t i o n r e s u l t s in accumulat ion o f p e r c h l o r a t e d i s c h a r g a b l e i o d i d e . The i r a c t i o n a l s o cannot be prevented by la rge doses of i od ide ( ingbar and Woeber, 1974). The e f f e c t s , o f a d m i n i s t e r i n g thionamides to animals have been noted f o r a long t ime . Astwood and B i s s e l l (1944) commented that a f t e r t h i o u r a c i l t rea tment , growth rate of ra ts was depressed, - 19 -t h y r o i d weight increased sharp l y and a marked compensatory h y p e r p l a s i a of the t h y r o i d g land can be observed . F u r t h e r m o r e , . t h e ' i od ine va lue w i t h i n the t h y r o i d gland was extremely low compared to those of c o n t r o l and f i n a1 l y , t h y r o i d hormone product ion was prevented. These observa t ions were conf i rmed by other i n v e s t i g a t o r s us ing r a b b i t s and ch i cks f ed . th iourea or th iourac i 1 (Bauman et_ aj_. , 1944; Mixner et a l . , 1944;. Larson e t^a j . , 1945) . There are numerous repor ts concern ing the i n h i b i t o r y e f f e c t of PTU and of t h i o u r a c i1 on thyro id :hormone b i o s y n t h e s i s . However, Wentworth and Mel 1 en (1961) coneluded from t h e i r exper imental f i n d i n g s that t h i o u r a c i1 d i d . n o t complete ly b lock t h y r o i d hormone synthes in ch icks even when the drug was inc luded in a d i e t g i yen ad 1 i b iturn. I n h i b i t i o n of t h e c a p a c i t y of t h y r o i d gland to o r g a n i f y the i n j e c t e d r a d i o i o d i n e has been observed (Larson et a 1., 1945a, b) i n d i c a t i that o r g a n i f i c a t i o n of iod ide is i n h i b i t e d . This has been conf i rmed l a t e r (Lazarus et_ a j_ . , 1975) - Moreover, L a b e l l e (1964) suggested that PTU may ac t on p i t u i t a r y g1 and to r e l e a s e t h y r o t r o p i n d i r e c t l y , but a c t i o n s on the t h y r o i d gland may a l s o be p o s s i b l e . However, by a d m i n i s t e r i n g graded doses of PTU to r a t s , i t has been shown that each step in hormone s y n t h e s i s is a f f e c t e d . The most s u s c e p t i b l e step being the c o u p l i n g of i o d o t y r o s i n e s to form iodothyron ines (Green, 1971; Kasai et_ a j_ . , 1976). l o d i n a t i o n of monoi odotyros i ne (MIT) to form d i i o d o t y r o s i n e i s less s e n s i t i v e w h i l e i o d i n a t i o n of t y r o s i n e to form MIT is the l e a s t s e n s i t i v e towards PTU treatments ( l i n o , 1961; SI i nger land et_ a j_ . , 1959; Green, 1971 ;• Richards and Ingbar, 1959). - 20 -Furthermore, PTU a l s o i n h i b i t s the e x t r a t h y r o i d a l convers ion of t h y r o x i n e (T^) to t r i i o d o t h y r o n i n e ( T^) (Bernal et_ a_l_. , 1974; Oppenheimer et^aj_. , 1972; Geff ner et^ a j_ . , 1975), t h u s , t h i s may account f o r the increased f r e e f r a c t i o n observed by M i c h a j l o v s k i j et a 1. Cl976). In a d d i t i o n to t h e i r a n t i t h y r o i d a c t i o n , some th ionamides , i n c l u d i n g t h i o u r a c i l and PTU, i n h i b i t the p e r i p h e r a l d e i o d i n a t i o n of t h y r o i d hormones (Launay, 1965; Gef fner e t aj_. , 1975). The c o n c e n t r a t i o n of c y c l i c AMP does not inc rease a f t e r PTU treatment in ra ts when -s t i m u l a t e d by t h y r o t r o p i n (TSH) ( Z a k a r i j a et_ a_l_. , 1975). An acute increase in TSH s e c r e t i o n a l s o occurs when PTU i s g iven to i n t a c t ra ts (Escobar del Rey et_ a_l_. , 1962; Mouriz et_ aj_. , 1966). Thionamides can i n h i b i t the o x i d a t i o n of va r ious s u b s t r a t e s i n c l u d i n g i o d i d e , by v a r i o u s p e r o x i d a s e s , i n c l u d i n g perox idases i s o l a t e d from t h y r o i d t i s s u e (A lexander , 1959; DeGroot and D a v i s , 1962; Hosoya, 1963; Mor r i s and Hager, 1966; Cova1 and Taurog, 1967; Taurog, 1970; Dempsey, 1944; Van Zyl et_ a_l_. , 1924; Nagasaka e_t aj_. , 1975; Taurog, 1976). The i n h i b i t i o n seems to be c o m p e t i t i v e w i t h iod ide (DeGroot and D a v i s , 1962; Cova1. and Taurog, 1967)• Green (1971) commented that the a c t i o n of the thionamides is based on the theory that the iod inated in te rmediates suggested as l + or I0H^+ (Tong, 1971), is a p r o t e i n s u l f e n y l i od ide (Maloof and Soodak, 1963) w i t h which thionamides react to form a mixed d i s u l f i d e (Cunningham, 1964). Thus, by d e s t r o y i n g the s u l f e n y l i o d i d e , thionamides would prevent t r a n s f e r of iod ine to t y r o s y l groups in the t h y r o g l o b u l i n . However, d i r e c t evidence fo r the r o l e of s u l f e n y l i od ide in p h y s i o l o g i c a l i o d i n a t i o n s i s needed. - 21 -Nagasaka and Hidaka (1976) compared the e f f e c t s of methimazole and p ropy1 th iourac i1 . on human t h y r o i d peroxidase. They found that PTU i n t e r a c t e d d i r e c t l y . w i t h the product of t h y r o i d i o d i d e p e r o x i d a s e , namely o x i d i z e d i o d i d e in the r e a c t i o n mixture wi thout s i g n i f i c a n t l y a f f e c t i n g the perox idase a c t i v i t y . The methimazole i n t e r a c t e d d i r e c t l y w i th the perox idase and i n h i b i t e d enzyme a c t i v i t y . Thus, PTU i n h i b i t i o n on the perox idase is r e v e r s i b l e w h i l e the i n h i b i t i o n by methimazole on the t h y r o i d perox idase is i r r e v e r s i b l e . AMINOHETEROCYCLIC COMPOUNDS The aminoheterocyc1 ic compounds are in general f a r l ess potent than the thionamides (Vagenakis and Braverman, 1976). The i r e f f e c t s on the t h y r o i d a re sometimes mani fested dur ing t h e i r use in the treatment of o ther d i s e a s e s . For example, para-ami n o - s a l i c y c l i c a c i d , used as an a n t i t u b e r c u l o s i s agent , i s g o i t r o g e n i c in r a t s , lowers t h y r o i d a l 1-131 uptake in man and o c c a s i o n a l l y produces g o i t e r w i th or w i thout hypothyro id ism (Komrower, 1951; McGregor and Somner, 1954; Ingbar and Woeber, 1974). The i r mode of a c t i o n is s i m i l a r to the thionamides (Edwards et_ aj_. , 1954). The hypoglycemic s u l f o n y l u r e a s decrease I —131 uptake in man a l though they are not s u f f i c i e n t l y potent to be g o i t r o g e n i c in man. The g o i t r o g e n i c e f f e c t of para -amino -s a l i c y c l i c a c i d and the s u l f o n y l u r e a s , l i k e that of th ionamides , is s l i g h t l y decreased by la rge amount of i o d i n e (Mackenzie , 1947; M i l n e and Greer , 1962; Brown and Solomon, 1958). An a d d i t i o n a l group of agents in t h i s c l a s s is su l fonamides . Al though they have not been shown to be g o i t r o g e n i c in man, they stand apar t in that - 22 -t h e i r a n t i t h y r o i d potency i s markedly p o t e n t i a t e d by iod ide (Green, 1971)- Th is and other ev idence i n d i c a t e s that the mechanism o f a c t i o n of sul fonamides d i f f e r s from that of the thionamides and of o ther aminoheterocyc1 i c compounds which i n h i b i t o r g a n i c b ind ing and c o u p l i n g r e a c t i o n in the t h y r o i d g l a n d s . IODIDE Iodine i s the b a s i c element requ i red f o r the s y n t h e s i s of the t h y r o i d hormones. P a r a d o x i c a l l y , hypothyro id ism w i t h or wi thout g o i t e r may be induced by the c h r o n i c a d m i n i s t r a t i o n of i od ine (Vagenakes and Braverman, 1976). Acute a d m i n i s t r a t i o n of iod ide can produce I n h i b i t i o n of t h y r o i d hormone f o r m a t i o n , the W o l f f -Cha iko f f e f f e c t . (Wolff and C h a i k o f f , 1948). Hence, the e f f e c t of a high iod ine in take on t h y r o i d f u n c t i o n is a combinat ion of the e f f e c t s of the pharmacological a c t i o n of iod ide and the e f f e c t s o f iod ide as a s u b s t r a t e f o r s y n t h e s i s of t h y r o i d hormone (Nagatak i , 1976). When graded doses of iod ide are g iven to r a t s , a b i p h a s i c e f f e c t on i o d i n a t i o n can be shown- Modest i od ide supplements lead to increased i o d i n a t i o n u n t i l a c r i t i c a l dosage i s reached above which i o d i n a t i o n s p r o g r e s s i v e l y d i m i n i s h (Nagataki and Ingbar, 1964) At t h i s s tage , iodothyron ine s y n t h e s i s i s more markedly i n h i b i t e d than i o d o t y r o s i n e s y n t h e s i s . H e n c e , i o d i d e resembles a n t i t h y r o i d agents in t h i s respect (Green, 1971) • I n h i b i t i o n c o r r e l a t e s best w i t h i n t r a t h y r o i d a l iod ide l e v e l s , hence, the iod ide t r a n s p o r t mechanism - 23 -may be i n v o l v e d . When the t r a n s p o r t mechanism is v e r y . a c t i v e , smal l doses of iod ide can produce i n h i b i t i o n which can be reversed by b l o c k i n g the t r a n s p o r t system by th iocyanate (Braverman and ingbar , 1963). The i n h i b i t o r y e f f e c t i s u s u a l l y t r a n s i e n t d e s p i t e a cont inuous high iod ine in take (Wolff et a l . 19^9). Th is escape from the W o l f f - C h a i k o f f e f f e c t may occur through a m o d i f i c a t i o n of e i t h e r iod ide t ranpor t o r r o r g a n i c b ind ing or both (Nagatak i , 1976). Th is change i s not dependent on a l t e r e d TSH s e c r e t i o n , and thus represents some autoregu1atory process in the t h y r o i d . iod ine a l s o I n h i b i t s the rap id hormone s e c r e t i o n in p a t i e n t s w i t h Graves' d i s e a s e and in TSH- t reated normal sub jec ts (Solomon, 1965)> the same e f f e c t may a l s o occur in uns t imulated normal sub jec ts (Mercer e t a l . , i960) and ra t (Onaya and H a l m i , 1967)- Th is i n h i b i t i o n can be accounted fo r by the decrease in the s t i m u l a t i o n of cAMP c o n c e n t r a t i o n (Rapoport et a l . , 1975; Sherwin and Tong, 1975)-Rapoport et a l . (1976) commented that there is an unknown o r g a n i c form of iod ine present that l i m i t s t h y r o i d adeny la te c y c l a s e responsiveness to t h y r o t r o p i n . Th is - "can e x p l a i n in part the g e n e r a l l y i n h i b i t i n g a c t i o n of iod ide in t h y r o i d a l f u n c t i o n s such as the iod ide t r a n s p o r t mechanism (Studer et a 1., 1976), o rgan ic i od ine s e c r e t i o n and t h y r o i d growth. Yamada (1976) on the other hand, commented that excess i od ide appeared to mani fes t i t s i n h i b i t o r y e f f e c t on t h y r o i d hormone s e c r e t i o n at a s i t e subsequent to generat ion of cAMP. - 24 -MISCELLANEOUS INHIBITORS There are m a n y o t h e r a n t i t h y r o i d compounds present i n c l u d i n g the s u b s t i t u t e d phenols which a l s o i n h i b i t the o r g a n i c b ind ing of i o d i d e . R e s o r c i n o l , a cutaneous a n t i s e p t i c , can produce g o i t r o u s hypothyro id ism in man (Ingbar and Woeber, 1974). An e a r l y study revealed that the g r e a t e s t a n t i t h y r o i d a c t i v i t y res ided in aromat ic compounds w i th hydroxyl groups meta to one another (Arnott and Doniach, 1952). The f a c t that these compounds are a l s o perox idase i n h i b i t o r s i s f u r t h e r i n d i r e c t ev idence that a perox idase mediates o rgan ic i o d i n a t i o n s in the t h y r o i d gland (Rosenburg, 1952). A number of l e s s e a s i l y c a t e g o r i z e d compounds have a n t i t h y r o i d a c t i v i t y . A m i n o t r i a z o l e (Mayberry, 1968) and t r i -cyanoami nopropene ( Ingbar , l'96l) i n h i b i t t h y r o i d a l iod ine uptake and produce g o i t e r in l a b o r a t o r y a n i m a l s . Greer et a l . (1964) pub l i shed an e x t e n s i v e survey on other m i s c e l l a n e o u s compounds w i t h a n t i t h y r o i d a c t i v i t y . NATURALLY OCCURRING GOITROGEN There i s another c l a s s of a n t i t h y r o i d compounds which i s n a t u r a l l y o c c u r r i n g and. which w i l l be reviewed below. GOITROGENIC SUBSTANCES IN RAPESEED MEAL Most p l a n t s of the C r u c i f e r a e f a m i l y that serve as sources of food and condiments belong to the B r a s s i c a genus. The seed meals - 25 -of rape ( B r a s s i c a Napus L.) a re high in p r o t e i n and can be used as a feed f o r l i v e s t o c k and p o u l t r y ( C l a n d i n i n and Robblee, 1966; C l a n d i n i n eJ^aJL , 1959; C l a n d i n i n and T a j c n a r , I 9 6 I ; C l a n d i n i n , 1967; Rutkowski , 1971)• However, rapeseed meal may c o n t a i n g l u c o s i n o l a t e s (Rutkowski , 1971) , which are s p e c i f i c components of p l a n t s in the C r u c i f e r a e f a m i l y (VanEtten, 1969; K j a e r , 1976) . The C r u c i f e r a e possess more than 60 g l u c o s i n o l a t e s w i t h i n the f a m i l y (K jaer , 1976) . W i t h i n the genus B r a s s i c a to which rape be longs , there are at l e a s t seven d i f f e r e n t g l u c o s i n o l a t e s known to occur and they a re d i s t r i b u t e d in almost a l l par ts of the p l a n t s ( J o s e f s s o n , 1967; Josefsson and Muhlenberg, 1968) . A group of enzymes, myrosinases i s present in the t i s s u e s of these g l u c o s i n o -l a t e - p r o d u c i n g p l a n t s ; however, these enzymes a re s e p a r a t e l y depos i ted w i t h i n the p l a n t s . B i o l o g i c a l l y i n a c t i v e g l u c o s i n o l a t e s are present in the seed o f B r a s s i c a p l a n t s . D e s t r u c t i o n o f the c e l l u l a r s t r u c t u r e a l lows the g l u c o s i n o l a t e s to come i n t o contact w i t h the myros inases . These enzymes hydro lyze the g l u c o s i n o l a t e s , l i b e r a t i n g g lucose and b i s u l p h a t e , w h i l e b i o l o g i c a l l y a c t i v a t i n g the compounds i n t o one of the three groups: g o i t r i n and r e l a t e d n i t r i l e s , o r g a n i c i s o t h i o -cyanates and ino rgan ic th iocyanate (Rutkowski , 1971; K j a e r , 1976; VanEt ten , 1969)-The problems of feed ing rapeseed meals to l i v e s t o c k have been s tud ied ( B e l l , 1955; B e l l , 1957: Manns and Bowland, 1963; Manns et_aj_. , 1963 , C l a n d i n i n and Robblee, V966; March et_aj_. , 1972; Leung and March, 1976) . Astwood e_t_aj_. (1949a ; b) and C a r r o l l (19^9) - 26 -have shown the presence of L - 5 - v i n y 1 - 2 - o x a z o l i d i nethione(goi t r i n) in seeds of rape and t u r n i p - r a p e . Th is compound can account f o r the g o i t r o g e n i c p r o p e r t i e s observed in rapeseed mea1. However, i t i s a h y d r o l y s i s product of i t s p r e c u r s o r s , p r o g o i t r i n and e p i -p r o g o i t r i n . G o i t r i n i s probably not pre- formed in the g1ucos ino la te molecule but a r i s e s from o x i d a t i o n a f t e r the a c t i o n of myrosinase ( P i t t - R i v e r s , 1950). The mode of a c t i o n of g o i t r i n on the t h y r o i d gland i s not we l l understood. However, i t i s known that g o i t r i n b locks the i r r e v e r s i b l e mechanism connected w i t h the o r g a n i c b ind ing of i od ine in the t h y r o i d , hence suppress ing the t h y r o i d hormone s y n t h e s i s p a r t i a l l y (Rutkowski , 1971; Lo and B e l l , 1972). However, Matsumoto et a l . (1968; I969) s ta ted that there i s no ev idence to i n d i c a t e that the i n c o r p o r a t i o n of i od ide i n t o the t h y r o i d is i n h i b i t e d by the g o i t r i n a l though i n h i b i t i o n of the s y n t h e s i s of t h y r o i d hormone i s observed . Both C l a n d i n i n and Bayly (i960) and Rutkowski (1971) desc r ibed the h i s t o p a t h o l o g i c a l p i c t u r e of the rapeseed mea l - fed ch icken t h y r o i d glands showing hypertrophy and h y p e r p l a s i a of parenchymal elements and almost complete loss of f o l l i c u l a r c o l l o i d . Photomicrographs of ch ickens fed rapeseed meal f o r four weeks a l s o showed desquamation of e p i t h e l i u m , compressed f o l l i c l e s in smal l r e s t r i c t e d a r e a s , and decrease in c o l l o i d (Rutkowski , 1971)-I so th iocyanates and th iocyanates can a l s o be found in rape and t u r n i p - r a p e seeds. The th iocyanates have c o n s i d e r a b l y lower goi t r o g e n i c e f f e c t s than the o x a z o l i d inethione* whii1e - 27 -i s o t h i o c y a n a t e s are c h a r a c t e r i z e d by s t i l l weaker g o i t r o g e n i c a c t i v i t y (Fertman and Curt 1s; 1951; Gmelin and V i r t a n e n , I960). However, t h e i r i n f l u e n c e on t h y r o i d gland i s s i m i l a r . They b lock the t r a p p i n g of i od ine by the t h y r o i d gland and set f r e e the iod ine accumulated in i t . By the a d d i t i o n of more iod ine to the r a t i o n , t h i s g o i t r o g e n i c e f f e c t of i s o t h i o c y a n a t e s and th iocyanates can be removed (Greer , 1957; Langer , I960; Rutkowski , 197 0 -It has been known f o r a long time that there are many g l u c o s i n o l a t e s in the rapeseed meal (Fertman and C u r t i s , 1951; B e l l and W i l l i a m s , 1953; Josefsson and Muhlenberg, 1968). Hence, a synerg ic e f f e c t of these g l u c o s i n o l a t e s may be p o s s i b l e s i n c e Langer (1966) showed that synerg ic e f f e c t s of g o i t r i n , a 11y1 isothiocyanate and th iocyanate on t h y r o i d glands of r a t s do e x i s t . G o i t r i n , th iocyanate and i s o t h i o c y a n a t e a l s o have s y n e r g i s t i c e f f e c t s on the t h y r o i d gland (Langer and Greer , 1968) . INFLUENCE OF L~5"VINYL-2-0XAZ0LIDINETHI ONE ON THE THYROID GLANDS OF CHICKENS The d i f f i c u l t i e s invo lved in the feed ing of rapeseed meal to p o u l t r y have been reviewed ( C l a n d i n i n and Robblee, 1965; Rutkowski , 1971; Witz et_a_j_., 1950; K l a i n et_ aj_. , 1956). Thyro id hypertrophy has been reported in domestic animals fed rapeseed meal ( K l a i n e t a l . , 1956; K rus ius and P e l t o l a , 1966; Langer, 1966; Cland i ni n et a l . , I966), and has been a t t r i b u t e d to the e f f e c t of - 28 -L _ 5 ~ v i n y l-2 - o x a z o l i d i n e t h i o n e (Astwood et_ aj_. :, 1949a, b; Car ro l 1 , 1949) and a l l y l or c r o t o n y l i s o t h i o c y a n a t e s (Dow and A l1 e n , 1954). G o i t r i n i s the most potent natura l g o i t r o g e n i c compound to have been i s o l a t e d . (Langer et • a 1 . , 1 9 7 0 -C l a n d i n j n et a l . (1966) s tud ied the e f f e c t s of feed ing s y n t h e t i c g o i t r i n on the t h y r o i d metabol ism of growing c h i c k s . They observed that i od ine uptake is reduced and the c o l l o i d s t o r e s in the t h y r o i d glands are dep leted i n i t i a l l y . Chick growth ra te i s depressed. Then rap id enlargement of the glands f o l l o w s wi th accompanying hypertrophy and h y p e r p l a s i a . Iodine uptake increases at the same t ime . Thus the c o l l o i d s to res re tu rn and the t h y r o i d takes on a more normal appearance. F i n a l l y , g l a n d u l a r a c t i v i t y comes in to balance at an inc rease t h y r o i d to body weight r a t i o s . Matsumoto et a l . (1968) s tud ied the g o i t r o g e n i c e f f e c t s of g o i t r i n , 1 -methy l-2 -mercapto imidazo le (methimazole) and 6 - m e t h y l -t r i o u r a c i l ( m e t h i o c i l ) in growing c h i c k s and reported that the c h i c k s r e c e i v i n g .05% g o i t r i n showed t h y r o i d enlargement, h ighest t h y r o i d a l uptake of r a d i o i o d i n e 131 ( 1 - 1 3 0 and a slow r e l e a s e of 1-131. However, these o b s e r v a t i o n s a re in c o n t r a s t to those on the methimazole or m e t h i o c i1 - f e d c h i c k s . They concluded that the i n c o r p o r a t i o n of blood 1-131 by the t h y r o i d gland is not i n h i b i t e d ; but a decrease in t h y r o i d hormone in the blood i s observed , because of depress ion of P BI -131 c o n t e n t . Hence, i t i s assumed from the slow r e l e a s e of 1-131 and the depressed t h y r o i d hormone in b l o o d , that some step of t h y r o i d hormone s y n t h e s i s and f u r t h e r s e c r e t i o n of the t h y r o i d hormone in to blood or d e i o d i n a t i o n of some iodoamino - 29 -a c i d s in t h y r o i d gland a r e . i n h i bi ted by treatment w i t h g o i t r i n . The d i f f e r e n t g o i t r o g e n i c e f f e c t s o f p r o p y l t h i o u r a c i l (PTU) and g o i t r i n in growing c h i c k s . w e r e f u r t h e r s tud ied by Matsumoto et_ a\_. (1969) and Akiba and Matsumoto (1971). They showed that the t h y r o i d glands of the PTU fed c h i c k s r e l e a s e the incorporated r — 131 i n t o the blood eas i1y whi1e the t h y r o i d in the g o i t r i n c h i c k s can main ta in a l a r g e r amount of incorporated I —131 and appears to i n h i b i t i t s r e l e a s e . Furthermore, the g o i t r i n - t r e a t e d c h i c k s have a lower percentage of iodothyron ines (T^ and T^) in t h e i r t h y r o i d glands and have a higher percentage of MIT and a h igher MIT/DIT r a t i o than do c o n t r o l b i r d s . It i s c o n c e i v a b l e that g o i t r i n does not i n h i b i t themonoiodinat ion of t y r o s i n e , but i t main ly i n h i b i t s the c o u p l i n g of MIT to s y n t h e s i z e DTT. It i s a l s o assumed that g o i t r i n i n h i b i t s the h y d r o l y s i s of t h y r o g l o b u l i n and r e l e a s e of t h y r o i d hormone to decrease the s e c r e t i o n of t h y r o i d hormone i n t o the b l o o d . The f i n d i n g of increased MIT percentage i s in agreement w i th r e s u l t s of o ther s t u d i e s in which r a t s are fed PTU (I i no et_ aj_. , 1961 ; Greer et_ a_l_. , 1962), and in b i r d s fed rapeseed meal (Leung and March, 1976). A f t e r the withdrawal of g o i t r i n from the d i e t of c h i c k s a f t e r twenty-one days of f e e d i n g , Ak iba and Matsumoto (1973) showed that there is a rap id r e s t o r a t i o n of normal t h y r o i d metabol ism and b i o s y n t h e s i s . However, hypertrophy of the t h y r o i d g lands p e r s i s t s f o r a r e l a t i v e l y longer p e r i o d . A f t e r the withdrawal of g o i t r i n , plasma PBI-131 l e v e l s i n c r e a s e d . Th is rebound phenomenon is s i m i l a r to that o c c u r r i n g upon g o i t r o g e n withdrawal in other l a b o r a t o r y animals such as guinea p i g s , r a t s and mice (D1 Angelo et_ a_l_. , 1951; 1954; L ipner et a l . , 1959; Studer and Greer , 1967; Langer , I968). It - 30 -can be explained by an o v e r s t i m u l a t i o n of TSH on the unblocked t h y r o i d gland a f t e r the wi thdrawa 1 of goitrogens (D'Angelo, l'96l ; 1969). The gradual decrease in t h y r o i d weight a f t e r g o i t r i n withdrawal i s a l s o observed a f t e r PTU withdrawal i n . c h i c k s CA k i ba et_ aj_. I97I) and guinea pigs (D1 Angel o et_ aj_. . 1954). B e l l (I955) commented that in p r a c t i c a l feeding of l i v e s t o c k , the percentage of rapeseed meal included should not exceed 10% because of i t s g o i t r o g e n i c p r o p e r t i e s . However, approaches to s e l e c t i o n of v a r i e t i e s of rapeseed low in g1ucosinolate content has been i n i t i a t e d (VanEtten 1969)- P r e s e n t l y , new c u l t i v a r s of rape have been developed in which the concentration of g l u c o s i n o l a t e s have been much reduced.Tower rapeseed meal contains only 0.5 " 1•5 mg isothiocyanate per gram of o i l - f r e e meal (McGregor and Downey, 1975). A recent low g1ucosinolate c u l t i v a r developed i s Candle. The g o i t r o g e n e i c i t y of Candle seed has yet to be evaluated. - 31 -Experiment 1: EFFECTS OF IODINE DEFICIENCY AND THIOURACIL ON THE THYROID HISTOLOGY OF WHITE LEGHORN HENS The f o l l o w i n g experiment was conducted to compare the e f f e c t s of i o d i n e d e f i c i e n c y and t h i o u r a c i l on the h i s t o l o g i c a l changes in the t h y r o i d glands of a d u l t b i r d s . Mater?a1s and Methods F i f t e e n White Leghorn hens (61 weeks o ld ) which had been fed a p r a c t i c a l d i e t were used in t h i s exper iment . They were housed in i n d i v i d u a l cages. Exper imental r a t i o n s (Table I) were fed ad 1? b i turn to groups o f f i v e b i r d s f o r 11 weeks. The body weight of each b i r d was recorded at the beginning and the end of the experiment when the t h y r o i d glands of each b i r d were exc i sed and weighed. The t h y r o i d glands were then f i x e d in t i s s u e f i x a t i v e (Baker ' s formal ca lc ium) f o r h i s t o l o g i c a l s t u d i e s . " The f i x e d glands were sec t ioned at 6 microns and s t a i n e d w i th haema-t o x y l i n and e o s i n . By us ing a l i g h t microscope f i t t e d w i t h an eyepiece micrometer , the s e c t i o n s taken across the m i d - p o r t i o n of the gland were chosen f o r measurement of the percentage of e p i t h e l i a l t i s s u e which was the t o t a l length of e p i t h e l i a l t i s s u e across both the long and shor t axes of the s e c t i o n r e l a t i v e to the combined - S e c t i o n i n g and s t a i n i n g of the t h y r o i d g1ands in t h i s and the f o l l o w i n g experiments were performed by the H i s t o l o g i c a l Labora to ry , Department o f Zoology , U n i v e r s i t y of B r i t i s h Columbia. - 32 -length of the two axes . Photomicrographs were t a k e n . The data of t h i s and the f o l l o w i n g experiments were subjected to ana 1ysis of v a r i a n c e and Student-Newman-Keu1s t e s t s (Zar , 1974). Resu l t s The t h y r o i d weights expressed as a percentage of body we ight , and the percentage of e p i t h e l i a l t i s s u e present in the t h y r o i d glands are shown in Tables II and III r e s p e c t i v e l y . Both t h i o u r a c i l a d m i n i s t r a t i o n and iod ine d e f i c i e n c y produced g o i t e r in the b i r d s . E p i t h e l i a l t i s s u e s were th ickened and the t h y r o i d weights of the t h i o u r a c i 1 - t r e a t e d b i r d s increased tremendously . However, the t h y r o i d weights of the b i r d s r e c e i v i n g the c o n t r o l d i e t or the i o d i n e - d e f i c i e n t d i e t were s i m i l a r , a l though the t o t a l amount of t h y r o i d e p i t h e l i a l t i s s u e was increased in i od ine d e f i c i e n c y . Photomicrographs of the t h y r o i d glands are shown in F igures 1 to 3-D i s c u s s i o n and Conc lus ions There was no s i g n i f i c a n t d i f f e r e n c e in the t h y r o i d weights between the b i r d s r e c e i v i n g an i o d i n e - d e f i c i e n t d i e t and those r e c e i v i n g the c o n t r o l . d i e t . Th is can be exp la ined by the f a c t that the b i r d s had been fed an i o d i n e - s u f f i c i e n t d i e t f o r 61 weeks p r e v i o u s l y . The t h y r o i d iod ine s t o r e was t h e r e f o r e h i g h . A f t e r - 33 -11 weeks on the exper imental d i e t , the glands of the b i r d s were s t i l l not depleted of i o d i n e . Furthermore, a minute q u a n t i t y o f i od ine can a l s o be obta ined :through the tap water and the other i n g r e d i e n t s in the d i e t . Thus, the d i e t fed was not a b s o l u t e l y f r e e of iod i ne. B i r d s t r e a t e d w i t h t h i o u r a c i l showed a tremendous inc rease in t h y r o i d we ight . Th is was due to the e f f e c t of t h i o u r a c i l which i s a potent i n h i b i t o r of t h y r o i d hormone b i o s y n t h e s i s . The feedback c o n t r o l system of the t h y r o i d - p i t u i t a r y a x i s was t h e r e f o r e a c t i v a t e d and subsequent ly more t h y r o t r o p i n was re leased ( L a b e l l e , 1964). Th is led to s t i m u l a t i o n of the t h y r o i d gland and an increase in t h y r o i d we ight . A l though the t h y r o i d weights of the i o d i n e - d e f i c i e n t b i r d s were not s i g n i f i c a n t l y d i f f e r e n t from those of the c o n t r o l , the p r o p o r t i o n of e p i t h e l i a l t i s s u e in the t h y r o i d s of t h i s group was s i g n i f i c a n t l y higher than in the c o n t r o l group. T h i s o b s e r v a t i o n , in c o n j u n c t i o n w i t h the photomicrographs in F igure 1 i n d i c a t e h y p e r p l a s i a . S t a t i s t i c a l a n a l y s i s showed no s i g n i f i c a n t d i f f e r e n c e between the i o d i n e - d e f i c i e n t and the t h i o u r a c i 1 - t r e a t e d b i r d s w i th respect to the percentage o f e p i t h e l i u m present in the t h y r o i d g lands of the b i r d s on the r e s p e c t i v e t reatments . F igure 1 showed that there were more e p i t h e l i a l c e l l s present in the i o d i n e - d e f i c i e n t b i r d s than in the c o n t r o l b i r d s . Th is photomicrograph corresponded to the h i s t o l o g i c a l p i c t u r e of a r e s t i n g a d u l t t h y r o i d gland proposed by Wilgus et a 1. (1953)- S ince the d i e t was on ly moderately d e f i c i e n t in i o d i n e , - 3k -t h i s compensatory h y p e r p l a s i a may enable the b i r d s to main ta in normal i od ine metabol ism in the t h y r o i d g l a n d . However, i f the experiment had been c o n t i n u e d , the degree of h y p e r p l a s i a would have increased as the t h y r o i d a l i od ine s t o r e became d e p l e t e d . The observed percentage of e p i t h e l i a l t i s s u e present in the t h y r o i d g lands in the b i r d s of the t h i o u r a c i 1 - t r e a t e d group was h igher than in those of the c o n t r o l and the i o d i n e - d e f i c i e n t group suggest ing s t i m u l a t i o n of the t h y r o i d gland l e a d i n g to hypertrophy and h y p e r p l a s i a . No s i g n i f i c a n t d i f f e r e n c e was observed , however, between the i o d i d e - d e f i c i e n t and the t h i o u r a c i 1 - t r e a t e d b i r d s . An increase in t h y r o i d a l e p i t h e l i u m has a l s o been reported in growing c h i c k s a f t e r t h i o u r a c i l t reatment by Larson et a 1., (1 9^5) • The photomicrograph (F igure 3) corresponded to the h i s t o l o g i c a l p i c t u r e of t h y r o i d a l h y p e r p l a s i a and hypertrophy in severe g o i t e r in the young b i r d s as proposed by Wi lgus et_ a_l_. (1953). Furthermore, l oss o f c o l l o i d occur red in the t h i o u r a c i 1 - t r e a t e d group w h i l e the t h y r o i d glands of the i o d i n e - d e f i c i e n t b i r d s conta ined much c o l l o i d . A c c o r d i n g l y , a s l i g h t degree o f compensatory h y p e r p l a s i a can compensate f o r a moderate s t a t e of iod ine d e f i c i e n c y in a d u l t b i r d s w h i l e severe h y p e r p l a s i a and hypertrophy o f t h y r o i d a re produced by a d m i n i s t e r i n g t h i o u r a c i l . The t h y r o i d a l enlargement cannot compensate f o r the e f f e c t o f t h i o u r a c i l because ' it i s an i n h i b i t o r of t h y r o i d hormone b i o s y n t h e s i s (Ingbar and Woebar, 1974). Thus, no matter how much more iod ine i s concent rated by the en larged t h y r o i d g l a n d , no hormone i s re leased and the p i t u i t a r y gland is c o n s t a n t l y - 35 -being s t i m u l a t e d to r e l e a s e t h y r o t r o p i n which in t u r n , leads to a s e v e r e l y o v e r s t i m u l a t e d t h y r o i d g l a n d . - 36 -Table I. Composit ion of d i e t s fed in Experiment 1 Ingred i ents Diet 1 % Diet 2 °/ Diet 3 9 Wheat Corn Oats Soybean meal A l f a l f a meal D i s t i l l e r ' s d r i e d s o l u b l e s Limestone Calc ium phosphate S a l t (non - iod i zed ) M i c r o n u t r i e n t s 43.15 15-00 10.00 16.00 2.00 1.25 5-50 1 .75 0.35 as d i e t 1 + 0.885 mg KlO^/kg as d i e t 2 + 0 . 1 * th iourac i1 - / k g : manganese su lphate 246 mg, v i t a m i n 3 meg, r i b o f l a v i n 0 .2 mg, f o l i c a c i d 1.1 mg, v i t a m i n A 5000 I .U . , v i t a m i n D^  1000 I .C .U . - 37 -Table I I . Average t h y r o i d a l weights at t e r m i n a t i o n of Experiment 1. Treatment A v<~- t h y r o i d weight mg/100 g body weight 1. Iodine d e f i c i e n t 7.56 s (4) -> 2. Contro l 10.43a(3) 3. T h i o u r a c i 1 - t r e a t e d 51.70b(4) * Number of c h i c k s per treatment ab values w i t h the same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t P < 0.01 - 38 -Table I I I . Average percentage of e p i t h e l i a l t i s s u e in the t h y r o i d gl'ands of b i r d s in Experiment 1 . Treatment Epi t h e l i a l t i ssue % of t o t a l t h y r o i d gland 1 . Iodine d e f i c i e n t 2. Contro l 3. T h i o u r a c i 1 - t r e a t e d 35.8 a(4)* 24.0 b(3) 42.8 a(4) Number of c h i c k s per treatment ab va lues w i t h the same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t P < 0.05. - 39 -( 4 0 0 x ) - 40 -F igure 3- Photomicrograph of t h y r o i d g land of t h i o u r a c i 1 - t r e a t e d b i r d (400x) - k] -Experiment 2. THE EFFECTS OF RAPESEED MEAL AND THIOURACIL, SINGLY AND IN COMBINATION ON THE THYROID GLANDS OF GROWING CHICKS. The e f f e c t s of t h i o u r a c i l on the t h y r o i d glands of a d u l t b i r d s were demonstrated in Experiment 1. The present experiment was conducted to study the e f f e c t s of t h i o u r a c i l on the t h y r o i d metabol ism of growing c h i c k s . The e f f e c t s of rapeseed meal and the combined e f f e c t s of rapeseed meal and t h i o u r a c i l on t h y r o i d metabol ism of the c h i c k s were a l s o examined. M a t e r i a l s and Methods One hundred and twenty, d a y - o l d New Hampshire c h i c k s were fed four exper imental d i e t s . The c o n t r o l d i e t conta ined soybean meal (SBM) as the source of supplementary p r o t e i n . A second d i e t conta ined rapeseed meal (RSM) as the source of supplementary p r o t e i n . Both d i e t s were fed w i t h and wi thout the a d d i t i o n of 0.05% of t h i o u r a c i l (TU) ( N u t r i t i o n a l B iochemica ls C o r p o r a t i o n , C l e v e l a n d , Oh io ) . The composi t ion of the d i e t s is shown in Table IV. In t h i s , and f o l l o w i n g experiments us ing c h i c k s , a c o c c i d i o s t a t (amproliurn*) was inc luded in the d i e t s . Each of the d i e t s and water were g i ven ad 1i bi turn. F i ve b i r d s from each d i e t were randomly s e l e c t e d and k i l l e d at the age of seven days. The i r - Amprol s u p p l i e d cour tesy of Merck Sharp and Dohme Canada L t d . - kl -t h y r o i d glands were e x c i s e d , weighed and f i x e d f o r h i s t o l o g i c a l s tudy . The glands were sec t ioned and s t a ined wi th haematoxyl i n and e o s i n . The p r o p o r t i o n of the t o t a l t h y r o i d s e c t i o n area occupied by e p i t h e l i a l t i s s u e was measured as desc r ibed under M a t e r i a l s and Methods f o r Experiment 1. At the age of two,weeks, the remaining c h i c k s from each d i e t were i n j e c t e d i n t r a p e r i t o n e a 1 1 y w i t h 0 . 5 uCi of r a d i o i o d i n e (1-131) in p h y s i o l o g i c a l s a l i n e . S i x c h i c k s from each treatment were k i l l e d at 4 , 8 , 16 and 32 hours a f t e r 1—131 i n j e c t i o n . The t h y r o i d glands were exc i sed and the r a d i o a c t i v i t y of each p a i r of glands was measured in a Nuclear Chicago 8166 w e l l - t y p e s c i n t i l l a t i o n d e t e c t o r . Resu1ts The t h y r o i d weights are expressed as t h y r o i d mg/lOOg body weight (Table V ) . The data i n d i c a t e d that c h i c k s fed RSM had heav ier t h y r o i d weights than c h i c k s fed SBM. Th is d i f f e r e n c e was not , however, s i g n i f i c a n t . With the i n t r o d u c t i o n of 0.05% TU in to the d i e t , t h y r o i d weight increased markedly . Th is inc rease was more pronounced in b i r d s fed RSM than in b i r d s fed SBM. Thus, the t h y r o i d weights of c h i c k s r e c e i v i n g both the RSM and TU were s i g n i f i c a n t l y d i f f e r e n t than those of b i r d s r e c e i v i n g SBM and TU, which in t u r n , were s i g n i f i c a n t l y d i f f e r e n t than those of b i r d s fed SBM or RSM a l o n e . - 43 -Est imates of the e p i t h e l i a l t i s s u e area expressed as a percentage of the t o t a l s e c t i o n area are shown in Table V I . However, the weights of the t h y r o i d glands v a r i e d among the d i f f e r e n t d i e t a r y t rea tments , hence, i t w i l l . b e more accura te to i n t e r p r e t the r e s u l t s when they are expressed in terms of the a b s o l u t e amount of the e p i t h e l i a l t i s s u e .present (Table V I ) . The data i n d i c a t e d that there was s i g n i f i c a n t l y more t h y r o i d a l e p i t h e l i a l t i s s u e in b i r d s r e c e i v i n g both TU and RSM than in the b i r d s on the other three t reatments . A l though the TU - t reated b i r d s conta ined more e p i t h e l i a l t i s s u e than b i r d s fed e i t h e r SBM or RSM wi thout t h i o u r a c i l , t h i s d i f f e r e n c e was not s i g n i f i c a n t . The RSM-fed c h i c k s showed on ly an i n s i g n i f i c a n t inc rease in the amount of e p i t h e l i a l t i s s u e compared w i th the c o n t r o l b i rds , fed a SBM d i e t . The data w i th respect to the t h y r o i d a l uptake of 1 —131 are shown in Table V l l and F igure 4. It i s obvious that b i r d s t r e a t e d with.SBM and TU or w i t h combined TU and RSM had much higher uptake than d id the c o n t r o l and ; the RSM-fed b i r d s wi thout TU. The d i f f e r e n c e s were s i g n i f i c a n t a t a l l t imes when r a d i o a c t i v i t y of the t h y r o i d was measured. Rapeseed m e a l - f e d c h i c k s had s l i g h t l y h igher t h y r o i d a l uptake of 1-131 than the c o n t r o l b i r d s at a l l t imes . However, t h i s inc rease was not s t a t i s t i c a l l y s i g n i f i c a n t . There were no r e s u l t s obta ined f o r the c o n t r o l b i r d s a t 16 hours , becauseQ6% of the b i r d s d ied from haemorrhage a f t e r - hk -the i n j e c t i o n . Hence, the r e s u l t s obta ined from the remaining b i r d s were d i scarded s i n c e they were not r e p r e s e n t a t i v e of the e n t i r e p o p u l a t i o n . B i rds r e c e i v i n g the combined TU and RSM d i e t showed a tremendous increase in the 1-131 uptake at 4 and 8 hours . Th is increase was s i g n i f i c a n t l y g r e a t e r than in the b i r d s t rea ted w i t h SBM+TU. The percentage .o f f — 131 present in the t h y r o i d gland decreased d r a s t i c a l l y a f t e r 8 hours and l e v e l l e d o f f a f t e r 16 hours . B i r d s t r e a t e d w i t h SBM+ TU showed a rap id uptake of I —131 at h hours . Subsequent ly , the r a t e of uptake d e c l i n e d w i th o n l y a gradual inc rease in uptake between h and 16 hours ; the 1-131 was re leased s low ly a f t e r that t i m e . D i s c u s s i o n and Cone 1 us ions The increase in weight of the t h y r o i d gland a f t e r t h i o u r a c i l or rapeseed meal feed ing has been observed p r e v i o u s l y (Mixner et a l . , ^^kh; C l a n d i n i n and Bay l y , I960). However, in the present exper iment , the t h y r o i d weights of the one -week -o ld c h i c k s fed RSM d i e t were not s i g n i f i c a n t l y heav ier than those of the c o n t r o l c h i c k s . Th is l a t t e r r e -s u l t may be due to the f a c t that the preformed t h y r o i d hormone in the RSM-fed c h i c k s had not been d e p l e t e d , hence no g o i t r o g e n i c e f f e c t was ev ident (Larson et a j_ . , 1945)- The f a c t that TU - t rea ted b i r d s showed a marked increase in t h y r o i d weight even at one week of age, i n d i c a t e d g reate r g o i t r o g e n i c i t y of TU at the l e v e l fed than of RSM. - 45 -l i n o (1961) reported s i m i l a r l y that TU i s more potent than g o i t r i n in RSM, in i t s a b i 1 i t y : t o ; i n h i b i t t h y r o i d hormone s y n t h e s i s . Langer (1966) commented that the more go i t rogens are admin is te red s imul taneous l y and the l a r g e r t h e i r doses , the g r e a t e r the a n t i t h y r o i d e f f e c t o b t a i n e d . The r e s u l t s of the present ex -periment c o n f i r m Langer 's o b s e r v a t i o n that c h i c k s which rece ived both TU and RSM had heav ie r t h y r o i d g lands than those which rece ived e i t h e r SBM -i- TU or RSM a l o n e . The amount of the e p i t h e l i a l t i s s u e was a l s o g rea te r in the c h i c k s fed both TU and RSM than in the c h i c k s fed e i t h e r TU or RSM s i n g l y . An inc rease in e p i t h e l i a l t i s s u e occur red in b i r d s fed TU. Th is inc rease was not s t a t i s t i c a 1 1 y d i f f e r e n t than in the b i r d s fed e i t h e r SBM or RSM. N e v e r t h e l e s s , the t h y r o i d weight of the TU-fed b i r d s was s i g n i f i c a n t l y d i f f e r e n t than in the other two groups, hence, inc rease in the amount of c o l l o i d was suggested. The e f f e c t of SBM- + TU., however, was not as powerful as the combined TU and RSM d i e t in caus ing t h y r o i d enlargement. Although h y p e r p l a s i a and hypertrophy of e p i t h e l i a l t i s s u e should account f o r the inc rease in the amount of the e p i t h e l i a l t i s s u e of rapeseed m e a l - f e d c h i c k s ( C l a n d i n i n and B a y l y , I960), t h i s was not ev ident in one -week -o ld c h i c k s . The data on ly showed a s l i g h t inc rease in e p i t h e l i a l t i s s u e in the rapeseed m e a l - f e d c h i c k s that was not s t a t i s t i c a l l y s i g n i f i c a n t . - 46 -Besides having the h ighest t h y r o i d weight and the most e p i t h e l i a l t i s s u e ^ the c h i c k s r e c e i v i n g both TU and RSM a l s o had the h ighest t h y r o i d a l uptake of I-131 • Th is showed that the iod ine t rapp ing mechanisms was extremely a c t i v e s i n c e almost 80% of the 1-131 i n j e c t e d was accumulated b y : t h e t h y r o i d g lands in c h i c k s r e c e i v i n g both TU and RSM. Th is o b s e r v a t i o n a l s o i n d i c a t e d that n e i t h e r TU nor RSM go i t rogens i n t e r f e r e d w i t h the i o d i n e t r a p p i n g mechanism of the t h y r o i d g l a n d . Th iourac i1•blocks o r g a n i c i o d i n a t i o n of the iod ine in the t h y r o i d gland ( L a z a r u s _ e t _ a j . , 1975), hence a high percentage of the inorgan ic iod ide accumulated i s re leased r a p i d l y as iod ide when TU i s a d m i n i s t r a t e d (Nadler and Leb lond , 1958). In the combined TU and RSM-fed c h i c k s , however, the 1 —131 remained high a f t e r the i n i t i a l r e l e a s e . Th is may have been due to the incomplete blockage by TU on the t h y r o i d hormone b i o s y n t h e s i s (Wentworth and M e l l e n , 1961). Thus some I —131 was incorporated i n t o the t h y r o i d gland as MIT or DIT wi thout being re leased in to the c i r c u l a t i o n . At the same t i m e , RSM does not enhance the s e c r e t i o n o f t h y r o i d hormone (Akiba and Matsumoto, 1971; Leung and March, 1976). Therefore , the I —131 which was incorporated was not re leased and a h igh percentage of r a d i o i o d i n e remained in the t h y r o i d even a f t e r an i n i t i a l phase of rap id r e l e a s e of i o d i d e . The t h i o u r a c i1 - t r e a t e d group a l s o showed rap id t h y r i o d a l uptake of I-I3I a t 4 hours . A f t e r that t ime there was a more gradual increase to 16 hours and a subsequent gradual r e l e a s e of i o d i n e . - 47 -The slow increase may due to the rap id r e l e a s e of i od ide -accumulated, hence, most of the accumulated t-I31 was r e l e a s e d , l e a v i n g a small inc rease in thyro ida1 I —131 when the r a d i o a c t i v i t y was measured. The slow r e l e a s e of 1-131 from the t h y r o i d may be exp la ined by the increase in TSH s e c r e t i o n under t h i o u r a c i 1 t reatment , which in t u r n , s t i m u l a t e d the t r a p p i n g of I —131 (Mouriz et a l . , 1966), thus a rap id r e l e a s e of 1-131 was not observed because r a d i o i o d i n e in the blood was c o n s t a n t l y concent rated i n t o the g l a n d . The t h y r o i d a l uptake ra te of I —131 in the RSM-fed c h i c k s was s l i g h t l y g reate r than in the c o n t r o l c h i c k s . However, t h i s d i f f e r e n c e was not s i g n i f i c a n t . Th is o b s e r v a t i o n can be exp la ined by the f a c t that the g o i t r o g e n i c e f f e c t of the rapeseed was j u s t beginning to mani fes t i t s e l f s i n c e the preformed t h y r o i d hormone stored in the c h i c k s was not complete ly d e p l e t e d . T h e r e f o r e , the e f f e c t of rapeseed meal in two weeks o l d c h i c k s was not very d i s t i n c t . On the bas i s of the three parameters s t u d i e d , the combined TU and RSM d i s p l a y e d the h ighest degree of g o i t r o g e n i c i t y . Whether t h i s inc rease in g o i t r o g e n i c i t y i s due to the s y n e r g i s t i c a c t i o n of TU and RSM go i t rogens or the enhancement in' the g o i t r o g e n i c e f f e c t o f one g o i t r o g e n towards the o ther i s not c l e a r . Furthermore, RSM go i t rogens at the l e v e l s f e d , were not as potent as TU in caus ing a n t i t h y r o i d e f f e c t s . Moreover, the e f f e c t s of the RSM are not f u l l y apparent u n t i l the stored t h y r o i d hormone in the growing c h i c k s i s d e p l e t e d . U n l i k e rapeseed g o i t r o g e n s , TU has a g o i t r o g e n i c - 48 -e f f e c t even though the growing c h i c k s s t i l l r e t a i n some t h y r o i d hormone. - 49 -Table IV. Composit ion of d i e t s fed in Experiment 2. Ingredients Diet 1 Diet 2 Diet 3 D iet 4 % % % % Wheat 31.4 23-5 as d i e t 1 as d i e t 2 + + Corn 31.4 23-5 0.05% 0.05% t h i o i i r a c i 1 ' t h i o u r a c i 1 Soybean meal 29.1 Rapeseed meal 45-6 T r i c a l c i u m phosphate 1.3 L i mestone 1.3 1-9 Iodized s a l t 0.5 0.5 D i s t i l l e r ' s d r i e d s o l u b l e s 2.0 2.0 Tal low 3-0 3-0 M i c r o n u t r i e n t s * ** »/kg: manganese su lphate 132 mg, z i n c ox ide 62 mg, r i b o f l a v i n 4 mg, f o l a c i n 0 .55 mg, c h o l i n e 492 mg, n i a c i n 27 mg, c a l c i u m pantothenate 9 .24 mg, v i t a m i n A 4400 I .U . , v i t a m i n D^  440 I . C . U . , v i t a m i n 13 meg - - / k g : as in "-/kg wi thout c h o l i n e - 50 -Table V. Average t h y r o i d weights of c h i c k s at 1 week of age in Experiment 2. Diet Ave. t h y r o i d wt. mg/100 g body weight 1. SBM l4 .7 a (5 ) -2. RSM 19.4a(5) 3. SBM + TU 39-0 b(5) k. RSM + TU 73-2 c(5) * Number of b i r d s per treatment abc va lues w i t h the same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t P <. 0.05 - 51 -Table V I . Average weight of e p i t h e l i a l t i s s u e of c h i c k s at 1 week of age in Experiment 2. % epi t h e l i a l Amount of t i s s u e of t o t a l e p i t h e l i a l t i s s u e (mg) Diet 1. SBM 34.2 (5)* 3 .9 3 (5)' 2. RSM 30.0 (5) 4 .2 a (5) 3. SBM + TU 28.2 (5) 7 .8 a (5) 4. RSM + TU 28.9 (5) 13-8 b (5) » Number of b i r d s per treatment ab va lues wi th the same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t P < 0.01 - 52 -Table V l l . Thyro ida l uptake of r a d i o i o d i n e -131 by c h i c k s at 2 weeks of age in Experiment 2. . % of i n j e c t e d dose of 1-131 uptake Hours a f t e r a d m i n i s t r a t i o n 4 8 16 32 1. SBM 21 . ,4 a(6)* 23. • l a(6) 21 . 2 a ( 4 ) * 2. RSM 25. •0 a(5) 32. •7 a(6) 23. 6 a(6) 27. 7 a(6) 3- SBM + TU 46. •7 b(6) 48. •6 b(6) 57. l b ( 6 ) 46. 7 b(6) 4. RSM + TU 72. •6 C(6) 76. •2 C(5) 54. 9 b(5) 56. l b ( 6 ) abc Number of c h i c k s per treatment values w i t h the same s u p e r s c r i p t w i t h i n each t ime i n t e r v a l are not s i g n i f i c a n t l y d i f f e r e n t P < 0.05-- 53 -1 0 2 0 3 0 H O U R S F igure h. Thyro ida l 1-131 uptake of c h i c k s at two weeks of age k, 8 , 16 and 32 hours a f t e r 1-131 a d m i n i s t r a t i o n . - 5h -Experiment 3- FACTORS AFFECTING THE GOITROGENICI.TY OF THIOURACIL IN THE CHICK. The e f f e c t s of i od ine d e f i c i e n c y and t h i o u r a c i l on the t h y r o i d h i s t o l o g y of a d u l t b i r d s were s tud ied in Experiment 1. Experiment 2 was conducted : to study the e f f e c t s , i n d i v i d u a l l y and t o g e t h e r , of two sources of g o i t r o g e n s , namely, rapeseed meal and t h i o u r a c i l on the t h y r o i d metabol ism of very young c h i c k s . The present experiment was designed to study the e f f e c t of t h i o u r a c i l on the t h y r o i d g lands of growing c h i c k s when (a) d i f f e r e n t amounts of exogenous t h y r o x i n e were admin is te red and (b) the d i e t conta ined d i f f e r e n t c o n c e n t r a t i o n s of i o d i n e . M a t e r i a l s and Methods Day -o ld White Leghorn male c h i c k s were randomly a l l o t t e d to s i x t reatments (Rl to R6) w i t h a d u p l i c a t e f o r each t reatment . The exper imental r a t i o n s were fed ad 1 ib l tum. The treatment imposed and the composi t ion of d i e t s a re shown in Tables VIII and IX r e s p e c t i v e l y . Body weights of the c h i c k s were recorded p e r i o d i c a l l y . At day 0, f i v e c h i c k s were s a c r i f i c e d to determine the t h y r o i d we ight . Ten c h i c k s were randomly s e l e c t e d , weighed and k i l l e d from each treatment a t the i n t e r v a l s shown in F igure 5-The t h y r o i d glands of these b i r d s were e x c i s e d . F i ve p a i r s of - 55 -t h y r o i d glands from each treatment were weighed w h i l e another f i v e p a i r s of glands were exc i sed and f i x e d immediately in Baker ' s formal c a l c i u m t i s s u e f i x a t i v e f o r h i s t o l o g i c a l s t u d i e s . S t a r t i n g from day 9 u n t i l day 21 , 0 .8 ug/day of D,L-T^ (ICN Pharmaceut ical I n c ^ C l e v e l a n d ) was i n j e c t e d i n t r a p e r i t o n e a 1 1 y i n t o the b i r d s in R5 and commencing on day 1 to 6 , the c h i c k s in R-6 rece ived 5-1 pg TSH/day. The dosage of TSH used was increased every week. Hence, from day 7 to 1 3 , . 1 0 ug TSH/day was a d m i n i s t e r e d , w h i l e 25 ug TSH/day was i n j e c t e d in to every c h i c k in R-6 from day 1 4 - 2 1 , and f i n a l l y 52 ug TSH/day was g iven from day 2 2 - 2 4 . When the experiment t e r m i n a t e d , the t h y r o i d glands that were f i x e d at day 20 were s e c t i o n e d , s t a i n e d w i t h haematoxyl in and e o s i n . The percentage of e p i t h e l i a l t i s s u e occupying the t o t a l t h y r o i d s e c t i o n area was measured as desc r ibed under M a t e r i a l s and Methods f o r Experiment 1. Resu l ts The average body weights of the c h i c k s are shown in Table X. The weights inc lude a l l the body weights recorded on days 9 , 13, 20 and 24 when ten c h i c k s were randomly s e l e c t e d from each treatment f o r t h y r o i d weights and h i s t o l o g i c a l s t u d i e s . The data showed that the growth ra tes of the c h i c k s on a l l t reatments were s i m i l a r u n t i l one week of age. Subsequent ly , the growth ra te of R-3 and R-4 b i r d s was depressed. The growth r a t e - 56 -of R^  b i r d s was suppressed m o r e . s e v e r e l y than those of R^  b i r d s . R5 b i rds showed an improvement i n the growth, r a t e a f t e r thyrox i ne supplementat ion w h i l e the R I , R2 and R6 b i r d s d i s p l a y e d a s l i g h t d i f -ference among themselves in t h e i r ra te of growth even at day 2 0 . The body weights o f the R3,Rk and R5 b i r d s , however, were s i g n i f i c a n t 1y d i f f e r e n t from each other at 20 days of age. The growth r a t e of the R3 b i r d s was s e v e r e l y suppressed whereas the i n h i b i t i o n of growth was l e s s s t rong in the Rk b i r d s . R5 b i r d s r e c e i v i n g T^ supp lementat ion , showed the l e a s t depressed growth ra te among the three groups. The t h y r o i d weights of the c h i c k s were expressed as a percentage of the body weight (Table XI and F igure 5 ) - The t h y r o i d weight increased r a p i d l y f o r Rk and R5 b i r d s , fo l l owed by those of R3 and RI b i r d s . The t h y r o i d weight of the R6 b i r d s increased s l i g h t l y and then decreased again whereas the c o n t r o l b i r d s e x h i b i t e d a r e l a t i v e l y constant t h y r o i d weight throughout the exper iment . E s t i m a t i o n of the area in the t h y r o i d gland occupied by the e p i t h e l i a l t i s s u e was expressed as a percentage of the t o t a l t h y r o i d g l a n d . The amount of the t h y r o i d e p i t h e l i u m and the amount of t h y r o i d e p i t h e l i u m wi th respect to body weight were a l s o e s t i m a t e d . These data are shown in Table X I I . The r e s u l t s showed tha t there were more t h y r o i d a l e p i t h e l i a l t i s s u e in RI , R3 and R5 b i r d s than those of Rk b i r d s , which in t u r n , possessed more e p i t h e l i a l t i s s u e than the b i r d s in R2 and R6. B i rds in R6 , however, showed a s l i g h t increase in t h y r o i d a l e p i t h e l i a l t i s s u e when compared to the c o n t r o l . - 57 -D i s c u s s i o n and Conc lus ions The growth ra te of the R3 b i r d s was the poorest among the b i r d s in a l l the t reatments , because both iod ine d e f i c i e n c y and t h i o u r a c i l depressed growth (Wilgus et a l . , 1953; Astwood and B i s s e l l , ]Skk). Rk b i r d s showed a b e t t e r growth r a t e s i n c e on ly t h i o u r a c i l was present in the d i e t . The growth r a t e of the R5 b i r d s showed improvement a f t e r the supplementat ion of D, L"T^ ^ r o m day 9 t o day 2 0 . N e v e r t h e l e s s , the r a t e of growth was not as rap id as those of the c o n t r o l b i r d s , suggest ing that the l e v e l of D, L-T^ used could not complete ly r e l i e v e the growth depresss ing e f f e c t of t h i o u r a c i l . A h igher dosage of D, L~T^ might have enabled the R5 b i r d s to ach ieve a b e t t e r growth r a t e . At day 20, no s i g n i f i c a n t d i f f e r e n c e was observed among the body weights of b i r d s in R I , R2 and R6, i n d i c a t i n g that the RI b i r d s were not a b s o l u t e l y iod ine d e f i c i e n t . Th is may be due to the f a c t that the d i e t fed to the RI b i r d s was not a b s o l u t e l y devoid of i o d i n e . "Therefore, the RI b i r d s cou ld main ta in a normal growth ra te at the expense of t h y r o i d a l enlargement to t rap more i o d i n e . The R6 b i r d s a l s o showed no s i g n i f i c a n t d i f f e r e n c e in the growth r a t e when compared to the c o n t r o l b i r d s i n d i c a t i n g exogenous TSH does not i n t e r f e r e w i t h the normal growth of the c h i c k e n s . - 58 -The t h y r o i d weights of the t h i o u r a c i1 - t r e a t e d group showed a marked inc rease a f t e r day 9- This e f f e c t had been observed in Experiment 2. One s u r p r i s i n g f a c t was that when t h i o u r a c i l -t reatment was coupled to iod ine d e f i c i e n c y , the glands were not as heavy as when the b i r d s were t r e a t e d w i th t h i o u r a c i l a l o n e . This o b s e r v a t i o n is in c o n t r a s t to the augmented e f f e c t s caused by the combinat ion of t h i o u r a c i l and rapeseed meal in Experiment 2. There was no s i g n i f i c a n t d i f f e r e n c e in the t h y r o i d weight of the Rl and R3 b i r d s at 20 days of age, g i v i n g f u r t h e r evidence that the t h y r o i d of the i o d i n e - d e f i c i e n t c h i c k s i s i n s e n s i t i v e to the a d d i t i o n a1 g o i t r o g e n i c e f f e c t s of t h i o u r a c i l . The t h y r o i d weights of the R4 and R5 c h i c k s were not s i g n i f i c a n t l y d i f f e r e n t than each other at 20 days of age, a l though the R5 c h i c k s rece ived 0.8 ug T^ per day. This r e s u l t i n d i c a t e d that the dosage of T^ used was not adequate to reduce t h y r o i d we ight . Mixner et_ aj_. (1944) suggested that at l e a s t 4.0 ug T^ per day was requ i red to reduce the t h y r o i d weight of c h i c k s t r e a t e d w i t h t h i o u r a c i1. TSH- t reated b i r d s (R6) were not s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l b i r d s in t h y r o i d s i z e a f t e r day 9- The lack of g o i t r o g e n i c e f f e c t may have been due to an inadequate dose of i n j e c t e d TSH. TSH should however, induce an increase in t h y r o i d a l we ight . (Keat ing et_ a_l_. , 1 945 ; Sasson and Rosenberg , 1963) • Furthermore, the dose of TSH may not be e n t i r e l y absorbed i n t o the c i r c u l a t o r y system of the c h i c k , t h e r e f o r e , some TSH d i d not reach the t h y r o i d - 59 -gland and consequent ly l i t t l e or no s t i m u l a t i o n was observed . If the TSH had been i n j e c t e d i n t r a v e n o u s l y rather than i n t r a p e r i t o n e a 1 1 y , a more d i s t i n c t response might have been apparent in the t h y r o i d g l a n d . There was a s t a t i o n a r y phase observed f o r R3, R4, and R5 b i r d s w i t h respect to t h y r o i d a l enlargement. For the R5 b i r d s , t h i s corresponded to the per iod of T^ a d m i n i s t r a t i o n . Hence, T^ may a l l e v i a t e the e f f e c t of t h i o u r a c i l on the t h y r o i d g l a n d . A f t e r a d m i n i s t r a t i o n , the t h y r o i d gland cont inued to e n l a r g e . The reason why a s t a t i o n a r y phase or a slow increase in the t h y r o i d weight was observed in R3 and R4 c h i c k s is not understood . Both the weight of the t h y r o i d a l e p i t h e l i a l t i s s u e and the photomicrographs (F igures 6-11) i n d i c a t e d that the amount of t h y r o i d e p i t h e l i u m increased in the iod ine d e f i c i e n t or t h i o u r a c i 1 - t r e a t e d b i r d s . The R6 b i r d s showed a small inc rease in t h y r o i d e p i t h e l i u m , but the d i f f e r e n c e was not s i g n i f i c a n t from the c o n t r o l . Th is i n s i g n i f i c a n t inc rease in t h y r o i d e p i t h e l i u m suggested that on ly a small amount of TSH i n j e c t e d cou ld reach the t h y r o i d s fo r s t i m u l a t i o n . R l , R3 and R5 b i r d s showed a marked inc rease in t h y r o i d a l e p i t h e l i a l t i s s u e . Th is inc rease may : be due to hypertrophy and/or h y p e r p l a s i a of the t h y r o i d a l e p i t h e l i a l c e l l s . R3 b i r d s showed exhaust ion atrophy of the t h y r o i d g land as d e f i n e d by Wi lgus et_ aj_. (1953) w h i l e Rl b i r d s d i s p l a y e d h y p e r p l a s i a . Both R4 and R5 b i r d s e x h i b i t e d hypertrophy of the t h y r o i d a l f o l l i c l e s and h y p e r p l a s i a . Th is can e x p l a i n the tremendous inc rease in t h y r o i d weight and t h y r o i d a l e p i t h e l i a l t i s s u e s observed . The increase in t h y r o i d e p i t h e l i u m - 60 -in the Rh b i r d s , however, was not as great as in the R5 b i r d s at 20 days of age. It has been shown that pretreatment w i t h would enhance the t h y r o i d a l response to TSH (Se i f et a l . , 1975). Moreover, the l e v e l of TSH increased a c u t e l y a f t e r t h i o u r a c i l t reatment ( L a b e l l e , 1964). These o b s e r v a t i o n s may e x p l a i n why a g reate r inc rease in e p i t h e l i a l t i s s u e was observed in the t h y r o i d glands of the R5 b i r d s than those of the Rh b i r d s . T h e r e f o r e , i t was observed that t h i o u r a c i l treatment produced hypertrophy and h y p e r p l a s i a in growing c h i c k s w h i l e i od ine d e f i c i e n c y or iod ine d e f i c i e n c y coupled wi th t h i o u r a c i l on l y showed severe h y p e r p l a s i a w i th the loss of c o l l o i d . Th is may account f o r the f a c t that the t h y r o i d weight in R3 b i r d s was not as h igh as those of Rh and R5 b i r d s . In the present exper iment , i t was found that the g o i t r o g e n i c a c t i v i t y of t h i o u r a c i l was increased when the b i r d s rece ived a small amount of exogenous T^. The g o i t r o g e n i c i t y of i od ine d e f i c i e n c y was weaker than that of t h i o u r a c i l , even when the drug was added to the iod ine d e f i c i e n t d i e t . Furthermore, a g o i t r o g e n i c e f f e c t of exogenous TSH was not apparent even a t a dosage of 52 ug per day. In Experiment 1, i t was found tha t the e f f e c t s of iod ine d e f i c i e n c y in a d u l t b i r d s were not apparent . The photomicrograph of an i o d i n e - d e f i c i e n t b i r d showed r e t e n t i o n of c o l l o i d and on ly a s l i g h t inc rease in the amount of e p i t h e l i a l t i s s u e , whereas the photomicrograph (F igure 6) of an i o d i n e - d e f i c i e n t c h i c k in t h i s experiment showed severe h y p e r p l a s i a and loss of c o l l o i d . Hence, - 61 -growing c h i c k s are,more : suscept i bl e to i od ine d e f i c i e n c y '. than are a d u l t b i r d s . The t h y r o i d h i s t o l o g i c a l p i c t u r e s of both the a d u l t b i r d s and the growing c h i c k s t r e a t e d w i t h t h i o u r a c i l i n d i c a t e d that h y p e r p l a s i a and hypertrophy occur red in the t h y r o i d g l a n d s . The growing c h i c k accumulated m o r e , c o l l o i d in the f o l l i c l e , but t h i s e f f e c t may due to the d i f f e r e n c e in the per iod of t h i o u r a c i l t reatment . Consequent ly , there was no apparent d i f f e r e n c e in the h i s t o l o g i c a l p i c t u r e s of the t h i o u r a c i 1 - t r e a t e d a d u l t b i r d s and growing c h i c k s . - 62 -Table V I M . Treatments employed in Experiment 3. Treatment (R) Experimental Cond i t ions Iodine d e f i c i e n t d i e t Cont ro l d i e t ( i o d i n e supplemented) Iodine d e f i c i e n t d i e t w i t h 0.1% th i ourac i1 Contro l d i e t w i t h 0.1% t h i o u r a c i l Contro l d i e t w i t h 0.1% t h i o u r a c i l and 0.8 meg/day D , L - t h y r o x i n e a d m i n i s t r a t i o n from day 9 to 20 Contro l d i e t w i t h exogenous TSH i n j e c t i o n from day 1 to 2k - 63 -Table IX. Composit ion of d i e t s fed in Experiment 3-Ingred ients Diet 1 Diet 2 Diet 3 D iet 4 D iet 5 Diet 6 Ot 0/ Oi O/ Oy O, 'o 'o 'o 'o 'a 'o Soybean o i1 Corn Soybean meal D i s t i 1 l e r s 1 Dried So lub les Dehydrated c e r e a l grass L imestone Calc ium phosphate S a l t (non- i od i zed) D,L-meth ion i ne Mic ronut r i ents 1. 0 as as as d i e t 1 d i e t 1 d i e t 2 68. 35 wi th wi th wi th 0 . 3 mg 0.1% 0.1% 27. 50 l/kg t h i o - t h i o -feed as urac i1 urac i1 2 . 0 K I 0 4 2. 0 1. 5 1 . 5 0 . 5 0. 12 as d i e t 4 as d i e t 2 - / k g : Manganese su lphate 132 mg, z i n c ox ide 62 mg, r i b o f l a v i n h mg, n i a c i n 10 mg, c h o l i n e 935 mg, v i t a m i n A 4400 I .U . , v i t a m i n D^  440 I . C . U . , v i t a m i n B 13 meg - 64 -T a b l e X . A v e r a g e b o d y w e i g h t s o f c h i c k s o f a l l t r e a t m e n t s i n E x p e r i m e n t 3-T r e a t m e n t s (R) Body W e i g h t (g) Days 0 7 9 i i 20 22 24 1 34 (6*0* 67(44) 80a(10) 107a(10) 1703(10) 197a(l4) 2l4 a e (10) 2 34(64) 69(44) 78a(10) 109a(10) I82A(10) 2 l4 b ( l4 ) 235b(10) 3 34(64) 67(44) 69 a (10) 89b(10) 106b(10) 111C(14) 117C(10) 4 33(64) 66(43) 72 a(10) 89b(10) 125°(10) 136D(13) I47d(10) 5 34(64) 68(44) 70 a(10) 95 b c(10) 155d(10) I84 e(l4) 200e(10) 6 33(64) 67(44) 72 a(10) i o i a c ( 1 0 ) I8l a(10) 206 a b ( l4) 224 a b(10) Number o f c h i c k s p e r t r e a t m e n t a b c d e v a l u e s w i t h t h e same s u p e r s c r i p t a r e n o t s i g n i f i c a n t l y d i f f e r e n t P < 0.05. - 65 -Table X I . Average t h y r o i d weights of c h i c k s of a l l t reatments at d i f f e r e n t time i n t e r v a l s in Experiment 3-- Ave. t h y r o i d w e i g h t s " Treatments (R) , , . . v ' mg/ OOg body weight Days 1 8.60 2 (day 0 c h i c k s not ^ fed e x p e r i -menta1 4 d i e t s ) 5 6 9 11 20 24 12.7a 16.8a 35-4 a 37.1 9.26a 8.20a 7.02 b 8.51 23.9 b c 22.9 a 37-9 a 44.6 I 6 . 6 a b «3.5 b k7.7a 82.6 27.0 C 46.0 b 45- l a 53-0 I 8 . 3 a b c 12.5a 9.77b 7.64 * Number of c h i c k s per treatment at each time i n t e r v a l are 10. abc va lues w i t h the same s u p e r s c r i p t w i t h i n the same time i n t e r v a l are not s i g n i f i c a n t l y d i f f e r e n t P < 0.05. - 66 -T a b l e X I I . C h i c k t h y r o i d e p i t h e l i a l t i s s u e s a t t h e a g e o f 20 d a y s i n E x p e r i m e n t 3-o . -ii. i. r c E s t i m a t e d mg T ^ ^ i*\ % e p i t h e 1iurn o f E s t i m a t e d a m o u n t o f , . , . , , . , T r e a t m e n t (R) _ r . . . , , . . , , . , _ . t \ t h y r o i d e p i t h e l i u m / t o t a l g l a n d - e p i t h e l i a l t i s s u e (mg)* ' . / . , , 3 K 3 OOg b o d y w e i g h t * ' 73.4a kk.k2a 26.133 2 45.6b 5 . 9 7 b 3.28b 3 72.4a 28.95° 27.31* * 48.9b 30.45° 24.36c 5 61.5° 42.96a 27.72a 6 48.7b 8.62d k . 7 7 b * 5 c h i c k s p e r t r e a t m e n t a b e d v a l u e s w i t h t h e same s u p e r s c r i p t a r e n o t s i g n i f i c a n t l y d i f f e r e n t P C 0.05. - 67 -0 9 13 20 24 D A Y S Figure 5. Increase in t h y r o i d weight d i f f e r e n t t reatments . in response to Figure 7-Photomicrograph of the t h y r o i d gland of the t h i o u r a c i 1 -t r e a t e d (R-4) b i r d s (kOOx). F igure 11. Photomicrograph of the t h y r o i d gland of the TSH-t r e a t e d (R-6) b i r d s (400x) . - 71 -Experiment 4 : EFFECTS OF ADMINISTRATION OF DIFFERENT AMOUNTS OF THYROXINE ON THE THYROID WEIGHTS OF CHICKS IN RESPONSE TO THIOURACIL. The f o l l o w i n g experiment was conducted to study the e f f e c t s of a d m i n i s t e r i n g graded doses of D, L - t h y r o x i n e (D,L-T^) on the t h y r o i d glands of the growing c h i c k s t r e a t e d w i t h t h i o u r a c i l . M a t e r i a l s and Methods Day -o ld White Leghorn c h i c k s were randomly d i s t r i b u t e d i n t o f i v e b a t t e r i e s . Chicks in one ba t te r y rece ived a soybean meal ( R - l ) d i e t w h i l e the other c h i c k s rece ived the same d i e t w i t h the a d d i t i o n of O . U of t h i o u r a c i l (R-2) (Table XI I I) ad 1 i b i t u m . A f t e r two weeks, the c h i c k s fed R-2 were d i v i d e d i n t o seven groups (R2-R8) , each r e c e i v i n g a d i f f e r e n t dose of D , L - t h y r o x i n e (ICN Pharmaceut ica l Company, C leveland) d a i l y f o r 10 days . The dosage of D, L""!" ,^ i n j e c t e d i n t r a p e r i t o n e a 11 y , ranged from Oug to 4ug per day per c h i c k in a t o t a l volume of 0.1 ml (Table XIV.,) . Chicks fed R - l were d i v i d e d i n t o three groups ( R l , R9 and RIO), two which was s h i f t e d to the R-2 d i e t two weeks a f t e r the experiment began (R9, RIO). One of these groups (Rl0) rece ived 0 .4 jjg D,L-T^ per c h i c k d a i l y w h i l e the other group (R-9) served as c o n t r o l . Ch icks in R - l , R - 2 , and R-9 rece ived no T^, but 0.1 ml d i s t i l l e d water was i n j e c t e d d a i l y . - 72 -Eleven days a f t e r the i n i t i a t i o n of the t reatment , the c h i c k s were k i l l e d and t h e i r t h y r o i d glands were exc i sed and weighed. Resu1ts Growth r a t e of the c h i c k s (Table XV) was s i g n i f i c a n t l y lower when t h i o u r a c i l was added to the d i e t . A f t e r D,L-T^ t reatment , improvement in the growth r a t e was observed which was in p r o p o r t i o n to the dosage of D,L-T^ a d m i n i s t e r e d . Growth r a t e of R~9 and R-10 b i r d s were a l s o depressed a l though they had been fed the R-2 d i e t f o r on ly 1-1/2 weeks. The average t h y r o i d weights expressed as a percentage o f body weight of the c h i c k s a re presented in Table XVI and F igure 12. The b i r d s r e c e i v i n g t h i o u r a c i l e x h i b i t e d an increase both in the s i z e and the weight of the t h y r o i d g l a n d . A f t e r T^ a d m i n i s t r a t i o n , two e f f e c t s were noted . With the s m a l l e r doses of T^, there was an augmented increase in the t h y r o i d weight in response to t h i o u r a c i l . As the dosage of T^ was increased f u r t h e r , the t h y r o i d weights decreased p r o g r e s s i v e l y . Thyro id weights of the c h i c k s fed R-2 a f t e r two weeks of age a l s o showed c o n s i d e r a b l e enlargement. - 73 -D i s c u s s i o n and Conc lus ions The treatment w i t h t h i o u r a c i l would be expected to depress growth ra te (Astwood and B i s s e l l , 1944). In the Group 9 and 10 c h i c k s , the growth r a t e a l s o decreased even though the c h i c k s were fed the R-2 d i e t f o r on ly 1-1/2 weeks, i n d i c a t i n g the r a p i d i t y of the response to 0.1% of t h i o u r a c i l (Mixner et a l . , 1944). Small amounts of D,L-T^ admin is te red to the b i r d s t r e a t e d w i t h t h i o u r a c i l increased the g o i t r o g e n i c e f f e c t of the t h i o u r a c i l , a l though the d i f f e r e n c e s in weight were not s i g n i f i c a n t . A s i m i l a r response has been observed when the t h y r o x i n e s e c r e t i o n r a t e in c h i c k s was determined by the g o i t e r - p r e v e n t i o n method. In t h i s method graded doses of T^ are i n j e c t e d i n t o the c h i c k s u n t i l a l e v e l i s reached whereby the t h y r o i d weights of the t h i o u r a c i 1 -t r e a t e d c h i c k s are the same as c o n t r o l ( S t u r k i e , 1965)- It has been noted that the lowest doses of f r e q u e n t l y r e s u l t in an increase in t h y r o i d we ight . With i n c r e a s i n g dosage of above 1.0 ug per day, t h y r o i d weight decreased p r o g r e s s i v e l y . In the present experiment 4 ug per day was adequate to prevent g o i t e r . Al though the % t h y r o i d / body weight in R-8 b i r d s was h igher than in the c o n t r o l b i r d s , t h i s d i f f e r e n c e was not s i g n i f i c a n t . Mixner et a l . (1944) a l s o commented that 4 . 0 ug per day was s u f f i c i e n t to prevent t h y r o i d enlargement above the normal l e v e l in the t h i o u r a c i 1 - t r e a t e d c h i c k s . - 74 -Two ug T^ supplementat ion a l s o reduced the t h y r o i d weight of t h i o u r a c i 1 -t rea ted c h i c k s . Th is reduc t ion was not as e f f e c t i v e as in the b i r d s r e c e i v i n g k.O ug T^ per day. But the d i f f e r e n c e in t h y r o i d weight between the R-7 and R-8 (2 and k ug T^ r e s p e c t i v e l y ) c h i c k s was not s i g n i f i c a n t . In s p i t e of the reduc t ion in the t h y r o i d weights of c h i c k s g iven 2.0 ug T^ per day, the t h y r o i d weights of t h i s group of c h i c k s were not s i g n i f i c a n t l y d i f f e r e n t than those c h i c k s r e c e i v i n g a s m a l l e r dosage of T^ per day. T h e r e f o r e , k.O T^ per day was the e f f e c t i v e dose in reducing t h y r o i d weights of c h i c k s t r e a t e d w i th t h i o u r a c i 1 ; w h e r e a s , s m a l l e r doses of T^ (0.2 - 1.0 ug) enhanced the g o i t r o g e n i c e f f e c t of t h i o u r a c i l l e a d i n g to a f u r t h e r inc rease in t h y r o i d we ight . In the R-10 b i r d s , the t h y r o i d weights were as high as in the R~9 b i r d s a l though O.k ug T^ was g iven d a i l y to each c h i c k . The t h y r o i d glands in R-10 b i r d s were approx imate ly h a l f the weight of those in the R-4 group because these b i r d s were o n l y on R-2 d i e t s f o r 1-1/2 weeks w h i l e the b i r d s in R-k were on the R-2 d i e t from the beginning of the exper iment . L a b e l l e (1964), s tud ied the t h y r o i d a l 1-131 r e l e a s e in the d a y - o l d c h i c k by a s i n g l e i n j e c t i o n of smal l amount of t h y r o x i n e or p r o p y l t h i o u r a c i l and found an enhancement in the r e l e a s e of 1-131 in both c a s e s . Maximum r e l e a s e of 1-131 was observed when T^ and p r o p y l t h i o u r a c i l were admin is te red s i m u l t a n e o u s l y . Based on these o b s e r v a t i o n s , L a b e l l e (1964) concluded that a smal l amount of T^ would s t i m u l a t e TSH r e l e a s e mechanism. A l s o , the t h y r o i d - 75 -s t i m u l a t o r y a c t i o n of small amount of and of PTU appeared to be a d d i t i v e , because both substances may ac t on p i t u i t a r y to r e l e a s e TSH d i r e c t l y . L a b e l l e (1964) f u r t h e r suggested that a c t i o n s of T^ and PTU on the t h y r o i d g land i t s e l f may be p o s s i b l e . Th is may e x p l a i n the increase in t h y r o i d a l enlargement from smal l doses of T^ admin is te red to t h i o u r a c i 1 - t r e a t e d c h i c k s . Moreover, small amounts of T^ can increase the r a t e of p r o t e i n s y n t h e s i s in the ta rget t i s s u e s (Evered, 1976), hence the T^ may be a b l e to ac t on the t h y r o i d gland i t s e l f to s t i m u l a t e p r o t e i n s y n t h e s i s lead ing to an increase in t h y r o i d we ight . - 76 -Table X I I I . Composit ion of d i e t s as fed in Experiment 4. Ingred ients Diet °/ 'a Diet 2 °/ 'o Soybean meal Her r ing meal Corn Wheat Dehydrated c e r e a l grass D i s t i l l e r ' s d r i e d s o l u b l e s Calc ium phosphate Limestone Iodized s a l t M i c r o n u t r i e n t s 17-0 5 .0 36.6 34 .2 2.0 2 .0 1.5 1.2 0 . 5 as d i e t 1 + 0.1% t h i o u r a c i 1 - / k g : manganese su lphate 132 mg, r i b o f l a v i n 3 -4 mg. n i a c i n 10 mg, v i t a m i n A 4400 I .U . , v i t a m i n D^  440 I .C .U . - 77 -Table XIV. Experimental t reatments imposed in Experiment 4. 1. Contro l 0 2. Contro l + TU 0 3. Contro l + TU 0.2 4. Contro l + TU 0.4 5. Contro l + TU 0.8 6. Cont ro l + TU 1.0 7- Contro l + TU 2.0 8. Contro l + TU 4.0 9. Contro l ( s h i f t e d to R-2 d i e t s 0 at 2 weeks) 10. Cont ro l ( s h i f t e d to R-2 d i e t s 0.4 . at 2 weeks) - 78 -Table XV. Growth ra tes of c h i c k s on a l l t reatments in Experiment 4. / r.\ Ave. body weight at tments (R) _, x 3-1/2 weeks (g) 1 243 ( I 2 ) " a 2 182 ( I 2 ) b 3 187 ( I 2 ) b 4 196 ( 1 2 ) b c 5 206 ( 1 2 ) c 6 206 (12) c 7 206 ( 1 2 ) C 8 211 ( 1 2 ) c 9 207 ( 1 2 ) c 10 213 ( H ) C Number of c h i c k s abc va lues w i th the same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t P < 0 . 0 1 . - 79 -Table XVI . Thyro id weights of c h i c k s in Experiment k. -p , , ,n\ Ave. t h y r o i d weight Treatment (R) / , „ „ , , mg/lOOg body weight 1 5.4 a ( l2)* 2 73-0 b c ( l2 ) 3 73.5 b C(12) 4 74.5 b c (12) 5 73.4 b c (12) 6 77.6C(12) 7 35.9 a b(12) 8 19.0a(12) 1 5.4a(12) 9 33.6b(12) 10 3 0 . 9 b ( l l ) Number of c h i c k s per treatment abc va lues w i th the same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t p < 0:01. - 80 -•08 0 1 0 2 ^ ~ 3 ^ 0 470 M I C R O G R A M O F D , L T 4 I N J E C T E D PER DAY F igure 12. Average t h y r o i d weight as a percentage of body weight in t h i o u r a c i 1 - t r e a t e d c h i c k s w i t h respect to i n c r e a s i n g dosage of D,L-T, . - 81 -Experiment 5- EFFECTS OF GRADED DOSES OF D,L-THYROXINE ON THE THYROID GLANDS OF THE GROWING CHICKS TREATED WITH THIOURACIL In Experiment k, i t was found that k.O ug T^/day can prevent the enlargement of the t h y r o i d gland in c h i c k s g iven t h i o u r a c i l . Small q u a n t i t i e s of T^, however, increased the g o i t r o g e n i c i t y of t h i o u r a c i l . The f o l l o w i n g experiment was conducted to study the t h y r o i d a l iod ine uptake and h i s t o l o g i c a l changes in the c h i c k s t r e a t e d w i t h t h i o u r a c i l when graded doses of D,L-T^ a re a d m i n i s t e r e d . M a t e r i a l s and Methods Day -o ld White Leghorn male c h i c k s were d i v i d e d in to 6 groups. The c h i c k s were then randomly d i s t r i b u t e d i n t o 12 cages . Feed and water were g iven ad 1 ib i turn. They were fed accord ing to the treatments a s s i g n e d . Composit ions of d i e t s are shown in Table XV I I . At day 8 , \k and 20, 5 b i r d s from each treatment were randomly s e l e c t e d and t h e i r t h y r o i d glands were e x c i s e d , weighed, and f i x e d q u i c k l y in B a k e r ' s formal c a l c i u m t i s s u e f i x a t i v e . They were sec t ioned at 6 urn and s t a i n e d w i t h haematoxyl in and e o s i n . The t o t a l area occupied by the e p i t h e l i a l t i s s u e in the t h y r o i d s e c t i o n was measured as d e s c r i b e d under M a t e r i a l s and Methods in Experiment 1. Graded doses of D,L-T. (ICN Pharmaceut ica l I n c . , - 82 -Cleve land) were admin is te red from day 9 to 20 and at day 2 1 , 0 . 5 uCi of rad io iod ine -131 was i n j e c t e d i n t r a v e n o u s l y i n t o the b i r d s from a l l t reatments . The t h y r o i d glands were removed from e i g h t c h i c k s of each treatment at k, 8, 12, 16, 2k and 32 hours a f t e r i n j e c t i o n . The r a d i o a c t i v i t y in the t h y r o i d glands was determined in a w e l l - t y p e s c i n t i l l a t i o n d e t e c t o r . Table XVIM o u t l i n e s the treatments employed and the l e v e l s of D,L-T^ admin is te red to each group. Resu l t s The growth ra te of the b i r d s r e c e i v i n g t h i o u r a c i l was increased when T^ was admin is te red (Table X IX ) . Even w i t h the h ighest l e v e l of T^, however, growth ra te was not s t i m u l a t e d to equal that w i t h the c o n t r o l d i e t . Whether h igher dosage of the T^ would support a b e t t e r growth r a t e was not c l e a r s i n c e the average body weight of the c h i c k s in the T^-supplemented group v a r i e d c o n s i d e r a b l y . The t h y r o i d weights of the c h i c k s in d i f f e r e n t t reatments changed w i t h time (Table XX) . The data obta ined from day 8 showed that the t h y r o i d weights as a percentage of body weight of the c h i c k s in the d i f f e r e n t t reatments were s i m i l a r . N e v e r t h e l e s s , the e f f e c t s of t h i o u r a c i l were beginning to show in the b i r d s of R2, 3, k and 6. - 83 -At day 14 and day 20 , that i s , a f t e r supp lementat ion , the r e s u l t s i n d i c a t e d that the c h i c k s r e c e i v i n g e i t h e r no supplementat ion or the s m a l l e s t amounts of (0 .5 and 1.0 ug T^/day) had c o n s i d e r a b l y l a r g e r t h y r o i d glands than the c o n t r o l b i r d s , or the b i r d s that rece i ved l a r g e r amount of supplement (3 .0 and 5 - 0 ug T^/day). At day 20, i t was shown that the t h y r o i d a l weights were i n v e r s e l y r e l a t e d to the dosage of admin is te red e x c l u d i n g the group of c h i c k s r e c e i v i n g no supplementat ion . Al though the t h y r o i d a l weights showed l i t t l e d i f f e r e n c e a t day 8, the amount of t h y r o i d e p i t h e l i a l t i s s u e of the t h i o u r a c i 1 - t r e a t e d c h i c k s increased in a l l groups (Table X X I ) . A f t e r T^ supplementat ion f o r 5 days , the amount of t h y r o i d a l e p i t h e l i a l t i s s u e decreased in the groups of c h i c k s g iven e i t h e r 3 . 0 or 5 -0 ug T^ per day whereas the groups of c h i c k s r e c e i v i n g no T^ supplementat ion or smal l amount of T^ i n j e c t i o n showed a f u r t h e r increase in the amount of t h y r o i d e p i t h e l i u m . At day 20, the % e p i t h e l i a l t i s s u e s of the t h y r o i d gland was r e l a t i v e l y constant in the R - l , R - 2 , and R-6 c h i c k s w h i l e those of R~3 and R-4 b i r d s was reduced. The t h y r o i d e p i t h e l i u m of the R~5 c h i c k s d i s p l a y e d a s l i g h t i n c r e a s e . The t h y r o i d a l uptake of 1-131 of c h i c k s a l s o v a r i e d w i t h the treatments imposed (Table XXII and F igure 13) . F igu re I3 showed that the uptake f o r R-5 and R-6 b i r d s was very much l e s s than the o ther groups. Both groups showed a maximum iod ine c o n c e n t r a t i o n at 8 hours a f t e r i n j e c t i o n of 1 -131. The r a t e of uptake in the R-6 b i r d s , however, was even smal le r than those of R-5 - 8k -b i r d s . The c o n t r o l b i r d s d i s p l a y e d a f a s t uptake of 1-131 and then a p la teau was observed i n d i c a t i n g constant r e c y c l i n g . The t h i o u r a c i 1 - t r e a t e d (R-2) b i r d s e x h i b i t e d tremendous v a r i a t i o n in the uptake of i o d i n e . However, t h i s group had the f a s t e s t and the h ighest t h y r o i d a l uptake of r a d i o i o d i n e . Maximum iod ine c o n c e n t r a t i o n occur red at approx imate ly 8 to 12 hours a f t e r i n j e c t i o n . The R-3 and R-k b i r d s showed a very s i m i l a r iod ine uptake and r e l e a s e graph except a t 32 hours a f t e r i n j e c t i o n . Both groups e x h i b i t e d a f a s t uptake of 1-131 i n i t i a l l y at k hours and then a gradual inc rease u n t i l maximum c o n c e n t r a t i o n of 1—131 occurred at 16 hours . Af terwards a f a s t r e l e a s e of r a d i o i o d i n e o c c u r r e d . Another i n t e r e s t i n g o b s e r v a t i o n was that in the R - l , R _ 3 , and R~5 b i r d s , a f u r t h e r inc rease in t h y r o i d a l 1-131 uptake was observed at 32 hours a f t e r i n j e c t i o n . D i s c u s s i o n and Conc lus ions The growth ra te of the c o n t r o l b i r d s was s i g n i f i c a n t l y b e t t e r than the b i r d s r e c e i v i n g T^ supplementat ion and the b i r d s r e c e i v i n g no supp lementat ion . The growth ra te of the t h i o u r a c i l -t rea ted b i r d s showed s i g n i f i c a n t improvement a f t e r T^ supp lementat ion . However, the ra te of growth of these T^ supplemented b i r d s v a r i e d c o n s i d e r a b l y . - 85 -In Experiment 4, i t was found that when a b s o l u t e t h y r o i d weight reached a maximum, f u r t h e r inc rease in treatment on ly reduced the t h y r o i d weight but had no e f f e c t on body weight g a i n . A l s o , the b i r d s r e c e i v i n g a h igher dosage than 0.8 ug T^/day showed b e t t e r growth than the c h i c k s r e c e i v i n g 0.6 pg T^/day or l e s s . In the present exper iment , c h i c k s r e c e i v i n g 3.0 ug T^/day d i s p l a y e d a poorer r a t e of growth than those b i r d s r e c e i v i n g 0.5 ug T^/day at 19 days o l d , but t h i s d i f f e r e n c e was not s i g n i f i c a n t . S t a t i s t i c a l a n a l y s i s showed no d i f f e r e n c e in the body weights of the b i r d s in t reatments 3, 4, 5 and 6. T h e r e f o r e , a b e t t e r growth ra te from higher doses of T^ supplementat ion was not observed in t h i s exper iment . Growth r a t e was depressed by t h i o u r a c i l (Astwood and B i s s e l l , 19^4; Mixner et a l . , 1944). In s p i t e of T^ supplementat ion from day 9 to day 20, the growth r a t e of t h i o u r a c i 1 - t r e a t e d c h i c k s in R~3, 4, 5, and 6 d i d not inc rease to equal tha t of the c o n t r o l b i r d s . Th is i n d i c a t e d that the growth depress ion in the t h i o u r a c i l -t r e a t e d b i r d s was not j u s t a s imple case of reduced t h y r o i d hormone p r o d u c t i o n . The t h y r o i d gland at day 8 showed l i t t l e v a r i a t i o n in we ight . R~5 b i r d s had the s m a l l e s t t h y r o i d glands w h i l e R-2 b i r d s had the l a r g e s t . There was not much d i f f e r e n c e among the other groups. Th is was not the case in Experiment 2 where b i r d s at 7 days of age t r e a t e d w i t h t h i o u r a c i l showed a s i g n i f i c a n t inc rease in t h y r o i d we ight . However, d i f f e r e n t breeds of b i r d s were - 86 -employed in these two exper iments . Leghorn c h i c k s were used in t h i s present exper iment , w h i l e New Hampshire c h i c k s were used in Experiment 2. There might be a breed d i f f e r e n c e in response s i n c e New Hampshire c h i c k s were reported by Premachandra et_ a_l_. (1957) to be h i g h l y respons ive to g o i t r o g e n i c e f f e c t of t h i o u r a c i l . A f t e r 5 days of T^ a d m i n i s t r a t i o n , s i g n i f i c a n t e f f e c t s were observed among the groups of b i r d s r e c e i v i n g more than k ug T^/day and the groups of c h i c k s r e c e i v i n g 1 ug T^ or l e s s d a i l y . R -2, R-3 and R-k b i r d s d i s p l a y e d an increase in t h y r o i d weight whereas the t h y r o i d weights of R-6 b i r d s were not s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l . R-5 b i r d s rece ived 3.0 ug T^/day. Th is amount of T^ was c l o s e to the d a i l y thy rox ine s e c r e t i o n r a t e of approx imate ly k.O pg T^ (Mel len and Wentworth, I960). Hence, the t h y r o i d gland d id not en la rge as much as the R -2, R-3 and R-k b i r d s . Only a s l i g h t inc rease in the t h y r o i d weight was observed and t h i s inc rease was not s i g n i f i c a n t when compared to the c o n t r o l . At day 20, the percentage of t h y r o i d in respect to body weight of the R -2, R-3 and R-k b i r d s cont inued to increase s i g n i f i c a n t l y whereas R - l , R-5 and R-6 b i r d s e x h i b i t e d a decrease in t h y r o i d we ight . A decrease in t h y r o i d weight has a l s o been observed by Raheja et a l . (1971), when high doses of T^ were g iven to c h i c k s . The increase in weight of the t h y r o i d gland in R -2, R-3 and R-k b i r d s may due to the increase in responsiveness of t h y r o i d to TSH s t i m u l a t i o n , s i n c e t h i s e f f e c t was observed when the animals are p re t rea ted w i t h T, (Se i f et_ a_l_., 1975). These i n v e s t i g a t o r s - 87 -f u r t h e r suggested that the enhancement in t h y r o i d response i s p r o p o r t i o n a l to the dosages of the thy rox ine g iven d a i l y , w h i l e a smal l dose of t h y r o x i n e would increase the s e n s i t i v i t y of the t h y r o i d g land towards TSH s t i m u l a t i o n , l ead ing to an inc rease in t h y r o i d we ight . A l a r g e dose of T^ would i n h i b i t f u r t h e r TSH r e l e a s e from the p i t u i t a r y gland through the negat ive feedback mechanism, r e s u l t i n g in a decrease in the t h y r o i d we ight . The R~5 b i r d s showed a decrease in the percentage of t h y r o i d weight w i t h respect to body w e i g h t , which may be exp la ined by the f a c t that the dosage of T^ used approximated the d a i l y T^ s e c r e t i o n r a t e . It was a c c o r d i n g l y p o s s i b l e that 3.0 ug T^/day was adequate to reduce t h y r o i d a l enlargement in some c h i c k s . In a d d i t i o n , the growth ra te improved a f t e r supp lementat ion , t h e r e f o r e , a lower t h y r o i d % body weight was a n t i c i p a t e d . The same e x p l a n a t i o n can account f o r the observed decrease percentage t h y r o i d body weight of R - l and R-6 b i r d s . Moreover, the d a i l y i n j e c t i o n o f 5.0 ug of T^ to the R-6 b i r d s i n h i b i t e d f u r t h e r r e l e a s e of TSH from the p i t u i t a r y and r e l e a s e of TRH from the hypothalamus (Synder and U t i g e r , 1972; Dupont et_ aj_. , 1976) w i t h the f i n a l r e s u l t that the t h y r o i d gland rece ived no f u r t h e r s t i m u l a t i o n . Al though the t h y r o i d weights v a r i e d among d i f f e r e n t t rea tments , h i s t o l o g i c a l examinat ion provided a c l e a r e r p i c t u r e . Chicks t r e a t e d w i th t h i o u r a c i l d i s p l a y e d an inc rease in the amount of t h y r o i d a l e p i t h e l i a l t i s s u e when compared to the c o n t r o l b i r d s a t day 8. Th is i n d i c a t e d that the t h i o u r a c i l began to mani fes t i t s a n t i t h y r o i d e f f e c t a l though the t h y r o i d weight d i d not - 88 -i n d i c a t e s i g n i f i c a n t e f f e c t of the t h i o u r a c i l t reatment . The doses of T^ s i g n i f i c a n t l y reduced the t h y r o i d e p i t h e l i u m of the R-5 and R-6 b i r d s to the extent tha t the amount of e p i t h e l i a l t i s s u e i s s i m i l a r to that of the c o n t r o l b i r d s , whereas the b i r d s r e c e i v i n g smal le r amounts of T^ showed a s i g n i f i c a n c e increase in the p r o p o r t i o n o f t h y r o i d e p i t h e l i u m . Th is o b s e r v a t i o n f u r t h e r s u b s t a n t i a t e s the hypothes is of L a b e l l e , (1964) and S i e f et a l . , (1975) that t h y r o i d a l responsiveness to TSH was enhanced. The f a c t that the R-5 b i r d s d i s p l a y e d a decrease in the amount of t h y r o i d e p i t h e l i u m may due to i n d i v i d u a l v a r i a t i o n s i n c e the group of c h i c k s s a c r i f i c e d a t each t ime were d i f f e r e n t , consequent l y , v a r i a t i o n would o c c u r . Th is may a l s o account f o r the inc rease observed at day 20. S ince the dosage used in R~5 was c l o s e to the normal s e c r e t i o n r a t e , whether the dosage was adequate or inadequate to suppress t h y r o i d enlargement depended on the i n d i v i d u a l c h i c k . The graph of i od ine uptake and r e l e a s e graph f o r the R-2 b i r d s showed c o n s i d e r a b l e f l u c t u a t i o n s due to v a r i a t i o n w i t h i n the group. N e v e r t h e l e s s , i t was apparent that t h i o u r a c i l d i d not a f f e c t t r a p p i n g of i o d i d e . The f a c t that t h i o u r a c i l d i d i n h i b i t o r g a n i c b i n d i n g led to a r a p i d d i s c h a r g e o f the accumulated i o d i d e . Hence a rap id r e l e a s e r a t e was observed in a d d i t i o n to a rap id a c c u m u l a t i o n . The uptake of 1-131 by the R~3 and R-4 b i r d s was rap id i n i t i a l l y , but increased more g r a d u a l l y a f t e r 4 hours u n t i l the maximum iod ide c o n c e n t r a t i o n was reached at 16 hours . Th is - 89 -o b s e r v a t i o n may be exp la ined by the f a c t that a small amount of s t i m u l a t e d TSH s e c r e t i o n which in t u r n , led to a f a s t e r i od ine t r a p p i n g r a t e . But s i n c e t h i o u r a c i l i n h i b i t e d the o r g a n i c b ind ing of i o d i d e , the trapped iod ide was re leased r a p i d l y , subsequent ly , on l y a smal l increase in 1-131 uptake was observed due to the balance between the rap id uptake and r e l e a s e of the I —131 accumulated in the t h y r o i d g l a n d . A f t e r the maximum c o n c e n t r a t i o n of 1-131, the r a d i o i o d i n e was re leased r a p i d l y because steps of t h y r o i d hormone b i o s y n t h e s i s were blocked by t h i o u r a c i l . . Fur thermore, a la rge amount of i od ide in the t h y r o i d was a l s o re leased wi thout being o x i d i z e d (Nadler and Leblond, 1958). The f a c t that R - l , R~3 and R~5 b i r d s e x h i b i t e d an inc rease in t h y r o i d a l 1-131 at 32 hours a f t e r i n j e c t i o n of I — 131 i s not e x p l a i n e d . From the parameters s t u d i e d , i t was found that at l e a s t 3 ug of DjL-T^ per day was requ i red to counteract the potent a n t i -t h y r o i d e f f e c t of t h i o u r a c i l as evidenced by the decrease in t h y r o i d we ight , t h y r o i d e p i t h e l i u m and accumulat ion of very l i t t l e I —131 -A small q u a n t i t y of t h y r o i d hormone would on ly aggravate the c o n d i t i o n s i n c e the e f f e c t of the TSH was enhanced. There was consequent ly evidence of increased t h y r o i d a c t i v i t y such as increase in t h y r o i d we ight , t h y r o i d e p i t h e l i u m and a much higher 1-131 uptake. Furthermore, even w i t h high T^ supp lementat ion , the growth r a t e of the c h i c k s t r e a t e d w i t h t h i o u r a c i l was s i g n i f i c a n t l y lower than the c o n t r o l b i r d s , i n d i c a t i n g the - 90 -depress ion of growth r a t e may not be e n t i r e l y exp la ined as s imply as by the i n h i b i t i o n of t h y r o i d hormone s y n t h e s i s . - 91 -Table XVI I . Composit ion of d i e t s fed in Experiment 5-Ingredients Diet 1 D iet 2 o, o, Corn oi1 1 . 0 Corn 63. 85 Soybean meal 27. 5 D i s t i l l e r ' s d r i e d solub1es 2. 0 Dehydrated c e r e a l grass 2. 0 Limestone 1 . 5 Calc ium phosphate 1 . 5 Iodized s a l t 0. 5 D,L-meth ion i ne 0. 1212 M i c r o n u t r i e n t s as d i e t 1 w i t h 0.1% th iourac i1 Die ts 3 - 6 a re the same as d i e t 2. -•"/kg: manganese s u l p h a t e 132 mg, z i n c ox ide 62 mg, r i b o f l a v i n k mg, n i a c i n 10 mg, v i t a m i n 13 meg, c h o l i n e 985 mg, v i t a m i n A kkOO I .U. , v i t a m i n D, hhO I .C .U . - 92 -Table XV I I I . D i f f e r e n t t reatments imposed in Experiment 5-Diet (R) Treatments d e s c r i p t i o n 1 a d m i n i s t r a t i o n of 0.1 ml d i s t i l l e d water/day 2 a d m i n i s t r a t i o n of 0.1 ml d i s t i l l e d water/day 3 a d m i n i s t r a t i o n of 0.5 meg T^/day in 0.1 ml p h y s i o l o g i c a l s a l i n e k a d m i n i s t r a t i o n of 1.0 meg T^/day in 0.1 ml p h y s i o l o g i c a l s a l i n e 5 a d m i n i s t r a t i o n of 3-0 meg T^/day in 0.1 ml p h y s i o l o g i c a l s a l i n e 6 a d m i n i s t r a t i o n of 5-0 meg T^/day in 0.1 ml p h y s i o l o g i c a l s a l i n e - 93 -Table XIX. Averaged body weight of c h i c k s at the age of 7, 13 and 19 days in Experiment 5-Treatments (R) Average body weight (g) Day _7_ 13 11 1 63 104 I63 a(50)* 2 63 87 H6 b (49) 3 65 98 I 4 l c d ( 5 0 ) 4 64 96 I48 c d (49) 5 59 87 138 C(5D 6 60 92 l47 C d (50) » Number of b i rds abcde va lues w i t h the same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t P < 0.01 . - 3k -Table XX. Thyro id weights of c h i c k s in d i f f e r e n t t reatments at days 8, ]k and 20 in Experiment 5. ~m. , , /n\ Ave. t h y r o i d weight Treatments (R) 0/ 7 . a mg 4, of body weight Day 8_ _T4_ 20_ 9.32a b(5)* 8.53a(5)^ 6.28a(5)* I4.7 b(5) 39-3 b(5) 56.8 b(5) 3 12.8 a b(5) Zk.kb{5) lk.2b{5) k 11.5ab(5) 43.2b(6) 67.7b(4) 5 8.74a(5) 11.2a(4) 9.53a(6) 6 13-2 a b(5) 12.5 a(5) 5.34 a(5) " Number of c h i c k s ab va lues w i t h the same s u p e r s c r i p t at each p a r t i c u l a r day are not s i g n i f i c a n t l y d i f f e r e n t P < 0.05-- 95 -Table XXI . Percentage e p i t h e l i a l t i s s u e of t o t a l t h y r o i d f o r d i f f e r e n t t reatments at d i f f e r e n t days in Experiment 5-Treatments (R) % e p i t h e l i a l t i s s u e of t o t a l Day 8 JA 20 1 36.2a(5)* 39.8a(4)^ 33-6a(5)-2 55.0b(5) 6l.6b(5) 60.2b(5) 3 57.3b(4) 63.5b(5) 51.8b c(5) k 60.3b(5) 64.4b(6) k3.Sbcd(k) 5 6l.2b(5) 39-5a(3) 45.0 c d(6) 6 6l.7b(4) 34.3a(4) 38.8a d(5) Number of c h i c k s per treatment abed va lues w i th the same s u p e r s c r i p t at each p a r t i c u l a r day are not s i g n i f i c a n t l y d i f f e r e n t P < 0.05. - 96 -T a b l e X X I I . T h y r o i d a l u p t a k e o f 1—131 f o r d i f f e r e n t t r e a t m e n t s a t d i f f e r e n t t i m e s a f t e r i n j e c t i o n i n E x p e r i m e n t 5. T r e a t m e n t s (R) T h y r o i d a l c o n c e n t r a t i o n o f i n j e c t e d d o s e T i m e ( h o u r s ) k_ 8 II IA 24 32 1 16.45ab(8)* 17.56ab(8) 17-38ab(8) 17-97a(8) l6.84a(8) 20.37ab(5) 2 36.23c(8) 31.60c(7) 39.59°(7) 20.39a(8) 29.67b(7) 28.04a(4) 3 24.12a(8) 24.85ac(8) 27.52a(8) 36-34b(8) 21.79ab(8) 27-59a(5) 4 23.78a(8) 25.0ac(8) 27.12a(8) 37-53b(8) 23.02ab(7) 17.89ab(5) 5 8.88bd(8) 11.4lbd(7) 9.08bd(5) 5.75°(8) 2.65c(8) 8.40bc(5) 6 3.24d(5) 2.95d(4) 2.91d(6) 1.44c(6) 0.42c(8) 0.59c(5) * Number o f c h i c k s t r e a t m e n t a t a p a r t i c u l a r t i m e a b e d v a l u e s w i t h t h e same s u p e r s c r i p t a r e n o t s i g n i f i c a n t l y d i f f e r e n t P < 0.05. - 97 -40 H O U R S A F T E R I N J E C T I O N igure 13 . Thy ro ida l 1-131 u p t a k e . o f c h i c k s at the age of 3 weeks in Experiment 5 . - 98 -Experiment 6: AUTORADIOGRAPHIC STUDIES OF THE UPTAKE AND RELEASE OF 1-125 IN THYROID GLANDS OF CHICKS TREATED WITH THIOURACIL OR FED WITH RAPESEED MEAL In the preceeding exper iments , i t was found that t h y r o i d a l iod ine uptake and r e l e a s e were a f f e c t e d by the feed ing of rapeseed meal or t h i o u r a c i l as a r e s u l t of t h e i r g o i t r o g e n i c a c t i v i t y . The present experiment was conducted to study f u r t h e r the means by which these go i t rogens may i n t e r f e r e w i th the iod ine metabol ism in the t h y r o i d g lands o f c h i c k s , employing autorad iography to f o l l o w the uptake and r e l e a s e of 1-125. M a t e r i a l s and Methods S e v e n t y - f i v e , d a y - o l d White Leghorn male c h i c k s were randomly d i s t r i b u t e d in to nine cages . Three groups of c h i c k s were ass igned to each of three t rea tments . The t reatments were R - l , a d i e t c o n t a i n i n g soybean meal as the source of supplementary p r o t e i n ; R -2, the R - l d i e t w i t h 0.1% t h i o u r a c i l added to i t ; and R~3, a d i e t c o n t a i n i n g rapeseed meal as the source of supplementary p r o t e i n (Table X X I I I ) . Feed and water were g iven ad 1 ibiturn. When the c h i c k s were 8 weeks o l d , 0.0k uCi of 1-125 in 0.1 ml of p h y s i o l o g i c a l s a l i n e (Radiochemical Cent re , Amersham) was i n j e c t e d i n t r a v e n o u s l y i n t o each of the b i r d s . S i x b i r d s from each treatment were k i l l e d at 5, 10, 15 and 20 hours a f t e r i n j e c t i o n w i th the except ion of R - l and R-2 where 7 b i r d s were k i l l e d a t - 99 -5 hours a f t e r i n j e c t i o n . The t h y r o i d glands were removed. The l e f t lobe of each gland was weighed and put in to a tube c o n t a i n i n g 3.0'ml of 2N NaOH fo r d i g e s t i o n . The r i g h t lobe of each gland was q u i c k l y f i x e d in Baker ' s formal c a l c i u m f o r a u t o r a d i o g r a p h i c stud i e s . The r a d i o a c t i v i t y present in the l e f t lobe of each t h y r o i d was measured. However, the w e l l ^ t y p e s c i n t i 1 l a t i o n de tec to r used was not very e f f i c i e n t in count ing 1-125, s i n c e i t is a gamma ray d e t e c t o r , t h e r e f o r e , l i q u i d s c i n t i 1 l a t i o n count ing was used i n s t e a d . The presence of 2N NaOH created problems because the la rge amount of water present rendered the samples immisc ib le w i t h the s c i n t i l l a t i o n f l u i d . Some t h y r o i d glands had a l ready d i s s o l v e d in the NaOH. Hence, the und isso l ved glands were t r a n s f e r r e d to the s c i n t i l l a t i o n v i a l s c o n t a i n i n g NCS* (I2x weight of gland) purchased from Amersham fo r d i g e s t i o n and then d e c o l o r i z e d by adding 30% H 2 0 2 CO.2 ml 30% H ^ / m l NCS used) s i n c e the NCS i s s l i g h t l y y e l l o w i s h in c o l o r . As f o r the t h y r o i d that had a l ready d i s s o l v e d in the NaOH, an A m b e r l i t e CG 120 type 2 r e s i n (F isher ) was added to the tube. The volume of r e s i n used was two times that of NaOH in order to remove a l l the sodium i o n s . The mix ture was l e f t overn ight f o r the r e s i n to s e t t l e and 1.0 ml of the c l e a r supernatant was taken out f o r count ing w i th 10 ml S c i n t i V e r s e complete c o c k t a i l ( F i s h e r ) . The NCS d i g e s t was counted w i t h 15 Tils of to luene s c i n t i l l a t i o n f l u i d . The samples were counted in a NCS is a t i s s u e s o l u b i l i z e r f o r l i q u i d s c i n t i l l a t i o n count ing - 100 -Un l lex II counter (Nuclear Ch icago ) , and the s e t t i n g s of the counter were f o r maximum c o u n t s . The d i s c r i m i n a t o r window f o r channel A was set at L-°° w h i l e those of channels B and C were set at L -U . The r a d i o a c t i v i t y present in the NCS d i g e s t and the supernatant was combined. The r i g h t lobe of each t h y r o i d was f i x e d and s e c t i o n e d . The glands were de-waxed and the s l i d e s were dipped in NTB-2 emulsion (Eastman-Kodak C o . , Rochester , New York) and s tored in b lack boxes a t h°C f o r 7 days . The s l i d e s were then developed f o r 2 minutes w i th Dektol (Kodak) d i l u t e d 1:1 w i t h water and then f i x e d in Kodak F i x e r fo r 10 minutes . The s l i d e s were then placed in water f o r another 10 minutes (S lonecker , 1971)- The a u t o -radiographs were s t a i n e d w i t h nuc lear f a s t red (Roboz S u r g i c a l Company, Washington, D.C.) and analyzed q u a l i t a t i v e l y . Photo -micrographs of radioautographs were taken . Resu l t s The l e f t lobe of each t h y r o i d gland was weighed and expressed as a percentage of body weight (.Table XXIV) . The r e s u l t s c l e a r l y showed that t h i o u r a c i l treatment induced g reate r t h y r o i d a l enlargement than the rapeseed meal t reatment . The t h y r o i d a l uptake of 1-125 (Table XXV and F igure ]k) by the l e f t lobe of the gland showed that the r a t e of iod ine uptake was h ighest in the t h i o u r a c i 1 - t r e a t e d group w i t h maximum c o n c e n t r a t i o n at 10 hours . B i r d s fed rapeseed meal a l s o showed a maximum - 101 -c o n c e n t r a t i o n of iod ine at 10 hours a f t e r i n j e c t i o n . The r a t e of uptake was, however, much slower than in the t h i o u r a c i 1 - t r e a t e d group. The uptake of 1-125 f o r the c o n t r o l b i r d s was r e l a t i v e l y c o n s t a n t . N e v e r t h e l e s s , there was marked v a r i a t i o n in the t h y r o i d a l uptake of i od ine among i n d i v i d u a l b i r d s w i t h i n the t rea tments , p a r t i c u l a r l y in the rapeseed m e a l - f e d b i r d s . The autoradiographs were s tud ied under the microscope . It was found that there were too many s i l v e r g r a i n s , even w i th 0.04 uCi of 1-125, to permit a v a l i d g r a i n count . Photomicrographs of autorad iographs were taken (F igures 15-26) which showed that the r a d i o i o d i n e taken up was g e n e r a l l y u n i f o r m l y d i s t r i b u t e d in the t h y r o i d glands of the b i r d s in a l l three t rea tments . The q u a n t i t y of r a d i o i o d i n e accumulated in to the t h y r o i d a l f o l l i c l e s of R - l b i r d s was s i m i l a r at the d i f f e r e n t t imes a f t e r i n j e c t i o n throughout the exper iment . In the R-2 b i r d s , the maximum c o n c e n t r a t i o n of 1-125 in the t h y r o i d a l f o l l i c l e s was observed a t 10 hours a f t e r i n j e c t i o n . By 20 hours a f t e r 1-125 a d m i n i s t r a t i o n , most of the r a d i o i o d i n e had been re leased from the t h y r o i d g l a n d s . In the R-3 b i r d s , the maximum c o n c e n t r a t i o n of iod ine accumulated i n t o the f o l l i c l e was a l s o observed at 10 hours a f t e r r a d i o i o d i n e i n j e c t i o n . On the other hand, at 15 and 20 hours a f t e r i n j e c t i o n , there appeared to be a moderate amount of r a d i o i o d i n e s t i l l present in the f o l l i c l e s , in c o n t r a s t to the R-2 b i r d s which showed a more rapid r e l e a s e of r a d i o i o d i n e as i n d i c a t e d in F igures 22, 23, 25 and 26. - 102 -Another o b s e r v a t i o n s made from the p h o t o m i c r o g r a p h s was t h a t t h e s i z e o f the f o l l i c l e s v a r i e d c o n s i d e r a b l y even w i t h i n t he same t r e a t m e n t and w i t h i n the same g l a n d . In a d d i t i o n t o the v a r i a t i o n i n the s i z e o f the f o l l i c l e s , the amount o f the e p i t h e l i a l t i s s u e p r e s e n t a l s o f l u c t u a t e d among i n d i v i d u a l b i r d s i n a l l t r e a t m e n t s . D i s c u s s i o n and C o n c l u s i o n s The t h y r o i d w e i g h t s o f the R-2 ( t h i o u r a c i 1 - t r e a t e d ) b i r d s were s i g n i f i c a n t l y d i f f e r e n t t h a n t h o s e o f the R - l ( c o n t r o l ) and R~3 (rapeseed meal-fed) b i r d s . There was no s i g n i f i c a n t d i f f e r e n c e between the t h y r o i d w e i g h t s o f the c h i c k s on t h e s e l a t t e r t r e a t m e n t s . When the t h y r o i d w e i g h t s of t h e R-l and R-3 b i r d s were a n a l y s e d s e p a r a t e l y , however, t h e r e was a s i g n i f i c a n t d i f f e r e n c e between them. The enlargement o f the t h y r o i d g l a n d a f t e r t h i o u r a c i l t r e a t m e n t and f e e d i n g o f rapeseed meal would be a n t i c i p a t e d . The d a t a on the t h y r o i d a l c o n c e n t r a t i o n o f 1-125 ( T a b l e XXV) showed t h a t t h e r e was a more r a p i d i n c r e a s e and d e c r e a s e i n R-2 b i r d s . At 15 hours a f t e r i n j e c t i o n o f 1-125, a s t a t i o n a r y phase was o b s e r v e d which may be e x p l a i n e d by t h e f a c t t h a t some r a d i o i o d i n e was r e t a i n e d i n the t h y r o i d g l a n d as MIT or DIT due t o i n c o m p l e t e b l o c k a g e o f t h y r o i d hormone b i o s y n t h e s i s by 0.1% t h i o u r a c i l (Wentworth and M e l l e n , 1961). A n o t h e r p o s s i b l e e x p l a n a t i o n f o r t h i s h i g h p e r c e n t a g e o f 1-125 - 103 -remaining in the t h y r o i d a f t e r the i n i t i a l rap id r e l e a s e of r a d i o i o d i n e was the tremendous i n i t i a l uptake of r a d i o i o d i n e . A s i m i l a r r a t e of uptake has a l s o been observed in q u a i l s (Howarth and Marks, 1973). Hence, a s i g n i f i c a n t amount of 1-125 was r e t a i n e d in the t h y r o i d even a f t e r the i n i t i a l r e l e a s e of r a d i o i o d i n e by the t h y r o i d g l a n d . The rapeseed m e a l - f e d c h i c k s a l s o d i s p l a y e d an increase in t h y r o i d a l r a d i o i o d i n e uptake r e l a t i v e to the c o n t r o l s . As in the R-2 b i r d s , the peak of accumulat ion was a t 10 hours a f t e r i n j e c t i o n of 1-125. A rap id r e l e a s e of 1-125 was noted a f t e r the peak of i od ine uptake . The percentages of 1-125 uptake in the rapeseed m e a l - f e d c h i c k s at 10-20 hours were h igher than in the c o n t r o l b i r d s but the d i f f e r e n c e s was not s i g n i f i c a n t l y d i f f e r e n t . The f a c t that the r a t e of r a d i o i o d i n e uptake was lower in the R~3 b i r d s than in the R-2 b i r d s suggested that the g o i t r o g e n i c ! of the rapeseed meal was weaker than that of t h i o u r a c i l but does not i n d i c a t e whether the e f f e c t i s a q u a l i t a t i v e or a q u a n t i t a t i v e one w i th respect to the go i t rogens p r e s e n t . The photomicrographs of the autoradiographs showed that the r a d i o i o d i n e concentrated was evenly d i s t r i b u t e d in the c o l l o i d of the i n d i v i d u a l t h y r o i d f o l l i c l e s a l though the c o n c e n t r a t i o n v a r i e d markedly among the f o l l i c l e s present in any one g l a n d . Th is uni form d i s t r i b u t i o n was observed in the t h y r o i d glands of c h i c k s in a l l t reatments at a l l t imes . By c o n t r a s t , l i t t l e r a d i o i o d i n e was observed in the e p i t h e l i a l c e l l s of the t h y r o i d . These o b s e r v a t i o n s - 104 -conf i rm the f i n d i n g s on the iod ine d i s t r i b u t i o n reported by other i n v e s t i g a t o r s (Nadler and Leb lond , 1955; N a d l e r , 1965; Simon and Droz , 1965; Doniach, 1967; Andros and Wollman, 1967)-The f a c t that the r a d i o i o d i n e had d i f f u s e d evenly through the c o l l o i d w i t h i n 5 hours a f t e r 1-125 i n j e c t i o n i n d i c a t e d that the r a d i o i o d i n e was r a p i d l y t ranspor ted across the c e l l s i n t o the f o l l i c u l a r lumen. A r i n g r e a c t i o n was not observed in the present ex -periment in c o n t r a s t to t h e o b s e r v a t i o n s of Nadler and Leblond •(1955) and Nadler (1965). Our f i r s t o b s e r v a t i o n s however, were made at 5 hours a f t e r 1-125 i n j e c t i o n , and by t h i s t i m e , any r i n g that had formed i n i t i a l l y may have d i f f u s e d evenly throughout the c o l l o i d . The photomicrographs of the t h y r o i d autoradiographs of R-2 c h i c k s showed that rap id d i s c h a r g e of r a d i o i o d i n e was o c c u r r i n g by 10 hours a f t e r a d m i n i s t r a t i o n . The d i s t r i b u t i o n of the 1-125, however, was s t i l l q u i t e un i form by 20 hours a f t e r i n j e c t i o n . Th is o b s e r v a t i o n suggested the r a d i o i o d i n e s tored near the c o l l o i d c e l l su r face was not re leased p r e f e r e n t i a l l y . in the autorad iographs of the c h i c k t h y r o i d s , i t was observed tha t a c t i v i t y of i n d i v i d u a l f o l l i c l e s v a r i e d . The s m a l l e r f o l l i c l e s appeared to be more a c t i v e and accumulated more r a d i o i o d i n e than the la rge r ones. A s i m i l a r o b s e r v a t i o n was made by Andros and Wollman (1967) on the l o c a l i z a t i o n of i od ine in the ra t t h y r o i d . They found that the image d e n s i t y over the lumen of the a c t i v e gland was denser over the c e l l s . Dens i ty over small f o l l i c l e s was u s u a l l y g reate r than over l a r g e ones a f t e r r a d i o i o d i n e i n j e c t i o n at short time i n t e r v a l s , but g e n e r a l l y was independent of f o l l i c l e diameter - 105 -a t l o n g time i n t e r v a l s a f t e r i n j e c t i o n . These f i n d i n g s by Andros and Wollman (1967) were a l s o o b s e r v e d i n our p h o t o m i c r o g r a p h s . I f i t may be assumed t h a t s m a l l e r , more a c t i v e f o l l i c l e s , i n a d d i t i o n t o a c c u m u l a t i n g i o d i n e a t a more r a p i d r a t e , l i k e w i s e d i s c h a r g e d i o d i n e more r a p i d l y than do l a r g e r f o l l i c l e s , the " l a s t come, f i r s t s e r v e d " p r i n c i p l e o f S c h n e i d e r (1964) i s e x p l a i n e d . The more a c t i v e s m a l l f o l l i c l e s would r e l e a s e t h e r e c e n t l y t r a p p e d i o d i n e f a s t e r than t he l a r g e r l e s s a c t i v e f o l l i c l e s , thus g i v i n g an i m p r e s s i o n t h a t the i o d i n e s t o r e d l a s t i n t h e g l a n d as a whole i s s e c r e t e d f i r s t . L o e w e n s t e i n and Wollman (1970) and Smeds et_ aj_. (1977) a l s o r e p o r t e d t h a t the l a r g e r f o l l i c l e s u s u a l l y r e t a i n the a d m i n i s t e r e d r a d i o i o d i n e f o r a l o n g e r p e r i o d o f t i m e t h a n the s m a l l f o l l i c l e s . T h i s i s i n agreement w i t h t h e o b s e r v a t i o n s made i n th e p r e s e n t e x p e r i m e n t , t h a t t h e r e was v a r i a t i o n i n a c t i v i t y i n the i n d i v i d u a l f o l l i c l e s . R a d i o i o d i n e was o b s e r v e d t o be d i s t r i b u t e d u n i f o r m l y i n the f o l l i c u l a r c o l l o i d , and i t was a l m o s t t o t a l l y a b s e n t w i t h i n the e p i t h e l i a l c e l l s . Matsumoto et_ aJL (1969) a l s o found t h a t t he t h y r o i d g l a n d c o n t a i n s a p p r e c i a b l e i n o r g a n i c i o d i n e f o r many hours a f t e r i t s a d m i n i s t r a t i o n . These o b s e r v a t i o n s do not s u p p o r t t he h y p o t h e s i s o f o t h e r i n v e s t i g a t o r s ( P i t t - R i v e r s , 1967; L i ss i t z k y e t a 1 . , 1969; C r o f t and P i t t - R i v e r s , 1971; Rapoport and DeGroot, 1971) t h a t i o d i n a t i o n o c c u r s a t the a p i c a l membrane o f the f o l l i c u l a r c e l l b o r d e r i n g the lumen. - 106 -The t h y r o i d a l iod ine may p e r s i s t in the iod ide form f o r some time depending upon the t h y r o i d s t a t e of the a n i m a l . Matsumoto et a l . (1969) found that at 4 hours a f t e r r a d i o i o d i n e a d m i n i s t r a t i o n to the PTU- t reated c h i c k s , over k0% of the iod ine present was s t i l l in the ino rgan ic form. In the present exper iment , much of the r a d i o i o d i n e that had been accumulated i n t o the t h y r o i d glands of the R-2 c h i c k s was d ischarged presumably as ino rgan ic i o d i d e , by 20 hours a f t e r 1-125 a d m i n i s t r a t i o n . The photomicrographs showed on ly a small amount of r a d i o i o d i n e in the t h y r o i d glands of the R-2 b i r d s a f t e r 20 h o u r s , a l t h o u g h , t h e r e were d i f f e r e n c e s among i n d i v i d u a l f o l l i c l e s . Nadler and Leblond (1958) found that a s u b s t a n t i a l f r a c t i o n of the r a d i o i o d i n e accumulated i n t o the t h y r o i d gland was returned to the c i r c u l a t i o n as i o d i d e . Hence, i t i s l i k e l y in the present experiment that r a d i o i o d i n e which was concent rated r a p i d l y by the t h y r o i d gland and t ranspor ted in to the c o l l o i d d i r e c t l y as inorgan ic i od ide was subsequent ly re leased as such from the g l a n d . The photomicrographs of the t h y r o i d of c h i c k s fed rapeseed meal (R~3) showed less r a d i o i o d i n e accumulat ion than those of c h i c k s g iven t h i o u r a c i l . Th is o b s e r v a t i o n i s in agreement w i t h the r e s u l t s of the de te rminat ions of t o t a l t h y r o i d a l uptake of r a d i o i o d i n e (F igure 14) in the c h i c k s on the two t reatments . Th is cou ld be due to the presence of i s o t h i o c y a n a t e and th iocyanate which b lock t rapp ing of iod ine (Rutkowski , 1971). The g o i t r o g e n i c e f f e c t s of these compounds a r e , however, r e l a t i v e l y weak. Consequent ly , - 107 -r a d i o i o d i n e accumulat ion would not be complete ly b locked by these g o i t r o g e n s . Lo and B e l l (1972) concluded that the go i t rogens in the rapeseed meal i n t e r f e r e w i t h the o r g a n i c b ind ing and the c o u p l i n g r e a c t i o n in t h y r o i d hormone s y n t h e s i s . It has been observed that g o i t r i n - t r e a t e d b i r d s have a h igher percentage of MIT and a lower percentage of T^ and T^ (Matsumoto e_t a j_ . , 1969); Akiba and Matsumoto, 1971; Leung and March, 1976). These i n v e s t i g a t o r s , t h e r e f o r e , concluded t h a t g o i t r i n does not i n h i b i t the monoiod inat ion of t y r o s i n e but main ly i n h i b i t s the c o u p l i n g r e a c t i o n . Hence, i t would appear that the r a d i o i o d i n e i n d i c a t e d in the photomicrographs of the R~3 b i r d s was l a r g e l y in MIT form and as ino rgan ic i o d i d e . Akiba and Matsumoto ( 1 9 7 1 ) showed a s lower r e l e a s e of the incorporated iod ide from the t h y r o i d glands of rapeseed mea l - fed c h i c k s . This was a l s o observed in photomicrographs of R~3 b i r d s taken at 20 hours when compared to the photomicrographs of R-2 b i r d s . Another o b s e r v a t i o n from the photomicrographs of the t h y r o i d glands of R-2 b i r d s at 20 hours was the presence of a small q u a n t i t y of r a d i o i o d i n e ; whereas, the r a d i o a c t i v i t y detected was much h igher than those of R - l and R~3 b i r d s , whose t h y r o i d photomicrographs e x h i b i t e d the presence of more r a d i o i o d i n e . Th is can be e x p l a i n e d by the f a c t that the t h y r o i d gland of the t h i o u r a c i 1 - t r e a t e d b i r d s , i n i t i a l l y accumulated a l a r g e q u a n t i t y of 1-125- Subsequent ly , there was s t i l l a l o t of 1-125 present in the t h y r o i d g l a n d s , d i s t r i b u t e d among the v a r i o u s f o l l i c l e s a f t e r the i n i t i a l r e l e a s e of i o d i n e . If the t h y r o i d r a d i o a c t i v i t y was expressed in terms of t h y r o i d we ight , the % uptake of 1-125 of the t h i o u r a c i 1 - t r e a t e d and the rapeseed m e a l - f e d b i r d s would be s m a l l e r than those of the c o n t r o l (Leung, 1975)• - 108 -The a n t i t h y r o i d a c t i v i t y of 0 .1% d i e t a r y t h i o u r a c i l was more potent than of the rapeseed meal d i e t as i n d i c a t e d by greater enlargement of the t h y r o i d gland. Both t h i o u r a c i l and rapeseed meal stimulated increased uptake of r a d i o i o d i n e . The e f f e c t s on r a d i o i o d i n e r e l e a s e , however, are not c l e a r in t h i s experiment. - 109 -Table X X I I I . Composit ion of d i e t s as fed in Experiment 6. 1ngred ien ts Diet 1 % Diet 2 9 Diet 3 % Wheat 31.4 as d i e t 1 23-5 + Corn 31.4 0.05% 23-5 th iourac i1 Soybean meal 29.1 Rapeseed meal (Span) 45 .6 Calcium phosphate 1.3 0 .6 L imestone 1.3 1 -3 Iodized s a l t 0 . 5 0 . 5 D i s t i l l e r ' s d r i e d s o l u b l e s 2 .0 2 .0 Ta11ow 3 - 0 3 - 0 Lys ine HC1 98% 0.1 D ,L -meth ion ine 99% 0.05 Cho i i ne 0.049 M i c ronut r i ents -'-/kg: manganese su lphate 132 mg, z i n c ox ide 62 mg, r i b o f l a v i n 4 mg, f o l a c i n 0 .55 mg, n i a c i n 27 mg, c a l c i u m pantothenate 9.3 mg, v i t a m i n D^  440 I .C .U . v i t a m i n 8 ^ 1 3 meg, v i t a m i n A 4400 I.U. - 110 -Table XXIV. Average t h y r o i d weights of c h i c k s at the age of 8 weeks ( l e f t lobe o n l y ) , in Experiment 6. /D\ Thyro id weight Treatments vK; /inn u J • mg/100 g body weight 1 Soybean meal 4.22 (25)* 2 Soybean + T .U . 43.7 (25) a R Rapeseed meal . 10.2 (23) * Number of c h i c k s ab va lues w i t h the same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t P < 0.05 AB s i g n i f i c a n t l y d i f f e r e n t by separate comparison P < 0.01 - I l l -Table XXV. Uptake of 1-125 by the l e f t lobe of the t h y r o i d glands of c h i c k s at the age of 8 weeks in Experiment 6. / n x Uptake of 1-125 Treatment (R) . 0/ c . . . . , % of i n j e c t e d dose 5 hours S . D . " 10 h r s . S .D . 15 h r s . S .D . 20 h r s . S .D . 1 7-73a(6)**- 3-95 6.33 a(6) 2.84 6.88a(6) 6.05 6.29 a(6) 2.02 2 25.32b(7) 16.01 40.22 b(5) 19-71 29.01b(6) 14.44 28.93b(6) 21.53 3 5.60a(5) 1-80 27-47 a b(6)31 -42 10.49a(6) 10.49 l4.49 a b (4) 9-82 Level of s i g n i f i c a n c e 5% 10% 5% 5% * Standard d e v i a t i o n ** Number of c h i c k s per treatment ab va lues w i th the same s u p e r s c r i p t w i t h i n the same time i n t e r v a l a re not s i g n i f i c a n t l y d i f f e r e n t . - 112 -o 5 To : rr~ 20 H O U R S F igure 14. Average percentage 1-125 uptake by t h y r o i d of c h i c k s at the age of 8 weeks. Rl Soybean mea R2 Soybean meal + O.U .T .U. , R3 Rapeseed meal . F igure 16. Autoradiograph of t h y r o i d gland of t h i o u r a c i 1 - t r e a t e d b i r d 5 hours a f t e r 1-125 i n j e c t i o n (400x) F igure 18. Autoradiograph of t h y r o i d g land of c o n t r o l b i r d 10 hours a f t e r 1-125 i n j e c t i o n (400x) F igure 20. Autoradiograph of t h y r o i d g land of rapeseed m e a l - f e d b i r d 10 hours a f t e r 1-125 i n j e c t i o n (400x) - 116 -F i g u r e 22. Autoradiograph of t h y r o i d gland of t h i o u r a c i 1 - t r e a t e d b i r d 15 hours a f t e r 1-125 i n j e c t i o n (400x) - 117 -F igure 2k. Autoradiograph of t h y r o i d gland of c o n t r o l b i r d 20 hours a f t e r 1-125 i n j e c t i o n (400x) - 118 -F igu re 26. Autoradiograph of t h y r o i d gland of rapeseed m e a l - f e d b i r d 20 hours a f t e r 1-125 i n j e c t i o n (400x) - 119 -Experiment 7- EFFECTS OF RAPESEED MEALS WITH HIGH OR LOW CONCENTRATIONS OF GLUCOSINOLATES ON THE THYROIDAL UPTAKE AND RETENTION OF IODINE. Rapeseed c o n t a i n s d i f f e r e n t amounts of at l e a s t seven g l u c o s i n o l a t e s depending upon the c u l t i v a r (Josefsson and Muhlenberg, 1968). These are p o t e n t i a l sources of a number of g o i t r o g e n i c compounds such as t h i o c y a n a t e , a 1 1 y l i s o t h i o c y a n a t e and g o i t r i n . S y n e r g i s t i c e f f e c t s of these compounds have been observed (Langer, 1966). The g o i t r o g e n i c d e r i v a t i v e s of the g1ucos ino la tes present in the rapeseed i n t e r f e r e w i t h the normal t h y r o i d metabol ism. New c u l t i v a r s of rape have been developed in which the g1ucos ino la te content has been much reduced. The experiment reported here was conducted to compare meals made from two such c u l t i v a r s , Tower and Candle , w i t h a high g l u c o s i n o l a t e c u l t i v a r , Span, on the uptake and r e t e n t i o n of i od ine by the t h y r o i d g l a n d . M a t e r i a l s and Methods The meals were fed to c h i c k s at l e v e l s of approx imate ly h0% in d i e t s formulated to c o n t a i n 20% of p r o t e i n (Table XXVI) . A soybean d i e t served as c o n t r o l . Each of the d i e t s was fed ad 1i bi turn to 30 - day -o ld White Leghorn male c h i c k s in b a t t e r y brooders . When the c h i c k s were 12 days o l d , 0.5 pCi of I —131 in p h y s i o l o g i c a l s a l i n e was admin is te red i n t r a v e n o u s l y to 12 c h i c k s - 120 -from each t reatment . The c h i c k s were returned to t h e i r cages and provided w i th feed and water . S i x b i r d s were k i l l e d from each treatment 5 hours a f t e r i n j e c t i o n . The remaining s i x i n j e c t e d b i r d s were k i l l e d 12 hours a f t e r i n j e c t i o n . The t h y r o i d glands were e x c i s e d and t h e i r r a d i o a c t i v i t y measured in a w e l l - t y p e s c i n t i l l a t i o n d e t e c t o r . When the remaining c h i c k s were 35 days o l d , they were weighed and i n j e c t e d as before w i t h 2 .0 uCi I —131 -B i rds from each treatment were k i l l e d at 5 , 10 and 20 hours r e s p e c t i v e l y a f t e r i n j e c t i o n and the r a d i o a c t i v i t y of the t h y r o i d glands determined. Resu 1ts The average body weights at 35 days of age (Table XXVI I) showed that the d i e t s c o n t a i n i n g the Span and the Tower meal supported growth rates s i m i l a r to that w i th soybean meal , whereas growth w i th the Candle meal was poorer . The t h y r o i d a l uptake and r e l e a s e ra tes of 1-131 in the t h y r o i d gland of c h i c k s at 12 days of age and 35 days of age are shown in Tables XXVIII and XXIX r e s p e c t i v e l y . It was found that at 12 days of age, the t h y r o i d a l uptake of 1-131 was approx imate ly the same fo r the Span and the Tower meal whereas a lower rate of uptake occur red when the d i e t conta ined Candle meal (Table XXV I I I ) . At 35 days of age, the Tower m e a l - f e d c h i c k s showed a f a s t e r ra te of 1-131 uptake in the t h y r o i d glands at 5 hours than the Span m e a l - f e d c h i c k s . Subsequent ly , the rate of r a d i o i o d i n e r e l e a s e approached the ra te of uptake in the Tower m e a l - f e d b i r d s so that by 10 hours a f t e r - 121 -1-125 i n j e c t i o n the t h y r o i d a l r a d i o a c t i v i t y was s i m i l a r in c h i c k s fed these two kinds of rapeseed meals as i n d i c a t e d in Table XXIX. Candle meal fed c h i c k s , however, d i s p l a y e d a slower uptake at 35 days of age. The r e t e n t i o n of r a d i o i o d i n e in the t h y r o i d glands of the Candle m e a l - f e d c h i c k s was higher than in the Span mea l - fed c h i c k s as shown by the slower r e l e a s e of i n j e c t e d 1-131 at 20 hours a f t e r i n j e c t i o n . These o b s e r v a t i o n s are summarized g r a p h i c a l l y in F igure 27. D i s c u s s i o n and Conc lus ions The growth r a t e of c h i c k s showed that the Candle meal d i d not support maximum growth r a t e . The d i f f e r e n c e between the Candle meal and the other three meals was s i g n i f i c a n t l y d i f f e r e n t . The data .on the turnover of the i n j e c t e d 1-131 in the t h y r o i d g land showed that the q u a n t i t i e s of r a d i o i o d i n e i n j e c t e d (0 .5 uCi at 12 days and 2 .0 uCi at 35 days) were c a l c u l a t e d as p r o -p o r t i o n a l to the body weights of the b i r d s at the r e s p e c t i v e t imes . The s i m i l a r i t y in the t h y r o i d a 1 uptake of 1-131 5 hours a f t e r i n j e c t i o n in the b i r d s of the d i f f e r e n t ages i n d i c a t e s that the s e l e c t e d doses were p r o p o r t i o n a l to the e x t r a t h y r o i d a l iod ine pools at these two t imes . From the data of the t r i a l conducted at 35 days of age, i t can be seen that the maximum c o n c e n t r a t i o n of r a d i o i o d i n e appeared in the t h y r o i d glands between 5 and 10 hours a f t e r i n j e c t i o n . More rap id uptake was a s s o c i a t e d w i t h more rap id r e l e a s e of r a d i o i o d i n e from the g l a n d s . E x t r a p o l a t i o n of the l i n e s i n d i c a t i n g uptake and - 122 -r e l e a s e ra te r e s p e c t i v e l y i n d i c a t e s that the time at which r a d i o i o d i n e c o n c e n t r a t i o n was maximum in the glands of the rapeseed m e a l - f e d c h i c k s a t 6 to 8 hours a f t e r i n j e c t i o n . B i rds fed Tower and Span rapeseed meals showed more rap id d i s c h a r g e as we l l as more rap id uptake of 1-131- The c o n t r o l b i r d s fed the soybean meal d i e t showed the lowest r a t e and t o t a l uptake of 1-131 f o l lowed by the b i r d s fed the Candle meal . The on ly s i g n i f i c a n t d i f f e r e n c e in uptake and turnover of 1—131 between the Span and the Tower m e a l - f e d b i r d s occurred in the d e t e r m i n a t i o n made 5 hours a f t e r i n j e c t i o n at 35 days of age when uptake by the Tower m e a l - f e d b i r d s was more r a p i d . There was a subsequent e a r l y r e l e a s e of 1-131 from the glands in these b i r d s , however, so that by 10 hours the r e t e n t i o n of I —131 in the gland of the Span and the Tower m e a l - f e d b i r d s was s i m i l a r . Thy ro ida l r e t e n t i o n of 1-131 was s i m i l a r in a l l the rapeseed m e a l - f e d b i r d s by 20 hours a f t e r i n j e c t i o n but was h igher than in the c o n t r o l b i r d s . Lo and B e l l (1972) concluded from t h e i r study on the e f f e c t s of g l u c o s i n o l a t e s in r a t s that the g l u c o s i n o l a t e products of rapeseed meal ( c o n t a i n i n g g o i t r i n and i s o t h i o c y a n a t e ) and Ye l low Sarson meal ( c o n t a i n i n g i s o t h i o c y a n a t e and 3~butenyl cyanides) exer t d i f f e r e n t e f f e c t s in the t h y r o i d g l a n d . G o i t r i n produced more enlargement of the gland and l e s s iod ine t r a p p i n g than the go i t rogens in Ye l low Sarson meal , because g o i t r i n in the rapeseed meal i s the prime iod ine b ind ing agent (Summers and Leeson, 1977)- C o n t r a d i c t o r y f i n d i n g s were observed by Matsumoto et a 1. (1968; 1969), who found that g o i t r i n d i d not i n t e r f e r e w i t h the i n c o r p o r a t i o n of i od ine i n t o - 123 -the t h y r o i d g l a n d s . Thyro id weight of c h i c k s (March, 1977 unpubl ished data) fed the same exper imental r a t i o n s as were used in the present experiment and reared under the same c o n d i t i o n s showed a marked increase in t h y r o i d weights in both the Span and Tower mea l - fed c h i c k s w h i l e the Candle m e a l - f e d c h i c k s showed s i g n i f i c a n t l y l e s s t h y r o i d enlargement. Moreover, supplementat ion w i t h iod ine d i d not modify the g o i t r o g e n i c e f f e c t . From these exper imental f i n d i n g s , i t i s suggested that both the Span and the Tower meal c o n t a i n g o i t r i n in a d d i t i o n to other weaker go i t rogens such as i s o t h i o c y a n a t e w h i l e the Candle meal has l e s s g o i t r o g e n i c a c t i v i t y s i n c e smal l or n e g l i g i b l e amounts of g o i t r i n may be p resent . - 124 -Table XXVI. Percentage compos i t ion of d i e t s fed in Experiment 7. . I • . D iet 1 D iet 2 D iet 3 D iet Ingredients 0 0 / o, o, 'Q 'O 'O 'O Wheat c o r n , 1:1 Soybean meal Rapeseed meal Calc ium phosphate Lys ine HC1 Cho i i ne Common i n g r e d i e n t s 64 .8 53 .0 29-1 41.6 (Span) 1.30 0 .60 0.10 0.049 54.6 50.8 40.0 43 .8 (Tower) (Candle) 0.60 0 .60 0.10 0 .10 " / k g ; t a l l o w 30g, l imestone 13 g , i od i zed s a l t 5 g , D ,L -meth ion ine 0.5 g , manganese su lphate 132 mg, z i n c ox ide 62 mg, r i b o f l a v i n 4 mg, f o l a c i n 0.55 mg, n i a c i n 27 mg, c a l c i u m pantothenate 9.25 mg, b i o t i n 0.088 mg, v i t a m i n 13 meg, v i t a m i n A 4400 I .U . , v i t a m i n D, 440 I . C U . - 125 -Table XXVII . Average body weights of c h i c k s at 35 days of age in Experiment 7-Diet P r o t e i n concent ra te Average body weight (g) 1 Soybean meal 388 a(l6) 2 Span meal 372 a(l8) 3 Tower meal 37V3 (17) k Candle meal 3z*8b(l6) " Number of b i r d s per treatment ab va lues w i t h the same s u p e r s c r i p t a re not s i g n i f i c a n t l y d i f f e r e n t P < 0 . 0 5 -- 126 -Table XXVI I I . Thy ro ida l uptake of 1-131 by c h i c k s at 12 days of age in Experiment 1. Diets Thy ro ida l uptake of 1-131 % of i n j e c t e d dose Time a f t e r I n j e c t i o n 5 h rs . 12 hrs Soybean meal 9.87 a(6)* 11.39 a(6) Span meal 18.02 D(6) 19-48D(6) Tower meal 17.75 D(6) 19.64 b(6) Candle meal 13.2V3 (6) 17.05 D(6) •> Number of b i r d s per treatment ab va lues w i th the same s u p e r s c r i p t at the same time i n t e r v a l are not s i g n i f i c a n t l y d i f f e r e n t P < 0.05-- 1 2 7 -Table XXIX. Thyro ida l uptake of 1-131 by c h i c k s at 35 days of age in Experiment 7-Diets Thy ro ida l uptake of 1—131 % of i n j e c t e d dose 5 hrs . Time a f t e r I n j e c t i o n 10 h r s . 20 hrs , 1 Soybean meal 6 . 9 2 a(5)* 11.60 a(6) 6.40 a(4) 2 Span meal 3 Tower meal k Candle mea 16.22 D(6) 25.63 b(6) 13-07 (6) 22.50^(5) 24.46 D(6) 16.48 D(5) 14.81 D (5) 15-93 a(6) 11.81 D(5) * Number of b i r d s per treatment abc va lues With the same s u p e r s c r i p t at the same time i n t e r v a l are not s i g n i f i c a n t l y d i f f e r e n t P < 0.05 - 1 2 8 -H O U R S F i g u r e 27. T h y r o i d a l u p t a k e of 1—131 a t 12 and 35 days o f age. Rl Soybean meal, R2 Span meal, R3 Tower meal and Rk Candle meal. - 129 -SUMMARY AND CONCLUSIONS The f o l l o w i n g c o n c l u s i o n s concern ing the e f f e c t s of feed ing g o i t r o g e n i c d i e t s to the ch ickens can be drawn from the data of the va r ious experiments d i scussed above. Exper iment 1: Feeding a d i e t wi thout iod ine supplementat ion fo r a per iod of 11 weeks to a d u l t hens d i d not cause t h y r o i d a l enlargement. The amount of e p i t h e l i a l t i s s u e present in the t h y r o i d g l a n d , however, was increased w i t h some p e r s i s t e n c e of c o l l o i d m a t e r i a l . These o b s e r v a t i o n s i n d i c a t e d that t h y r o i d f u n c t i o n was not seve re l y a f f e c t e d s i n c e h y p e r p l a s i a can be compensatory by i n c r e a s i n g the t r a p p i n g of iod ine from a d i e t that i s on ly moderately d e f i c i e n t in i o d i n e . By c o n t r a s t , feed ing a d i e t c o n t a i n i n g 0.1% t h i o u r a c i l to a d u l t b i r d s caused severe g o i t e r as i n d i c a t e d by the marked increase in t h y r o i d we ight , amount of e p i t h e l i a l t i s s u e and loss of c o l l o i d . Experiment 2: Rapeseed meal may have a g o i t r o g e n i c e f f e c t as the r e s u l t of i t s content of v a r i o u s g o i t r o g e n i c compounds. The m a n i f e s t a t i o n of the e f f e c t s of rapeseed meal was not as rap id as w i t h 0.05% t h i o u r a c i l . Growing c h i c k s fed rapeseed meal fo r one week showed no s i g n i f i c a n t inc rease in t h y r o i d weight or in the amount of t h y r o i d a l e p i t h e l i a l t i s s u e . Even a f t e r two weeks, rapeseed m e a l - f e d - 130 -c h i c k s , showed no a p p r e c i a b l e inc rease in t h y r o i d a l 1-131 uptake compared w i th c o n t r o l b i r d s . On the other hand, t h i o u r a c i 1 - t r e a t e d c h i c k s showed s i g n i f i c a n t increases in a l l three parameters of t h y r o i d a c t i v i t y s t u d i e d . When both t h i o u r a c i l and rapeseed meal were fed to the b i r d s , the g o i t r o g e n i c e f f e c t s were g reate r than feed ing c h i c k s t h i o u r a c i l or rapeseed meal s i n g l y as i n d i c a t e d by f u r t h e r inc reases in t h y r o i d we ight , t h y r o i d e p i t h e l i u m and r a d i o i o d i n e uptake. It cou ld not be concluded from the data whether the response to the combinat ion of t h i o u r a c i l and rapeseed meal was s y n e r g i s t i c or add i t i v e . Experiment 3' Feeding of a d i e t w i thout i od ine supplementat ion to growing c h i c k s caused severe h y p e r p l a s i a and loss of c o l l o i d in the t h y r o i d gland in c o n t r a s t to the f i n d i n g s w i th a d u l t b i r d s in Experiment 1. T h i o u r a c i l treatment a l s o produced a tremendous inc rease in both the t h y r o i d weight and t h y r o i d e p i t h e l i u m in the growing c h i c k s . These o b s e r v a t i o n s i n d i c a t e d that the s u s c e p t i b i l i t y to an iod ine d e f i c i e n t d i e t was more severe in the growing c h i c k s than in the a d u l t b i r d s because of the r e l a t i v e l y s m a l l e r s t o r e of iod ine in the t h y r o i d glands of the growing c h i c k s . The e f f e c t of t h i o u r a c i l , however, appeared to be independent of age. A d m i n i s t r a t i o n of 0 . 8 pg T^ per day f o r 10 days enhanced the g o i t r o g e n i c i t y of t h i o u r a c i l as shown by a f u r t h e r increase in the amount of t h y r o i d a l e p i t h e l i u m over that present in the c h i c k s r e c e i v i n g t h i o u r a c i l a l o n e . - 131 -Enhancement of the g o i t r o g e n i c e f f e c t of t h i o u r a c i l was not observed when t h i o u r a c i l was added to an iod ine d e f i c i e n t d i e t . It i s deduced that the growing c h i c k s in the iod ine d e f i c i e n t s t a t e were not s e n s i t i v e to the e f f e c t s of t h i o u r a c i l . Th is coupled w i t h the f i n d i n g that a smal l q u a n t i t y of exogenous T^ enhances the g o i t r o g e n i c e f f e c t of t h i o u r a c i l suggests that some minimal amount of t h y r o i d hormone i s requ i red fo r the response of t h y r o i d gland to TSH and fo r the s y n t h e s i s or s e c r e t i o n of TSH. Experiments 4 and 5: D a i l y a d m i n i s t r a t i o n of 4 . 0 ug T^ to b i r d s fed a d i e t c o n t a i n i n g 0.1% of t h i o u r a c i l reduced the e f f e c t s of t h i o u r a c i l on t h y r o i d we ight , t h y r o i d a l h i s t o l o g i c a l changes and r a d i o i o d i n e uptake. Small doses of exogenous T^, 1.0 ug per day or l e s s , on the other hand, enhanced the g o i t r o g e n i c i t y of the t h i o u r a c i l r e s u l t i n g in g rea te r inc reases in t h y r o i d we ight , t h y r o i d e p i t h e l i u m and r a d i o i o d i n e uptake , thus c o n f i r m i n g the r e s u l t s obta ined in Experiment 3 -The growth r a t e of c h i c k s g iven high doses of t h y r o i d hormone, in c o n j u n c t i o n w i th d i e t a r y t h i o u r a c i l , on l y increased to a c e r t a i n e x t e n t , and the c h i c k s d id not a t t a i n the r a t e of growth e x h i b i t e d by the c o n t r o l b i r d s . Th is o b s e r v a t i o n suggests that the growth depress ing e f f e c t of t h i o u r a c i l may invo lve more than a s imple reduc t ion in t h y r o i d hormone s e c r e t i o n and has the f u r t h e r i m p l i c a t i o n that increased t h y r o i d a l a c t i v i t y in b i r d s fed n a t u r a l sources of g o i t r o g e n s , such as rapeseed meal , a l though f u l l y - 132 -compensatory w i t h respect to hormone s e c r e t i o n , may not t o t a l l y overcome the adverse e f f e c t s of the g o i t r o g e n s . Experiment 6: The e f f e c t s of t h i o u r a c i l and of rapeseed meal go i t rogens on t h y r o i d a l iod ide uptake were compared on the b a s i s of both t o t a l uptake by the gland and a u t o r a d i o g r a p h i c s t u d i e s . Both t h i o u r a c i l and rapeseed meal enhanced the t h y r o i d a l uptake of 1-125. Never-t h e l e s s , t h i o u r a c i l was a more potent go i t rogen and r e s u l t e d in a higher accumulat ion of t h y r o i d a l 1-125 than d i d rapeseed meal . A u t o r a d i o g r a p h i c s t u d i e s showed that the r a d i o i o d i n e accumulated was evenly d i s t r i b u t e d in the c o l l o i d w i t h i n 5 hours a f t e r i n j e c t i o n of 1-125 a l though the c o n c e n t r a t i o n v a r i e d markedly among the f o i l i c l e s p r e s e n t in one g l a n d . Th is un i form d i s t r i b u t i o n of r a d i o i o d i n e was observed in a l l t reatments at a l l t imes . The f a c t that uni form d i s t r i b u t i o n occur red so q u i c k l y i n d i c a t e d that the r a d i o i o d i n e was t ranspor ted r a p i d l y i n t o and across the f o l l i c u l a r c e l l s to the lumen. The autorad iographs a l s o showed that there was a d i f f e r e n c e in the a c t i v i t y of i n d i v i d u a l f o l l i c l e . The smal le r f o l l i c l e s concentrated more r a d i o i o d i n e than the l a r g e r ones . The o b s e r v a t i o n s that r a d i o i o d i n e was r a p i d l y and u n i f o r m l y d i s t r i b u t e d in the f o l l i c u l a r c o l l o i d , together w i t h the almost t o t a l absence of r a d i o i o d i n e w i t h i n the e p i t h e l i a l c e l l s , and the f i n d i n g s reported in the l i t e r a t u r e that an a p p r e c i a b l e amount of ino rgan ic i od ide is present in the t h y r o i d a f t e r i t s a d m i n i s t r a t i o n , - 133 -do not support the hypothes is advanced by i n v e s t i g a t o r s that i o d i n a t i o n occurs at the a p i c a l membrane of the f o l l i c u l a r c e l l s border ing the lumen. Experiment 1: Rapeseed meal is manufactured from the seed of a number of c u l t i v a r s of rape. 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Weight and P. Ways. 1952. The e f f e c t of c e r t a i n anions upon the accumulat ion and r e t e n t i o n of iod ide by the t h y r o i d g l a n d . E n d o c r i n o l . 50: 537-549-Yamada, T. 1976. Contro l of t h y r o i d hormone s e c r e t i o n . Pharmac. Ther . C. 1: 3~l8. Yamamoto, K. and L . J . DeGroot. 1975- P a r t i c i p a t i o n of NADPH-cytochrome c reductase in t h y r o i d hormone b i o s y n t h e s i s . E n d o c r i n o l . 96: 1022-1029. Yukimura, Y . , K. I k e j i r i , A. Kojima and T. Yamada. 1976. E f f e c t s of excess iod ide and other anions on t h y r o i d hormone s e c r e t i o n in normal or hypophysectomized r a t s t r e a t e d w i t h graded doses of t h y r o i d hormone. E n d o c r i n o l . 99: 541-548. Z a k a r i j a , M. and J . M . Mackenzie . 1975- C y c l i c AMP in the t h y r o i d of the rat fed PTU: in v i t r o unresponsivensss to t h y r o t r o p i n . Endocr ine Research Communications 2: 4l9 -429. Zar , J . H . 1974. Bi .ostat i s t i c a l a n a l y s i s . P r e n t i c e - H a l l I n c . , Englewood C l i f f s , N . J . - 152 -APPENDIX 6 - 153 -Table I (A ) . Experiment 1: S t a t i s t i c a l a n a l y s i s comparing the average weights of t h y r o i d glands in White Leghorn hens at t e r m i n a t i o n of Exper iment 1. i . A n a l y s i s of v a r i a n c e : Source of v a r i a t i o n df MS Among groups Wi th in group 2350.7 35-82 (.0005 65.62 i i . Student-Newman-Keu1s m u l t i p l e range t e s t Rank of sample means ( i ) Ranked sample means (X . , in mg/lOOg B.W.) 7-56 10.43 51.70 Conc lus ion (P < 0.01) - 154 -T a b l e I I(A) Experiment 1: S t a t i s t i c a l a n a l y s i s comparing th e a v e r a g e p e r c e n t a g e s o f e p i t h e l i a l t i s s u e i n t h e t h y r o i d g l a n d s o f b i r d s . i . A n a l y s i s o f v a r i a n c e ; S ource o f v a r i a t i o n df MS Among groups W i t h i n groups 304.73 9-94 <0.01 30.65 Student-Newman-Keu1s m u l t i p l e range t e s t : Ranks o f sample means ( i ) 1 Ranked sample means (X., i n % o f t o t a l t h y r o i d g l a n d ) 24.0 35-8 42.8 C o n c l u s i o n (P < 0.05) b a - 155 -T a b l e I I I(A). Expe r i m e n t 2: S t a t i s t i c a l a n a l y s i s comparing th e a v e r a g e w e i g h t s o f t h y r o i d g l a n d s o f c h i c k s a t 1 week o f age. i . A n a l y s i s o f v a r i a n c e : S ource o f v a r i a t i o n df MF Among groups W i t h i n groups 16 3534.91 27.50 128.53 <0.0005 i i . Student-Newman-Keu1s m u l t i p l e range t e s t : Ranks o f sample means ( i ) Ranked sample means (X. , i n mg/lOOg B.W.) 14.7 19-4 39-0 73.2 C o n c l u s i o n (P < 0.05) - 156 -Table IV(A) . Experiment 2 : S t a t i s t i c a l a n a l y s i s comparing the average weights of e p i t h e l i a l t i s s u e of c h i c k s at 1 week of age. A n a l y s i s of v a r i a n c e : Source of v a r i a t i o n df MS Among groups Wi th in groups 16 105-53 13-01 8.11 < 0.0005 i i . Student-Newman-Keu1s m u l t i p l e range t e s t : Ranks of sample means ( i ) 1 Ranked sample means (X . , mg) 3-9 4 .2 7.8 13-8 Conclus ion (P < 0.01) - 157 -Table V(A) Experiment 2: S t a t i s t i c a l a n a l y s i s comparing t h y r o i d a l uptake of r a d i o i o d i n e -131 by c h i c k s at 4, 8, 16 and 32 hours a f t e r i n j e c t i o n . A n a l y s i s of v a r i a n c e : a . 4 hours a f t e r i n j e c t i on Source of v a r i a t i o n df Among groups 3 With in groups 19 MS 3248.72 87-55 F 37.11 < 0.0005 8 hours a f t e r i n j e c t i o n Source of v a r i a t i o n df Among groups 3 With in groups 19 MS 2890.36 88.24 F 32.76 < 0.0005 c . 16 hours a f t e r i n j e c t i o n Source of v a r i a t i o n Among groups Wi th in groups df 2 14 MS 2047.03 126.28 F 16.21 <0.0005 32 hours a f t e r i n j e c t i o n Source of v a r i a t i o n df Among groups 3 With in groups 18 MS 1381.84 165-51 F 8.349 P < 0.005 - 158 -Student-Newman-Keu1s m u l t i p l e range: 4 hours a f t e r i n j e c t i o n Ranks of sample means (j) Ranked sampl e jneans (X . , in % t h y r o i d a l 1-131) Conc lus ion (P < 0.05) 1 2 21.4 25.0 3 4 46.7 72.6 8 hours a f t e r i n j e c t i o n Ranks of sample means ( i ) Ranked sample means (X . , in % t h y r o i d a l 1-131) Conc lus ion (P < 0.05) 23 -1 2 3 4 32.7 48.6 76.2 c . 16 hours a f t e r i n j e c t i o n Ranks of sample means ( i ) Ranked sample means (X . , in % t h y r o i d a l 1-131) Conc lus ion (P '< 0.05) 23.6 2 54.9 3 57.1 d. 32 hours a f t e r i n j e c t i o n Ranks of sample means ( i ) 131) Ranked sample means ( X . , in % t h y r o i d a l Conc lus ion (P < 0.05) 21.2 2 27-7 3 46.7 4 56.1 - 159 -T a b l e V I ( A ) . Experiment 3 : S t a t i s t i c a l a n a l y s i s comparing the avera g e body w e i g h t s o f c h i c k s i n d i f f e r e n t t r e a t m e n t s a t d i f f e r e n t ages. i . A n a l y s i s o f v a r i a n c e Day 9 Source o f v a r i a t i o n d f MS F P Among groups 5 186.56 2.194 0.10 W i t h i n groups 54 85.03 Day 13 Source o f v a r i a t i o n d f MS F P Among groups 5 763.23 9-734 0.0005 W i t h i n groups 54 78.41 Day 20 Source o f v a r i a t i o n d f MS F P Among groups 5 9886.7 57-08 0.0005 W i t h i n groups " 54 173-2 Day 22 Source o f v a r i a t i o n d f MS F P Among groups 5 24050.96 83.17 0.0005 W i t h i n groups 77 289.18 Day 24 Source o f v a r i a t i o n d f MS F P Among groups 5 22269-65 70.99 0.0005 W i t h i n groups 54 313-66 - 160 -i i . Student-Newman-Keuls m u l t i p l e range t e s t . Day 13 Ranks of sample means ( i ) 1 2 3 4 5 Ranked sample means ' 89 95 101 107 109 (X:.,- in g) Conc lus ion (P 0.05) b be ac a a Day 20 Ranks of sample means ( i ) 1 2 3 4 5 6 Ranked sample means 106 125 155 170 181 182 (X . , in g) Conc lus ion (P 0.05) b e d a a a Day 22 Ranks o f sample means ( i ) ;1 2 3 4 5 6 Ranked sample means 111 136 184 197 206 214 (X . , in g) Conc lus ion (P 0.05) c d e a ab b Day 24 Ranks of sample means ( i ) 1 2 3 . 4 5 6 Ranked sampled means 117 147 200 214 224 235 (X . , in g) Conc lus ion (P 0.05) c d e ae ab b - 161 -Table VI I(A) Experiment 3: S t a t i s t i c a l a n a l y s i s comparing the percentages t h y r o i d body weight of c h i c k s at day 9, 13 and 20. i . A n a l y s i s of v a r i a n c e a . Day 9 Source of v a r i a t i o n Among groups Wi th in groups df 5 24 MS 225.7 31.5 F 7.165 <0.0005 Day 13 Source of v a r i a t i o n Among groups Wi th in groups df 5 2k MS 1294.78 189.94 F 6.817 < 0.0005 Day 20 Source of v a r i a t i o n df MS Among groups 5 1569-6 Wi th in groups 24 285-56 F 5.497 < 0.0005 Student-Newman-Keu1s m u l t i p l e range t e s t a . Day 9 Ranks of sample means ( i ) Ranked sample means ( X . , in % t h y r o i d body weight) Conclus ion (P < 0.05) 1 2 9-26 12.7 3 4 5 6 16.6 18.3 23.9 27.0 ab abc be c b. Ranks of sample means ( i ) Ranked sample means (X . , in % t h y r o i d body weight) Conc lus ion (P < 0.05) c . Ranks of sample means ( i ) Ranked sample means (X . , in % t h y r o i d body weight) Conc lus ion (P < 0.05) 162 -Day 13 1 2 3 8.20 12.5 16.8 a a a Day 20 1 2 3 7-02 9.77 35.4 b b a 4 5 6 22.9 43.5 46.0 a b b 4 5 6 37-9 45.1 47.7 - 163 -Table VI I I(A) Experiment 3'- S t a t i s t i c a l a n a l y s i s comparing the percentages of e p i t h e l i u m of t o t a l t h y r o i d gland of c h i c k s at 20 days of age. i . A n a l y s i s of v a r i a n c e Source of v a r i a t i o n df MS Among groups Wi th in groups 24 779-99 0.710 80.33 <0.0005 i i . Student-Newman-Keu1s m u l t i p l e range t e s t Ranks of sample means ( i ) 1 Ranked sample means (X . , in % t h y r o i d body weight) 45-6 48.7 48.9 61.5 72.4 73-4 Conc lus ion (P< 0.05) b b b c a a - 164 -Table IX(A) Experiment 3: S t a t i s t i c a l a n a l y s i s comparing the est imated weights of t h y r o i d a l e p i t h e l i a l t i s s u e s of c h i c k s at the age of 20 days . i . A n a l y s i s of v a r i a n c e Source of v a r i a t i o n df MS Among groups W i t h i n groups 24 1354.01 2.434 556.29 <0.0005 i i . Student-Newman-Keu1s m u l t i p l e range t e s t Ranks of sample means ( i ) 1 5 6 Ranked sample means (X . , in mg) 5.97 8.62 28.95 30.45 42.96 44. Conclus ion (P < 0.05) - 165 -Table X (A) . Experiment 3: S t a t i s t i c a l a n a l y s i s comparing the est imated weights of t h y r o i d e p i t h e l i u m per 100 g body weight of c h i c k s at 20 days of age. i . A n a l y s i s of v a r i a n c e Source of v a r i a t i o n df MS F P Among groups 5 675-21 483.29 <0.0005 W i t h i n groups 24 1.397 i i . Student-Newman-Keu1s m u l t i p l e range t e s t Ranks of sample means ( i ) 1 2 3 k 5 6 Ranked sample means 3-28 4.77 24.36 26.13 27-31 27-72 (X . , i n mg t h y r o i d epithel ium/100 g body weight) Conc lus ion (P < 0.05) b b c a a a - 166 -Table X I ( A ) . Experiment h: S t a t i s t i c a l a n a l y s i s f o r comparing the average body weights of c h i c k s at 3~ l/2 weeks of age. Source of v a r i a t i o n Among groups Wi th in groups i . Analys i s df 9 109 of v a r i a n c e MS F 3391.01 15-35 220.91 P ^0.0005 i i . Student-Newman-Keuls m u l t i p l e range t e s t Ranks of sample means ( i ) 1 2 3 4 5 6 7 8 9 Ranked sample means 182 187 196 206 206 206 207 211 213 CX., i n g ) Conc lus ion b b b c c c c c c c (P ^ 0.01) - 167 -Table XI I ( A ) . Experiment k: S t a t i s t i c a l a n a l y s i s f o r comparing the t h y r o i d weights of c h i c k s in R - l to R-8 a t 3-1/2 weeks of age. Source of v a r i a t i o n Among groups W i t h i n groups A n a l y s i s of v a r i a n c e df MS 17 10186.3 89 771.5 F P 13.20 <0.0005 i i . Student-Newman-Keu1s m u l t i p l e range t e s t Ranks of sample means ( i ) 1 2 3 4 5 6 7 8 Ranked sample means 5-4 19-0 35-9 73-0 73-4 73-5 7 .^5 77-6 CX., mg/lOOg B.W.) Conc lus ion a a ab be be be be c CP < 0.01) - 168 -Table XI I I(A) Experiment k: S t a t i s t i c a l a n a l y s i s fo r comparing the t h y r o i d weights of c h i c k s in R - l , R-9 and R-10 at 3" l/2 weeks of age. i . A n a l y s i s of v a r i a n c e Source of v a r i a t i o n df MS Among groups Wi th in groups 32 2713.70 25-39 106.86 <0.0005 1 1 . Student-Newman-Keu1s m u l t i p l e range t e s t Ranks of sample means ( i ) Ranked sample means CX., mg/lOOg. B.W.) 5.4 30.9 33.6 Conc lus ion (P < 0.01) - 169 -Table .XIV (A) . Experiment 5: S t a t i s t i c a l a n a l y s i s f o r comparing the average body weights of c h i c k s at the age of 19 days . i . A n a l y s i s of v a r i a n c e Source of v a r i a t i o n df MS Among groups W i t h i n groups 293 11603.14 64.97 <0.0005 178.61 i i . Student-Newman-Keu1s m u l t i p l e range t e s t Ranks of sample means ( i ) 1 2 Ranked sample means (X . , in g) 116 138 141 147 148 163 Conc lus ion (P < 0.01) cd cd cd - 170 -Table XV.(A) Experiment 5'- S t a t i s t i c a l a n a l y s i s fo r comparing the average t h y r o i d weights of c h i c k s at day 8, 14 and 20. A n a l y s i s of v a r i a n c e a . Day 8 Source of v a r i a t i o n df Among groups 5 With in groups 24 MS 26.51 8.879 F 2.986 P <0.05 Day ]k Source of v a r i a t i o n df MS Among groups . 5 1263.5 W i t h i n groups 2k 96.51 F ' 13-09 <0.0005 c . Day 20 Source of v a r i a t i o n df MS Among groups 5 5342.63 W i t h i n groups 2k 166.58 F P 32.07 <0.0005 i i . Student-Newman-Keu1s m u l t i p l e range t e s t Day 8 Ranks of sample means ( i ) Ranked sample means (X . , i n mg % of body weight) Conc lus ion (P < 0.05) 1 2 3 8.74 9-32 11.5 a ab ab k 5 12.8 13-2 14 ab ab b. Ranks of sample means ( i ) Ranked sample means (X . , mg % of body weight) Conc lus ion (P < 0.05) c . Ranks of sample means ( i ) Ranked sample means (X . , mg % of body weight) Conc lus ion (P < 0.05) 171 " Day 14 1 2 3 8.53 11.2 12.5 a a a Day 20 1 2 3 5.34 6.28 9-53 4 5 6 34.4 39-3 43.2 b b b 4 5 6 56.8 67.7 74.2 b b b - 172 -Table XVI (A) . Experiment 5: S t a t i s t i c a l a n a l y s i s f o r comparing the percentage e p i t h e l i a l t i s s u e of t o t a l g land at day 8, 14 and 20. A n a l y s i s of v a r i a n c e Day 8 Source of v a r i a t i o n Among groups W i t h i n groups df 5 22 MS 458.77 66.89 F 6.858 P <0.001 b. Day 14 Source of v a r i a t i o n Among groups Wi th in groups df 5 21 MS 865.81 31.94 F 27.111 t 0.0005 Day 20 Source of v a r i a t i o n Among groups Wi th in groups df 5 24 MS 452.06 45-75 F 9.882 <0.0005 i i Student-Newman-Keu1s m u l t i p l e range t e s t a . Day 8 Ranks of sample means ( i ) Ranked sample means (X . , in % e p i t h e l i a l t i s s u e of t o t a l ) Conc lus ion (P < 0.05) 36.2 2 55.0 3 57.3 4 60.3 5 61.2 6 61.7 - 173 -b. Day 14 Ranks of sample means ( i ) 1 2 3 4 5 6 Ranked sample means 34.3 39-5 39.8 61.6 63.5 64.4 (X . , In % e p i t h e l i a l t i ssue of t o t a l ) Conc lus ion (P < 0.05) a a a b b b c . Day 20 Ranks of sample means ( i ) 1 2 3 4 5 6 Ranked sample means 33-6 38.8 45.0 49.8 51.8 60.2 (X . , in % e p i t h e l i a l t i ssue of t o t a l ) Conc lus ion (P < 0.05) a ad cd bed be b - 174 -Table XVI I (A ) . Source of v a r i a t i o n Among groups Wi th in groups Source of v a r i a t i o n Among groups Wi th in groups Source of v a r i a t i o n Among groups Wi th in groups Source of v a r i a t i o n Among groups Wi th in groups Experiment 5- S t a t i s t i c a l a n a l y s i s f o r comparing the percentages of i n j e c t e d dose in c h i c k s at va r ious times a f t e r a d m i n i s t r a t i o n at 1-131 i . A n a l y s i s of v a r i a n c e a . 4 hours a f t e r i n j e c t i o n df MS F P 5 969-71 13-15 <0.0005 39 73-73 b. 8 hours a f t e r i n j e c t i o n df MS F P 5 610.95 7.085 <0.0005 36 86.23 c . 12 hours a f t e r i n j e c t i o n df MS F P 5 1161.79 14.78 <0.0005 36 78.61 d . 16 hours a f t e r i n j e c t i o n df MS F P 5 1658.65 13.58 <0.0005 40 122.12 175 -Source of v a r i a t i o n Among groups Wi th in groups Source of v a r i a t i o n Among groups Wi th in groups e . 24 hours a f t e r i n j e c t i o n df MS F P 5 1054.13 22.42 <0.0005 40 47.02 f . 32 hours a f t e r i n j e c t i o n df MS F P 5 564.80 6.574 <0.001 23 85.92 D - 176 -i . Student-Newman-Keu1s m u l t i p l e range t e s t 4 hours a f t e r i n j e c t i o n Ranks of sample means ( i ) Ranked sample means (X . , in % i n j e c t e d dose) Conclus ion (P < 0.05) 1 2 3 4 5 6 3.24 8.88 16.45 23-78 24.12 36.23 bd ab 8 hours a f t e r i n j e c t i o n Ranks of sample means ( i ) Ranked sample means (X . , in % i n j e c t e d dose) Conc lus ion (P < 0.05) 2-95 2 3 4 5 6 11.41 17-56 24.85 25.0 31.60 bd ab ac ac 12 hours a f t e r i n j e c t i o n Ranks of sample means ( i ) Ranked sample means (X . , in % i n j e c t e d dose) Conc lus ion (P < 0.05) 1 2 3 4 5 6 2.91 9-08 17.38 27.12 27.52 39-59 d bd ab a a c 16 hours a f t e r i n j e c t i o n Ranks of sample means ( i ) Ranked sample means (X . , i n . % i n j e c t e d dose) Conclus ion (P < 0.05) 1 .44 2 3 4 5 6 5.75 17.97 20.39 36.34 37-53 24 hours a f t e r i n j e c t i o n Ranks of sample means ( i ) Ranked sample means (X . , in % i n j e c t e d dose) Conc lus ion (P < 0.05) 1 2 3 4 5 6 0.42 2.65 16.84 21.79 23-02 29.67 ab ab - 177 -f . Ranks of sample means ( i ) Ranked sample means (X . , in % i n j e c t e d dose) Conc lus ion (P * 0.05) 32 hours a f t e r i n j e c t i o n 1 2 3 4 0.59 8.40 17.89 20.37 c be ab ab 5 6 27.59 28.04 - 178 -Table XVI I I (A ) . Experiment 6: S t a t i s t i c a l a n a l y s i s f o r comparing the average weights of t h y r o i d l e f t lobe of c h i c k s at the age of 8 weeks. Source of v a r i a t i o n Among groups Wi th in groups A n a l y s i s of v a r i a n c e df MS 2 11233-89 70 9010.27 F P 87.28 <0.0005 i i . Student-Newman-Keuls m u l t i p l e range t e s t Ranks of sample means ( i ) 1 2 3 Ranked sample means 4.22 10.2 43.7 ( X . , in mg/lOOg body weight) Conc lus ion (P < 0.05) a a b - 179 -Table X IX(A) . Experiment 6: S t a t i s t i c a l a n a l y s i s fo r comparing the average weights of t h y r o i d l e f t lobe of c o n t r o l and rapeseed mea l - fed c h i c k s at the age of 8 weeks. i . A n a l y s i s of v a r i a n c e Source of v a r i a t i o n df MS F P Among groups 1 423.09 77.17 <0.0005 With in groups 46 5.48 - 180 -Table XX(A) Experiment 6: S t a t i s t i c a l a n a l y s i s fo r comparing the percentage uptake of 1-125 in the t h y r o i d l e f t lobes of c h i c k s at 8 weeks of age. i . A n a l y s i s of v a r i a n c e 5 hours a f t e r i n j e c t i o n Source of v a r i a t i o n Among groups Wi th in groups df 2 15 MS 743.19 108.63 F 6.841 P <0.0T b. 10 hours a f t e r i n j e c t i o n Source of v a r i a t i o n df MS Among groups 2 1630.59 With in groups 14 466.60 F 3.495 P <0.10 c . 15 hours a f t e r i n j e c t i o n Source of v a r i a t i o n df MS F P Among groups 2 845-96 7-258 <0.01 With in groups 15 116.56 d . 20 hours a f t e r i n j e c t i o n Source of v a r i a t i o n df Among groups 2 With in groups 13 MS 783.68 202.05 F 3.879 P *0.05 - 181 -i i . Student-Newman-Keuls m u l t i p l e range t e s t a . 5 hours a f t e r i n j e c t i o n Ranks of sample means(i) Ranked sample means (X . , % of i n j e c t e d dose) Conc lus ion (P < 0.05) 5-60 2 7-73 ab 3 25-32 b. 10 hours a f t e r i n j e c t i o n Ranks of sample means ( i ) Ranked sample means ( X . , % of i n j e c t e d dose) Conc lus ion (P < 0.05) 1 2 3 6.33 27-47 40.22 b ab b c . 15 hours a f t e r i n j e c t i o n Ranks of sample means (i.) Ranked sample means (X . , % of i n j e c t e d dose) Conc lus ion (P <- 0.05) 1 2 3 6.88 10.49 29.01 a a b d . Ranks of sample means ( i ) Ranked sample means (X.,.% of i n j e c t e d dose) Conc lus ion (P < 0.05) 20 hours a f t e r i n j e c t i o n 1 2 3 6.29 14.49 28.93 a ab b - 182 -Table XXI (A) . Exp. the i . Source of v a r i a t i o n Among groups W i t h i n groups i i . Ranks of sample means (i Ranked sample means CX., in g) Conc lus ion (P ^ 0.05) riment 7' S t a t i s t i c a l average body weights of A n a l y s i s of v a r i a n c e df MS 3 4530.75 63 890.20 Student-Newman-Keu 1 s ) 1 2 348 372 b a a n a l y s i s f o r comparing c h i c k s at 5 weeks of age. F P 5.090 <0.005 m u l t i p l e range t e s t 3 4 374 388 a a - 183 -Table XXI I (A) . Experiment 7: S t a t i s t i c a l a n a l y s i s f o r comparing the percentages of t h y r o i d a l 1-131 uptake by c h i c k s at the age of 12 days. A n a l y s i s of v a r i a n c e a . 5 hours a f t e r i n j e c t i o n Source of v a r i a t i o n Among Groups Wi th in groups df 3 20 MS 91 .53 10.15 F 9.018 P ^0.001 12 hours a f t e r i n j e c t i o n Source of v a r i a t i o n Among groups Wi th in groups df 3 20 MS 89.09 15.11 F 5.898 P <0.005 i i . Student-Newman-Keu1s m u l t i p l e range t e s t a . 5 hours a f t e r i n j e c t i o n Ranks of sample means ( i ) Ranked sample means (X . , in % of i n j e c t e d dose) Conc lus ion (P <0.05) 9-87 2 13-24 3 17.75 4 18.02 12 hours a f t e r i n j e c t i o n Ranks of sample means ( i ) Ranked sample means (X . , in % of i n j e c t e d dose) Conc lus ion (P < 0.05) 11.39 17.05 3 19-48 4 19-64 - 184 -Table XXI I I (A) . Experiment 7: S t a t i s t i c a l a n a l y s i s f o r comparing the percentages of t h y r o i d a l 1-131 uptake by c h i c k s at 35 days of age. i . A n a l y s i s of v a r i a n c e a . 5 hours a f t e r i n j e c t i o n Source of v a r i a t i o n df Among groups 3 With in groups 17 b. Source of v a r i a t i o n df Among groups 3 With in groups 20 c . Source of v a r i a t i o n df Among groups 3 With in groups 16 MS F P 205.19 23.63 <0.0005 8.68 10 hours a f t e r i n j e c t i o n MS F P 274.54 13.97 <0.0005 19-65 20 hours a f t e r i n j e c t i o n MS F P 77.13 5.59 <0.01 13.79 i i . Student-Newman-Keu1s m u l t i p l e range t e s t a . 5 hours a f t e r i n j e c t i o n Ranks of sample means ( i ) 1 2 3 4 Ranked sample means 6.92 14.81 16.22 22.50 (X., in % of i n j e c t e d dose) Conc lus ion (P<0.05) a b b e - 185 -b Ranks of sample means ( i ) Ranked sample means (X . , in % of i n j e c t e d dose) Conclus ion (P < 0.05) c Ranks of sample means ( i ) Ranked sample means (X . , in % of i n j e c t e d dose) Conc lus ion (P < 0.05) 10 hours a f t e r i n j e c t i o n 1 2 3 4 11.60 15.93 24.46 25.63 a a b b 20 hours a f t e r i n j e c t i o n 1 2 3 4 6.40 11.81 13.07 16.48 a b b b - 186 -Table XXIV(A). C a l c u l a t e d crude p r o t e i n c o n c e n t r a t i o n f o r a l l r a t i o n s in d i f f e r e n t exper iments : Experiment Crude P r o t e i n % Diets 1 2 3 4 5 6 1 16.75 16.75 16.75 2 21.04 21.03 21.04 21.03 3 19-69 19.69 19.69 19.69 19.69 19.69 4 20.1 20.1 5 19.31 19.31 19.31 19.31 19.31 19.31 6 21.0 21.0 21.0 7 20.75 20.80 20.79 20.76 

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