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Effects of chronic and early goitrogenic stimulation on thyroidal metabolism in the chicken and in the.. 1975

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EFFECTS OF CHRONIC AND EARLY GOITROGENIC .STIMULATION ON THYROIDAL METABOLISM I N THE CHICKEN AND IN - - " THE PROGENY. . • BY PETER CHUNG KWOR LEUNG A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department o f i POULTRY SCIENCE We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA Augus t , 1975 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for s c h o l a r l y purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or p u b l i c a t i o n of th is thes is for f inanc ia l gain sha l l not be allowed without my wri t ten permiss ion. Department of P o u l t r y S c i e n c e The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date August 15, 1975 ABSTRACT White Leghorn p u l l e t s were f e d g o i t r o g e n i c r a p e s e e d meal o r soybean meal d u r i n g e i t h e r o r both t h e growing p e r i o d and the l a y i n g p e r i o d . Rapeseed meal was f e d a t a l e v e l o f 17.5 p e r c e n t o f t h e s t a r t e r - g r o w e r d i e t and 19.0 p e r c e n t o f the l a y e r d i e t . C o n t r o l r a t i o n s c o n t a i n e d 11.0 and 13.1 p e r c e n t o f soybean m e a l , r e s p e c t i v e l y . The r e s p o n s e s t o the d i e t a r y t r e a t m e n t s were s t u d i e d when t h e b i r d s were o v e r 90 weeks o f age. P a r a m e t e r s measured i n c l u d e d : w e i g h t s , h i s t o l o g y and i o d i n e c o n t e n t s o f t h e t h y r o i d g l a n d s , t h y r o i d a l 1-131 u p t a k e a t d i f f e r e n t t ime i n t e r v a l s a f t e r a d m i n i s t r a t i o n o f the r a d i o - i o d i n e , d i s t r i b u t i o n o f i n j e c t e d doses o f r a d i o i o d i n e i n i d d i n a t e d s u b s t a n c e s i n t h e t h y r o i d g l a n d s , i o d i n e c o n t e n t s o f egg y o l k s , and t h e t h y r o i d a l c h a r a c t e r i s t i c s o f the progeny o f t h e s e b i r d s a t h a t c h i n g . B i r d s which were b e i n g f e d r a p e s e e d meal a t the t i m e t h e t h y r o i d g l a n d s were examined showed e f f e c t s which v a r i e d i n magnitude a c c o r d i n g t o the l e n g t h o f t i m e o v e r which r a p e s e e d meal had been f e d . T h y r o i d w e i g h t s , f o l l i c l e d i a m e t e r s , amounts o f e p i t h i u m and i o d i n e c o n t e n t s o f t h e t h y r o i d g l a n d s o f r a p e s e e d m e a l - f e d b i r d s i n c r e a s e d whereas c o u p l i n g e f f i c i e n c y d e c l i n e d w i t h t i m e . The e f f e c t s o f o v e r s t i m u l a t i o n o f t h e t h y r o i d g l a n d i n d u c e d by t h e f e e d i n g o f g o i t r o g e n i c r a p e s e e d meal d u r i n g t h e growing p e r i o d p e r s i s t e d f o r as l o n g as 75 weeks a f t e r w i t h d r a w a l o f the s o u r c e o f g o i t r o g e n f r o m t h e d i e t . T h y r o i d w e i g h t s , t h y r o i d i o d i n e c o n t e n t s and r a d i o i o d i n e uptake were a l l i n c r e a s e d i n b i r d s which had r e c e i v e d g o i t r o g e n d u r i n g the growing p e r i o d . F o l l i c l e d i a m e t e r s were g r e a t e r - i i i - and h e i g h t s o f e p i t h e l i a l c e l l s were lower in::the t h y r o i d g l a n d s o f t h e s e b i r d s t h a n i n t h o s e o f t h e c o n t r o l . Eggs from r a p e s e e d m e a l - f e d b i r d s were low i n i o d i n e . The t h y r o i d g l a n d s o f n e w l y - h a t c h e d progeny from dams which had been f e d g o i t r o g e n i c r a p e s e e d meal were o b s e r v e d t o be h y p e r t r o p h i c . S i n c e a n a l y s i s o f the eggs f o r g o i t r i n was n e g a t i v e and a n a l y s i s f o r i s o - t h i o c y a n a t e s i n d i c a t e d s i m i l a r c o n c e n t r a t i o n s i n t h e eggs from b i r d s f e d r a p e s e e d meal and from c o n t r o l b i r d s f e d soybean m e a l , t h e hypo- t h e s i s was t h a t the low c o n c e n t r a t i o n o f i o d i n e i n t h e eggs o f the g o i t r o u s b i r d s was due t o d i v e r s i o n o f a l a r g e p r o p o r t i o n o f c i r c u l a t i n g i o d i n e i n t o the t h y r o i d g l a n d s w i t h the r e s u l t t h a t t h e amounts r e a c h i n g t h e d e v e l o p i n g ova were r e d u c e d . - i v - TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS v i i i INTRODUCTION i x REVIEW OF LITERATURE BIOSYNTHESIS AND RELEASE OF THYROID HORMONES 1 EFFECTS OF GOITROGENS ON THYROID HORMONE BIO-SYNTHESIS 6 RAPESEED MEAL AS A SOURCE OF GOITROGEN 8 EFFECTS OF GOITRIN ON THYROID METABOLISM ON THE CHICKEN 10 EFFECTS OF LONG-TERM ADMINISTRATION OF RAPESEED MEAL ON BIRDS AND PROGENY ,13 EXPERIMENTAL E x p e r i m e n t 1: EFFECTS OF CHRONIC AND EARLY STIMU-LATION BY GOITROGENIC ,RSM ON WEIGHT AND HISTOLOGY OF THYROID GLAND 15 E x p e r i m e n t 2: EFFECTS OF CHRONIC AND EARLY STIMU-LATION BY GOITROGENIC-RSM ON TOTAL IODINE CONTENT OF THE THYROID GLAND 23 E x p e r i m e n t 3: EFFECTS ON UPTAKE OF A SINGLE DOSE OF 1-131 BY THE THYROID GLAND 6 HOURS AND 22 HOURS AFTER ADMINISTRATION OF THE RADIOIODINE 29 E x p e r i m e n t 4: THE DISTRIBUTION OF A SINGLE DOSE OF 1-131 IN IODINATED SUBSTANCES IN THYROID GLANDS OF SBM- OR RSM-FED BIRDS 33 - V - E x p e r i m e n t 5: EitEECTSi,OF GOITROGENIC RSM ON SUS-CEPTIBILITY TO HYDROLYSIS OF THYROGLOBULIN 39 Ex p e r i m e n t 6A: EFFECTS OF GOITROGENIC RSM ON THE IODINE CONTENT OF EGG YOLK 42 Exp e r i m e n t 6B: EFFECTS OF INCREASED DIETARY IODINE INTAKE ON IODINE DEPOSITION IN EGG YOLKS OF SBM-AND RSM-FED BIRDS 46 Ex p e r i m e n t 7: OVARIAN UPTAKE OF A SINGLE DOSE OF 1-131 BY DEVELOPING FOLLICLES 50 Exp e r i m e n t 8: EFFECTS OF CHRONIC AND EARLY GOI-TROGENIC STIMULATION BY RSM IN THE DAM ON EMBRYONIC THYROID DEVELOPMENT 55 DISCUSSION THYROIDAL RESPONSE TO CHRONIC GOITROGENIC STIMU-LATION BY RSM 60 PERSISTENCE OF EFFECTS OF EARLY GOITROGENIC STIMU-LATION 67 EFFECTS OF ALTERATIONS IN MATERNAL THYROID META-BOLISM ON EMBRYONIC THYROID DEVELOPMENT IN THE CHICK 70 SUMMARY AND CONCLUSIONS 72 BIBLIOGRAPHY 75 APPENDIX LIST OF TABLES T a b l e Page 1 P e r c e n t a g e c o m p o s i t i o n o f d i e t s f e d i n e x p e r i m e n t 1 and RSM g o i t r o g e n i c i t y t e s t 19 2 Comparison o f a v e r a g e t h y r o i d w e i g h t s o f White Leghorn c o c k e r e l s f e d SBM- o r RSM-supplemented d i e t s a t 1 week o r 3 weeks o f t r e a t m e n t from h a t c h i n g 20 3 Average w e i g h t s o f t h y r o i d g l a n d s i n b i r d s f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s o f t i m e i n e x p e r i m e n t 1 21 4 H i s t o l o g i c a l measurements on t h y r o i d g l a n d s from b i r d s ( S h a v e r s t r a i n ) i n e x p e r i m e n t 1 a t 91 weeks o f age 22 5 I o d i n e c o n c e n t r a t i o n i n t h y r o i d g l a n d s o f b i r d s f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s o f t i m e 27 6 A n a l y z e d i o d i n e c o n c e n t r a t i o n s i n SBM- and RSM-supple-mented d i e t s f e d t o b i r d s i n e x p e r i m e n t s 1 and 6B 28 7 C o n c e n t r a t i o n s o f r a d i o i o d i n e i n t h e t h y r o i d g l a n d and l i v e r 6 and 22 hours a f t e r a d m i n i s t r a t i o n 32 8 D i s t r i b u t i o n , 22 hours a f t e r a d m i n i s t r a t i o n , o f a s i n g l e dose o f r a d i o i o d i n e i n t o t h y r o i d a l compounds 37 9 D i s t r i b u t i o n , 22 hours a f t e r a d m i n i s t r a t i o n , o f a s i n g l e dose o f r a d i o i o d i n e i n t o t h y r o i d a l compounds measured i n h y d r o l y ^ a t e s a f t e r d i f f e r e n t t i m e s o f p r o n a s e d i g e s t i o n . . 41 10 I o d i n e c o n c e n t r a t i o n s i n egg y o l k s from b i r d s on d i f f e r e n t d i e t a r y t r e a t m e n t s i n e x p e r i m e n t 6A 45 11 I o d i n e c o n c e n t r a t i o n s i n egg y o l k s from b i r d s on d i f f e r e n t d i e t a r y t r e a t m e n t s i n e x p e r i m e n t 6B 49 12 R a d i o i o d i n e c o n c e n t r a t i o n s i n t h e t h y r o i d g l a n d s and d e v e l o p i n g o v a r i a n f o l l i c l e s 22 hours a f t e r a d m i n i s t r a t i o n o f a s i n g l e dose o f r a d i o i o d i n e t o b i r d s which had been f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s o f time 53 13 T h y r o i d w e i g h t s a t h a t c h i n g o f progeny b i r d s f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s i n e x p e r i m e n t 8 57 14 . T h y r o i d a l c h a r a c t e r i s t i c s i n n e w l y - h a t c h e d progeny o f b i r d s f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s i n e x p e r i m e n t 8. 58 15 Comparison o f t h y r o i d a l c h a r a c t e r i s t i c s o f dams and p r o -geny on d i f f e r e n t d i e t a r y t r e a t m e n t s 59 - v i i - TABLE OF FIGURES F i g u r e Page 1 Paper chromatograms o f t h y r o i d a l m e t a b o l i s m o f b i r d s on d i f f e r e n t d i e t a r y t r e a t m e n t s 38 2 R e g r e s s i o n o f 1-131 u p t a k e on s u r f a c e a r e a o f o v a r i a n f o l l i c l e s o f b i r d s f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s . . . 54 - v i i i - ACKNOWLEDGEMENTS The a u t h o r g r a t e f u l l y acknowledges t h e s u p e r v i s i o n and encouragement o f P r o f e s s o r B.E. March i n a c c o r d w i t h h e r p a r t i c u l a r c o n t r i b u t i o n t o my academic development (leading t o t h e c o m p l e t i o n o f t h i s work, and a l s o h e r many h e l p f u l a d v i c e s i n a r e a s o u t s i d e t he academic f i e l d . A f u l l measure o f a p p r e c i a t i o n i s a l s o e x t e n d e d t o the o t h e r members o f my T h e s i s Committee: Dr. W.D. K i t t s Chairman, Departments o f Animal S c i e n c e and P o u l t r y S c i e n c e Dr. R i C . Fitzsimmons Department o f P o u l t r y S c i e n c e Dr. C.R. K r i s h n a m u r t i Department o f Animal S c i e n c e Dr. A.M. P e r k s Department o f Z o o l o g y In p a r t i c u l a r , Dr. A.M. Perks must r e c e i v e s p e c i a l m e n t i o n i n a c c o r d w i t h h i s h e l p and c o n c e r n i n my programme. S p e c i a l a p p r e c i a t i o n i s g i v e n t o Dr. J . B i e l y ( R e s e a r c h P r o f e s s o r , Department o f P o u l t r y S c i e n c e ) f o r h i s many i n t e r e s t i n g and s t i m u l a t i n g i d e a s t h a t p r o v i d e d f o o d f o r t h o u g h t , and t o Mr. R ^ S o o n g ( P o u l t r y N u t r i t i o n L a b o r a t o r y ) f o r h i s f r e q u e n t a s s i s t a n c e when needed. - I X - INTRODUCTION The t h y r o i d g l a n d i s one o f t h e f i r s t e n d o c r i n e organs s t u d i e d by man, and the e f f e c t s o f i t s hormones on man and a n i m a l s have l o n g been known. Many a s p e c t s o f i t s m e t a b o l i s m , however, s t i l l remain u n c l e a r . A l t h o u g h many i n v e s t i g a t i o n s have been made on the f o r m a t i o n o f t h e t h y r o i d hormones, both i n v i t r o and i n v i v o , the e x a c t mechanism whereby t h y r o i d hormonogenesis t a k e s p l a c e under 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 . The p i c t u r e becomes more c o m p l i c a t e d when the t h y r o i d g l a n d i s i n h i b i t e d by s u b s t a n c e s such as g o i t r o g e n s . T h y r o i d f u n c t i o n and hormone b i o s y n t h e s i s i s e i t h e r a f f e c t d d o r b l o c k e d , dependent on the mode o f a c t i o n o f t h e g o i t r o g e n c o n c e r n e d . W h i l e numerous s t u d i e s have been made on t h e e f f e c t s o f t h e a n t i t h y r o i d compounds on t h y r o i d f u n c - t i o n s o f e x p e r i m e n t a l a n i m a l s , o n l y a few o f t h e s e i n v e s t i g a t i o n s have examined t h e e f f e c t s o f c h r o n i c g o i t r o g e n i c s t i m u l a t i o n on t h e t h y r o i d g l a n d . Much l e s s i s known o f any p o s s i b l e p e r s i s t e n c e o f e f f e c t s o f e a r l y s t i m u l a t i o n ? by g o i t r o g e n s when t h e y a r e l a t e r withdrawn. In t h e p r e s e n t s t u d y , r a p e s e e d meal was f e d t o c h i c k e n s e i t h e r j u s t f o r t h e growing p e r i o d o r o v e r a l o n g p e r i o d o f t i m e , and t h e e f f e c t s o f t h e d i e t a r y t r e a t m e n t s on the t h y r o i d g l a n d s were examined. Rapeseed i s one o f t h e most v a l u a b l e o i l s e e d c r o p s i n Canada, both f o r i t s p o t e n t i a l i n t h e p r o d u c t i o n o f e d i b l e and i n d u s t r i a l o i l s and i n the m a n u f a c t u r e o f h i g h p r o t e i n meals s u i t a b l e f o r s u p p l e m e n t i n g animal d i e t s . I t has been i n d i c a t e d i n t h e l i t e r a t u r e t h a t the mode o f a c t i o n o f t h e r a p e s e e d meal g o i t r o g e n s i s d i f f e r e n t from t h a t o f o t h e r a n t i - -x- t h y r o i d compounds. T h e r e f o r e , a l t h o u g h i t i s now p o s s i b l e t o m a n u f a c t u r e r a p e s e e d meals w i t h ^ a n e g l i g i b l e g o i t r o g e n c o n t e n t , t h e e f f e c t s o f c h r o n i c s t i m u l a t i o n by g o i t r o g e n i c r a p e s e e d meal on t h y r o i d h o r monogenesis, compared t o o t h e r a n t i t h y r o i d compounds such as p r o p y l t h i o u r a c i l and t h i o u r e a , a r e s t i l l w orthy o f i n v e s t i g a t i o n from t h e academic s t a n d p o i n t . In a d d i t i o n , t h e p o s s i b i l i t y t h a t e f f e c t s o f e a r l y s t i m u l a t i o n o f t h e t h y r o i d g l a n d may p e r s i s t v a f t e r w i t h d r a w a l o f t h e g o i t r o g e n w a r r a n t s f u r t h e r s t u d i e s i n i t s own r i g h t . - 1 - REVIEW OF LITERATURE BIOSYNTHESIS AND RELEASE OF THYROID HORMONES The i m p o r t a n c e o f i o d i n e i n the p r e v e n t i o n o f g o i t e r has l o n g been r e c o g n i z e d ( H a r t and S t e e n b e c k , 1918; K a l k u s , 1920; Welch, 1928) and i o d i n e m e t a b o l i s m i n t h e body has been t h o r o u g h l y r e v i e w e d ( W i l g u s e t a l , 1953; C h a i k o f f and T a u r g g , 1948; Hemken, 1970; K o u t r a s , 1972). In t h e a l i m e n t a r y t r a c t , d i e t a r y i o d i d e i s r e a d i l y a b s o r b e d i n t o t h e c i r c u l a t i o n and i s m a i n l y c o n c e n t r a t e d w i t h i n t h e t h y r o i d . The r o l e o f the t h y r o i d , t h e n , i s t o i n c o r p o r a t e i n o r g a n i c i o d i d e i n t o t h y r o i d hormones and t o m a i n t a i n a s u f f i c i e n t r e l e a s e o f t h e s e hormones t o t h e p e r i p h e r y t i s s u e s . I n t r a t h y r o i d a l b i o s y n t h e s i s o f t h y r o i d hormones has been ex- t e n s i v e l y r e v i e w e d ( P l a s k e t t et_ al _ , 1963; Rapoport and DeGroot, 1971; D a v i e s , 1972; Ermans e t a l _ , 1972; DeGroot e t a l _ , 1972; Mason and W i l k i n s o n , 1973). T h e r e f o r e i n the p r e s e n t d i s c u s s i o n , we w i l l r e v i e w o n l y t h e more r e c e n t developments i n the p h y s i o l o g y o f t h y r o i d hormone b i o s y n t h e s i s , w i t h s p e c i a l a t t e n t i o n t o such a r e a s t h a t may perhaps be a f f e c t e d by g o i t r o g e n i c a g e n t s . I t i s g e n e r a l l y a g r e e d t h a t f o r m a t i o n o f t h y r o i d hormones i n - v o l v e s t h e f o l l o w i n g i n t e r m e d i a r y s t e p s (DeGroot e t aj_, 1972): 1) t r a n s - p o r t and c o n c e n t r a t i o n o f i o d i d e i n t o t he t h y r o i d , 2) s y n t h e s i s o f t h y r o - g l o b u l i n , 3) g e n e r a t i o n o f an o x i d i z i n g a g e n t , p r o b a b l y hydrogen p e r o x i d e , 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 l o b u l 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 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 , and 8) r e l e a s e o f t h y r o i d hormones t o t h e c i r c u l a t i o n . - 2 - I o d i d e t r a p p i n g by t h e t h y r o i d i s dependent on adquate p o t a s s i u m c o n c e n t r a t i o n ( W o l f f and H a l m i , 1963) and i s b l o c k e d by i n h i b i t o r s o f o r a g ents t h a t u n c o u p l e o x i d a t i v e p h o s p h o r y l a t i o n such as 2 , 4 - d i n i t r o p h e n o l , c y a n i d e , c a r d i a c g l y c o s i d e s and a n o x i a (Wyngaarden e t a l _ , 1953; F r e i n k e l and I n g b a r , 1955; W o l f f , 1964). T h e r e f o r e i t i s p r o b a b l y an a c t i v e p r o c e s s r e q u i r i n g e n e r g y r e l a t e d w i t h t h e Na-,K +-ATPase-dependent t r a n s p o r t s ystem ( T u r k i n g t o n , 1962). Much a t t e n t i o n has been f o c u s s e d on t h e s i t e o f t h i s i o d i d e pump mechanism, which i s p r o b a b l y a membrane r e c e p t o r s i t e l o c a t e d i n : t h e p e r i p h e r a l b o r d e r o f t h e f o l l i c u l a r c e l l s o f t h e t h y r o i d ( H a l m i , 1961; P i t t - R i v e r s and T r o t t e r , 1953; Tong e t a l _ , 1962). I t has been s u g g e s t e d t h a t a l e c i t h i n p h o s p h o l i p i d i s a c o n s t i t u e n t o f t h e r e c e p - t o r s i t e ( V i l k k i and Jaakonmaki, 1966) and a r o l e o f F e + + has a l s o been i m p l i c a t e d (Dhopeshwarkar, 1968). T h y r o g l o b u l i n , l i k e o t h e r p r o t e i n s , i s s y n t h e s i z e d i n the p o l y r i b o s o m e s o f the e n d o p l a s m i c r e t i c u l u m o f t h e t h y r o i d c e l l s ( N a d l e r et_ a l _ , 1964; Thomson and G o l d b e r g , 1968); i t t h e n t r a v e l s down t o t h e a p i - c a l r e g i o n o f t h e c e l l s t o be a c c u m u l a t e d i n t h e c o l l o i d (Whur e t a l _ , 1969). I t i s a g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f a b o u t 650,000 ( G o l d b e r g and L e e d , 1965; Robbins and R a i l , 1960; R a i l e t a l _ , 1960) and a s e d i m e n t a t i o n c o e f f i c i e n t o f 19S when i o d i n a t e d . The s i t e o f i t s i o d i n a t i o n has been t h e t o p i c o f much e x t e n s i v e s t u d i e s ( E d e l h o c h , 1962; Nunez, 1966; L i s s i t z k y , e t a l _ , 1969), and i t i s p r o b a b l y a t t h e b o r d e r o f the a p i c a l membrane o f t h e t h y r o i d c e l l and t h e c o l l o i d . The d i s t r i b u t i o n o f v a r i o u s iodoamino a c i d s i n t h e t h y r o i d t i s s u e i s l a r g e l y d e t e r m i n e d by t h e i o d i n a t i o n l e v e l o f the t h y r o g l o b u l i n (DeCrombrugghe e t a l , 1967; Ermans e t a l , 1968; V a l e n t a e t a l _ , 1968). - 3 - Many q u e s t i o n s p e r t a i n i n g t o t h e mechanism o f t h y r o i d hormone f o r m a t i o n a r e s t i l l u n s o l v e d ; t h e y have r e c e n t l y have d i s c u s s e d t h o r o u g h l y by DeGroot e t a l _ (1972). I t i s w i d e l y a c c e p t e d t h a t i n v i v o i o d i n a t i o n s r e q u i r e g e n e r a t i o n , o f H,,02 w h i c h , t o g e t h e r w i t h 0 2 a r e t h e o n l y b i o l o g i c a l o x i d i z i n g a gents w i t h a n . j D x i d a t i o n ^ r e d u c t i o n p o t e n t i a l above t h a t o f the 2 I " ^ I 2 c o u p l e , i . e . +0.535 v o l t s ( B e r n a r d and DeGroot, 1969; Ahn and Rosenberg, 1970). A l t h o u g h c y t o p l a s m i c DPNH o x i d a s e ( R o s s i e t a l _ , 1969) and monoamine o x i d a s e ( F i s c h e r et_ a l _ , 1966, 1968) a c t i v i t y have been s u g g e s t e d t o be t h e o r i g i n o f H 2 0 2 , t h e c o f a c t o r NADPH i s l i k e l y t o be i n v o l v e d i n t h y r o i d a l H 2 0 2 p r o d u c t i o n , p o s s i b l y v i a r e a c t i o n w i t h NADPH- cytochrome C r e d u c t a s e (DeGroot and D a v i s , 1961; Nagasaka e t a l _ , 1971). The p a r t i c u l a t e - b o u n d enzyme, p e r o x i d a s e , has been i s o l a t e d from t h y r o i d t i s s u e s o f a number o f s p e c i e s ( A l e x a n d e r , 1959; Hosoya and M o r r i s o n , 1967; Mahoney and Igo, 1966; L j u n g g r e n and Adeson, 1968; Naga- saka and DeGroot, 1971). I t i s a heme p r o t e i n c o n t a i n i n g 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 (Taurog e t a l _ , 1970). In a d d i t i o n t o u t i l i z i n g H 2 0 2 f o r p e r o x i d a t i o n o f i o d i d e t o i o d i n e — p r o b a b l y t o i t s f r e e r a d i c a l f o r m , I 0 ( K l e b a n o f f e t a l _ , 1962; Y i p and H a d l e y , 1966) — p e r o - x i d a s e has a l s o been h e l d r e s p o n s i b l e f o r c a t a l y z i n g t h e o r g a n i f i c a t i o n 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 l o b u l i n . DeGroot e t a l (1972) e n v i s a g e a "ping-pong" mechanism o f o r g a n i f i c a t i o n , which presumably i n v o l v e s f o r - m a t i o n o f f r e e r a d i c a l s o f i o d i d e and t y r o s i n e by p e r o x i d a s e complexed w i t h H 2 0 2 . Union o f the i o d i n e and t y r o s i n e f r e e r a d i c a l s t o form i o d o t y r o s i n e r e t u r n s the enzyme 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 s y n t h e s i s o f i o d o t i y r o s i n e s i s a r a p i d p r o c e s s (DeGroot and D a v i s , 1961). D i s t r i b u t i o n o f r a d i o i o d i n e between m o n o i o d o t y r o s i n e (MIT) and d i i o d o t y r o s i n e (DIT) r e a c h e s a c o n s t a n t l e v e l a f t e r v a r i a b l e l e n g t h s - 4 - o f t i m e , and t h e c o n s t a n t MIT/DIT r a t i o i n 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 c o n s o n a n t w i t h a p r e c u r s o r - p r o d u c t r e l a t i o n s h i p between t h e two i o d o t y r o - s i n e s ( P l a s k e t t e t a l _ , 1963a). When th e i o d i n e c o n t e n t o f t h e t h y r o i d t i s s u e i s r e d u c e d , however, t h e r e a p p e a r s a marked i n c r e a s e i n t h e MIT/DIT r a t i o ( Q u e r i d o e t a l _ , 1957; Ermans e t a l _ , 1961), w h i c h , as m entioned e a r l i e r , i s dependent on the d e g r e e o f i o d i n a t i o n o f t h y r o g l o b u l i n (Ermans e t a l _ , 1968). The 1 coy p i l i n g 1 o f i o d o t y r o s i n e s t o i n t r a t h y r o g l o b u l i n i o d o - t h y r o n i n e s , as f i r s t p r o p o s e d by H a r i n g t o n and B a r g e r (1927), i s a much s l o w e r p r o c e s s ( P i t t - R i v e r s , 1962; P l a s k e t t e t al_, 1963b). A l t h o u g h i t has been a c h i e v e d i n v i t r o ( P i t t - R i v e r s , 1948; Y i p and K l e b a n o f f , 1952), i t s i n v i v o mechanism i s s t i l l u n c e r t a i n . The f a c t t h a t f r e e t h y r o n i n e i s n o t found i n n a t u r e l e a v e s two p o s s i b l e r o u t e s whereby i o d o t h y r o n i n e s can be formed i n t h y r o g l o b u l i n (DeGroot e t a l _ , 1972): 1) by i n t r a m o l e c u l a r r e a r r a n g e m e n t , i . e . c o n f o r m a t i o n a l changes w i t h i n t h e t h y r o g l o b u l i n which a l l o w an i o d o t y r o s y l f r e e r a d i c a l ( p r o d u c e d by an o x i d i z i n g agent such as p e r o x i d a s e - I " ) t o be t r a n s f e r r e d t o a n o t h e r i o d o t y r o s y l group t o form i o d o - t h y r o n i n e ; o r 2) by o x i d a t i v e - d e a m i n a t i o n o f DIT t o form h y d r o x y p h e n o l - p y r u v a t e ( D I H P P A ) , p e r o x i d a t i o n o f the e n o l form o f which l e a d s t o the f o r m a t i o n o f a DIHPPA-peroxide, which 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 form t h y r o x i n e ( T o i e t a l _ , 1963, 1965). D e s p i t e e v i d e n c e o f the p r e s e n c e o f n e c e s s a r y enzymes i n the t h y r o i d t i s u s e (Igo e t a l _ , 1968; B l a s i e t a l _ , 1969a,b), t h e e x t e n t o f t h e s e pathways In v i v o t h y r o i d hormone f o r m a t i o n i s s t i l l u n c e r t a i n . T h u s , t h y r o i d hormone i s s t o r e d i n t h e f o l l i c u l a r lumen, as a c o n s t i t u e n t o f t h e t h y r o g l o b u l i n p o l y p e p t i d e . Recent e l e c t r o n m i c r o g r a p h i c a l d a t a show, i n 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 - 5 — t y p e s o f v e s i c l e s i n the a p i c a l r e g i o n s 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 l _ , 1970): e n d o c y t o t i c and e x o c y t o t i c . E x o c y t o t i c v e s i c l e s may be i n v o l v e d i n the t r a n s p o r t o f newly s y n t h e s i z e d t h y r o g l o b u l i n i n t o the f o l l i c l e lumen f o r s t o r a g e , w h i l e 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 a n c e o f t h y r o g l o b u l i n i n t o t h e 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 . F u r t h e r m o r e , t h e r e i s e v i d e n t f o r t h e " 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 i o d i n e t u r n o v e r i n the t h y r o i d pool ( S c h n e i d e r , 1964), which i s s u p p o r t e d by t h e s t r u c t u r a l and f u n c t i o n a l h e t e r o g e n e i t y among t h e f o l l i c l e s ( N a d l e r , 1954; S t u d e r e t a l _ , 1972). Once r e - e n t e r e d i n t o t h e t h y r o i d c e l l , r e l e a s e o f t h e t h y r o - g l o b u l i n i s a c h i e v e d by p r o t e o l y s i s by a c i d i c p r o t e a s e s d a r i d ' p e p t i d a s e s c o n t a i n e d i n t h e l y s o s o m e s ( W o l l m a n e t a l , 1964; S e l j e l i d , 1968). The l i b e r a t e d i o d o t y r o s i n e s a r e d e i o n a t e d by i o d o t y r o s i n e d e i o d i n a s e w i t h i n the t h y r o i d c e l l , w h i l e the i o d o t h y r o n i n e s a r e s e c r e t e d i n t o t h e c i r c u - l a t i o n t o a f f e c t t a r g e t t i s s u e s . A l m o s t e v e r y i n t e r m e d i a r y s t e p i n t h y r o i d hormone b i o s y n t h e s i s and r e l e a s e i s r e g u l a t e d one way o r the o t h e r by t h y r o t r o p i n (TSH) from t h e a d e n o h y p o p h y s i s . An e x c e l l e n t r e v i e w o f the a c t i o n o f t h y r o t r o p i n on t h y r o i d m e t a b o l i s m i s g i v e n by Dumont (1971). In a d d i t i o n t o the e x t r a - t h y r o i d a l c l a s s i c a l n e g a t i v e f e e d b a c k c o n t r o l by t h y r o i d hormones and t h e hypothalamic;\ppn,tr fp:lcby T S H - r e l e a s i n g hormone (TRF) v i a t h e hypothalamo- hypophyseal p o r t a l system ( S c h a l l y e t a l _ , 1969; Burgus and G u i l l e m i n , 1970), t h e r e seems t o be an i n t r a t h y r o i d a l autonomous mechanism whereby t h y r o i d a l i o d i n e m e t a b o l i s m i s r e g u l a t e d ( W o l f f and C h a i k o f f , 1948; Socolow e t a l , 1968; B a r a k a t and I n g b a r , 1965; Golman e t a^, 1966; S t u d e r and G r e e r , 1968). I t a l s o a p p e a r s t o a l t e r t h y r o i d hormone s e c r e t i o n , i n d e p e n d e n t l y o f TSH s u p p l y (Green and I n g b a r , 1962; H a l m i , 1961; Onaya and H a l m i , 1967). S o l o - mon and Dowling (1960) h y p o t h e s i z e d t h a t a u t o r e g u l a t i o n o f t h y r o i d a l i o d i d e - 6 - t r a n s p o r t a l l o w s an i o d i d e - d e f i c i e n t a nimal t o m a i n t a i n 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 acc u m u l a t e d s u p p l y may then be r e l e a s e d by t h e e x t r a TSH s e c r e t e d i n r e s p o n s e t o lo w e r e d i o d o t h y r o n i n e l e v e l s i n t h e c i r c u l a t i o n . A n a l y s e 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 t h e mammalian g l a n d s ( S h e l l a b a r g e r and P i t t - R i v e r s , 1958; M e l l e n and Wentworth, 1959). Hence a d i v e r s i t y o f mechanisms has n o t been r e c o g n i z e d f o r t h y r o i d hormone b i o - s y n t h e s i s i n the d i f f e r e n t s p e c i e s . However, i t s h o u l d be no t e d t h a t w h i l e i n mammals L - t r i i o d o t h y r o n i n e (Tg) has a h i g h e r p o t e n c y t h a n an e q u i m o l a r q u a n t i t y o f L - t h y r o x i n e ( T ^ ) , t h e i o d o t h y r o n i n e s have d i f f e r e n t r e l a t i v e p o t e n c i e s i n b i r d s ( S h e l l a b a r g e r , 1955; H u t c h i n s and Newcomer, 1966; New- comer, 1957; Robbins and R a i l , 1957). F u r t h e r m o r e , t h e f i n d i n g by T a t a and S h e l l a b a r g e r (1959) t h a t t h y r o x i n e - b i n d i n g g l o b u l i n i s a b s e n t i n c h i c k e n b l o o d p r o b a b l y e x p l a i n s the f a i l u r e o f p r o t e i n - b o u n d i o d i n e (PBI) as a c r i t e r i o n o f measurement o f t h y r o i d hormone m e t a b o l i s m i n the b i r d . EFFECTS OF GOITROGENS ON THYROID HORMONE BIOSYNTHESIS A n t i t h y r o i d a l s u b s t a n c e s and n a t u r a l g o i t r o g e n s have been ex- t e n s i v e l y r e v i e w e d ( V a n E t t e n , 1969). The f o r m e r i n c l u d e , among o i t h e r s , i n o r g a n i c i o n s s u c h as i o d i d e , ( l a r g e doses o f which i n h i b i t t h y r o i d h o r - mone s y n t h e s i s i n the r a t -- the s o - c a l l e d W o l f f - C h a i k o f f e f f e c t ) , t h e t h i o n a m i d e d r u g s such as t h i o u r e a and t h i o u r a c i l , and the s u l f o n a m i d e s such as p a r a - a m i n o b e n z o i c a c i d and s u l f a n i l a m i d e (Aswood, 1943; Astwood e t a l _ , 1945). Among t h e n a t u r a l l y o c c u r r i n g g o i t r o g e n s , ( - ) - 5 - v i n y l - 2 - t h i o - o x a z o l i d o n e i s o f p a r t i c u l a r i m p o r t a n c e because o f i t s p o s s i b l e r o l e i n t h e p a t h o g e n e s i s o f endemic g o i t e r and w i l l be d i s c u s s e d i n more d e t a i l . - 7 - D i s t u r b a n c e s o r i n h i b i t i o n o f t h y r o i d hormone b i o s y n t h e s i s due t o d e f e c t s o f i o d i n e m e t a b o l i s m c a n be c l a s s i f i e d as f o l l o w s ( B l i z z a r d , 1960): 1) ^ d e f e c t i n t r a p p i n g o f i n o r g a n i c i o d i d e from t h e c i r c u l a t i o n ; 2) i n a b i l i t y t o o x i d i z e i o d i d e t o an a c t i v e form; 3) i n a b i l i t y t o h a l o - genate t y r o s i n e i n p r e s e n c e o f e l e m e n t a l i o d i n e ; 4) i n a b i l i t y t o c o u p l e i o d i n a t e d t y r o s i n e s t o form i o d o t h y r o n c i n e s ; 5) i n a b i l i t y t o h y d r o l y z e t h y r o g l o b u l i n ; 6) abnormal f o r m a t i o n o r r e l e a s e o f t h y r o g l o b u l i n o r " t h y r o g l o b u l i n - l i k e " s u b s t a n c e s ; 7) i n a b i l i t y t o d e i o d i n a t e f r e e i o d b - t y r o s i n e s f o r r e u t i l i z a t i o n . One o r more o f t h e s e i n t e r m e d i a t e s t e p s o f t h y r o i d hormone b i o s y n t h e s i s can be i n h i b i t e d by a g o i t r o g e n . F o r example, the t h i o n a m i d e s such as t h i o u r a c i l a p p e a r t o i n t e r f e r e w i t h the o r g a n i c b i n d i n g o f i n t r a t h y r o i d a l i o d i d e ( l i n o , 1961), t h u s competing w i t h the i o d i d e w h i l e the p e r o x i d a s e i s a c t i n g (DeGroot and D a v i s , 1962). The s u l f o n a m i d e s i n t e r f e r e w i t h o r g a n i f i c a t i o n o f i o d i n e w i t h o u t i n h i b i t i n g t h e t r a p p i n g o f i o d i d e ( M i l n e and G r e e r , 1962). T h a t t h e i r mechanisms o f a c t i o n a r e d i f f e r e n t i s e v i d e n t by t h e o b s e r v a t i o n t h a t a d d i t i o n o f i o d i n e r e d u c e s g o i t e r s i z e i n a n i m a l s t r e a t e d w i t h t h i o n a m i d e s b u t i n c r e a s e t h y - r o i d h y p e r t r o p h y i n a n i m a l s t r e a t e d w i t h s u l f o n a m i d e s (MacKenzie and M a c K e n z i e , 1943). In a s y s t e m a t i c s t u d y i n which N - i o d o s u c c i n i m d d e was used as a model f o r the a c t i v e i o d i n e o f the t h y r o i d g l a n d ( J i r o u s e k and Soodak, 1973), a l l c l a s s e s o f g o i t r o g e n s t e s t e d were f o u n d t o r e a c t . The e f f e c t s o f t h e v a r i o u s g o i t r o g e n s on i n v i t r o b i o s y n t h e s i s o f t h y r o i d hormones have been d e l i n e a t e d ( l i n o , 1961). T h e i r a b i l i t y t o c o m p l e t e l y i n h i b i t DIT f o r m a t i o n i n d e c r e a s i n g o r d e r a r e : l - m e t h y l - 2 - m e r c a p t o i m i d a z o l e (MIA); t h i o u r a c i l ( T U ) ; p r o p y l t h i o u r a c i l (PTU) and ( ) - ) - 5 - v i n y l - 2 - t h i o - o x a z o l i d o n e ( g o i t r i n ) ; t h i o u r e a and m e t h y l t h i o u r a c i l (MTU). T h e i r a b i l i t y t o i n h i b i t - 8 - MIT f o r m a t i o n i n d e c r e a s i n g o r d e r a r e : MIA; PTU, MTU, TU and t h i o u r e a ; g o i t r i n . A l l o f them seem t o show s i m i l a r mechanisms o f a c t i o n e x c e p t g o i t r i n . T h i s i s i n agreement w i t h e v i d e n c e p r e s e n t e d i n a f o l l o w i n g s e c t i o n w i t h r e g a r d t o g o i t r o g e n i c ! ' t y o f r a p e s e e d meal. Many s t u d i e s have been f o c u s e d on t h e enzyme systems i n the t h y r o i d g l a n d , n o t a b l y on t h y r o i d a l p e r i d a s e ( S e r i f and Kirkwood, 1958) and i t s p o s s i b l e i n t e r a c t i o n w i t h a n t i t h y r o i d compounds. Though p e r - c h l o r a t e a p p e a r s t o have no e f f e c t on t h e enzyme, i t has been shown t h a t a z i d e , c y a n i d e , t h i o u r a c i l , t h i o c y a n a t e , p-aminobenzoate, and 3-amono- 1 , 2 , 4 - t r i a z o l e a r e p o t e n t i n h i b i t o r s o f i o d i d e p e r o x i d a s e (Alemander, 1959), and t h a t some n a t u r a l l y o c c u r r i n g g o i t r o g e n s i n c l u d i n g g o i t r i n may a l s o p o s s e s s i n h i b i t o r y a c t i v i t y on t h e v a r i o u s t h y r o i d enzymes such as p e r o x i d a s e , cytochrome C o x i d a s e and DPNH-linked i o d i n a t i n g a c t i v i t y ( L a n g e r and M i c h a j l o v s k i j , 1972). I n s u f f i c i e n t p r o t e o l y s i s o f t h e t h y r o g l o b u l i n and s u p p r e s s i o n o f l y s o s o m a l enzyme a c t i v i t i e s have a l s o been r e p o r t e d as causes o f g o i t e r i n man ( B e c k e r s and D e V i s s c h e r , 1962) and m i c e ( I t k i a w a and Kawada, 1974). RAPESEED MEAL AS A SOURCE OE GOITROGEN The seed meals from rape ( B r a s s i c a napus L.) and t u r n i p r a p e (B. c a m p e s t r i s L . ) , a f t e r t h e o i l s have been e x p e l l e d o r s o l v e n t - e x t r a c t e d , a r e h i g h p r o t e i n - c o n t a i n i n g m a t e r i a l s and used i n animal f e e d s t u f f s (Bowland e t a l _ , 1965; C l a n d i n i n and R o b b l e e , 1966; C l a n d i n i n , 1967; B e l l and B e l z i l e , 1965; R utkowski, 1971). - 9 - P r a c t i c a l l y a l l p l a n t s o f the f a m i l y C r u c i f e r a e , t o which t h e genus B r a s s i c a b e l o n g s , which have been i n v e s t i g a t e d c o n t a i n g l u c o s i n o l a t e s ( V a n E t t e n 1969). In B r a s s i c a p l a n t s , t w e l v e d i f f e r e n t g l u c o s i n o l a t e s a r e known t o o c c u r and a r e d i s t r i b u t e d i n a l m o s t a l l p a r t s o f t h e p l a n t s ( J o s e f s s o n , 1967). M y r o s i n a s e i s p r e s e n t i n the t i s s u e s o f t h e s e 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 as w e l l as i n g a s t r o i n t e s t i n a l t r a c t o f a n i m a l s ( G r e e r , 1962 a , b ) . I t i s p r o b a b l y an -SH dependent enzyme s i n c e i t i s i n a c t i v a t e d by i n h i b i t o r s o f t h a t c h e m i c a l r a d i c a l (Sandberg and H o l l y , 1932). G l u c o s i n o l a t e s a r e b i o l o g i c a l l y i n a c t i v e . D e s t r u c t i o n o f t h e p l a n t c e l l u l a r s t r u c t u r e a l l o w s t h e m y r o s i n a s e enzyme ( o r enzyme system) t o h y d r o l y z e t h e 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 l u c o s e and b i s u l p h a t e , w h i l e a c t i v a t i n g t h e compounds i n t o one o f t h r e e g r o u p s : 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 c y a n a t e s ; i n o r g a n i c t h i o c y a n a t e ( E t t l i n g e r and Lundeen, 1957). The g o i t r o g e n i c p r o p e r t i e s o f r a p e s e e d meal a r e l a r g e l y a t t r i - b uted t o ( - ) - 5 - v i n y l - o x a z o l i d i n e t h i o n e ( g o i t r i n ) , a h y d r o l y s i s p r o d u c t o f 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 . I t i s p r o b a b l y n o t p r e f o r m e d i n the g l u c o s i n o l a t e m o l e c u l e but a r i s e s from c y c l i z a t i o n f o l l o w i n g myro- s i n a s e a c t i o n ( C l a n d i n i n e_t a l _ , 1959). The mechanism o f g o i t r i n i n f l u e n c e on t h e t h y r o i d g l a n d i s f a r from b e i n g u n d e r s t o o d . Rutkowski (1971) s t a t e d t h a t g o i t r i n b l o c k s t h e i r r e v e r s i b l e mechanism c o n n e c t e d w i t h o r g a n i c b i n d i n g o f i o d i n e i n t h e t h y r o i d , t h u s p a r t i a l l y s u p p r e s s i n g t h y r o i d hormone b i o s y n t h e s i s . T h i s , however, i s n o t borne o u t by r e p o r t s from o t h e r i n v e s t i g a t o r s (Matsumoto e t a l , 1969; A k i b a and Matsumoto, 1971). C l a n d i n i n e t i l (1961) d e s c r i b e d -10- a h i s t o p a t h o l o g i c a l p i c t u r e o f the r a p e s e e d m e a l - t r e a t e d c h i c k e n t h y r o i d g l a n d , which shows c o n s i d e r a b l e h y p e r t r o p h y and h y p e r p l a s i a o f p a r e n c h y - mal." e l e m e n t s and a l m o s t c o m p l e t e l o s s o f f o l l i c u l a r c o l l o i d . T h i s i s f u r t h e r s u p p o r t e d by t h e p h o t o m i o r o g r a p h i c f i n d i n g s o f Rutkowski ( 1 9 7 1 ) , i n which desquamation o f e p i t h e l i u m compressed f o l l i c l e s i n s m a l l r e s t r i c t e d a r e a s and d e c r e a s e i n c o l l o i d c o u l d be r e a d i l y o b s e r v e d i n t h y r o i d s o f c h i c k e n s a f t e r f o u r weeks o f r a p e s e e d meal f e e d i n g . The i s o t h i o c y a n a t e s and t h i o c y a n a t e s have a c o n s i d e r a b l y lower g o i t r o g e n i c e f f e c t than the o x a z o l i d i n e t h i o n e s (Fertman and C u r t i s , 1951; Gmelin and V i r t a n e n , 1960). They b l o c k i o d i n e a c c u m u l a t i o n i n t o t h e t h y - r o i d g l a n d , but t h e i r e f f e c t c o u l d be removed by a d d i t i o n o f i o d i n e t o t h e r a t i o n ( R u t k o w s k i , 1971). A s y n e r g e t i c i n f l u e n c e o f t h e o x a z o l i d i n e - t h i o n e s , i s o t h i o c y a n a t e s and t h i o c y a n a t e s may be p o s s i b l e ( t h e s o - c a l l e d " B r a s s i c a f a c t o r " ) , however, s i n c e i t has been shown ( L a n g e r , 1966) t h a t t h e e f f e c t o f the m i x t u r e o f g o i t r o g e n s was c o n s i d e r a b l y h i g h e r than t h e sum o f r e s u l t s o b t a i n e d i n a d m i n i s t r a t i n g them i n d i v i d u a l l y . EFFECTS OF GOITRIN ON THYROID METABOLISM OF THE CHICKEN The problems i n v o l v e d i n t h e f e e d i n g o f rapesteed meal t o l i v e - s t o c k have been e x t e n s i v e l y reviewed ( B e l l , 1955, 1957; Manns and Bowland, 1963; Manns e j t a l _ , 1963). T h y r o i d h y p e r t r o p h y has i n v a r i a b l y been r e p o r t e d i n d o m e s t i c a n i m a l s f e d r a p e s e e d meal ( T u r n e r , 1946;Bla k e l y and A n d e r s o n , 1948 a,b'; W i t z e t al, 1950; K l a i n e t a l _ , 1956) and has been a s c r i b e d t o t h e e f f e c t o f t h e known g o i t r o g e n i c compounds o f r a p e s e e d : ( > - ) - 5 - v i n y l - 2 - t h i o - o x a z o l i d o n e (Astwood e t a l _ , 1949; C a r r o l l , 1949) and a l l y l o r 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 l l e n , 1954). -11- ( _ ) _ 5 - v i n y l - 2 - t h i o - o x a z o l i d o n e ( g o i t t r i m ) i s t h e most p o t e n t n a t u r a l g o i t r o g e n i c compound i s o l a t e d so f a r ( L a n g e r e t ^ a l _ , 1971). I t was f i r s t i d e n t i f i e d i n r a p e s e e d by Astwood ejt a l _ (1949 a,b) and C a r r o l l (1949) as L - 5 - v i n y l - 2 - t h i o o x a z o l i d o n e and l a t e r renamed ( - ) - 5 - 2 - o x a z o l i d i n e - t h i o n e . I t s s y n t h e t i c a n a l o g u e , , ( + ) - 5 - v i n y l - 2 - o x a z o l i d i n e t h i o n e , was f i r s t made a v a i l a b l e by E t t l i n g e r (1950). The work o f C l a n d i n i n (1962) and C l a n d i n i n e t _ a l _ (1966) i n the e f f e c t s o f s y n t h e t i c g o i t r i n i n t h y r o i d m e t a b o l i s m i n the growing c h i c k s u g g e s t s t h e f o l l o w i n g sequence o f e v e n t s : i n i t i a l l y , i o d i n e uptake i s g r e a t l y r e d u c e d and c o l l o i d s t o r e s i n t h e g l a n d s a r e r a p i d l y d e p l e t e d . C h i c k growth r a t e i s d e p r e s s e d . Rapid e n l a r g e m e n t o f the g l a n d s f o l l o w s w i t h accompanying h y p e r t r o p h y and h y p e r p l a s i a . I o d i n e u p t a k e i n c r e a s e s . C o l l o i d s t o r e s r e t u r n and t h e g l a n d t a k e s on a more normal a p p e a r a n c e . Hence i t would a p p e a r t h a t c h i c k s f e d t h e g o i t r o g e n e v e n t u a l l y r e a c h p h y s i o l o g i c a l e q u i l i b r i u m a t i n c r e a s e d t h y r o i d - t o - b o d y w e i g h t r a t i o s . When comparing t h e g o i t r o g e n i c e f f e c t s o f g o i t r i n , 1-methyl - 2 - m e r c a p t o i m i d a z o l e ( m ethimazole) and 6 - m e t h y l - t h i o u r a c i l ( m e t h i o c i l ) i n g r o wing c h i c k s , Matsumoto e t al_ (1968) r e p o r t e d t h a t t h e c h i c k s w hich r e c e i v e d 0.05% g o i t r i n showed t h y r o i d e n l a r g e m e n t , the h i g h e s t t h y r o i d a l u ptake o f 1-131 and t h e slow r e l e a s e o f 1-131, which was i n marked c o n t r a s t t o the m e t h i m a z o l e - o r m e t h i o c i l - f e d c h i c k s . They c o n c l u d e d t h a t t h e i n c o r p o r a t i o n o f b l o o d 1-131 by the t h y r o i d g l a n d i s n o t i n h i b i t e d and t h a t , from t h e s l o w r e l e a s e o f 1-131 and the d e p r e s s e d t h y r o i d hormone i n b l o o d , some s t e p o f t h y r o i d hormone b i o s y n t h e s i s and f u r t h e r m o r e s e c r e t i o n o f t h y r o i d hormone i n t o b l o o d o r d e i o d i n a t i o n o f some iodoamino a c i d s i n t h y r o i d g l a n d may be i n h i b i t e d by t h e t r e a t m e n t w i t h g o i t r i n . -12- T h a t the g o i t r o g e n i c e f f e c t s o f g o i t r i n a r e d i f f e r e n t from t h o s e o f p r o p y l t h i o u r a c i l (PTU) i n the growing c h i c k was f u r t h e r s t u d i e d by Matsumoto e t al_ (1969) and A k l b a and Matsumoto (1971). T h e s e i n v e s t i - g a t o r s showed t h a t w h i l e t h e PTU c h i c k s do n o t m a i n t a i n i n c o r p o r a t e d 1-131 i n t h e t h y r o i d g l a n d b u t r e l e a s e i t i n t o t h e b l o o d e a s i l y , the g o i t r i n - f e d c h i c k ; i s promoted t o m a i n t a i n a l a r g e amount o f i n c o r p o r a t e d 1-131 i n t h e g l a n d and a p p e a r s t o i n h i b i t i t s r e l e a s e . F u r t h e r m o r e , s i n c e the p e r c e n t a g e o f i o d o t h y r o n i n . e s (Tg and T^) i n t h i s l a t t e r group a l s o d e c r e a s e d t o o n e - t h i r d o f the c o n t r o l c h i c k s i n t h e i r d i s t r i b u t i o n o f r a d i o a c t i v e i o d i n a t e d s u b s t a n c e s found i n t h y r o i d homogenates, and t h a t t h e h i g h e s t p e r c e n t a g e was o b s e r v e d i n MIT (65%) and t h e MIT/DIT r a t i o was h i g h e r than t h a t o f the c o n t r o l group, i t i s q u i t e p o s s i b l e t h a t g o i t r i n a l m o s t n e v e r i n h i b i t s t h e m o n o i o d i n a t i o n o f t y r o s i n e , but i t i n h i b i t s the c o u p l i n g o f MIT t o s y n t h e s i z e DIT. The f i n d i n g o f i n c r e a s e d MIT p e r - c e n t a g e i s i n agreement w i t h r e s u l t s o f l i n o e t a l _ (1961) and G r e e r e t a l (1962) i n r a t s g i v e n s m a l l d oses o f PTU. R e p o r t s on t h e e f f e c t o f PTU o r t h i o u r a c i l on t h e i n t r a t h y r o i d a l m e t a b o l i s m o f 1-131 a r e numerous. F o r example, t h i o u r a c i l i n h i b i t s t h e o r g a n i c b i n d i n g o f i o d i n e i n r a t t h y r o i d i n v i v o (Astwood and B i s s e l , 1944) and i n v i t r o , ( F r a n k l i n e t a l _ , 1944), PTU b l o c k s the i o d i n a t i o n o f t y r o s i n e ( P i t t - R i v e r s , 1948; Mayberry and Astwood, 1960) and i n h i b i t s t h e s y n t h e s i s and c o u p l i n g o f DIT ( R i c h a r d and I n g b a r , 1959). Thus i t a p p e a r s t h a t PTU i n h i b i t s t h e s e s t e p s o f t h y r o i d hormone b i o s y n t h e s i s : o x i d a t i o n o f i o d i d e , i o d i n a t i o n o f t y r o s i n e , and i o d o t h y r o n i n e s y n t h e s i s from i o d o t y r o s i n e s . The most d i s t i n c t d i f f e r e n c e between e f f e c t s o f g o i t r i n and PTU i s t h a t the amount o f i o d o t h y r o n i n e s s y n t h e s i z e d i n the g l a n d i s markedly d e p r e s s e d -13- i n PTU-fed c h i c k s but i s i n c r e a s e d i n g o i t r i n - f e d c h i c k s w h i l e the plasma l e v e l o f t h y r o i d hormone i s d e p r e s s e d i n both groups ( A k i b a and Matsumoto, 1971). I t i s s u g g e s t e d t h a t t h e h y d r o l y s i s o f t h y r o g l o b u l i n and s e c r e t i o n o f t h y r o i d hormone from t h e g l a n d may be d e c r e a s e d o r a t l e a s t n o t enhanced by g o i t r i n t r e a t m e n t i n growing c h i c k s . M o r e o v e r , i n t h e i r s t u d y o f t h y r o i d f u n c t i o n o f c h i c k s a f t e r w i t h d r a w a l o f g o i t r i n from the d i e t f o l l o w i n g twenty-one days o f f e e d i n g , A k i b a and Matsumato (1973) r e p o r t e d a r a p i d r e s t o r a t i o n o f normal t h y r o i d m e t a b o l i s m and hormone b i o s y n t h e s i s , d e s p i t e h y p e r t r o p h y o f t h y r o i d g l a n d which p e r s i s t s f o r i r e l a t i v e l y l o n g e r t i m e . A f t e r w i t h d r a w a l o f g o i t r i n , plasma PBI-131 was r a d i c a l l y e l e v a t e d and a l i t t l e i n c r e a s e c o u l d be d e m o n s t r a t e d . T h i s s o - c a l l e d rebound phenomenon i s s i m i l a r t o g o i t r o g e n w i t h d r a w a l i n o t h e r l a b o r a t o r y a n i m a l s such as m i c e , r a t s and g u i n e a p i g s (D'Angelo e t a l _ , 1951, 1954; L i p n e r e t a l _ , 1959; S t u d e r and G r e e r , 1967; L a n g e r, 1968) and can be e x p l a i n e d by an o v e r s t i m u l a t i o n o f TSH on t h e u n b l o c k e d t h y r o i d g l a n d a f t e r r e l e a s e o f g o i t r o g e n i c e f f e c t s by g o i t r o g e n w i t h d r a w a l (D'Angelo, 1961, 1969). The g r a d u a l i n v o l u t i o n i n t h y r o i d w e i g h t a f t e r g o i t r i n w i t h d r a w a l i s i n agreement w i t h t h a t r e p o r t e d f o r PTU w i t h d r a w a l i n c h i c k s ( A k i b a e t al, 1971), r a t s (Shimoda, 1960) and g u i n e a p i g s (D'Angelo e t al, 1954). EFFECTS OF LONG-TERM ADMINISTRATION OF RAPESEED MEAL ON BIRDS AND PROGENY A l t h o u g h a c o n s i d e r a b l e amount o f work has been c a r r i e d o u t on the f e e d i n g o f r a p e s e e d meal t o young a n i m a l s , ' r e l a t i v e l y few e x p e r i m e n t s have been performed w h e r e i n the g o i t r o g e n s a r e f e d c h r o n i c a l l y ( P e l t o l a , -14- 1965; Summers e t a l _ , 1969; Vogt e t al_, 1969; C l a n d i n i n and Robblee, 1970; March e t a l _ , 1972). In an e x p e r i m e n t where r a p e s e e d meal was used as a p r o t e i n supplement i n the d i e t s o f l a y i n g hens f o r 252 days ( J a c k s o n , 1969), the h i s t o l o g i c a l p i c t u r e o f t h e t h y r o i d g l a n d s was n o t i d e n t i c a l w i t h t h a t o b t a i n e d i n growing c h i c k s ( C l a n d i n i n and B a y l y , 1960). No e v i d e n c e o f c e l l u l a r i n f i l t r a t i o n was o b t a i n e d , thus s u p p o r t i n g t h e view t h a t t h e i n i t i a l e f f e c t o f the r a p e s e e d g o i t r o g e n s a r e compensated f o r by t h y r o i d e n l a r g e m e n t , and the c h r o n i c a l l y - t r e a t e d b i r d s have normal p h y s i o l o g i c a l f u n c t i o n w i t h r e g a r d t o t h e e g g - l a y i n g p r o c e s s . Some i n v e s t i g a t o r s r e p o r t e d t h e p r e s e n c e o f s o - c a l l e d Psammoma b o d i e s i n t h e t h y r o i d s o f c h r o n i c a l l y s t i m u l a t e d a n i m a l s ( A x e l r a d and L e b l o n d , 1955; Wollman, 1961; Grimm e _ t a l _ , 1970), which a r e p r o b a b l y dense a g g r e g a t i o n s o f t h y r o p r o t e i n r e l a t e d t o t h e o b s e r v a t i o n o f r a d i o a c t i v i t y w i t h a v e r y l o n g b i o l o g i c a l h a l f - l i f e i n t h y r o i d s d e s c r i b e d by Van M i d d l e s - worth (1965). F o l l i s ( 1 9 6 5 ) , however, c o u l d n o t f i n d t h e s e Psammoma b o d i e s i n t h y r o i d s o f hamster, mice o r monkeys g i v e n g o i t r o g e n s f o r p r o l o n g e d p e r i o d s . I t a p p e a r s t h a t g o i t r i n i s n o t t r a n s f e r r e d f o r d e p o s i t i o n i n t h e egg ( R u t k o w s k i , 1971; March et^ a l _ , 1972). The t h y r o i d g l a n d s o f new l y - h a t c h e d progeny of.dams f e d rapes'eed m e a l , however, showed h y p e r t r o p h y , which was g r e a t e r when the d i e t c o n t a i n e d t h e h i g h e r l e v e l o f r a p e s e e d meal (March e t ^ a l . , 1972). A l s o t h e s e c h i c k s a t h a t c h i n g were s l i g h t l y s m a l l e r t h a n t h o s e from b i r d s f e d soybean meal c o n t r o l d i e t . T h i s i s i n agreement w i t h t h e a d v e r s e e f f e c t s o f r a p e s e e d meal i n progeny o f o t h e r s p e c i e s such as g i l t s ( B e l l and B e l z i l e , 1965). -15- E x p e r i m e n t 1: EFFECTS OF CHRONIC AND EARLY STIMULATION BY GOITROGENIC RSM ON WEIGHT AND HISTOLOGY OF THYROID GLAND I t has l o n g been known t h a t the f e e d i n g o f RSM t o the fowl c a u s e s t h y r o i d h y p e r t r o p h y and h y p e r p l a s i a . However, s i n c e most o f t h e h i s t o l o g i c a l d a t a a r e on growing c h i c k s , l i t t l e i s known about t h e a l t e r a t i o n o f c h r o n i c RSM s t i m u l a t i o n on t h y r o i d h i s t o l o g y . F u r t h e r m o r e , t h e e x t e n t t o which a d a p t a t i o n o f t h e t h y r o i d g l a n d i s p o s s i b l e v i a t h e mechanism o f compen- s a t i o n has not been e s t a b l i s h e d . When compensatory f u n c t i o n s o f the h y potha- l a m u s - p i t u i t a r y - t h y r o i d a x i s i n the fowl c e a s e and a t r o p h y o f the g l a n d b e g i n s have n o t been a c c u r a t e l y a s s e s s e d . I t i s n o t c l e a r i f i n v o l u t i o n does o c c u r i n t h e e a r l y - s t i m u l a t e d t h y r o d d g l a n d s when g o i t r o g e n i c s t i m u l a t i o n i s a l l e v i a t e d . Hence t h e f o l l o w i n g l o n g - t e r m e x p e r i m e n t was c o n d u c t e d t o e l u c i d a t e t h e s e e f f e c t s on t h y r o i d w e i g h t and h i s t o l o g y . M a t e r i a l s and Methods: In t h i s and a l l t h e f o l l o w i n g e x p e r i m e n t s ( w i t h t h e e x c e p t i o n s o f e x p e r i m e n t s 5 and 6B), the b i r d s used were on an e x p e r i m e n t d e s i g n e d t o compare p r o d u c t i o n and m o r t a l i t y o f b i r d s f e d soybean meal (SBM) and r a p e s e e d meal (RSM) as the s o u r c e o f s u p p l e m e n t a r y p r o t e i n i n g r owing and/or l a y i n g d i e t s (March e t a l , 1975). D a y - o l d White Leghorn c h i c k s o f two s t r a i n s ( Shaver c h i c k s and DeKalb c h i c k s ) were r e a r e d i n b a t t e r i e s and f e d s t a r t i n g and growing d i e t s c o n t a i n i n g e i t h e r SBM o r RSM as i n d i c a t e d i n T a b l e 1. The s t a r t i n g d i e t s were f e d from 0-8 weeks and t h e growing d i e t s from 8-23 weeks. The b i r d s were v a c c i n a t e d a g a i n s t Marek's d i s e a s e , N e w c a s t l e d i s e a s e and i n f e c t i o u s b r o n c h i t i s . Causes o f m o r t a l i t y were r e c o r d e d . -16- A t 23 weeks o f age t h e b i r d s were t r a n s f e r r e d t o community cages and f e d the l a y i n g d i e t s ( T a b l e 1) c o n t a i n i n g e i t h e r SBM o r RSM. The l a y i n g d i e t s were f e d a c c o r d i n g t o the f o l l o w i n g c r o s s - o v e r d e s i g n . One h a l f t h e b i r d s were c o n t i n u e d on t h e p r o t e i n supplement which t h e y had r e c e i v e d d u r i n g t h e growing p e r i o d and the o t h e r h a l f were t r a n s f e r r e d t o the a l t e r n a t e p r o t e i n supplement. The t r e a t m e n t s were d e s i g n a t e d as S-S, R-S, R-R and S-R, t h e f i r s t l e t t e r o f each p a i r i n d i c a t i n g soybean meal o r r a p e s e e d meal i n t h e growing d i e t and t h e s e c o n d l e t t e r i n d i c a t i n g soybean meal o r r a p e s e e d meal i n t h e l a y i n g d i e t . S i x hundred b i r d s were d i s t r i b u t e d i n t o 24 l o t s o f 25 b i r d s e a c h , i n community cages (2 s t r a i n s x 4 t r e a t m e n t s x 3 r e p l i c a t e s ) . A t 91 weeks, f o r t y b i r d s o f t h e Shaver s t r a i n (10 from each t r e a t m e n t ) were weighed and k i l l e d . The t h y r o i d g l a n d s were d i s s e c t e d f r e e from c o n n e c t i v e t i s s u e and f a t , weighed, and i m m e d i a t e l y f i x e d i n b u f f e r e d 10% f o r m a l i n f o r s u b s e q u e n t h i s t o l o g i c a l p r e p a r a t i o n ( H i s t o l o g y L a b o r a t o r y , Z o o l o g y Department, U.B.C.). The g l a n d s were embedded i n p a r a f f i n , s e c t i o n e d a t a t h i c k n e s s o f 5 j u , and e v e r y 20th s e c t i o n o f t h e l e f t g l a n d was s t a i n e d w i t h h a e m a t o x y l i n and e o s i n . The c e n t r a l s e c t i o n s o f each g l a n d were chosen f o r measurements o f e p i t h e l i a l h e i g h t and volume p r o p o r t i o n o f t h y r o i d a l t i s s u e s u s i n g a m i c r o m e t e r f i t t e d t o the l i g h t m i c r o s c o p e . A t 99 weeks o f age, f o r t y b i r d s o f t h e DeKalb s t r a i n were i n - d i v i d u a l l y weighed and k i l l e d , and t h e i r t h y r o i d g l a n d s were removed f o r measurement o f w e i g h t s o n l y . A l l e x p e r i m e n t a l d a t a o f t h i s and the f o l l o w i n g e x p e r i m e n t s were s u b j e c t e d t o a n a l y s i s o f v a r i a n c e and Duncan's m u l t i p l e range t e s t s ( S n e d e c o r and C o c h r a n , 1956). -17- G o i t r o g e n i c i t y o f the RSM f e d i n the e x p e r i m e n t a t the time t h e b i r d s were k i l l e d was d e t e r m i n e d by a s e p a r a t e e x p e r i m e n t , i n which d a y - o l d White Leghorn c o c k e r e l s were f e d e i t h e r a SBM- o r RSM-supplemented d i e t ( T a b l e 1) s i m i l a r i n c o m p o s i t i o n t o t h o s e f e d i n e x p e r i m e n t 1. S i x c h i c k s from each t r e a t m e n t a t 1 week and t w e l v e c h i c k s from each t r e a t m e n t a t 3 weeks were k i l l e d t o d e t e r m i n e t h e w e i g h t o f t h e t h y r o i d g l a n d s . R e s u l t s : G o i t r o g e n i c i t y o f t h e p r e s e n t l o t o f RSM (Span) was e s t a b l i s h e d by the h y p e r t r o p h y o f t h e t h y r o i d g l a n d s shown b y : t h e RSM-fed c h i c k s a t 1 week o r 3 weeks o f t r e a t m e n t ( T a b l e 2 ) . The a v e r a g e t h y r o i d w e i g h t s (mg/100 g body w e i g h t ) o f the S h a v e r and DeKalb b i r d s a t 91 and 99 weeks o f age a r e p r e s e n t e d i n T a b l e 3 S -The R-R b i r d s showed the most t h y r o i d a l e n l a r g e m e n t o v e r t h a t o f t h e c o n t r o l (10 t o 13 t i m e s i n c r e a s e ) , w h i l e t h e R-S b i r d s a l s o had d o u b l e t h e t h y r o i d s i z e o f t h e S-S b i r d s . H i s t o l o g i c a l d a t a o f t h e t h y r o i d g l a n d s o f t h e Shaver b i r d s a t 91 weeks a r e p r e s e n t e d i n T a b l e 4. T o t a l p e r c e n t a g e t h y r o i d e p i t h e l i u m i n the R-R b i r d s was l e s s than t h a t i n the c o n t r o l (15.1% vs 2 4 . 4 % ) , w h i l e t h e a v e r a g e e p i t h e l i a l h e i g h t o f the R-R t h y r o i d s (10.65 ja) was h i g h e r than t h a t (7.13>i) f o r t h e c o n t r o l . S i m i l a r l y , t h e R-S t h y r o i d s showed a d e c r e a s e i n p e r c e n t a g e e p i t h e l i u m (14.8%) but t h e a v e r a g e e p i t h e l i a l h e i g h t (5.61 ja) was a l s o l o wer than t h a t o f t h e c o n t r o l . -18- C o n s i s t e n t w i t h t he appearance o f e n l a r g e d g l a n d s , t he a v e r a g e f o l l i c l e d i a m e t e r s o f t h e RSM-treated t h y r o i d g l a n d s were a l s o i n c r e a s e d o v e r t h a t o f the c o n t r o l , t h e l a r g e s t f o l l i c l e s b e i n g i n t h e R-R t h y r o i d s . A g a i n , the a v e r a g e f o l l i c l e d i a m e t e r o f t h e R-S t h y r o i d s was a l s o g r e a t e r than t h a t o f t h e c o n t r o l (0.84 mm vs 0.54 mm). I t s h o u l d be noted t h a t , w i t h a l l t h e h i s t o l o g i c a l p a r a m e t e r s measured, t h e S-R t h y r o i d s were c o n s i s t e n t l y i n t e r m e d i a t e between t h e R-R and t h e c o n t r o l g l a n d s . Under l i g h t m i c r o s c o p y , t h e f o l l i c l e ilumerisof t h e t h y r o i d s i n the f o u r t r e a t m e n t s were f i l l e d w i t h s t a i n e d c o l l o i d . The R-R and S-R g l a n d s showed the appearance o f c o l l o i d g o i t e r . Desquamated c e l l s were i n f r e q u e n t but d i d o c c u r i n the t h y r o i d g l a n d s o f t h e c h r o n i c a l l y RSM-fed b i r d s . No e v i d e n c e o f a t r o p h y was seen i n t h e s e g l a n d s . O n l y i n one o r two o c c a s i o n s had t h y r o i d c y s t s been found i n the c h r o n i c a l l y R SM-stimulated b i r d s . -19- T a b l e 1: P e r c e n t a g e c o m p o s i t i o n o f d i e t s f e d i n e x p e r i m e n t 1 and RSM g o i t r o g e n i c i t y t e s t E x p e r i m e n t 1 RSM-Goitro- g e n i c i t y S t a r t e r - G r o w e r L a y e r T e s t Code L e t t e r S R S R S R I n g r e d i e n t Soybean meal 11.0 - 13.1 - 26.3 7.8 Rapeseed meal - 17.5 - 19.0 - 30.0 Ground wheat 66.5 60.0 60.6 53.5 53.6 42.6 Ground c o r n 15.0 15.0 6.0 6.0 10.0 10.0 Ground o a t s - - 10.0 10.0 - - Dehydrated c e r e a l g r a s s 2.0 2.0 2.0 2.0 2.0 2.0 Bone meal 2.0 2.0 2.5 2.5 2.0 2.0 Limestone 1.0 1.0 4.75 4.75 1.3 1.9 I o d i z e d s a l t 0.5 0.5 0.25 0.25 0.5 0.5 F e e d i n g t a l l o w 2.0 2.0 0.5 2.0 3.0 3.0 DL-Methionine - - 0.1 - 0.1 - L - L y s i n e - - - - - 0.1 T r i c a l c i u m phosphate - - - - 1.2 0.1 M i c r o n u t r i e n t s * * ** ** * * */kg: manganese s u l p h a t e 132 mg, c h o l i n e c h l o r i d e 1320 mg, n i a c i n 27 mg, r i b o f l a v i n 4.0 mg, c a l c i u m p a n t o t h e n a t e 9.2 mg, f o l a c i n 0.55 mg, p y r i d o x i n e HC1 2.9 mg, b i o t i n 0.09 mg, v i t a m i n B . 2 13.0 meg, v i t a m i n A 4400 I.U., v i t a m i n D 3 660 I.U., Zn b a c i t r a c i n 9.7 mg, o l e a n d o -mycin 11.0 mg. * * / k g : manganese s u l p h a t e 198 mg, r i b o f l a v i n 3.3 mg, v i t a m i n B , 2 3.3 meg, v i t a m i n A 6600 I . I L , v i t a m i n D 0 660 I.II. -20- T a b l e 2: Comparison o f a v e r a g e t h y r o i d w e i g h t s o f White Leghorn c o c k e r e l s f e d SBM- o r RSM-supplemented d i e t s a t 1 week o r 3 weeks o f t r e a t m e n t f r o m h a t c h i n g T r e a t m e n t s Average t h y r o i d w e i g h t s (mg/100 g.3tiipdy?wgtght) 1 week 3 weeks SBM - d i e t 5.8 ( 6 ) * 5.1 (12) RSM - d i e t 9.4 (6) 9.9 (12) * No. o f d e t e r m i n a t i o n s -21- T a b l e 3: Average w e i g h t s o f t h y r o i d g l a n d s i n b i r d s f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s o f time i n e x p e r i m e n t 1 Ave. t h y r o i d wt. D i e t a r y a t 58 weeks o f age* t r e a t . mg/100 g body wt. EXPERIMENT 1 S t r a i n A Ave. t h y r o i d wt. a t 91 weeks o f age mg/100 g body wt. S t r a i n B Ave. t h y r o i d wt. a t 99 weeks o f age mg/100 g body wt. S-S R-S R-R S-R 1 1 . 0 a A ( 1 2 ) * * 27.1° (12) 67.8° (12) 4 4 . 9 C (12) 1 0 . 7 a A ( 1 0 ) 2 3 . 5 a B ( 1 0 ) 112.5° (10) 84.9° (10) 1 1 . 2 a A ( 1 0 ) 2 8 . 4 a B ( 1 0 ) 149.4 C (10) 103.4° (10) T h i o u r a c i l i n s t a r t i n g d i e t u n t i l a K D / i o \ 4 wks o f a g e : 1 7 . 7 a D B U ^ L e v e l o f s i g n i f i c a n c e 5% 1% 1% * u n p u b l i s h e d d a t a from an e a r l i e r e x p e r i m e n t i n t h i s l a b o r a t o r y ** number o f d e t e r m i n a t i o n s p er t r e a t m e n t a D c 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 . AB S i g n i f i c a n t l y d i f f e r e n t a t t h e 1% l e v e l o f s i g n i f i c a n c e by s e p a r a t e c o m p a r i s o n . -22- T a b l e 4: H i s t o l o g i c a l measurements on t h y r o i d glands, from b i r d s ( S h a v e r s t r a i n ) i n e x p e r i m e n t 1 a t 91 weeks o f age. Ave.* Ave. Ave. % Wt. Ave. no. D i e t a r y t h y r o i d e p i t h e l i a l f o l l i c l e e p i t h e l i u m e p i t h e l i u m f o l l i c l e s t r e a t m e n t wt. g, h t . / i diam. mm mg a c r o s s gland S-S 0 . 1 9 a A 7.13 a 0.108 a 24.4 C 4 1 . l a 67 R-S 0 . 5 2 a B 5.72 a 0.167° 14.8 a 7 6 . 5 a 51 R-R 2.30 c 10.65° 0.227 c 1 5 . 1 a b 334.6° 58 S-R 1.39° 10.11° 0 . 1 9 6 ° C 2 0 . 1 a b c 277.4° 67 L e v e l o f s i g n i f i c a n c e 1% 1% 5% 5% 1% * f i v e g l a n d s examined per t r e a t m e n t a D c 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 . AB s i g n i f i c a n t l y d i f f e r e n t a t the 1% l e v e l o f s i g n i f i c a n c e by s e p a r a t e comparison, -23- E x p e r i m e n t 2: EFFECTS OF CHRONIC AND EARLY STIMULATION BY GOITROGENIC RSM ON TOTAL IODINE CONTENT OF THE THYROID GLAND S i n c e the h i s t o l o g i c a l p i c t u r e o f t h e R-R and S-R t h y r o i d s showed t h e h e a l t h y a p p e a r a n c e o f c o l l o i d g o i t e r i n s t e a d o f some degree o f a t r o p h y due t o c h r o n i c RSM s t i m u l a t i o n , t h e f o l l o w i n g e x p e r i m e n t was c o n d u c t e d t o d e t e r m i n e t h e t o t a l i o d i d e c o n t e n t o f t h e t h y r o i d t i s s u e as a f f e c t e d by the t r e a t m e n t s . M a t e r i a l s and Methods: F i v e Shaver b i r d s o f each o f the f o u r d i e t a r y t r e a t m e n t s (S-S, R-S, R-R and S-R as i n e x p e r i m e n t 1) were k i l l e d and t h e i r t h y r o i d g l a n d s removed and weighed. Each p a i r o f g l a n d s was homogenized a P o t t e r - E l v e h j e m g l a s s - h o m o g e n i z e r t o g i v e a 25 ml s u s p e n s i o n w i t h d e i o n i z e d w a t e r . The a n a l y t i c a l method f o r t h e measurement o f t o t a l i o d i n e c o n t e n t was an a d a p t a t i o n o f the B a r k e r p r o c e d u r e f o r serum ( B a r k e r , 1948). The c o n c e n t r a t i o n o f i o d i n e was d e t e r m i n e d by m e a s u r i n g i t s c a t a l y t i c e f f e c t on t h e r a t e o f r e a c t i o n between e e r i e i o n and a r s e n i o u s a c i d , which i n v o l v e d t h e c o n v e r s i o n o f t h e y e l l o w e e r i e i o n t o the c o l o r l e s s c e r o u s i o n . The c o l o r o f t h e e e r i e i o n was d e t e r m i n e d p h o t o m e t r i c a l l y twenty m i n u t e s a f t e r t h e r e a c t i o n s t a r t e d , and t h e i o d i n e c o n e n t r a t i o n was r e l a t e d i n v e r s e l y t o the l o g a r i t h m o f t h e o p t i c a l d e n s i t y . 1 ml o f t h e t h y r o i d t i s s u e homogenate was weighed i n t o Kimax 16 x 125 mm c u l t u r e t u b e s . 1 ml o f 4N sodium c a r b o n a t e was added t o each tube and t h e c o n t e n t was mixed t h o r o u g h l y by a s h a k e r . The t u b e s were d r i e d i n n -24- an oven a t 100°C t o complete d r y n e s s , a f t e r which t h e y were ashed i n a m u f f l e f u r n a c e a t 625 +_ 25°C f o r 4 h o u r s . The tubes were then removed and a l l o w e d t o c o o l . Four ml o f mixed a c i d s o l u t i o n (7N h^SC^ + 2N HC1; 1:1) were added t o each t u b e . The c o n t e n t s were mixed and a l l o w e d t o s t a n d f o r a p p r o - x i m a t e l y 10 m i n u t e s t o d i s s o l v e t h e a s h . T h r e e ml o f d e i o n i z e d water were added, t h e c o n t e n t s o f the tube a g a i n mixed. 10>ul o f t h i s m i x t u r e was added t o 8 ml o f d e i o n i z e d water i n a n o t h e r s e t o f 12 ml g l a s s - s t o p p e r e d t u b e s . A b l a n d and t h r e e i o d i n e s t a n d a r d s (.01, .05 and .10 jug) were a n a l y z e d i n d u p l i c a t e w i t h each g r o u p o f samples. A l l t h e t u b e s ( s t a n d a r d , b l a n k and sample) c o n t a i n e d 8 ml o f s o l u t i o n . To each g l a s s - s t o p p e r e d t u b e ( b l a n k s , s t a n d a r d s and samples) was added 0.5 ml o f 0.1N sodium a r s e n i t e s o l u t i o n . The tubes were p l a c e d i n a s h a k i n g water b a t h a t 39 +_ 1°C f o r 10 m i n u t e s t o a l l o w f o r t h e r m a l e q u i l i b r i u m . C e r i c ammonium s u l f a t e s o l u t i o n (.02N) was a l s o warmed i n the same water b a t h . U s i n g a C e n t a u r m i c r o l i t e r p i p e t t e , 1 ml o f c e r i c ammonium s u l f a t e s o l u t i o n was added t o each g l a s s - s t o p p e r e d tube a t p r e c i s e l y 1 minute i n t e r v a l s . A s t o p watch was used t o e n s u r e a c c u r a t e t i m i n g . The c o n t e n t o f t h e tube was i m m e d i a t e l y shaken t o e n s u r e t h o r o u g h m i x i n g . Twenty mi n u t e s a f t e r t h e c e r i c ammonium s u l f a t e s o l u t i o n was added t o t h e f i r s t t u b e , measurements o f o p t i c a l d e n s i t y a t 420 nm were begun, u s i n g an Unicam SP1800 s p e c t r o p h o t o m e t e r . These r e a d i n g s were made a t i n t e r - v a l s o f 1 minute i n o r d e r t h a t the l a p s e d t i m e f o r a l l o f t h e samples were c o n s t a n t . Measurements o f the o p t i c a l d e n s i t y o f t h e b l a n k and t h e s t a n d a r d i o d i n e s o l u t i o n s were p l o t t e d on s e m i l o g paper. The c o n c e n t r a t i o n s o f i o d i n e i n t he unknown samples were r e a d from t h e graph and m u l t i p l i e d by t h e d i l u t i o n -25- f a c t o r o f 700 ( t o compensate f o r the 10 u l a l i q u o t t a ken from t h e 7 ml a c i d s o l u t i o n o f t h e ashed s a m p l e ) . T o t a l i o d i n e c o n t e n t i n the p a i r o f t h y r o i d g l a n d s = i o d i n e c o n c e n t r a t i o n i n t h e sample x 25. The i o d i n e c o n c e n t r a t i o n s i n the d i e t s were a l s o a n a l y z e d by t h e same p r o c e d u r e , as m o d i f i e d by Sunderman (1963). 0.1 t o 0.2 g o f t h e sample was weighed i n t o c u l t u r e t u b e s . A f t e r a d d i t i o n o f 1 ml o f 4N sodium c a r - bonate and subsequent d r y i n g a t 100 C, the samples were ashed a t 625 + 25 C f o r 12-16 h o u r s . Upon c o o l i n g and m i x i n g w i t h 4 ml o f mixed a c i d s o l u t i o n ,; and 3 ml o f d e i o n i z e d w a t e r , the c o n t e n t s o f the tubes were passed t h r o u g h a Pyrex h i g h form f r i t t e d g l a s s f i l t e r o f F ( f i n e ) p o r o s i t y t o remove any i n s o l u b l e m a t e r i a l . A 3 ml a l i q u o t o f the f i l t r a t e from each ashed sample was p i p e t t e d i n t o a n o t h e r s e t o f g l a s s - s t o p p e r e d tubes t o which 5 ml o f d e i o n i z e d were added t o make up a t o t a l o f 8 ml i n each t u b e . O p t i c a l den- s i t i e s o f t h e unknown samples were r e a d o f f from a s t a n d a r d graph and mul- t i p l i e d by a d i l u t i o n f a c t o r o f 7/3 t o compensate f o r t h e 3 ml a l i q u o t t a k e n from t h e 7 ml a c i d s o l u t i o n o f t h e ashed sample. R e s u l t s : The a v e r a g e a b s o l u t e g l a n d w e i g h t s , i o d i n e c o n c e n t r a t i o n and t o t a l i o d i n e c o n t e n t o f t h e t h y r o i d s o f t h e b i r d s a f t e r c h r o n i c d i e t a r y t r e a t m e n t s a r e g i v e n i n T a b l e 5. The a b s o l u t e t h y r o i d w e i g h t s were i n the same o r d e r as the r e l a t i v e w e i g h t s ; the l a r g e s t a g a i n b e i n g t h e R-R g l a n d s (2.08 g) which were 10 t i m e s the s i z e o f t h e c o n t r o l . -26- The i o d i n e c o n c e n t r a t i o n (mg p e r g t h y r o i d t i s s u e ) was l o w e s t i n R-R b i r d s (0.89) compared t o 2.65 mg/g o f t h e c o n t r o l . However, because o f t h e much e n l a r g e d s i z e , the R-R t h y r o i d s had an a v e r a g e t o t a l i o d i n e c o n t e n t o f 1.72 mg i n the p a i r o f g l a n d s , w h i l e t h a t i n t h e c o n t r o l was o n l y 0.55 mg. L i k e w i s e , t h e R-S t h y r o i d s had an a c c u m u l a t i o n o f 1.25 mg i o d i n e i n a p a i r o f g l a n d s , which was more than d o u b l e t h a t o f the c o n t r o l . A g a i n , t h e i o d i n e c o n c e n t r a t i o n o r t o t a l i o d i n e c o n t e n t o f t h e S-R t h y r o i d s were i n t e r m e d i a t e between t h o s e o f t h e R-R and S-S g l a n d s . The i o d i n e c o n c e n t r a t i o n s o f t h e i . r e s p e c t i v e / i d i e t s a r e shown i n T a b l e 6. Both SBM- and RSM-supplemented r a t i o n s had i o d i n e v a l u e s above t h a t o f 0.3 mg/kg, which i s t h e NRC s t a t e d r e q u i r e m e n t f o r i o d i n e by p o u l t r y . -27- T a b l e 5: I o d i n e c o n c e n t r a t i o n i n t h y r o i d g l a n d s o f b i r d s f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s o f timei; Ave. i o d i n e c o n c e n t r a t i o n D i e t a r y Ave. wt. o f i n g l a n d t r e a t m e n t t h y r o i d g l a n d s * g mg/g S-S 0.20 a 2.65 a 0.55 a R-S 0.47 a 2.73 a 1.25 b R-R 2.08 c 0.89 b 1.72 c S-R 1.51 b 1.08 b 1.65 c L e v e l o f s i g n i f i c a n c e 5% 1% 5% * 5 d e t e r m i n a t i o n s per t r e a t m e n t . abed v a l u e s w i t h the 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 . T o t a l i o d i n e i n g l a n d mg \ -28- T a b l e 6: A n a l y z e d i o d i n e c o n c e n t r a t i o n s i n SBM- and RSM-supplemented d i e t s f e d t o b i r d s i n e x p e r i m e n t s 1 and 6B. A n a l y z e d i o d i n e c o n c e n t r a t i o n s R a t i o n s mg/kg In e x p e r i m e n t s 1 and 6B: SBM-supplemented 0.85 RSM-supplemented 0.91 In e x p e r i m e n t 6B o n l y : SBM-supplemented + K I 0 3 1.10 RSB-supplemented + KIO., 1.35 -29- E x p e r i m e n t 3: EFFECTS ON UPTAKE OF A SINGLE DOSE OF 1-131 BY THE THYROID GLAND 6 HOURS AND 22 HOURS AFTER ADMINISTRATION OF THE RADIOIODINE R a d i o i d d i n e has l o n g been used i n a s s e s s i n g t h y r o i d i o d i n e me- t a b o l i s m ( C h a i k o f f and T a u r o g , 1948; T u r n e r e t a l _ , 1959; P i t t - R i v e r s and R a i l , 1961). S i n c e t h e g o i t r o u s t h y r o i d g l a n d s o f t h e c h r o n i c a l l y RSM- s t i m u l a t e d b i r d s a p p e a r e d t o be a c t i v e l y a c c u m u l a t i n g i o d i n e (which c o u l d be t h r e e t i m e s as much as i n the c o n t r o l g l a n d s ) , i t would be o f i n t e r e s t t o compare the p a t t e r n s o f t h y r o i d a l r a d i o i o d i n e u p t a k e among the b i r d s o f \ the d i f f e r e n t t r e a t m e n t s . The f o l l o w i n g e x p e r i m e n t s was c o n d u c t e d t o d e t e r m i n e the p e r c e n t a g e uptake o f a dose o f 1-131 by the t h y r o i d g l a n d 6 and 22 hours a f t e r a d m i n i s t r a t i o n . M a t e r i a l s and Methods: (A) 1-131 u p t a k e a t 6 hours a f t e r a d m i n i s t r a t i o n : F i v e b i r d s ( S h a v e r s t r a i n ) o f each o f t h e f o u r t r e a t m e n t s as i n e x p e r i m e n t 1 were moved t o i n d i v i d u a l cages i n a s e p a r a t e room r e s e r v e d f o r r a d i o i s o t o p e s t u d i e s . The b i r d s were g i v e n t h e i r r e s p e c t i v e d i e t s and w a t e r ad l i b i t u m . 1-131 was p u r c h a s e d from Atomic Energy o f Canada, Ottawa, 131 i n t h e f o r m o f Na I i n sodium s u l f a t e s o l u t i o n . 0 . 5 / i C i o f 1-131 i n 0.5 ml p h y s i o l o g i c a l s a l i n e was i n j e c t e d i n t o the l e f t b r a c h i a l v e i n o f each b i r d . The,time o f each i n j e c t i o n was n o t e d . A t e x a c t l y 6 hours a f t e r each 1-131 a d m i n i s t r a t i o n , t h e b i r d s were k i l l e d . The t h y r o i d g l a n d s were removed-; and weighed, and c o u n t e d f o r r a d i o a c t i v i t y i n a N u c l e a r - C h i c a g o 8166 w e l l - t y p e s c i n t i l l a t i o n c o u n t e r . The c o u n t r a t e s (cpm) were c o r r e c t e d f o r back- ground and c o i n c i d e n c e l o s s a c c o r d i n g t o t h e e q u a t i o n i n Wang and W i l l i s ( 1 9 6 5 ) : -30- 1 - mT where n i s the c o r r e c t e d cpm, m i s t h e o b s e r v e d cpm, and T i s t h e r e s o l v i n g t i m e o f t h e c o u n t i n g assembly. The c o r r e c t e d cpm was c a l c u l a t e d back t o i n j e c t i o n ( z e r o ) t i m e by c o r r e c t i o n w i t h t h e decay c o n s t a n t o f 1-131, and e x p r e s s e d as a p e r c e n t a g e o f t h e c o r r e c t e d c o u n t r a t e o f a s t a n d a r d o r i g i n a l dose ( 0 . 5 / i C i ) . (B) 1-131 uptake a t 22 hours a f t e r a d m i n i s t r a t i o n : T h i s p a r t o f t h e e x p e r i m e n t was c a r r i e d o u t i n c o n j u n c t i o n w i t h e x p e r i m e n t 4. A n o t h e r twenty White Leghorn l a y e r s ( S h a v e r ) , f i v e o f each t r e a t m e n t , from t h e same s t o c k were s e p a r a t e l y caged. 50 u C i o f 1-131 i n normal s a l i n e was i n j e c t e d i n t o t h e l e f t wing v e i n o f each b i r d and t h e ti m e o f i n j e c t i o n was r e c o r d e d . The b i r d s were g i v e n t h e i r r e s p e c t i v e d i e t s and water ad l i b i t u m . Twenty-two hours a f t e r 1-131 a d m i n i s t r a t i o n , t h e b i r d s were k i l l e d , and t h e i r t h y r o i d s were removed and homogenized i n a P o t t e r - Elvehjem. g j a s s - h o m o g e n i z e r i n T r i s - H C l b u f f e r t o g i v e a s o l u t i o n o f t i s s u e c o n c e n t r a t i o n 40 mg/ml. 100 u l o f t h i s s u s p e n s i o n was ta k e n f o r c o u n t i n g o f r a d i o a c t i v i t y i n a w e l l - t y p e s c i n t i l l a t i o n c o u n t e r . A g a i n , t h e c o u n t r a t e s were c o r r e c t e d f o r background, c o i n c i d e n c e l o s s and d e c a y , and f i n a l l y e x p r e s s e d as p e r c e n t a g e s o f t h e c o r r e c t e d cpm o f t h e o r i g i n a l dose ( 5 0 y u C i ) o f r a d i o i o d i n e . In a d d i t i o n t o t h e t h y r o i d g l a n d s , p o r t i o n s o f t h e l i v e r o f each b i r d s were e x c i s e d , weighed and c o u n t e d f o r r a d i o a c t i v i t y . -31- R e s u i t s : The a v e r a g e a b s o l u t e t h y r o i d w e i g h t s , t o t a l p e r c e n t a g e t h y r o i d u p t a k e , p e r c e n t a g e u p t a k e p e r 100 mg t h y r o i d t i s s u e , and e s t i m a t e d p e r c e n t a g e uptake p e r u n i t e p i t h e l i a l w e i g h t o f 1-131 f o r both 6 h and 22 h i n t e r v a l s a r e p r e s e n t e d i n T a b l e 7. The marked i n c r e a s e o f a b s o l u t e t h y r o i d 1-131 uptake by t h e c h r o n i c a l l y RSM-fed b i r d s were c o m p a t i b l e w i t h t h e i r enhanced a c c u m u l a t i o n o f t h y r o i d a l i o d i d e as d e t e r m i n e d by c h e m i c a l a n a l y s i s . When the' * I-131 u p t a k e / g t h y r o i d ' d a t a were c a l c u l a t e d w i t h t h e 'average p e r c e n t a g e e p i t h e l i u m ' o f t h e r e s p e c t i v e g l a n d s as d e t e r m i n e d i n e x p e r i m e n t 1, e s t i m a t e d v a l u e s o f '1-131 u p t a k e / w e i g h t e p i t h e l i u m ' were o b t a i n e d . F u r t h e r m o r e , i t can be n o t e d t h a t w h i l e t o t a l t h y r o i d 1-131 uptake had dropped from 6 hours t o 22 hours i n the S-S and R-S b i r d s , i t remained h i g h i n t h e R-R and S-R g r o u p s . T h e r e d i d n o t a p p e a r t o be any d i f f e r e n c e i n t h e l i v e r 1-131 c o n c e n t r a t i o n 22 hours a f t e r a d m i n i s t r a t i o n o f t h e r a d i o i o d i n e ( T a b l e 7) among the b i r d s from t h e d i f f e r e n t d i e t a r y t r e a t m e n t s . -32- T a b l e 7: C o n c e n t r a t i o n s o f r a d i o i o d i n e i n the t h y r o i d g l a n d and 1 l i v e r 6 and 22 hours a f t e r a d m i n i s t r a t i o n . T o t a l t h y r o i d T h y r o i d E s t i m a t e d t h y . r a d i o a c t i v i t y r a d i o a c t i v i t y r a d i o a c t i v i t y L i v e r D i e t a r y % i n j e c t e d dose % i n j e c t e d dose % i n j e c t e d dose r a d i o a c t i v i t y t r e a t m e n t /100 mg t h y r o i d /mg e p i t h e l i u m a t 22 h 6 h 22 h 6 h 22r.h 6 h 22 h cpm/g S-S 9.1 a 7.8 a 4.7 b 4.2 b c .194 .172 556 R-S 13.6 3 10.9 a 2.3 a 3.6 b c .156 .247 397 R-R 35.4 b 40.6 b 1.5 a 2.2 a .104 .143 694 S-R 30.1 b 42.4 b 2.7 a 3.0 a b .134 .147 382 L e v e l o f s i g n i f i c a n c e 1% 1% 1% 5% v a l u e s w i t h t he 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 . -33- Experiment 4: THE DISTRIBUTION OF A SINGLE DOSE OF 1-131 IN IODINATED SUBSTANCES IN THYROID GLANDS OF SBM- OR RSM-FED BIRDS S i n c e t h e c h r o n i c a l l y R SM-stimulated t h y r o i d g l a n d s showed such marked enhancement o f 1-131 uptake when compared t o t h e c o n t r o l , i t might be p e r t i n e n t t o know how t h i s a c c u m u l a t e d r a d i o i o d i n e i s d i s t r i b u t e d among t h e v a r i o u s iodo-compounds i n the t h y r o i d t i s s u e . The d e t e r m i n a t i o n o f l a b e l e d iodoamino a c i d d i s t r i b u t i o n i n t h y r o g l o b u l i n s i s c u r r e n t l y a common measure o f t h y r o i d hormone b i o s y n t h e s i s under p h y s i o l o g i c a l o r p a t h o l o g i c a l c o n d i t i o n s ( M e l l e n and Wentworth, 1959; G a t t e r e a u e t a l _ , 1971; Kobayashi and G r e e r , 1971, 1973; B u r g i e_t al_, 1974; A k i b a and Matsumoto, 1973; S o r i m a c h i and U i , 1974). The p r e s e n t e x p e r i m e n t was c a r r i e d o u t t o d e t e r m i n e t h e e f f e c t s o f c h r o n i c and e a r l y g o i t r o g e n i c s t i m u l a t i o n by RSM on t h e d i s t r i b u t i o n o f 1-131 r a d i o a c t i v i t y i n t h y r o i d h y d r o l y s a t e s a f t e r p ronase d i g e s t i o n . M a t e r i a l s and Methods: F i v e b i r d s (same as i n e x p e r i m e n t 3B) o f each o f f o u r t r e a t m e n t s (S-S, R-S, R-R and S-R) f e d e x p e r i m e n t a l d i e t s as i n e x p e r i m e n t 1 were s e - p a r a t e d from t h e r e s t o f the p o p u l a t i o n . 50,uCi o f 1-131 (Atomic Energy, Ottawa) i n 0.5 ml normal s a l i n e was i n j e c t e d i n t r a v e n o u s l y t o each b i r d s . Twenty-two hours a f t e r i n j e c t i o n , the t h y r o i d g l a n d s were removed. The g l a n d s were weighed and homogenized i n a P o t t e r - E l v e h j e m . g l a s s - h o m o g e n i z e r i n i c e - c o l d T r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e - H C l b u f f e r , pH 8.6 (Lamas and DeEscobar, 1972), c o n t a i n i n g 10 M t h i o u r a c i l ( S t u d e r and G r e e r , 1968), t o g i v e a s o l u t i o n o f t i s s u e c o n c e n t r a t i o n 40 mg/ml. -34- The method f o r t h e measurement o f I - 1 3 1 - l a b e l e d m e t a b o l i t e s i n t h y r o i d t i s s u e f o l l o w e d was a m o d i f i c a t i o n o f t h a t o f Agerbaek (1972). Two ml o f w e l l - m i x e d homogenate, t i s s u e c o n c e n t r a t i o n 40 mg/ml, was p l a c e d i n a Thunberg tube and 4 mg Pronase ( s u b t i l o p e p t i d a s e - A , K and K F i n e C h e m i c a l s ) p l u s 2 drops o f t o l u e n e as b a c t e r i o s t a t i c agent were added. The tubes were e v a c u a t e d and f l u s h e d w i t h p u r i f i e d N 2 s e v e r a l t i m e s , n i t r o g e n f i n a l l y b e i n g a l l o w e d t o remain i n t h e tube a t a t m o s p h e r i c p r e s s u r e . D u r i n g t h e e v a c u a t i o n p r o c e d u r e i t was n e c e s s a r y t o t a p t h e t u b e b r i s k l y t o c o n t r o l f r o t h i n g . The d i g e s t i o n t o o k p l a c e i n t h e s t o p p e r e d tube under n i t r o g e n p l a c e d i n a s h a k i n g water bath a t 37 t fC f o r 21-24 h o u r s . F o r t y / J 1 o f d i g e s t s u s p e n s i o n was removed i m m e d i a t e l y a f t e r v i g o r o u s s h a k i n g o f the d i g e s t i o n tube and was a p p l i e d on Whatmann 3 MM chromatography paper s t r i p s (4.5;.em w i d e ) . A p p l i c a t i o n t o o k p l a c e i n a s t r e a m o f c o l d , a t m o s p h e r i c a i r a f t e r which the chromatography paper was suspended i n an a i r p r o o f and t e m p e r a t u r e i s o l a t e d j a r where i t was e q u i l i b r a t e d w i t h s o l v e n t f o r about 12 hours b e f o r e chromatography was s t a r t e d . F r e s h l y p r e p a r e d chromatography s o l v e n t n - b u t a n o l ( r e d i s t i l l e d ) - e t h a n o l - a m m o n i a IN (^5:1:2) was used. The a s c e n d i n g chromatogram was d e v e l o p e d f o r about 16 hours a t room t e m p e r a t u r e i n the d a r k (Lamas and D e E s c a r b a r , 1972). The chromatogram was then s p r a y e d l i g h t l y w i t h f e r r i c h l o r i d e - p o t a s s i u m f e r r i c y a n i d e - a r s e n i c a c i d (Gmelin and V i r t a n e n , 1959) so t h a t t h e c h r o m a t o g r a p h i c p r o c e s s c o u l d be v i s u a l l y m o n i t o r e d . The s e c t i o n s o f t h e chromatograms c o r r e s p o n d i n g t o MIT, DIT, T^ and T 3 > t o g e t h e r w i t h the o r i g i n and i n t e r m e d i a t e a r e a s , were e x c i s e d and c o u n t e d i n a w e l l - t y p e s c i n t i l l a t i o n c o u n t e r . O r i g i n 1-131, as used i n t h i s s t u d y , r e f e r r e d not o n l y t o r a d i o a c t i v i t y r e m a i n i n g a t the s i t e : , -35- o f a p p l i c a t i o n , but t o a l l 1-131 r a d i o a c t i v i t y up t o t h e DIT band. The 1-131 c o r r e s p o n d i n g t o each i n d i v i d u a l iodoamino a c i d was e x p r e s s e d as a p e r c e n t a g e o f t h e t o t a l 1-131 r a d i o a c t i v i t y on t h e chromatogram. A r e f e r e n c e chromatogram was a l s o o b t a i n e d by s p o t t i n g 1 u l o f a s t a n d a r d s o l u t i o n ( a p p r o x i m a t e l y 0.6%) o f s t a b l e K I , MIT, DIT, T^ and T^ (Sigma C h e m i c a l s ) t o g e t h e r w i t h a s m a l l amount o f d i g e s t . The p a t t e r n o f e l u t i o n o f t h e s e compounds i n the chromatogram was i d e n t i c a l t o t h a t g i v e n by Agerbaek (1972). S e v e r a l d i f f e r e n t s o l v e n t systems were t e s t e d such as n - b u t a n o l - a c e t i c a c i d - w a t e r (4:1:5) used by Kobayashi and G r e e r ( 1 9 7 1 ) , c o l l i d i n e - 3 N ammonium h y d r o x i d e (3;1) used by Inoue and T a u r o g ( 1 9 6 7 ) , n - b u t a n o l - a b s o l u t e e t h a n o l - 0 . 2 5 N ammonium h y d r o x i d e (5:1:2) used by Kobayashi and G r e e r ( 1 9 7 1 ) , and n - b u t a n o l - e t h a n o l - e t h a n o l - a i n r n o n i a 0.4N (5:1:2) used by Agerbaek ( 1 9 7 2 ) , b e f o r e a f i n a l d e c i s i o n was made on n - b u t a n o l - e t h a n - 1 N ammonia (5:1:2) which had been used by M o u r i z e t al_ (1966) and Lamas and DeEscobar (1972). Moreover, i t was found n e c e s s a r y t o r e d i s t i l l n - b u t a n o l and t o use a f r e s h l y - p r e p a r e d s o l v e n t system f o r each chromatography r u n . R e s u l t s : E f f e c t s o f t h e d i f f e r e n t d i e t a r y t r e a t m e n t s upon t h e d i s t r i b u t i o n o f r a d i o a c t i v e i o d i n a t e d s u b s t a n c e s found i n t h y r o i d g l a n d s 22 hours a f t e r i n j e c t i o n o f 1-131 a r e p r e s e n t e d i n T a b l e 8. A q u a n t i t a t i v e l y r e v e r s e d r e l a t i o n s h i p between t h e p e r c e n t a g e s o f r a d i o a c t i v i t y i n the i o d o t y r o s i n e s o f t h e c h r o n i c a l l y R S M - s timulated t h y r o i d t i s s u e s was e v i d e n t : In the S-S and R-S t h y r o i d s , t h e m a j o r i t y o f t h e 1-131 was f o u n d i n the DIT band, whereas i n t he R-R and S-R chromatograms, most o f t h e r a d i o a c t i v i t y was l o c a t e d a t t h e -36- MIT band. T h e r e was a c c o r d i n g l y an i n c r e a s e i n t h e MIT/DIT r a t i o s o f the RSM- s t i m u l a t e d t h y r o i d s . F u r t h e r m o r e , i t appeared t h a t RSM-stimulated b i r d s had more r a d i o a c t i v i t y r e s i d i n g i n t h e i o d i d e band and a t t h e o r i g i n , which p r o b a b l y r e p r e s e n t e d d e g r a d a t i v e l o s s o r h e t e r o g e n e i t y o f l a b e l e d i o d o p r o t e i n w i t h r e g a r d t o h y d r o l y s i s (Inoue and T a u r o g , 1967; Kobayashi and G r e e r , 1975). Upon s p r a y i n g w i t h f e r r i c h l o r i d e - p o t a s s i u m f e r r i c y a n i d e - a r s e n i c a c i d , t h e bands i n t h e chromatograms a p p e a r e d c l e a r l y v i s i b l e e x c e p t f o r Tg. D e s p i t e q u e s t i o n s as t o t h e p r e s e n c e o f t r i i o d o t h y r o n i n e i n t h y r o g l o b u l i n o f th e aves (Vlig'm, 1958; S o r i m a c h i and U i , 1974), 1-131 r a d i o a c t i v i t y was d e f i n i t e l y l o c a t e d i n the r e g i o n c o r r e s p o n d i n g t o t h e Tg band o f t h e r e f e r e n c e chromatogram. The amounts o f Tg and T^ p r e s e n t were, however, r e l a t i v e l y s m a l l and have t h e r e f o r e been combined i n t h e t a b l e ( T a b l e 8 ) . Th e r e appeared t o be an a d d i t i o n a l band i n a l l t h e chromatograms o f t h e S-S and R-S g r o u p s , which was a b s e n t from t h e R-R and S-R p a t t e r n s ^ ( F i g u r e 1 ) . T h i s band was c l o s e b u t d i s t i n c t l y s e p a r a t e d from t h e I " band, between t he I " and t h e DIT l e v e l s . In t h e absence o f i n d e n t i f i c a t i o n , i t was d e s i g n a t e d by (?) i n T a b l e 8. I t mi g h t be a h i t h e r t o u n r e c o g n i z e d iodoamino a c i d ( d i i o d o t h y r o n i n e , T2?) i n the chromatogram o r a more e x p e r i m e n t a l a r t e - f a c t o f t h e chromatography system. C o u p l i n g e f f i c i e n c y , as c a l c u l a t e d by Tg + T^/ ( M I T / D I T ) , was lower f o r t h e RSM-stimulated b i r d s . Once a g a i n , as c o n s i s t e n t w i t h t h e o t h e r p a r a m e t e r s o f t h y r o i d m e t a b o l i s m measured, t h e o r d e r o f i n h i b i t i o n i n t h y r o i d hormone b i o s y n t h e s i s , as r e p r e s e n t e d by t h e p a t t e i t i o f d i s t r i b u t i o n o f l a b e l e d m e t a b o l i t e s i n t h e t h y r o i d t i s s u e s , appeared i n t h e f o l l o w i n g o r d e r : R-R>S-R>R-S>S-S. -37- T a b l e 8: D i s t r i b u t i o n , 22 hours a f t e r a d m i n i s t r a t i o n , o f a s i n g l e dose o f r a d i o i o d i n e . i n t o t h y r o i d a l compounds. D i e t a r y % o f t o t a l t h y r o i d a l a c t i v i t y T3+T4 t r e a t m e n t O r i g i n * I " MIT DIT ? T 3 & T 4 MIT/DIT MIT/DIT S-S 5.0 1.2 40.7 47.9 1.9 3.3 0.85 3.89 R-S 5.3 1.4 42.7 45.3 2.0 3.3 0.95 3.43 R-R 8.3 2.8 49.1 36.8 - 3.0 1.34 2.28 S-R 6.4 2.1 49.0 39.5 - 2.9 1.27 2.39 R e p r e s e n t s t o t a l r a d i o a c t i v i t y from l i n e o f a p p l i c a t i o n t o band o f DIT. -38- F i g u r e 1: Paper chromatograms o f t h y r o i d a l m e t a b o l i t e s o f b i r d s on d i f f e r e n t d i e t a r y t r e a t m e n t s . C h r o m a t o g r a p h i c s o l v e n t : n-butanol-ethanol-ammonia IN (5:1:2). A b b r e v i a t i o n s used: 0 ( o r i g i n ) , DIT ( 3 , 5 - d i i o d o t y r o s i n e ) , MIT ( 3 - m o n o i o d o t y r o s i n e ) , S - S R - S R - R S - R -39- E x p e r i m e n t 5: EFFECTS OF GOITROGENIC RSM ON SUSCEPTIBILITY TO HYDROLYSIS OF THYROGLOBULIN H e t e r o g e n e i t y o f t h e t h y r o i d f o l l i c l e s has o f t e n been d i s c u s s e d i n t h e l i t e r a t u r e w i t h r e g a r d t o t h e r a t e o f i o d i n e t u r n o v e r ( S n e i d e r , 1964), and h e t e r o g e n e i t y o f t h e i o d o p r o t e i n s i n the t h y r o i d g l a n d have l o n g been r e c o g n i z e d ( T a r u t a n i and U i , 1969a). More r e c e n t l y , i t has been s u g g e s t e d t h a t t h y r o g l o b u l i n s s t o r e d i n t h e c o l l o i d may perhaps a l s o be heterogenous w i t h r e g a r d t o the r e l e a s e o f t h y r o i d hormones (Simon e t a l _ , 1966; T a r u t a n i and U i , 1969b; Nunez e t al_, 1966; Koyayashi and G r e e r , 1971; K o h l e r e t a l _ , 1971; Rosenberg et_ a l _ , 1964). S i n c e o u r f i n d i n g ( e x p e r i m e n t 4) o f a l a r g e r p e r c e n t a g e o f 1-131 r a d i o a c t i v i t y r e s i d i n g i n the I " and o r i g i n r e g i o n s o f t h e chromato- grams o f t h e R S M - s t i m u l a t e d t h y r o i d t i s s u e s , i t was c o n s i d e r e d r e l e v e n t t o t e s t whether t h e r e was i n d e e d a d i f f e r e n c e i n the s u s c e p t i b i l i t y t o p r o t e o l y s i s among t h e i o d o p r o t e i n s o f t h e v a r i o u s t r e a t m e n t g r o u p s . The f o l l o w i n g e x p e r i - ment was d e s i g n e d t o d e m o n s t r a t e any h e t e r o g e n e i t y i n the t h y r o i d t i s s u e s o f RSM- and SBM-fed b i r d s w i t h r e f e r e n c e t o the r a t e o f h y d r o l y s i s by p r o n a s e . M a t e r i a l s and Methods: White Leghorn c o c k e r e l s , 8 weeks o f age, f e d t h e SBM- o r RSM- s upplemented d i e t s same as t h o s e f o r t h e RSM g o i t r o g e n i c i t y t e s t i n e x p e r i m e n t 1 were used. S i x b i r d s each o f t h e two t r e a t m e n t s (SBM and RSM) were put i n s e p a r a t e cages and g i v e n w a t e r and t h e i r r e s p e c t i v e d i e t s ad 1 i b i t u m . 5 0 / j C i o f 1-131 i n 0.5 ml normal s a l i n e was i n j e c t e d i n t o t h e l e f t b r a c h i a l v e i n o f e a c h . Twenty-two 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 , t h r e e b i r d s -40- from each t r e a t m e n t were k i l l e d and t h e i r t h y r o i d s removed. Each p a i r o f g l a n d s was homogenized i n t h e same manner as i n e x p e r i m e n t 4, and s u b j e c t e d t o pronase d i g e s t i o n i n a water bath a t 37°C. Four hours a f t e r i t s commence- ment h y d r o l y s i s o f s i x samples was s t o p p e d ( t h r e e from each t r e a t m e n t ) and 4 0 / j l o f the d i g e s t s u s p e n s i o n was t r a n s f e r r e d f o r chromatography as d e s c r i b e d i n e x p e r i m e n t 4. The o t h e r s i x samples were s i m i l a r l y t r e a t e d a f t e r 24 hours o f pronase d i g e s t i o n . R e s u l t s : P e r c e n t d i s t r i b u t i o n o f r a d i o a c t i v i t y i n t h y r o i d t i s s u e s o f SBM- o r RSM-treated c o c k e r e l s a f t e r f o u r o r t w e n t y - f o u r hours o f p r o n a s e d i g e s t i o n a r e g i v e n on T a b l e 9. T h a t d i g e s t i o n was n o t c o m p l e t e by 4 hours was e v i d e n t by t h e h i g h p e r c e n t a g e o f r a d i o a c t i v i t y r e m a i n i n g a t t h e o r i g i n o f the chromatograms. However, t h e r e was no d i f f e r e n c e i n the p e r c e n t a g e s o f r a d i o a c t i v i t y r e m a i n i n g a t t h e o r i g i n . A f t e r 24 hours o f h y d r o l y s i s , t h e MIT/DIT r a t i o o f t h e RSM-fed b i r d s was m a r k e d l y i n c r e a s e d o v e r t h a t o f t h e c o n t r o l . T h e r e a l s o d i d n o t appear t o be any d i f f e r e n c e i n t h e p e r c e n t a g e s o f 1-131 r a d i o a c t i v i t y c o r r e s p o n d i n g t o t h e i o d o t h y r o n i n e s o r the ' o r i g i n ' bands i n the chromatograms o f t h e t h y r o i d t i s s u e o f t h e RSM- o r SBM-fed b i r d s . The d i s t r i b u t i o n o f r a d i o a c t i v i t y i n i o d i n a t e d s u b s t a n c e s i n t h e t h y r o i d t i s s u e o f young c o c k e r e l s i n the p r e s e n t e x p e r i m e n t was comparable t o t h a t r e p o r t e d f o r growing c h i c k s by o t h e r i n v e s t i g a t o r s ( e . g . A k i b a and Matsumoto, 1973). -41- T a b l e 9: D i s t r i b u t i o n , 22 hours a f t e r a d m i n i s t r a t i o n , o f a s i n g l e dose o f r a d i o i o d i n e i n t o t h y r o i d a l compounds measured i n h y d r o l y z a t e s a f t e r d i f f e r e n t times o f p r o n a s e d i g e s t i o n . D i e t a r y % o f t o t a l t h y r o i d a l r a d i o a c t i v i t y t r e a t m e n t O r i g i n I " MIT DIT T 3 & T 4 MIT/DIT A f t e r 4 hours pronase h y d r o l y s i s : SBM 16.2 6.1 29.7 41.3 6.7 0.72 RSM 15.0 6.5 43.8 24.3 10.4 1.81 A f t e r 24 hours p r o n a s e h y d r o l y s i s : SBM 7.0 6.0 33.2 46.8 9.0 0.72 RSM 8.1 7.2 44.3 28.2 12.2 1.58 -42- E x p e r i m e n t 6A: EFFECTS OF GOITROGENIC RSM ON THE IODINE CONTENT OF EGG YOLK A d i r e c t r e l a t i o n s h i p between t h e i o d i n e c o n t e n t o f t h e dam's d i e t and t h e i o d i n e c o n c e n t r a t i o n i n t h e egg has l o n g been known (Hercus and R o b e r t s , 1927; Simpson and S t r a n d , 1930; W i l d e r et_ a l _ , 1933; Asmundson ejt a l _ , 1936) and the e f f e c t s o f i o d i n e d e f i c i e n c y on embryo development and h a t c h a b i l i t y have been c l e a r l y e s t a b l i s h e d ( H o l l a n d e r and R i d d l e , 1943, 1946; Wil g u s e t a l _ , 1953; B e n n e t t and Adams, 1952; V i d a l , 1952; Adams and Buss, 1952; K i n g s b u r y e t a l _ , 1955; Romanoff and L a u f e r , 1956; Perdomo et_ al _ , 1966). T h y r o i d h i s t o l o g y o f t h e n e w l y - h a t c h e d progeny showed h y p e r t r o p h y o f t h e e p i - t h e l i u m and a l a c k o f c o l l o i d , which c o u l d be r e v e r s e d by a d d i t i o n o f p o t a s s i u m i o d i d e t o t h e dam's d i e t . T h y r o i d a l h y p e r t r o p h y was r e p o r t e d from t h i s l a b o r a t o r y i n t h e progeny from dams c h r o n i c a l l y f e d a RSM-supplemented d i e t (March et_ a l _ , 1972). The g o i t r o u s c o n d i t i o n was a l l e v i a t e d when the young c h i c k s were f e d a d i e t n o r m a l l y supplemented w i t h i o d i n e . The resemblance between t he progeny from the c h r o n i c a l l y RSM-treated b i r d s t o t h o s e from dams f e d an i o d i n e - d e f i c i e n t d i e t l e d t o the assumption o f an i o d i n e d e f i c i e n c y i n the eggs o f the f o r m e r group. T h i s was f u r t h e r s u p p o r t e d by t h e f a i l u r e t o d e m o n s t r a t e any d e p o s i t i o n o f g o i t r i n i n the eggs o f the RSM-fed b i r d s o r any d i f f e r e n c e i n i s o t h i o - c y a n a t e c o n t e n t o f t h e eggs o f t h e s e b i r d s when compared t o c o n t r o l (March e t a l , 1972). A n a l y s i s o f i o d i n e c o n t e n t o f t h e d i e t s , however, d i d not r e v e a l any d e f i c i e n c y o f t h i s e l e m e n t i n t h e RSM-supplemented r a t i o n ( e x p e r i m e n t 1 ) . S i n c e the egg p r o d u c t i o n and h a t c h a b i l i t y o f t h e s e b i r d s f e d c h r o n i c a l l y w i t h -43- the RSM-supplemented d i e t were a f f e c t e d (March et_ al_, 1975) and t h e progeny from them c o n s i s t e n t l y showed t h y r o i d h y p e r t r o p h y a t h a t c h i n g ( e x p e r i m e n t 8 ) , t h e f o l l o w i n g e x p e r i m e n t was c o n d u c t e d t o d e t e r m i n e i o d i n e c o n c e n t r a t i o n i n the eggs from t h e d i f f e r e n t t r e a t m e n t s and h o p e f u l l y t o e l u c i d a t e t h e materno-'emb.r'yonic-rela.tionsh.ip. :i<n the" iodi.ne/me'tabolisnvof t h e c h r o n i c a l l y s t i m u l a t e d b i r d s . M a t e r i a l s and Methods: F i v e eggs from each o f two r e p l i c a t e pens o f t h e b i r d s f e d t h e d i e t s o f e x p e r i m e n t 1 were c o l l e c t e d and t h e i r y o l k s were p o o l e d . Q u a d r i - p l i c a t e samples (0.1 - 0.2 g) o f t h e y o l k p o o l s were ashed w i t h 1 ml 4N sodium c a r b o n a t e and a s s a y e d f o r i o d i n e a c c o r d i n g t o the m o d i f i e d B a r k e r p r o c e d u r e as d e s c r i b e d i n e x p e r i m e n t 2 f o r t h e a s s a y o f i o d i n e i n t h e d i e t s . d D e s p i t e l o n g hours o f a s h i n g a t 625 + 25 C, t h e ash i n t h e tubes s t i l l a p p e a r e d g r a y i n s t e a d o f w h i t e . T h i s was e x p e c t e d ( p r o b a b l y due t o t h e h i g h l i p i d c o n t e n t o f t h e y o l k ) and t h e i n c i n e r a t i o n was deemed adequate t o put a l l o f t h e o r g a n i c i o d i n e i n t o e x t r a c t a b l e i n o r g a n i c form ( B a r k e r e t a l , 1951). I t was found n e c e s s a r y , however, t o f i l t e r t h e ash and a c i d m i x t u r e t o e l i m i n a t e f l u c t u a t i o n s i n t h e o p t i c a l d e n s i t y r e a d i n g s due t o u n d i s s o l v e d p a r t i c l e s . T h i s c o u l d be e f f i c i e n t l y c a r r i e d o u t i n a f r i t t e d - g l a s s f i l t e r c o n n e c t e d t o a w a t e r pump. I f t h i s was n o t a v a i l a b l e and f i l t e r paper was u s e d , i t would be n e c e s s a r y t o c o r r e c t f o r t h e a b s o r b a n c e due t o t h e f i l t r a t e . P r e c i s i o n o f the a s s a y system depended l a r g e l y on how t h i s problem wasoyercomed. The d e c i s i o n t o a n a l y z e o n l y t h e y o l k o f eggs f o r i o d i n e and n o t t h e albumin was based on p r e l i m i n a r y f i n d i n g s i n t h i s l a b o r a t o r y ( u n p u b l i s h e d d a t a ) t h a t i o d i n e was m o s t l y c o n c e n t r a t e d i n t h e y o l k ( A l s o : Romanoff and -44- Romanoff, 1949). T p t a l i o d i n e c o n t e n t was p r e f e r r e d t o p r o t e i n - b o u n d i o d i d e ( P B l ) measurement because t h e l a t t e r m i g h t n o t be an a p p r o p r i a t e p a rameter i n e s t i m a t i n g a v i a n i o d i n e m e t a b o l i s m . R e s u l t s : I o d i n e c o n c e n t r a t i o n s i n the y o l k s o f eggs from b i r d s on the d i f f e r e n t t r e a t m e n t s a r e shown i n T a b l e 10. The egg y o l k s from t h e c o n t r o l b i r d s c o n t a i n e d 0.27/jg/g o f i o d i n e . T h i s v a l u e i s i n agreement w i t h a v e r a g e v a l u e s g i v e n i n t h e l i t e r a t u r e (Romanoff and Romanoff, 1949). The d a t a c l e a r l y e s t a b l i s h e d t h a t t h e c h r o n i c a l l y RSM-treated b i r d s produce eggs w i t h a lower i o d i n e c o n c e n t r a t i o n . The y o l k s o f R-R eggs c o n t a i n e d l e s s than 0.09yug/g i o d i n e , a v a l u e which was e s t i m a t e d from t h e lower l i m i t o f t h e w o r k i n g range o f the a s s a y under t h e d e s c r i b e d e x p e r i m e n t a l c o n d i t i o n s . A l a r g e r y o l k sample from t h i s p ool would n o t be a p p r o p r i a t e because o f the d i f f i c u l t y t h a t m i g h t be e n c o u n t e r e d d u r i n g t h e i n c i n e r a t i o n p r o c e s s . A g a i n t h e i o d i n e c o n c e n t r a t i o n i n t h e S-R egg y o l k s was i n t e r - m e d i a t e between t h a t o f the R-R and S-S egg y o l k s . The c o n c e n t r a t i o n i n the R-S eggs was not s t a t i s t i c a l l y d i f f e r e n t from t h a t o f t h e c o n t r o l . -45- T a b l e 10: I o d i n e c o n c e n t r a t i o n s i n egg y o l k s from b i r d s on d i f f e r e n t d i e t a r y t r e a t m e n t s i n e x p e r i m e n t 6A. D i e t a r y I o d i n e c o n c e n t r a t i o n i n t r e a t m e n t egg y o l k , ppb S-S 273 a R-S 286 a R-R < 85 S-R ' 144 b 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 t a t i s t i c a l l y d i f f e r e n t , p<0. -46- E x p e r i m e n t 6B: EFFECTS OF INCREASED DIETARY IODINE INTAKE ON IODINE DEPOSITION IN EGG YOLKS OF SBM- AND RSM-FED BIRDS The f i n d i n g s t h a t the c h r o n i c a l l y RSM-stimulated b i r d s produced eggs w i t h a s i g n i f i c a n t l y l o w e r i o d i n e c o n t e n t ( e x p e r i m e n t 6A), d e s p i t e a p p a r e n t l y comparable d i e t a r y i o d i n e i n t a k e ( e x p e r i m e n t 1) compared t o the c o n t r o l , l e d t o t h e h y p o t h e s i s t h a t t h e p e r i p h e r a l b l o o d c i r c u l a t i o n o f t h e s e b i r d s were a l s o low i n i o d i n e c o n c e n t r a t i o n . S i n c e measurement o f serum PBI c o n t e n t would n o t be an a p p r o p r i a t e parameter i n t h e a v e s , i t was pos- t u l a t e d t h a t by i n c r e a s i n g t h e d i e t a r y i n t a k e o f i o d i n e , the RSM-stimulated b i r d s would show a c o r r e s p o n d i n g i n c r e a s e i n t h e d e p o s i t i o n o f t h i s element i n t h e i r eggs. The f o l l o w i n g e x p e r i m e n t was c o n d u c t e d t o e l u c i d a t e t h i s p o i n t . M a t e r i a l s and Methods: One hundred and n i n e t y - s i x Babcock l a y e r s , 44 weeks o f age, were d i v i d e d i n t o f o u r groups and i n d i v i d u a l l y caged. They were g i v e n one o f t h e f o l l o w i n g e x p e r i m e n t a l d i e t s : R - l : SBM-supplemented d i e t as i n e x p e r i m e n t 1; R-2: R - l p l u s p o t a s s i u m i o d a t e (0.3 mg i o d i n e / k g ) ; R-3: RSM-supplemented d i e t as i n e x p e r i m e n t 1; R-4: R-2 p l u s p o t a s s i u m i o d a t e (0.3 mg i o d i n e / k g ) . When t h e b i r d s had been on e x p e r i m e n t f o r 3 weeks, s i x eggs from each t r e a t m e n t were c o l l e c t e d from b i r d s o f comparable l a y i n g p e r f o r m a n c e . The y o l k s were weighed a n d ; i n d i v i d u a l l y a s s a y e d f o r i o d i n e by t h e B a r k e r -47- p r o c e d u r e as d e s c r i b e d i n e x p e r i m e n t 6A w i t h one j f i u p t h e r ? m 6 d T f i c a t i o n : s i x - m l a l i q u o t s f r o m t h e 7 ml mixed a c i d s o l u t i o n s c o n t a i n i n g t h e ashed samples were p i p e t t e d f o r t h e a s s a y i n s t e a d o f 3 ml a l i q u o t s , i n o r d e r t h a t t h e presumably low i o d i n e c o n c e n t r a t i o n o f t h e RSM-treated y o l k s c o u l d a l s o be measured. The i o d i n e c o n t e n t o f the d i e t s were a g a i n d e t e r m i n e d . Results:,: E f f e c t s o f a f u r t h e r a d d i t i o n o f 0.3 mg I/kg r a t i o n t o t h e SBM- and RSM-supplemented d i e t s a r e shown i n T a b l e 11. V a l u e s ^ f o r both i o d i n e c o n c e n t r a t i o n and t o t a l i o d i n e c o n t e n t i n t h e y o l k s a r e g i v e n . Even i n t h i s s h o r t - t e r m " f e e d i n g s t u d y , l e s s i o d i n e was d e p o s i t e d i n the eggs o f t h e RSM-fed b i r d s - t h a n i n t h o s e o f the SBM-fed b i r d s (41 ng/g vs 105 n g / g ) . When th e d i e t a r y i o d i n e l e v e l was locbeasledyby :?iarjdadd4-t 1tohal 0.3 mg p e r kg r a t i o n , i n t h e f o r m o f p o t a s s i u m i o d a t e , t h e r e s p e c t i v e i o d i n e c o n c e n t r a t i o n o f the SBM- o r RSM-treated egg y o l k s were a l s o e l e v a t e d c o r r e s p o n d i n g l y . The d i f f e r e n c e between the t o t a l i o d i n e c o n t e n t o f t h e R-2 and R-4 eggs was h i g h l y s i g n i f i c a n t (p<0.01), i n d i c a t i n g t h a t even a t an e l e v a t e d d i e t a r y i o d i n e u p t a k e , w h i l e the SBM-fed b i r d s showed a d r a s t i c i n c r e a s e i n t h e i r egg i o d i n e a c c u m u l a t i o n , a s i g n i f i c a n t l y s m a l l amount o f t h i s e l e m e n t was d e p o s i t e d i n the eggs o f t h e RSM-stimulated b i r d s . The i o d i n e c o n c e n t r a t i o n s o f t h e d i f f e r e n t d i e t s a r e a l s o shown i n T a b l e 6. The a d d i t i o n o f 0.3 mg I/kg r a t i o n i n t h e f o r m o f p o t a s s i u m i o d a t e was r e c o v e r e d d u r i n g t h e i o d i n e a s s a y , t h u s v a l i d a t i n g the a n a l y t i c a l - 4 8 - p r o c e d u r e used i n t h i s and the p r e v i o u s e x p e r i m e n t s . I t s h o u l d be n o t e d t h a t s m a l l samples (0 .1 - 0 .2 g) o n l y were used f o r the a s s a y p r o c e d u r e , so t h a t f u r t h e r m o d i f i c a t i o n s o f t h e method ( s u c h as a d d i t i o n o f more t h a n 1 ml 4N sodium c a r b o n a t e ) would n o t be n e c e s s a r y ^ t h a t m i g h t ^ i n t e r f e r e w i t h /\ t - h e - p r e c i s i o n o f the, assay.^ -49- T a b l e 11: I o d i n e c o n c e n t r a t i o n s i n egg y o l k s from b i r d s on d i f f e r e n t d i e t a r y t r e a t m e n t s i n e x p e r i m e n t 6B. D i e t a r y I o d i n e c o n c e n t r a t i o n s i n t r e a t m e n t egg y o l k , ppb SBM - d i e t 105 a A SBM - d i e t p l u s K I 0 3 242 b RSM « d i e t 41 a B RSM - d i e t p l u s K I 0 3 73 a a b v a l u e s w i t h t he same s u p e r s c r i p t a r e n o t s t a t i s t i c a l l y d i f f e r e n t , p<0.01, AB s i g n i f i c a n t l y d i f f e r e n t by s e p a r a t e c o m p a r i s o n , p<0.01. -50- E x p eriment 7: OVARIAN UPTAKE OF A SINGLE DOSE OF 1-131 BY DEVELOPING FOLLICLES The growth o f t h e avdan o v a r i a n f o l l i c l e has been e x t e n s i v e l y s t u d i e s ( e . g . S t i e v e , 1918; Warren and Conrad, 1939; Romanoff, 1943). E x p e r i - ments w i t h i s o t o p i c t r a c e r s have shown t h a t the l e v e l s o f d i e t a r y i o d i n e c o u l d a f f e c t t h e development o f ova o f l a y i n g hens ( M a r c i l e s e ejt a j , 1968). S i n c e t h e t o t a l i o d i n e c o n t e n t was f o u n d t o be l o wer i n t h e RSM-fed b i r d s ( e x p e r i - m e n t s ^ and 6B), t h e f o l l o w i n g e x p e r i m e n t was c o n d u c t e d t o d e t e r m i n e the r e l a t i v e r a d i o i o d i n e uptake o f t h e s e b i r d s as compared t o t h e c o n t r o l . M a t e r i a l s and Methods: The same b i r d s o f e x p e r i m e n t 4 were employed. Twenty-two hours a f t e r the a d m i n i s t r a t i o n o f 5 0 / i C i o f 1-131, the b i r d s were k i l l e d and the f i v e l a r g e s t ova were removed. P r e l i m i n a r y t e s t s i n which the l a b e l e d ova were p l u n g e d i n t o b o i l i n g w a t e r , c u t up and c o u n t e d p i e c e by p i e c e r e v e a l e d t h a t most o f t h e r a d i o a c t i v i t y o f t h e n e w l y - d e p o s i t e d 1-131 was c o n c e n t r a t e d t o a r i n g towards the p e r i p h e r y o f t h e ovum. T h e r e f o r e i n the p r e s e n t ex- p e r i m e n t where a hundred (5 ova x 5 b i r d s x 4 t r e a t m e n t s ) o v a r i a n f o l l i c l e s were i n v o l v e d , each ovum was mixed t h o r o u g h l y by r e p e a t e d s t i r r i n g w i t h a g l a s s r o d , and 0.1 t o 0.5 g a l i q u o t s o f t h e mixed y o l k s were p i p e t t e d t o t u b e s f o r c o u n t i n g i n a w e l l - t y p e s c i n t i l l a t i o n c o u n t e r . I n d i v i d u a l w e i g h t s o f t h e ova were r e c o r d e d , and t o t a l 1-131 uptake was e x p r e s s e d as a p e r c e n t a g e o f t h e c o u n t r a t e o f the o r i g i n a l d o s e. Moreover, p e r m e a b i l i t y o f t h e ova t o 1-131 was a l s o c a l c u l a t e d , a c c o r d i n g t o t h e e q u a t i o n o f Smith (1959) which e x p r e s s e d t h e a c t i v i t y o f t h e y o l k r e l a t i v e t o t h e s u r f a c e a r e a o f t h e f o l l i c l e and t h e time s i n c e i n j e c t i o n , i . e . p e r m e a b i l i t y = njuCi/cm /h. -51- R e s u l t s : The v a l u e s f o r t o t a l p e r c e n t 1-131 uptake by t h e f i v e l a r g e s t ova i n an a v e r a g e o f 5 b i r d s : p er t r e a t m e n t a r e p r e s e n t e d i n T a b l e 12. S i n c e t h e n e t t r a n s f e r r a t e o f y o l k m a t e r i a l s i n t o t h e d e v e l o p i n g y o l k was n o t l i n e a r , t h e d a t a on the p e r m e a b i l i t y t o 1-131 and the s u r f a c e a r e a o f the o v a r i a n f o l l i c l e s were t r a n s f o r m e d t o t h e l o g a r i t h m i c f u n c t i o n and a n a l y z e d by m u l t i p l e r e g r e s s i o n t o g i v e t h e p l o t shown i n F i g u r e 2. The r e g r e s s i o n e q u a t i o n s and R v a l u e s were computed as f o l l o w s : T reatments R e g r e s s i o n E q u a t i o n s R_ S-S log g Y = -1.1958 + 2.7086 l o g X - 0.0983 X 0.78 R-S l o g Y = -0.9731 + 2.1800 l o g X - 0.0785 X 0.75 R-R l o g Y = -1.4432 + 2.7948 l o g X - 0.1093 X 0.83 S-R l o g Y = -1.2304 + 2.1641 l o g X - 0.0857 X 0.63 T a b l e 12 a l s o compares t h e d a t a o b t a i n e d when l a y i n g b i r d s were i n j e c t e d w i t h r a d i o i o d i n e and the r a d i o a c t i v i t y o f t h e t h y r o i d g l a n d s and the f i v e l a r g e s t ova i n t h e o v a r y measured a f t e r 22 h o u r s . R a d i o i o d i n e c o n c e n - t r a t i o n s i n t h e t h y r o i d g l a n d s o f t h e RSM-fed b i r d s were a p p r o x i m a t e l y f o u r t i m e s t h o s e i n t h e SBM-fed b i r d s . Average r a d i o i o d i n e c o n c e n t r a t i o n appeared h i g h e r i n the t h y r o i d s o f t h e SBM-fed b i r d s t h a t had been f e d RSM d u r i n g t h e growing p e r i o d than i n the b i r d s t h a t were f e d SBM t h r o u g h o u t l i f e . 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 lower i n t h e ova from t h e RSM-fed b i r d s than i n the SBM-fed b i r d s . The ova from SBM-fed b i r d s c o n t a i n e d l e s s r a d i o i o d i n e when th e b i r d s had been f e d RSM d u r i n g t h e growing p e r i o d and ova f r o m t h e RSM-fed b i r d s c o n t a i n e d l e s s r a d i o i o d i n e when t h e b i r d s had been f e d SBM d u r i n g t h e growing p e r i o d . The amounts o f r a d i o i o d i n e t r a n s p o r t e d i n t o t h e i n d i v i d u a l -52- ova r e l a t i v e t o the s u r f a c e a r e a o f t h e ova i n t h e b i r d s on the d i f f e r e n t t r e a t m e n t s a r e shown i n F i g u r e 2. The t r a n s f e r r a t e o f 1-131 per u n i t o f f o l l i c l e s u r f a c e a r e a was maximal i n the 10 t o 12 cm s t a g e and d e c r e a s e d m o r e - o r - l e s s l i n e a r l y u n t i l o v u l a t i o n . T h i s i s i n agreement w i t h changes i n the f u n c t i o n a l c a p a c i t y o f t h e f o l l i c u l a r w a l l , which i s n o t u n i f o r m but r e a c h e s an e a r l y maximum and then d e c l i n e s ( S m i t h , 1959). The r a t e o f uptake o f 1-131 by d e v e l o p i n g ova o f l a y i n g hens was r e c e n t l y d e t e r m i n e d by Pena e t a l _ ( 1 9 7 5 ) , which conformed w e l l w i t h o u r p r e s e n t f i n d i n g . ,53- T a b l e 12: R a d i o i o d i n e c o n c e n t r a t i o n s i n t h e t h y r o i d g l a n d s and d e v e l o p i n g o v a r i a n f o l l i c l e s 22 hours a f t e r a d m i n i s t r a t i o n o f a s i n g l e dose o f r a d i o i o d i n e t o b i r d s which had been f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s o f t i m e . D i e t a r y t r e a t m e n t Ave. r a d i o i o d i n e i n t h y r o i d g l a n d % o f i n j e c t e d dose Ave. t o t a l r a d i o - i o d i n e i n 5 l a r g e s t o v a r i a n f o l l i c l e s it % o f i n j e c t e d dose S-S 7.8 3 6.7 a R-S 10.9 a 5.8 a R-R 40.6 b . 2.8 b S-R 42.4 b 2.6 b * a v e r a g e o f d e t e r m i n a t i o n s on f i v e b i r d s o f each t r e a t m e n t . v a l u e s w i t h the 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<0.01. -54- 4 - O r - F i g u r e 2: R e g r e s s i o n o f 1-131 uptake on s u r f a c e a r e a o f o v a r i a n f o l l i c l e s o f b i r d s f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s 1 - 0 2 0 OVUM 5 U R F A C E AREA -55- E x p e r i m e n t 8: EFFECTS OF CHRONIC AND EARLY GOITROGENIC STIMULATION BY RSM IN THE DAM ON EMBRYONIC THYROID DEVELOPMENT S i n c e t h e i o d i n e c o n c e n t r a t i o n s i n eggs from RSM-stimulated b i r d s were f o u n d t o be lower than i n eggs from the c o n t r o l b i r d s ( e x p e r i m e n t 6A), and t h a t t h e r a t e s o f d e p o s i t i o n o f t h i s element i n t h e d e v e l o p i n g ova o f t h e same b i r d s were lower than o f t h o s e i n the S-S b i r d s as shown by the r a d i o i s o t o p e s t u d y ( e x p e r i m e n t 6B), i t would be r e a s o n a b l e t o p o s t u l a t e t h y r o i d h y p e r t r o p h y when t h e progeny from t h e s e b i r d s were h a t c h e d . The t h y r o i d g l a n d s o f n e w l y - h a t c h e d progeny from dams f e d g o i t r o g e n i c RSM have been p r e v i o u s l y r e p o r t e d t o be g o i t r o u s from t h i s l a b o r a t o r y (March e t a l , 1972). The p r e s e n t e x p e r i m e n t was performed t o c o n f i r m t h i s p r e v i o u s f i n d i n g and a l s o t o e l u c i d a t e any d i f f e r e n c e s i n t h e h i s t o l o g i c a l p i c t u r e o f the t h y r o i d g l a n d s o f t h e progeny from t h e c h r o n i c a l l y - o r e a r l y - R S M n S t i m u l a t e d dams. M a t e r i a l s and Methods: S e v e n t y - f i v e b i r d s each o f t h e f o u r t r e a t m e n t s , 91 weeks o f age, f e d t h e same d i e t s o f e x p e r i m e n t 1 were a r t i f i c a l l y i n s e m i n a t e d a n d , a f t e r 4 days,eggs were c o l l e c t e d and i n c u b a t e d . A t h a t c h i n g , 20 c h i c k s from each t r e a t m e n t were k i l l e d and t h e i r t h y r o i d g l a n d s removed and weighed. T h y r o i d g l a n d s from a n o t h e r 5 c h i c k s p e r t r e a t m e n t were f i x e d i n b u f f e r e d 10% f o r m a l i n f o r h i s t o l o g i c a l s t u d y as i n e x p e r i m e n t 1. -56- R e s u l t s : T h y r o i d w e i g h t s o f the progeny from each t r e a t m e n t a r e g i v e n i n T a b l e 13. A l t h o u g h n o t s t a t i s t i c a l l y s i g n i f i c a n t , t h e w e i g h t s were i n t h e same o r d e r o f magnitude as i n t h e p r e v i o u s t e s t c o n d u c t e d on progeny o f dams 77 weeks o f age. In t h i s f o r m e r t e s t , t h e t h y r o i d g l a n d s from t h e progeny o f the R-R and S-R groups were s i g n i f i c a n t l y l a r g e r than t h o s e o f th e c o n t r o l (p<0.01). The h i s t o l o g i c a l c h a r a c t e r i s t i c s o f t h e t h y r o i d g l a n d s o f newly- h a t c h e d progeny o f b i r d s f e d RSM- o r SBM-supplemented d i e t s f o r d i f f e r e n t p e r i o d s a r e g i v e n i n T a b l e 14. The p e r c e n t a g e s o f e p i t h e l i u m i n t h e t h y r o i d s o f progeny from t h e RSM-fed dams were g r e a t e r than from t h e progeny o f the SBM-fed dams. The v a l u e s f o r a b s o l u t e e p i t h e l i a l w e i g h t s were c a l c u l a t e d by m u l t i p l y i n g p e r c e n t a g e s e p i t h e l i u m w i t h t he ave r a g e a b s o l u t e t h y r o i d w e i g h t s o f the r e s p e c t i v e g r o u p s . The a v e r a g e a b s o l u t e w e i g h t o f t h e t h y r o i d a l e p i t h i a l t i s s u e was g r e a t e r i n the progeny o f t h e SBM-fed dams when t h e b i r d s had been f e d RSM d u r i n g t he growing p e r i o d . F u r t h e r m o r e , t he a v e r a g e a b s o l u t e w e i g h t o f t h e 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 was lower i n t h e progeny o f t h e RSM-fed dams when the b i r d s had been f e d SBM d u r i n g t h e growing p e r i o d . The i n c r e a s e d a v e r a g e f o l l i c u l a r d i a m e t e r w i t h l e s s e r number o f f o l l i c l e s a c r o s s t h e g l a n d i n the t h y r o i d s o f t h e progeny from t h e c h r o n i c a l l y R SM-stimulated dams i n d i c a t e d f o l l i c l e h y p e r t r o p h y i n t h e s e g l a n d s . The h i s t o l o g i c a l c h a r a c t e r i s t i c s i n the t h y r o i d g l a n d s o f newly- h a t c h e d progeny and t h e i r dams a r e compared i n T a b l e 15. -57- T a b l e 13: T h y r o i d w e i g h t s a t h a t c h i n g o f progeny b i r d s f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s i n e x p e r i m e n t 8. D i e t a r y t r e a t m e n t Ave. t h y r o i d wt. o f progeny when dams were 77 wks, o f age mg/100 body wt. Ave. t h y r o i d wt. o f progeny when dams were 91 wks, o f age ( e x p t . 8) mg/100 body wt. S-S 11.7 a ( ( 7 4 ) 13.8 a (20) R-S 12.6 d (75) 14.1 a (20) R-R 16.-1 15.9 a (20) S-R 14.3 c (73) 14.0 a (20) u n p u b l i s h e d d a t a from an e a r l i e r d e t e r m i n a t i o n on the same p o p u l a t i o n , number o f d e t e r m i n a t i o n s i n p a r e n t h e s i s . 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 O . 0 1 . -58- T a b l e 14: T h y r o i d a l c h a r a c t e r i s t i c s i n n e w l y - h a t c h e d progeny o f b i r d s f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s i n e x p e r i m e n t 8. Ave. no. Ave. h t . Ave. wt. f o l l i c l e s Ave. f o l l i c l e D i e t a r y % e p i t h e l i u m e p i t h e l i u m a c r o s s t h e d i a m e t e r t r e a t m e n t e p i t h e l i u m AI mg g l a n d / j S-S 4 3 . 0 b ; ( 5 ) * ' 3.86 a 2.55 57 31.7 R-S 4 9 . 2 a b (5) 4.76 a b 3.10 48 35.1 R-R 5 5 . 8 a (5) 4.71 a b 4.03 47 39.6 S-R 5 5 . l a (5) 5.09 b 3.41 51 31.9 number o f t h y r o i d g l a n d s examined h i s t o l o g i c a l l y . v a l u e s w i t h the 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. -59- T a b l e 15: Comparison o f t h y r o i d a l c h a r a c t e r i s t i c s o f dams and progeny on d i f f e r e n t d i e t a r y t r e a t m e n t s . D i e t a r y Ave. a b s o l u t e % Ave. wt. Ave. h t . t r e a t m e n t t h y r o i d wt e p i t h e l i u m e p i t h e l i u m e p i t h e l i u m Dam Progeny Dam Progeny Dam Progeny Dam Progeny g mg % % mg mg p / i S-S 0.19 5.94 R-S 0.52 6.30 R-R 2.30 7.21 S-R 1.39 6.19 24.4 43.0 41.1 14.8 49.2 76.5 15.1 55.8 334.6 20.1 55.1 277.4 2.55 7.13 3.86 3.10 5.72 4.76 4.03 10.65 4.71 3.41 10.11 5.09 -60- DISCUSSION THYROIDAL RESPONSE TO CHRONIC GOITROGENIC STIMULATION BY RSM The l o n g - t e r m e f f e c t s o f c o n t i n u o u s f e e d i n g o f g o i t r o g e n i c RSM r e s u l t e d i n c o n t i n u o u s i n c r e a s e i n the s i z e o f t h e t h y r o i d g l a n d and i n d i s - r u p t i o n o f t h e e p i t h e l i a l t i s s u e s . The t h y r o i d g l a n d s o f t h e c h r o n i c a l l y RSM- f e d b i r d s were h e a v i e r when measurements were made a t 91 and 99 weeks o f age ( e x p e r i m e n t 1) than when t h e measurements were made a t o n l y 58 weeks o f age i n a p r e v i o u s e x p e r i m e n t ( T a b l e 3 ) . A l t h o u g h t h e b i r d s were a b l e t o m a i n t a i n p r o d u c t i o n and s e c r e t i o n o f t h y r o i d hormone t h e compensatory i n c r e a s e i n t h y - r o i d s e c r e t i o n was n o t a c c o m p l i s h e d w i t h a f i x e d number o f c e l l s . The l a r g e amount o f c o l l o i d p r e s e n t i n t h e g l a n d s o f t h e RSM-fed b i r d s s h o u l d n o t be c o n s i d e r e d c o n t r a r y t o the f i n d i n g s o f C l a n d i n i n e t al_ (1966) o r Matusmoto e t al_ (1968) s i n c e i n t h e s e e x p e r i m e n t s t h e b i r d s had been f e d g o i t r i n f o r a few weeks o n l y when h i s t o l o g i c a l s t u d i e s were made. I t has l o n g been known t h a t under c o n t i n u o u s s t i m u l a t i o n t h e e p i t h e l i a l c e l l s a r e u n a b l e t o m a i n t a i n t h e h y p e r a c t i v e s t a t e and e v e n t u a l l y become d i s o r g a n i s e d i n t h e f o l l i c l e w i t h desquamation and d i s i n t e g r a t i o n b e i n g e v i d e n t ( M a r i n e , 1935). G e n e r a l l y t he amount o f c o l l o i d i n such o v e r - s t i m u l a t e d g l a n d s i s v e r y s m a l l and t h e f o l l i c l e s become r e d u c e d i n s i z e as c e l l s e v e n t u a l l y d i e . However, t h i s r e s u l t f o l l o w s because t h e cause o f o v e r a c t i v i t y i s u s u a l l y a d e f i c i e n c y o f i o d i n e o r t h e i n a b i l i t y o f t h e t h y r o i d t o t r a p t h e i o d i n e o r t o produce t h e hormones. T h i s a p p a r e n t l y i s n o t t h e c a s e i n t h e p r e s e n t s t u d y o f t h e c h r o n i c g o i t r o g e n i c e f f e c t s o f RSM. -61- I t has a l r e a d y been mentioned t h a t d e f e c t s o f t h y r o i d a l i o d i n e m e t a b o l i s m may be ; caused by g o i t r o g e n i c d i s t u r b a n c e s a t any i n t e r m e d i a r y s t e p o r s t e p s o f t h y r o i d hormone b i o s y n t h e i s and/or s e c r e t i o n . T h y r o i d a l i o d i n e uptake seems t o be enhanced i n t h e RSM-fed b i r d s when compared t o S-S c o n t r o l s . T h i s i s s u p p o r t e d by t h e i o d i n e c o n c e n t r a t i o n s i n t h e t h y r o i d g l a n d s o f t h e s e b i r d s , measurement o f t h y r o i d a l 1-131 u p t a k e a t d i f f e r e n t t i m e i n t e r v a l s a f t e r a d m i n i s t r a t i o n o f t h e r a d i o i o d i n e , as w e l l as by t h e h i s t o l o g i c a l a p p e a r a n c e o f t h e s e t i s s u e s . A l t h o u g h the a v e r a g e i o d i n e c o n c e n t r a t i o n i n t h e t h y r o i d s o f t h e c h r o n i c a l l y RSM-fed b i r d s i s l e s s than h a l f o f t h a t o f t h e c o n t r o l (0.89 mg/g i n R-R ;anH 1.08 mg/q in.S-.R g r o u p ) , .-.••the a b s o l u t e i o d i n e c o n t e n t o f t h e s e g o i t r o u s g l a n d s a r e more th a n t h r e e t i m e s t h a t o f t h e c o n t r o l ( e x p e r i m e n t 2 ) . S i m i l a r l y , t h e r e l a t i v e t h y r o i d 1-131 u p t a k e ( t h y r o i d r a d i o a c t i v i t y p e r c e n t a g e o f i n j e c t e d dose/lOOgm t h y r o i d ) i s low f o r the RSM-fed b i r d s , but t h e t o t a l 1-131 uptake i n t h e t h y r o i d g l a n d s o f t h e l a t t e r groups i s d r a m a t i c a l l y i n c r e a s e d t o three-•or/', fdwRrfold*>'(e*-re*• • p e r i m e n t 3 ) . T h i s conforms w i t h t h e f i n d i n g o f Matsumoto e t a l _ (1968) pra the e f f e c t s o f g o i t r i n i n growing c h i c k s . A c c o r d i n g t o t h e s e i n v e s t i g a t o r s , i n a d d i t i o n t o m a i n t a i n i n g a l a r g e amount o f i n c o r p o r a t e d 1-131 i n t h e t h y r o i d g l a n d , the g o i t r i n - f e d c h i c k s a l s o show a slow t h y r o i d a l r e l e a s e o f t h e i n - j e c t e d r a d i o i o d i n e . T h i s may perhaps e x p l a i n our f i n d i n g t h a t i n b i r d s ' o f c h r o n i c RSM t r e a t m e n t s uptake o f a s i n g l e dose o f r a d i o i o d i n e a t 22 hours was g r e a t e r t h a n a t 6 h o u r s . T h i s was i n c o n t r a s t t o t h e b i r d s on t r e a t m e n t s S-S and R -S-in which r a d i o a c t i v i t y had, by 22 h o u r s , d e c l i n e d from t h e amount p r e s e n t 6 hours a f t e r i n j e c t i o n . The t ime o f maximum r a d i o a c t i v i t y , f o l l o w i n g , a d m i n i s t r a t i o n o f r a d i o i o d i n e , which i s d e t e r m i n e d by t h e r a t e o f r e l e a s e o f -62- i o d i n e r e l a t i v e t o the r a t e o f u p t a k e , o c c u r r e d l a t e r i n t h e RSM-fed b i r d s t h a n i n the SBM-fed b i r d s . R e l a t e d t o t h e a c t i v e p r o c e s s o f i o d i n e a c c u m u l a t i o n o f t h e t h y r o i d g l a n d o f RSM-fed b i r d s i s i t s h i s t o l o g i c a l p i c t u r e . The f u n c t i o n a l h y p e r a c t i v i t y i s d e m o n s t r a t e d by t h e m a s s i v e c o n t e n t o f e p i t h e l i u m i n t h e i r h y p e r t r o p h i c f o l l i c l e s . The a v e r a g e e p i t h e l i a l h e i g h t o f t h e s e g l a n d s i s a l s o s i g n i f i c a n t l y h i g h e r t h a n t h a t o f t h e c o n t r o l . The c o l l o i d i n t h e f o l l i c u l a r lumen o f t h e s e g l a n d s appear t o be b r i g h t l y s t a i n e d , which i s i n agreement w i t h t h e i r h i g h i o d i n e c o n t e n t . A l t h o u g h t h e o c c u r r e n c e o f e p i t h e - l i a l c e l l s s l o u g h i n g o f f i n t o the lumen a r e h i g h e r i n the c h r o n i c a l l y RSM-fed b i r d s t h a n i n the c o n t r o l , no s i g n o f f o l l i c u l a r a t r o p h y can be f o u n d . Thus i t can be s a i d t h a t t h e s e b i r d s a r e h y p e r a c t i v e i n the t h y r o i d a l c l e a r a n c e o f i o d i n e from t h e b l o o d c i r c u l a t i o n . G o i t r o g e n i c i n h i b i t i o n o f c o n v e r s i o n o f i o d i d e a c c u m u l a t e d by t h e t h y r o i d i n t o i t s a c t i v e f o r m and f u r t h e r t o MIT and DIT has been e x t e n s i v e l y i n v e s t i g a t e d ( S t a n b u r y e t a l _ , 1955; Haddad and S i d b u r y , 1959; G l a y t o n e t a l , 1958; F l o y d e t a l _ , i 9 6 0 ; P a r k e r and B e i e r w a l t e s , 1961; B a s c h i e r i et_ a l _ , 1963). A c c o r d i n g t o t h e s e i n v e s t i g a t o r s , such an i o d i d e o r g a n i z a t i o n d e f e c t i s c h a r a c t e r i z e d by a predominance o f i n o r g a n i c i o d i n e and s c a r c i t y o f i o d o - amino a c i d s when the t h y r o i d t i s s u e h y d r o l y z e d , p o s s i b l y due t o f a i l u r e o f i o d i n a t i o n o f t y r o s i n e by t h y r o i d a l p e r o x i d a s e . In our p r e s e n t i n v e s t i g a t i o n i n which t h e p r e - l a b e l e d t h y r o i d g l a n d s o f t h e c h r o n i c a l l y RSM-fed b i r d s were h y d r o l y z e d and the iodoamino a c i d s f r a c t i o n a t e d ( e x p e r i m e n t 4 ) , t h e f r a c t i o n s o f MIT and DIT were l a r g e and t h e r e was l i t t l e i n o r g a n i c i o d i n e 22 hours a f t e r a d m i n i s t r a t i o n o f r a d i o i o d i n e . Thus t h e i o d i d e o r g a n i z a t i o n p r o c e s s i s p r o - b a b l y n o t d i s t u r b e d i n the RSM-fed b i r d s . -63- T h a t t h y r o i d hormone b i o s y n t h e s i s i s i n d e e d i n h i b i t e d i n the RSM-fed b i r d s i s e v i d e n t by t h e r e s u l t s o b t a i n e d on f r a c t i o n a t i o n o f t h e iodoamino a c i d s i n t h y r o i d h y d r o l y s a t e s . The d i s t r i b u t i o n o f 1-131 r a d i o - a c t i v i t y among v a r i o u s t h y r o i d m e t a b o l i t e s shows t h a t t h e p r o p o r t i o n ' o f r a d i o i o d i n e p r e s e n t as MIT, 22 hours a f t e r a d m i n i s t r a t i o n , i s g r e a t e r i n t h e RSM-fed b i r d s . The su b s e q u e n t i n c r e a s e i n MIT/DIT r a t i o i s h e l d commonly as an i n d i c a t i o n o f d e f e c t ( s ) i n t h y r o i d hormonogenesis. C o u p l i n g e f f i c i e n c y i . e . Tg + T 4 / ( M I T / D I T ) , as a measure o f t h e combined r a t e s a t which d i o d o - t y r o s i n e and t h y r o i d hormones a r e pro d u c e d i s , i n o r d e r , SS>RS>SR>RR and i s i n v e r s e l y p r o p o r t i o n a l t o t h e t o t a l amount o f i o d i n e i n t h e t h y r o i d g l a n d . However, s i n c e t h e p e r c e n t a g e s o f t h y r o i d i o d o t h y r o n i n e s i n the RSM-fed b i r d s a r e n o t lower t o any s i g n i f i c a n t e x t e n t when compared t o t h e c o n t r o l , i t i s no t l i k e l y t h a t t h e c o u p l i n g mechanism o f MIT and DIT t o i o d o t h y r o n i n e s i s i n h i b i t e d p e r se by t h e f e e d i n g o f g o i t r o g e n i c RSM. T h i s seems t o be i n agreement w i t h t h e f i n d i n g o f A k i b a and Matsumoto (1971) t h a t a l t h o u g h t h e s y n t h e t i c a c t i v i t y o f t h e i o d o t h y r o n i n e s i n g o i t r i n - f e d c h i c k s i s ab o u t two- t h i r d o f the c o n t r o l a t 24 hours a f t e r dosage o f r a d i o i o d i n e , t h e amount o f i o d o t h y r o n i n e s s y n t h e s i z e d i n t h e s e c h i c k s i s about two times t h a t o f t h e c o n t r o l . In the p r e s e n t s t u d y , t h e s l i g h t l y l o w e r p e r c e n t a g e s o f t h y r o i d a l i o d o t h y r o n i n e s i n the RSM-fed birds,,;:when m u l t i p l i e d by t h e 10- t o 1 3 - f o l d i n c r e a s e i n the w e i g h t s o f t h e s e g l a n d s o v e r t h e c o n t r o l , w i l l g i v e e s t i m a t e d i o d o t h y r o n i n e v a l u e s l a r g e r t h a n t h a t o f t h e c o n t r o l . An i n h i b i t o r y s i t e would a p p e a r t o be a t t h e f o r m a t i o n l e v e l o f i o d o t y r o s i n e s , s i n c e i t i s shown t h a t t h e MIT/DIT r a t i o i s i n c r e a s e d i n the t h y r o i d s o f t h e RSM-fed b i r d s , w h i l e t h e c o u p l i n g d e f i c i e n c y i n t h e R-R and S-R groups i s a l s o d e p r e s s e d . Whatever t h e s i t e o f i n h i b i t i o n , however, i t ap p e a r s t h a t t h e s e c h r o n i c a l l y s t i m u l a t e d b i r d s have f u l l y compensated f o r t h e d e f e c t by t h y r o i d h y p e r t r o p h y and h y p e r p l a s i a , most l i k e l y i n v o l v i n g t h e -64- s e c r e t i o n s o f t h e h y p o t h a l a m u s - p i t u i t a r y - t h y r o i d a x i s . An u n r e p o r t e d ex- p e r i m e n t u s i n g t h e c h i c k method o f TSH b i o a s s a y ( B a t e s , 1962) and h i s t o l o g i c a l e x a m i n a t i o n o f the p i t u i t a r y g l a n d s o f t h e s e b i r d s f a i l e d t o be c o n c l u s i v e i n t h i s r e s p e c t . F u r t h e r i n v e s t i g a t i o n s i n t h i s a r e a a r e n e c e s s a r y b e f o r e the neurohormonal r e l a t i o n s h i p s c o n t r o l l i n g t h y r o i d m e t a b o l i s m o f t h e RSM- f e d b i r d s w i l l be e l u c i d a t e d . In the normal t h y r o i d , s y n t h e s i s and s e c r e t i o n a r e t a k i n g p l a c e under " s t e a d y s t a t e " c o n d i t i o n s n e a r t h e b o r d e r o f t h e e p i t h e l i a l c e l l i n t h e f o l l i c l e s . T h i s e q u i l i b r i u m i s d i s t u r b e d a l m o s t i m m e d i a t e l y a f t e r t h e i n t r o - d u c t i o n o f a g o i t r o g e n . Under t h e i n f l u e n c e o f t h e e a r l y r i s e i n TSH, new f r a c t i o n s o f t h y r o g l o b u l i n t a k e p a r t i n t h e hormonal exchange and t h e 1-131 mixes w i t h a d i f f e r e n t pool than i n t h e " r e s t i n g " , i o d i n e - r i c h t h y r o i d s . T h y r o i d s e c r e t i o n under t h e s e c o n d i t i o n s o f i o d i n e d e f i c i e n c y c o u l d t h u s f o l l o w r a t h e r c o m p l i c a t e d rules-. T h e r e f o r e , a more p r o b a b l e s i t e o f i n h i b i t i o n i s a t the r e l e a s e and s e c r e t i o n 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 , and t h i s i s s u p p o r t e d by t h e work o f A k i b a and Matsumoto (1971) on the e f f e c t o f g o i t r i n on the t h y - r o i d hormone b i o s y n t h e s i s i n growing c h i c k s . P a r t i c u l a r l y , i t i s presumed t h a t t h e s e c r e t i o n o f t h y r o i d hormone from t h e g l a n d by h y d r o l y s i s o f t h y r o g l o b u l i n i s d e p r e s s e d o r t h a t t h e r e i s no i n c r e m e n t o f h y d r o l y s i s o f t h y r o g l o b u l i n by t h e a p p a r e n t i n c r e a s e d s e c r e t i o n o f TSH from t h e p i t u i t a r y caused by t h e f e e d i n g o f g o i t r i n . Under t h e s e c i r c u m s t a n c e s the t h y r o i d g l a n d s were c a p a b l e o f c o n t i n u o u s h y p e r p l a s i a f o r the d u r a t i o n o f t h e e x p e r i m e n t . The s i t u a t i o n may be s i m i l a r t o t h a t when a s m a l l amount o f t h y r o x i n e i s a d m i n i s t e r e d t o b i r d s i n which t h y r o i d hormone p r o d u c t i o n i s b l o c k e d by a d m i n i s t r a t i o n o f t h i o u r a c i l . S h u l t z e and T u r n e r (1945) found t h a t t h e g o i t r o g e n i c i t y o f t h i o u r a c i l i s en- hanced when a s m a l l amount o f t h y r o x i n e i s a d m i n i s t e r e d s i m u l t a n e o u s l y w i t h t h e t h i o u r a c i l . In t h e p r e s e n t s t u d y , t h e h y d r o l y s i s r a t e by e x t r a n e o u s -65- p r o n a s e does n o t appear t o be any s l o w e r i n the t h y r o i d l o b e s o f the RSM-fed b i r d s t h a n the c o n t r o l ( e x p e r i m e n t 5 ) . How t h i s can be a p p l i e d t o endogenous 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 by t h y r o i d enzymes, however, i s u n d e t e r m i n e d . Hence the i n h i b i t o r y e f f e c t o f g o i t r o g e n i c RSM on the r e l e a s e o f hormones from t h y r o g l o b u l i n and s ubsequent s e c r e t i o n from t h e t h y r o i d g l a n d remains a p o s s i - b i l i t y . Due t o a d e f e c t i n i o d o t y r o s i n e d e i c d i n a s e , MIT o r DIT may n o t be d e i o d i n a t e d and a l a r g e amount o f i o d o t y r o s i n e may be l o s t i n t h e u r i n e , r e - s u l t i n g i n a d i s t u r b a n c e i n r e u t i l i z a t i o n o f i n o r g a n i c i o d i n e i n i o d i d e o r - g a n i z a t i o n ( S t a n b u r y , 1957; S t a n b u r y and M o r r i s , 1958). Such a p o s s i b l e e f f e c t by g o i t r o g e n i c RSM on t h e d e i o d i n a t i o n p r o c e s s has n o t been i n v e s t i g a t e d i n the p r e s e n t s t u d y . A l t h o u g h y c o u n t i n g o f e x c i s e d p i e c e s o f l i v e r 22 hours a f t e r a d m i n i s t r a t i o n o f r a d i o i o d i n e has n o t r e v e a l e d any n o t a b l e d i f f e r e n c e i n t h e 1-131 c o n c e n t r a t i o n o f t h e l i v e r s o f t h e b i r d s on t h e d i f f e r e n t d i e t a r y t r e a t - ments, t h e r e l a t i v e r a t e s o f removal o f i o d i n a t e d compounds by t h e s e organs c a n n o t be known from t h e s e d a t a . However, a d e f e c t i n t h e d e i o d i n a t i o n p r o c e s s due t o the f e e d i n g o f g o i t r o g e n i c RSM i s u n l i k e l y , a t l e a s t f o r t r i i o d o t h y r o n i n e . U n p u b l i s h e d d a t a from t h i s l a b o r a t o r y has shown t h a t when a s i n g l e dose o f 1-131-1abeled Tg was a d m i n i s t e r e d i n t o RSM-fed b i r d s , t h e r a t e o f 1-131 t u r n - o v e r , measured by r a d i o a c t i v i t y o f t h e e x c r e t a 24 hours o r 48 hours a f t e r i n j e c t i o n , i s n o t d i f f e r e n t from t h a t o f t h e c o n t r o l . Hence t h e d e i o d i n a t i o n c a p a c i t y i n t h e RSM-fed b i r d s does n o t a p p e a r t o be i n h i b i t e d . In c h r o n i c e x p e r i m e n t s w i t h hamster, i t was f o u n d t h a t i n s e v e r e i o d i n e d e f i c i e n c y a p a r t o f the s t o r e d r a d i o a c t i v i t y i n t h e t h y r o i d c o u l d n o t be d i s c h a r g e d even a f t e r TSH s t i m u l a t i o n and PTU t r e a t m e n t . T h i s i n d i c a t e d a l t e r e d , a b n o r m a l l y s t r o n g , p r o t e i n b i n d i n g o f t h e i n t r a t h y r o i d a l iodoamino a c i d s under c e r t a i n c o n d i t i o n s (Van M i d d l e s w o r t h , 1965; F o l l i s , 1965). In the p r e s e n t c a s e , t h e p o s s i b i l i t y t h a t t h e d e f e c t s i n t h y r o i d f u n c t i o n due t o -66- f o r m a t i o n o f abnormal i o d o p r o t e i n i n t h e t h y r o i d g l a n d i s u n l i k e l y . T h i s abnormal i o d o p r o t e i n o t h e r t h a n t h y r o g l o b u l i n i n t h e t h y r o i d i s w a t e r - s o l u b l e 'and b u t a n o l - i n s o l u b l e , so t h a t on paper chromatography w i t h b u t a n o l , i t w i l l n o t m i g r a t e from t h e o r i g i n ( S t a n b u r y and M c G i r r , 1957; DeGroot e t a l _ , 1958; DeGroot and S t a n b u r y , 1959; Van Wyk ejt a l _ , 1962). No such p r o t e i n was d e t e c t e d i n the t h y r o i d gliands o f the p r e s e n t s t u d y . The p e r c e n t a g e s o f r a d i o a c t i v i t y r e m a i n i n g a t the o r i g i n on t h e chromatograms o f t h e t h y r o i d h y d r o l y s a t e s a r e s i m i l a r and w i t h i n normal ranges f o r a l l b i r d s on the f o u r d i e t a r y t r e a t m e n t s . I t i s t h e r e f o r e h y p o t h e s i z e d t h a t c h r o n i c f e e d i n g o f g o i t r o g e n i c RSM t o b i r d s may i n t e r f e r e w i t h t h y r o i d f u n c t i o n , p r o b a b l y a t an i n t e r m e d i a r y s t e p i n v o l v i n g t h e f o r m a t i o n o f i o d o t y r o s i n e s and/or t h e r e l e a s e o r s e c r e t i o n o f t h e t h y r o i d hormones. One o r more enzyme systems such as t h y r o i d p e r o x i d a s e i n t h e t h y r o i d g l a n d may be i n v o l v e d . D i s o r d e r s o f t h e s e systems have been c o n s i d e r e d t o cause d i s t u r b a n c e s o f t h y r o i d hormone p r o d u c t i o n and provoke compensatory e n l a r g e m e n t o f t h e t h y r o i d w i t h h i g h u p t a k e o f i o d i n e (Fukase e t a l _ , 1967). Thus, i n t h e c h r o n i c a l l y RSM-fed b i r d s , a d i s t u r b a n c e o f t h y - r o i d hormone b i o s y n t h e s i s and/or r e l e a s e due t o an i n t r i n s i c enzyme a l t e r a t i o n i s s t r o n g l y s u g g e s t e d . The h i s t o l o g i c a l p i c t u r e and r a d i o i o d i n e m e t a b o l i s m i n the t h y r o i d s o f t h e s e b i r d s s u g g e s t h y p e r a c t i v i t y , and t h i s conforms w i t h t h e h y p o t h e s i s t h a t i n c r e a s e s i n enzyme a c t i v i t i e s t e n d t o o c c u r when t h e enzymes a r e s t i m u l a t e d o v e r a p e r i o d o f time (such as t h a t may o c c u r i n c h r o n i c RSM t r e a t m e n t ) , and t h a t once t h e enzyme p a t t e r n c h anges, t h e p a t t e r n s o f s u b s t r a t e d i s p o s i t i o n t e n d t o become f i x e d (Chance ejt a l _ , 1965). T h i s i d e a on t h e " b i o c h e m i c a l i m p r i n t i n g " o f m e t a b o l i c e x p e r i e n c e i n c e l l s s i g n i f i e s a d a p t i v e i n c r e a s e s i n enzyme a c t i v i t i e s t h a t o c c u r a l o n g s p e c i f i c m e t a b o l i c pathways when t h e r e i s a l a r g e and s u s t a i n e d i n c r e a s e i n s u b s t r a t e t r a f f i c t h r o u g h them, and may w e l l e x p l a i n t h e h y p e r f u n c t i o n o f t h e t h y r o i d o f t h e -67- R-R and S-R b i r d s which c o n t i n u o u s l y a c c u m u l a t e i o d i n e o f the c i r c u l a t i o n f o r o r g a n i f i c a t i o n . The e x p e r i e n c e o f a c comodating l a r g e amounts o f s u b s t r a t e i s o f t e n r e f l e c t e d i n a c h a r a c t e r i s t i c i m p r i n t on the enzyme p a t t e r n o f t h e c e l l . M o r eover, changes i n enzyme p a t t e r n p r o b a b l y c o n s t i t u t e a l o n g - r a n g e m e t a b o l i c c o n t r o l d e v i c e which i s super-imposed on o t h e r r e g u l a t o r y mechanisms such as the h y p o t h a l a m u s - p i t u i t a r y - t h y r o i d a x i s o r t h e f e e d b a c k mechanisms, which o p e r a t e w i t h i n a much s h o r t e r time s c a l e . PERSISTENCE OF EFFECTS OF EARLY GOITROGENIC STIMULATION The v a r i o u s p arameters o f t h y r o i d a c t i v i t y show t h a t t h e e f f e c t s o f e a r l y g o i t r o g e n i c s t i m u l a t i o n o f t h e t h y r o i d g l a n d remained a p p a r e n t i n l a t e r l i f e . Thus t h y r o i d g l a n d s remained e n l a r g e d i n b i r d s which had been f e d RSM d u r i n g t h e growing p e r i o d and i n b i r d s g i v e n t h i o u r a c i l f o r o n l y t h e f i r s t f o u r weeks o f l i f e ( T a b l e 3 ) . T h y r o i d g l a n d s examined from b i r d s 67 weeks a f t e r w i t h d r a w a l o f RSM from t h e d i e t had t h e h i s t o l o g i c a l a p p e a r a n c e o f c o l l o i d g o i t e r . In t h e s e b i r d s the t h y r o i d f o l l i c l e s were l a r g e r and the h e i g h t o f t h e e p i t h e l i a l c e l l s l o w e r . T h i s p e r s i s t e n c e o f e f f e c t o f e a r l y o v e r - s t i m u l a t i o n o f t h e t h y r o i d i s i n agreement w i t h t h e f i n d i n g s o f Bakke et_ a T (1970) i n t h e r a t . In t h e r a t b r i e f p e r i n a t a l h y p o t h y r o i d i s m i n d u c e d by a d m i n i s t r a t i o n o f p r o p y l t h i o u r a c i l t o t h e p r e g n a n t dam and/or t o t h e neonate r e s u l t e d i n a p e r s i s t e n t l y e n l a r g e d t h y r o i d g l a n d w i t h l a r g e r f o l l i c l e s and l o w e r e p i t h e l i a l h e i g h t . These i n v e s t i g a t o r s r e p o r t e d a l s o t h a t p e r i n a t a l h y p o t h y r o i d i s m c a u s e d a d e l a y i n p u b e r t y . E a r l i e r e x p e r i m e n t s i n t h i s l a b o - r a t o r y has r e v e a l e d no e v i d e n c e t h a t t h e i n g e s t i o n o f g o i t r i n by c h i c k e n s up t o 24 weeks o f age a f f e c t e d t h e time o f s e x u a l m a t u r i t y o r r e p r o d u c t i v e c a p a c i t y when RSM was withdrawn a t t h i s t i m e (March e t _ a l _ , 1975). T h y r o i d a l e n l a r g e m e n t was r e l a t e d t o t h e l e n g t h o f t i m e o v e r which t h e g o i t r o g e n i c RSM -68- was f e d . The f o l l i c l e s i z e and p r o p o r t i o n o f e p i t h e l i a l t i s s u e were l o w e r i n t h e t h y r o i d g l a n d s o f t h e R-R b i r d s t h a n i n t h e S-R b i r d s a t 91 weeks o f age. The t o t a l amount o f i o d i n e i n the g l a n d o f t h e R-S b i r d s was g r e a t e r than t h a t i n the c o n t r o l S-S b i r d s b ut t h e c o n c e n t r a t i o n p er u n i t w e i g h t o f g l a n d was s i m i l a r ( e x p e r i m e n t 2 ) . C o n s i s t e n t w i t h t h i s o b s e r v a t i o n was t h e g r e a t e r t o t a l u p t a k e o f a s i n g l e dose o f r a d i o i o d i n e by t h e t h y r o i d g l a n d s o f t h e R-S b i r d s b u t a l o w e r uptake when e x p r e s s e d r e l a t i v e t o t h e w e i g h t o f g l a n d ( e x p e r i m e n t 3 ) . T h e r e f o r e i t can be s a i d t h a t , a l t h o u g h t he s i z e o f t h e t h y r o i d g l a n d i s p e r m a n e n t l y a f f e c t e d by e a r l y g o i t r o g e n i n t a k e , t h e t h y r o i d a c t i v i t y i s n o r m a l . In t h e e n l a r g e d g l a n d t h e e p i t h e l i a l t i s s u e r e sponds n o r m a l l y t o t h y r o t r o p i n and t h e c a p a b i l i t y f o r t h y r o i d hormone s y n t h e s i s t o meet t h e r e q u i r e m e n t s o f t h e b i r d s i s n o t a d v e r s e l y a f f e c t e d . The p r e s e n c e o f i n - c r e a s e d numbers o f e p i t h e l i a l c e l l s i n t h e t h y r o i d g l a n d s o f t h e b i r d s f e d g o i t r o g e n i c RSM d u r i n g t h e growing p e r i o d makes i t p o s s i b l e , once t h e i n h i - b i t i o n o f t h y r o i d hormone p r o d u c t i o n and/or s e c r e t i o n i s withdrawn, f o r t h e h y p e r p l a s t i c g l a n d t o meet t h e r e q u i r e m e n t s o f t h e body f o r t h y r o i d hormone a t a lower l e v e l o f a c t i v i t y ( i . e . a t a lower l e v e l o f t h y r o t r o p i n s t i m u l a t i o n ) . I t has been shown t h a t t h y r o i d a l e p i t h e l i a l c e l l s , f o l l o w i n g e l i m i n a t i o n o f t h y r o t r o p i n s t i m u l a t i o n , c o n t i n u e t o t r a p i o d i n e and t o s e c r e t e t h y r o i d hormone i n t o t h e f o l l i c u l a r lumen f o r some t i m e a t l e a s t , whereas t h e r e l e a s e o f t h y - r o i d hormone i n t o t he c i r c u l a t i o n s t o p s a l m o s t i m m e d i a t e l y (Bjorkman e t a l , 1974). The f a c t t h a t t o t a l i o d i n e was h i g h e r and t h a t t h e r e were more t h y - r o n i n e s p r e s e n t i n t h e g l a n d s o f RSM-fed b i r d s than i n SBM-fed b i r d s i n d i c a t e s t h a t e n d o c y t o s i s and r e l e a s e 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 were r e t a r d e d i n the f o r m e r . In R-S b i r d s w i t h d r a w a l o f g o i t r i n a t 24 weeks o f age -69- was f o l l o w e d by c o l l o i d g o i t e r because t h e i o d i n e c o n c e n t r a t i o n by t h e g l a n d c o n t i n u e d a t a f a s t e r r a t e than d i d t h e r e l e a s e o f i o d i n e from t h e g l a n d . T h i s i s d i f f e r e n t from t h e f i n d i n g o f A k i b a and Matsumoto (1973) on t h e complete r e c o v e r y o f t h y r o i d f u n c t i o n w i t h i n a s h o r t time a f t e r w i t h d r a w a l o f g o i t r i n i n growing c h i c k s . A p e r s i s t e n t d i f f e r e n c e i n i o d i n e m e t a b o l i s m i n d u c e d by t h e f e e d i n g o f RSM t o b i r d s d u r i n g t h e growing p e r i o d was s t i l l e v i d e n t more t h a n a y e a r l a t e r i n t h e l e s s e r amount o f a s i n g l e dose o f r a d i o i o d i n e d e p o s i t e d i n the d e v e l o p i n g ova o f t h e s e b i r d s compared t o c o n t r o l b i r d s which had neven been f e d a g o i t r o g e n i c d i e t . T h i s a s p e c t o f t h e s t u d y ( t o be d i s c u s s e d l a t e r ) p r o v i d e s a d d i t i o n a l e v i d e n c e t h a t the t h y r o i d g l a n d m a i n t a i n s a h e i g h t e n e d c a p a c i t y f o r uptake o f i o d i n e as a r e s u l t o f e a r l y o v e r - s t i m u l a t i o n . The mechanism o f the p e r s i s t e n c e o f e a r l y s t i m u l a t i o n j-by g o i t r o - gens o f t h e t h y r o i d g l a n d i s unknown. The W o l f f - C h a i k o f f e f f e c t , which com- monly e x p l a i n s t h e b l o c k i n g e f f e c t o f i n t r a t h y r o i d a l i o d i d e o n i b i o s y n t h e s i s o f t h e t h y r o i d hormones when i o d i n e - d e f i c i e n t r a t s a r e r e - f e d a h i g h i o d i n e d i e t , i s c h a r a c t e r i z e d by a v e r y h i g h i o d i d e p e r c e n t a g e o f i n t r a t h y r o i d a l r a d i o a c t i v i t y , h i g h l a b e l e d MIT/DIT r a t i o , and absence o f l a b e l e d i o d o t h y r o n i n e s . N o r m a l l y t h i s b l o c k i n g e f f e c t o f l a r g e c o n c e n t r a t i o n s o f i o d i d e on hormone s y n t h e s i s i s a t r a n s i e n t phenomenon. T h e r e i s p r o b a b l y an autonomous a d a p t a - t i o n o f the t r a n s p o r t mechanism t o t h e h i g h l e v e l o f i o d i d e i n which t h e r e i s a r a p i d l o s s o f t h y r o i d a l c a p a c i t y t o c o n c e n t r a t e i o d i d e . In t h e R-S b i r d s i n t h e p r e s e n t s t u d y , i s i t p o s s i b l e t h a t t h i s a d a p t a t i o n mefchanism i s i m p a i r e d by g o i t r o g e n i c RSM so t h a t c o l l o i d g o i t e r r e s u l t s ? A l t h o u g h t h e W p l f f - C h a i k o f f e f f e c t i s not e v i d e n t i n t h e t h y r o i d h y d r o l y z a t e s o f t h e s e b i r d s , d e f e c t s i n t h e a u t o r e g u l a t i o n system i n t h e s e g l a n d s may s t i l l r emain a pos- s i b l e mechanism whereby e f f e c t s o f e a r l y g o i t r o g e n i c s t i m u l a t i o n m a y . p e r s i s t . -70- I t i s t h e r e f o r e c o n c l u d e d t h a t t h e e f f e c t s o f e a r l y g o i t r o g e n i c s t i m u l a t i o n o f t h e t h y r o i d g l a n d p e r s i s t a f t e r w i t h d r a w a l o f t h e g o i t r o g e n . The t h y r o i d g l a n d which has been o v e r - s t i m u l a t e d c o n t a i n s l a r g e r t h a n normal numbers o f e p i t h e l i a l c e l l s and a c c o r d i n g l y l e s s a c t i v i t y o f i n d i v i d u a l c e l l s i s r e q u i r e d t o m a i n t a i n normal l e v e l s o f hormone s e c r e t i o n . EFFECTS OF ALTERATIONS IN MATERNAL THYROID METABOLISM ON EMBRYONIC THYROID DEVELOPMENT IN THE CHICK S i n c e eggs l a i d by b i r d s f e d RSM c o n t a i n e d l e s s i o d i n e than when SBM was f e d ( e x p e r i m e n t 6A), t h e h y p o t h e s i s was t e s t e d t h a t , i n t h e g o i t r o u s b i r d s , the i n c r e a s e d t h y r o i d a l uptake o f d i e t a r y i o d i n e from t h e c i r c u l a t i o n l i m i t e d t h e amount a v a i l a b l e f o r d e p o s i t i o n i n the d e v e l o p i n g o v a . To e l i m i n a t e t h e p o s s i b i l i t y t h a t some component o f RSM i n t e r f e r e d w i t h a b s o r p - t i o n o f d i e t a r y i o d i n e from the i n t e s t i n e t h e d i s t r i b u t i o n o f i n j e c t e d r a d i o i o d i n e between t h e o v a r i a n f o l l i c l e s and the t h y r o i d g l a n d was compared i n t h e b i r d s f e d t h e SBM and RSM d i e t s ( e x p e r i m e n t 7 ) . The d a t a i n T a b l e 12 c l e a r l y show t h a t the d e v e l o p i n g ova i n the RSM-fed b i r d s c o n t a i n e d l e s s o f t h e i n j e c t e d r a d i o i o d i n e t h a n ova i n the SBM-fed b i r d s . I t i s a p p a r e n t from F i g u r e 2 t h a t t h e r a t e o f i o d i n e u p t a k e by t h e d e v e l o p i n g f o l l i c l e i s p r o - p o r t i o n a l t o t h e t o t a l r a t e o f t r a n s f e r o f a l l y o l k components from t h e c i r c u l a t i o n i n t o the f o l l i c l e . The amount o f i o d i n e p r e s e n t i n t h e f o l l i c l e i s t h e r e f o r e a f u n c t i o n o f both t h e r a t e o f t h e f o l l i c u l a r growth and t h e c o n c e n t r a t i o n o f i o d i n e i n t h e plasma. In c o n t r a s t t o t h e d e v e l o p i n g o v a , t h e t h y r o i d g l a n d s o f t h e RSM-fed b i r d s c o n t a i n e d more r a d i o i o d i n e 22 hours a f t e r i n j e c t i o n than d i d t h o s e o f t h e SBM-fed b i r d s . D e t a i l s o f the d i s t r i - b u t i o n o f the r a d i o a c t i v i t y among d i f f e r e n t i o d i n a t e d t h y r o i d a l compounds have a l r e a d y been d i s c u s s e d . - 7 1 - P r e l i m i n a r y t e s t s i n t h i s l a b o r a t o r y ( u n p u b l i s h e d d a t a ) have i n d i c a t e d t h a t most o f the i o d i n e p r e s e n t i n eggs i s i n i t s i n o r g a n i c f o r m . I f t h i s i s t r u e , i t w i l l be i n agreement w i t h t h e p o s t u l a t i o n t h a t when more i o d i n e i s t r a p p e d by t h e t h y r o i d g l a n d from t h e e x t r a t h y r o i d a l i n o r g a n i c p o o l , l e s s o f t h i s element w i l l be a v a i l a b l e f o r d e p o s i t i o n i n the d e v e l o p i n g ova. To t e s t t h i s h y p o t h e s i s , i t i s t h o u g h t t h a t i f the d i e t a r y i o d i n e i n t a k e o f t he RSM-fed b i r d s i s i n c r e a s e d t o o v e r t h a t can be a c c u m u l a t e d by t h e t h y r o i d g l a n d , t h e n e t d e p o s i t i o n o f t h i s element i n t h e eggs w i l l be f u r t h e r enhanced d e s p i t e t he i n h i b i t o r y e f f e c t o f t h e g o i t r o g e n i n t h y r o i d m e t a b o l i s m . R e s u l t s f r o m e x p e r i m e n t 6B seem t o b e a r t h i s o u t . T h y r o i d w e i g h t s a t h a t c h i n g were compared i n t h e progeny o f c h i c k e n s t h a t had been f e d g o i t r o g e n i c RSM and SBM r e s p e c t i v e l y i n the growing and/or l a y i n g d i e t s ( e x p e r i m e n t 8). The t h y r o i d g l a n d s were h e a v i e s t i n the progeny o f b i r d s which had been f e d RSM i n both t h e growing and t h e l a y i n g d i e t s . T h e r e was, however, a c a r r y o v e r o f t h e e f f e c t o f f e e d i n g RSM d u r i n g t h e growing p e r i o d when t h e b i r d s were s h i f t e d t o a SBM d i e t a t m a t u r i t y . A l t h o u g h t h e a v e r a g e w e i g h t s o f t h e g l a n d s o f t h e progeny o f dams r e c e i v i n g SBM i n t h e l a y i n g and f e d RSM d u r i n g t h e growing p e r i o d were n o t s i g n i f i c a n t l y h e a v i e r than i n t h o s e from dams f e d SBM t h r o u g h o u t l i f e , t h e p r o p o r t i o n o f e p i t h e l i u m i n t h e g l a n d s was g r e a t e r from t he RSM-fed b i r d s . In o t h e r words, t h e s t i m u l u s t o the t h y r o i d g l a n d was e v i d e n c e d by an i n c r e a s e i n t h e r a t i o j o f e p i t h e l i a l t i s s u e t o c o l l o i d w i t h i n t h e t h y r o i d g l a n d s but was n o t s u f f i c i e n t t o r e s u l t i n i n c r e a s e d t h y r o i d w e i g h t . On the o t h e r hand, progeny from dams f e d RSM d u r i n g t he l a y i n g p e r i o d o n l y , i had s m a l l e r t h y r o i d g l a n d s than d i d t h o s e f e d RSM t h r o u g h o u t l i f e . The p e r c e n t a g e o f e p i t h e l i a l t i s s u e was s i m i l a r i n t h e g l a n d s o f the progeny from t h e s e two t r e a t m e n t s . -72- SUMMARY AND CONCLUSIONS From t h e d a t a o f t h e v a r i o u s : e x p e r i m e n t s d i s c u s s e d above, t h e f o l l o w i n g c o n c l u s i o n s c o n c e r n i n g t h e c h r o n i c e f f e c t s o f f e e d i n g g o i t r o g e n i c RSM t o b i r d s can be drawn. A n a l y s e s . o f J h e 7 , t h y r o i d g l a n d s showed t h a t t h e i o d i n e c o n t e n t o f t h e RSM-fed b i r d s ( e x p e r i m e n t 2) was s i g n i f i c a n t l y h i g h e r than t h a t i n t h e c o n t r o l . The c h r o n i c a l l y s t i m u l a t e d (R-R and S-R) b i r d s had a t h r e e - f o l d i n c r e a s e i n t h e i r t h y r o i d a l i o d i n e c o n t e n t w h i l e even t he R-S group (which had o n l y been f e d g o i t r o g e n i c RSM d u r i n g t h e growing p e r i o d ) had o v e r t w i c e the t h y r o i d a l i o d i n e c o n t e n t o f the c o n t r o l b i r d s . T h i s was m a i n l y due t o t h e g o i t r o u s s t a t e o f the RSM-stimulated g l a n d s . The c h r o n i c a l l y - s t i m u l a t e d t h y r o i d g l a n d s showed a s e v e n - t o t e n - f o l d h y p e r t r o p h y , w h i l e t h e R-S group had an a v e r a g e t h y r o i d w e i g h t d o u b l e t h a t o f t h e c o n t r o l ( e x p e r i m e n t 1 ) . H i s t o l o g i c a l d a t a ( e x p e r i m e n t 1) c o n f i r m e d t h e a c t i v e l y f u n c - t i o n i n g s t a t e o f t h e t h y r o i d g l a n d s o f t h e b i r d s on c h r o n i c RSM and/or SBM t r e a t m e n t s . The t h y r o i d s o f the R-R and S-R b i r d s showed c o l l o i d g o i t e r r a t h e r t h a n a t r o p h y t h a t would sometimes be e x p e c t e d from c h r o n i c g o i t r o g e n i c t r e a t m e n t s , c o u p l e d w i t h i n c r e a s e d a v e r a g e e p i t h e l i a l h e i g h t s and f o l l i c l e d i a m e t e r s 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 n t h e s e g r o u p s . T h i s gave s u p p o r t t o t h e h y p o t h e t i c a l p i c t u r e o f a s t i m u l a t e d t h y r o i d g l a n d o f the c h r o n i c a l l y R S M - s t imulated b i r d s a c t i v e l y a c c u m u l a t i n g i o d i n e from t he b l o o d . On-the.etfcer hand, t h e t h y r o i d g l a n d s o f the b i r d s f e d c h r o n i c a l l y w i t h g o i t r o g e n i c RSM were found t o be a c c u m u l a t i n g r a d i o i o d i n e , when 1-131 upt a k e was measured a t d i f f e r e n t t ime i n t e r v a l s a f t e r a d m i n i s t r a t i o n o f a -73- s i n g l e dose i n t r a v e n e o u s l y ( e x p e r i m e n t 3). T h i s f o u r - t o f i v e - f o l d i n c r e a s e i n t h y r o i d a l r a d i o i o d i n e u p t a k e c o u l d a c c o u n t f o r t h e d e c r e a s e i n i o d i n e d e p o s i t i o n i n t h e eggs o f the RSM-fed b i r d s . The s i t e o f i n h i b i t i o n by g o i t r o g e n i c RSM on i n t r a t h y r o i d a l i o d i n e m e t a b o l i s m o f t h e c h r o n i c a l l y t r e a t e d b i r d s was i n v e s t i g a t e d ( e x p e r i - ment 4). I t was f o u n d t h a t a l t h o u g h t h e r e a p p e a r e d t o be i n h i b i t i o n a t an i n t e r m e d i a r y s t e p ( s ) i n t h y r o i d hormone b i o s y n t h e s i s as d e m o n s t r a t e d by an a l t e r a t i o n o f the i o d o t y r o s i n e r a t i o , t h e p r o d u c t i o n o f i o d o t h y r o n i n e s was e s t i m a t e d t o be adequate as j u d g e d from t h e i r p e r c e n t a g e s i n t h e g o i t r o u s t h y r o i d g l a n d s o f the RSM-fed b i r d s . A more p r o b a b l e i n h i b i t o r y s i t e m i g ht be a t the r e l e a s e mechanism o f t h e s e hormones from the t h y r o g l o b u l i n . An e x p e r i m e n t ( e x p e r i m e n t 5) t o d e t e r m i n e t h e r e s p e c t i v e r a t e s o f t h y r o g l o - b u l i n h y d r o l y s i s by t h e e x t r a n e o u s enzyme pronase f a i l e d t o r e v e a l any d i f f e r e n c e between the SBM- and RSM-fed b i r d s . However, the p o s s i b i l i t y t h a t t h e r e m i g h t be a d i f f e r e n c e i n t h e t h y r o g l o b u l i n p r o t e o l y s i s r a t e s by endogenous t h y r o i d enzymes between the two groups o f b i r d s i s s t i l l open t o f u r t h e r i n v e s t i g a t i o n . From t h e above d a t a , i t was h y p o t h e s i z e d t h a t t h e g o i t r o u s t h y r o i d g l a n d s o f the c h r o n i c a l l y RSM-fed b i r d s were a c t i v e l y a c c u m u l a t i n g i o d i n e from the c i r c u l a t i o n because o f a p o s s i b l e i n h i b i t o r y s t e p i n t h y r o i d hormone b i o s y n t h e s i s and/or r e l e a s e , so t h a t l e s s o f t h e element was a v a i l a b l e f o r d e p o s i t i o n t o t h e eggs. The y o l k o f eggs from c h r o n i c a l l y RSM-fed b i r d s was i n d e e d f o u n d t o be s i g n i f i c a n t l y l o w er i n t o t a l i o d i n e c o n t e n t than t h o s e from t h e c o n t r o l ( e x p e r i m e n t 6A). S i n c e t h e i o d i n e c o n c e n t r a t i o n i n t h e a l b u m i n was r e g a r d e d as t o o low t o be o f s i g n i f i c a n c e i n the p r e s e n t i s s u e , i t was c o n c l u d e d t h a t -74- the c h r o n i c a l l y RSM-fed b i r d s d e p o s i t e d l e s s i o d i n e i n t h e i r eggs. T h i s was f u r t h e r s u p p o r t e d by a r a d i o i o d i n e s t u d y ( e x p e r i m e n t 7) which demons- t r a t e d a d e c r e a s e i n the d e p o s i t i o n o f 1-131 i n t h e d e v e l o p i n g o v a r i a n f o l l i c l e s o f t h e s e b i r d s compared t o the c o n t r o l . S i n c e t h e r e was no i n h i b i t i o n by g o i t r o g e n i c RSM on i o d i n e a b s o r p t i o n i n t h e g a s t r o i n t e s t i n a l t r a c t because t h e r a d i o i o d i n e was a d m i n i s t e r e d i n t r a v e n o u s l y , t h i s d i f f e r e n c e i n i o d i n e d e p o s i t i o n i n the eggs o f t h e RSM- and SBM-fed b i r d s c o u l d n o t be due t o a d i f f e r e n c e i n i o d i n e l e v e l p i i r r r i d i e t a r y i n t a k e . The RSM- supplemented d i e t a c t u a l l y c o n t a i n e d a s l i g h t l y h i g h e r c o n c e n t r a t i o n o f i o d i n e t h a n t h e SIBM-supplemented r a t i o n f e d t o t h e b i r d s ( e x p e r i m e n t 2 ) . T h a t t h i s d e c r e a s e o f i o d i n e d e p o s i t i o n i n the eggs o f the RSM-fed b i r d s c o u l d be due t o a more r a p i d e x c r e t i o n o f t h i s e l e m e n t was a l s o u n l i k e l y , s i n c e u n p u b l i s h e d d a t a from t h i s l a b o r a t o r y showed t h a t t h e r e was p r a c t i c a l l y no d i f f e r e n c e i n the d e i o d i n a t i o n o r e x c r e t i o n r a t e J . when e x t r a n e o u s r a d i o a c t i v e t r i i o d o t h y r o n i n e was a d m i n i s t e r e d t o t h e SBM- o r RSM-fed b i r d s . T h y r o i d a l h y p e r t r o p h y and a l t e r e d t h y r o i d a l c h a r a c t e r i s t i c s were found i n t h e progeny o f the RSM-fed dams. I t i s c o n c l u d e d t h e r e f o r e t h a t t h e t h y r o i d a l e n l a r g e m e n t o b s e r v e d i n the progeny o f c h i c k s from dams f e d g o i t r o g e n i c RSM i s due t o a low c o n c e n t r a t i o n o f i o d i n e i n t h e eggs. 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The i o d i n a t i o n o f t y r o s i n e by myelo-p e r o x i d a s e and b e e f t h y r o i d s . The p o s s i b l e i n v o l v e m e n t o f f r e e r a d i c a l s . B i o c h i m B i o p h y s A c t a 122:406. Y i p , C and S . J . K l e b a n o f f , 1952. S y n t h e s i s o f t h y r o x i n e by L-amino a c i d o x i d a s e . Endocr 70:931. -94- A P P E N D I X -95- T a b l e 1 ( A ) . E x p e r i m e n t 1: S t a t i s t i c a l a n a l y s i s comparing t h 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 i n White Leghorn p u l l e t s (Shaver s t r a i n ) f e d RSM and/or SBM, 91 weeks o f age. i ) A n a l y s i s o f v a r i a n c e : S o u r c e o f v a r i a t i o n d f MS F P Among groups 3 23719.7 48.0 < 0 . 0 l W i t h i n groups 36 493.9 T o t a l 39 i i ) D u n c a n ! s m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t S-S R-S S-R R-R Ave. t h y r o i d wt. mg/100 body wt. 10.7 23.5 84.9 112.5 Duncan's comparison P < 0 . 0 1 ! a a b c i i i ) S e p a r a t e comparison o f S-S and R-S g r o u p s : Source o f v a r i a t i o n d f MS F P Among groups 1 819.2 39.4 < 0 . 0 i t W i t h i n groups 18 20.8 T o t a l 19 -96- T a b l e 2 ( A ) . E x p e r i m e n t 1: S t a t i s t i c a l a n a l y s i s comparing t h 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 i n White Leghorn p u l l e t s ( D e K a l b - s t r a i n ) f e d RSM and/or SBM, 99 weeks o f age. i ) A n a l y s i s o f v a r i a n c e : S o u r ce o f v a r i a t i o n d f MS F P Among groups 3 35005.0 20.7 <0.01 W i t h i n groups 36 1693.0 T o t a l 39 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t S-S R-S S-R R-R Ave. t h y r o i d wt. mg/100 g body wt. 11.2 28.4 103.4 149.4 Duncan's co m p a r i s o n P < 0 . 0 l „ a a b c i i i ) S e p a r a t e comparison o f S-S and R-S g r o u p s : Source o f v a r i a t i o n d f MS F P Among groups 1 1477.5 69.7 * 0 . 0 L W i t h i n groups 18 21.2 T o t a l 19 -97- T a b l e 3 ( A ) . E x p e r i m e n t 1: S t a t i s t i c a l a n a l y s i s comparing t h e av e r a g e t h y r o i d w e i g h t s (g) o f White Leghorn p u l l e t s ( S h a v e r s t r a i n ) a t 91 weeks o f age. i ) A n a l y s i s o f v a r i a n c e : S o u r c e o f v a r i a t i o n d f MS F P Among groups 3 4.5067 44.4 <0.0J.. W i t h i n groups 16 0.1016 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t S-S R-S S-R R-R Ave. t h y r o i d w t . ( g j 0.1877 0.5175 1.3930 2.3027 Duncan's c o m p a r i s o n P < 0.0'J : a a b c i i i ) S e p a r a t e comparison o f S-S and R-S g r o u p s : S o u r c e o f v a r i a t i o n d f MS F P Among groups 1 0.2720 11.29 <0.0-L W i t h i n groups 8 0.0241 T o t a l 9 -98- T a b l e 4 ( A ) . Ex p e r i m e n t 1: S t a t i s t i c a l a n a l y s i s comparing t h e a v e r a g e e p i t h e l i a l h e i g h t s i n t h y r o i d g l a n d s o f White Leghorn p u l l e t s a t 91 weeks o f age. i ) A n a l y s i s o f v a r i a n c e : S o u r c e o f v a r i a t i o n d f MS F P Among groups 3 28.050 11.53 <0.01 W i t h i n gorups 16^ 2.433 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : Di e t a r y t r e a t m e n t R-S S-S S-R R-R Ave. e p i t h e l i a l h e i g h t s , ja 5.61) 7.13 10.11 10.65 Duncan's comparison P < 0 . 0 i : a a b b -99- T a b l e 5 ( A ) . Ex p e r i m e n t 1: S t a t i s t i c a l a n a l y s i s comparing t h e aver a g e f o l l i c l e d i a m e t e r i n t h y r o i d g l a n d s o f White Leghorn p u l l e t s a t 91 weeks o f age. i ) A n a l y s i s o f v a r i a n c e : Source o f v a r i a t i o n d f MS F P Among groups 3 12905 11.44 <0.0i»! W i t h i n g roups 16_ 1128 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t S-S R-S S-R R-R Ave. f o l l i c l e d i a m e t e r , mm .108 .167 .196 .227 Duncan's comparison P< 0.05'.". a b bc c -100- T a b l e 6 ( A ) . E x p e r i m e n t 1: S t a t i s t i c a l a n a l y s i s comparing t h e p e r c e n t a g e e p i t h e l i u m i n t h y r o i d g l a n d s o f White Leghorn p u l l e t s a t 91 weeks o f age. i ) A n a l y s i s o f v a r i a n c e : S o u r c e o f v a r i a t i o n d f MS F P Among groups 3 111.44 2.99 ^ 0 . 0 5 W i t h i n groups 16_ 37.32 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t R-S R-R S-R S-S P e r c e n t a g e e p i t h e l i u m 14.2 15,1 20.1 24.4 Duncan's comparison a ab abc c P< O.OB: -101- T a b l e 7 ( A ) . Ex p e r i m e n t 1: S t a t i s t i c a l a n a l y s i s comparing t h e aver a g e e p i t h e l i a l w e i g h t s i n t h y r o i d g l a n d s o f White Leghorn p u l l e t s a t 91 weeks o f age. i ) A n a l y s i s o f v a r i a n c e : S o u r ce o f v a r i a t i o n d f MS F P Among groups 3 105615 18.92 <0.0-lI W i t h i n groups li5 5580 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : Di e t a r y t r e a t m e n t S-S R-S S-R R-R Weight o f e p i t h e l i u m , mg 41.1 76.5 277.4 334.6 Duncan's comparison a a b b P^O.OII -102- T a b l e 8 ( A ) . E x p e 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 t h e aver 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 White Leghorn p u l l e t s f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s o f t i m e . i ) A n a l y s i s o f v a r i a n c e : S o u r ce o f v a r i a t i o n : d f MS F P Among groups 3 3.8623 23.09 < 0 . 0 l W i t h i n groups 16_ 0.1673 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t S-S R-S S-R R-R Ave. t h y r o i d w e i g h t s , g 0.2039 0.4733 1.5115 2.9781 Duncan's co m p a r i s o n P<0.0'5 a a b c -103- T a b l e 9 ( A ) . E x p e 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 t h e a v e r a g e i o d i n e c o n c e n t r a t i o n s i n t h y r o i d g l a n d s o f White Leghorn p u l l e t s . i ) A n a l y s i s o f v a r i a n c e : S o u r ce o f v a r i a t i o n d f MS F P Among groups 3 4.879 29.25 <0.01 W i t h i n groups 16^ 0.1668 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t R-R S-R S-S R-S Ave. i o d i n e c o n c e n t r a t i o n , mg/g 0.89 1.08 2.65 2.73 Duncan's comparison P< 0.0.1':, a a b b -104- T a b l e 1 0 ( 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 e t o t a l i o d i n e c o n t e n t s i n t h y r o i d g l a n d s o f White Leghorn p u l l e t s . i ) A n a l y s i s o f v a r i a n c e : S o u r c e o f v a r i a t i o n d f MS F P Among groups 3 1.4305 25.8 < 0 . 0 l W i t h i n groups 16^ 0.0554 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t S-S R-S S-R R-R T o t a l i o d i n e i n g l a n d , mg 0.55 1.25 1.65 1.72 Duncan's c o m p a r i s o n P^O.OSc a b c c -105- T a b l e 1 1 ( A ) . E x p e r i m e n t 3: S t a t i s t i c a l a n a l y s i s comparing t h e t o t a l t h y r o i d r a d i o a c t i v i t y 6 hours a f t e r a d m i n i s t r a t i o n o f t h e r a d i o i o d i n e . i ) A n a l y s i s o f v a r i a n c e : S o u r c e o f v a r i a t i o n d f MS F P Among groups 3 851.00 18.4 < 0.01 W i t h i n groups 16 46.26 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t S-S R-S S-R R-R P e r c e n t a g e o f i n j e c t e d dose 9.08 13.55 30.14 35.36 Duncan's comparison P<0.01' a a b b -106- T a b l e 1 2 ( A ) . E x p e r i m e n t 3: S t a t i s t i c a l a n a l y s i s comparing t h e t o t a l t h y r o i d r a d i o a c t i v i t y 22 hours a f t e r a d m i n i s t r a t i o n o f t h e r a d i o i o d i n e . i ) A n a l y s i s o f v a r i a n c e : S o u r ce o f v a r i a t i o n d f MS F P Among groups 3 1731.54 40.5 <.0.01 W i t h i n groups 16. 42.72 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t S-S R-S R-R S-R P e r c e n t a g e p f i n j e c t e d dose 7.83 10.87 40.57 42.40 Duncan's comparison P < 0 . 0 i : a a b b -107- T a b l e 1 3 ( A ) . Experiment 3: S t a t i s t i c a l a n a l y s i s comparing t h e t h y r o i d r a d i o a c t i v i t y (% i n j e c t e d dose/g t h y r o i d ) 6 hours a f t e r a d m i n i s t r a t i o n o f t h e r a d i o i o d i n e . i ) A n a l y s i s o f v a r i a n c e : S o u r c e o f v a r i a t i o n d f MS F P Among groups 3 933.54 10.40 <0.01 W i t h i n groups 16 89.73 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t R-R R-S S-R S-S % i n j e c t e d dose per g t h y r o i d 15.41 23.13 26.98 47.18 Duncan's comparison P<0.01-' a a a b -108- T a b l e 1 4 ( A ) . E x p e r i m e n t 3: S t a t i s t i c a l a n a l y s i s comparing the t h y r o i d r a d i o a c t i v i t y {% i n j e c t e d dose/g t h y r o i d ) 22 hours a f t e r a d m i n i s t r a t i o n o f t h e r a d i o i o d i n e . i ) A n a l y s i s o f v a r i a n c e : S o u r c e o f v a r i a t i o n d f MS F P Among groups 3 376.34 3.88 <0.G5 W i t h i n groups _16 96.91 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t R-R S-R R-S S-S % i n j e c t e d dos>e per g t h y r o i d 21.61 29.55 35.58 41.96 Duncan's comparison P < 0 . 0 5 ; a ab bc bc -109- T a b l e 1 5 ( A ) . E x p e r i m e n t 6A: S t a t i s t i c a l a n a l y s i s comparing t h e i o d i n e c o n c e n t r a t i o n s i n egg y o l k s from b i r d s on d i f f e r e n t d i e t a r y t r e a t m e n t s . i ) A n a l y s i s o f v a r i a n c e : S o u r ce o f v a r i a t i o n d f MS F P Among groups 2 50473 22.02 <0.01... W i t h i n g roups 18_ 2292 T o t a l 20 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t S-R S-S R-S I o d i n e cone, i n egg y o l k , ppb 144 273 286 Duncan's comparison P < 0 . 0 l - a b b -110- T a b l e 1 6 ( A ) . E x p e r i m e n t 6B: S t a t i s t i c a l a n a l y s i s comparing t h e i o d i n e c o n c e n t r a t i o n s i n egg y o l k s from b i r d s on d i f f e r e n t d i e t a r y t r e a t m e n t s . i ) A n a l y s i s o f v a r i a n c e : S o u r c e o f v a r i a t i o n d f MS F P Among groups 3 43093 4.321 <0.05 W i t h i n g r o u p s 20_ 9973 T o t a l 23 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t RSM RSM+I SBM SBM+I I o d i n e cone, i n egg y o l k , ppb 41 73 105 241 Duncan's c o m p a r i s o n P<0.01 a a a b i i i ) S e p a r a t i o n c o m p a r i s o n o f SMB and RSM g r o u p s : S o u r c e o f v a r i a t i o n d f MS F P Among groups 1 12352 10.78 < 0 . 0 l W i t h i n groups l f j 1145 T o t a l 11 T a b l e 1 7 ( A ) . E x p e r i m e n t 7: S t a t i s t i c a l a n a l y s i s comparing t h e av e r a g e t o t a l r a d i o i o d i n e i n 5 l a r g e s t o v a r i a n f o l l i c l 22 hours a f t e r a d m i n i s t r a t i o n . i ) A n a l y s i s o f v a r i a n c e : S o u r c e o f v a r i a t i o n d f MS F P Among groups 3 21.5 39.09 <0.01 W i t h i n groups 16_ 0.55 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t S-R R-R R-S S-S % i n j e c t e d dose i n 5 l a r g e s t o v a r i a n f o l l i c l e s 2.624 2.830 5.796 6.717 Duncan's comparison P<0.01 a a b b i-3-ttIHDS—PERMCA3IL IT V A?i|) f-CUl. ICuL A'* 317F R E G R E S S I O N A N A L Y S I S , T H L OFPE'M.'C'. f V A ^ I A H I F IS L O T , PERM CONt»rri!|t . / f iM TO T IPLE REGRfcSSI 0" I »:r-l'.(;LM)i M V A I' I A h L f; Hr GRFSSI ON CCEhT J C U NT S T A N D A R D E R R O R Of- T H L : RtGR. C O E F F . " • P i - T T i l . Cut f F I C 11 'N r O F DfcTfcRuI'NATIOM VARIANCE RATIO (K} S.AKTA L (.1 r; A W r. A •0.982Sn3F-01 0.2708SSK+01 INTERCEPT STANDARD ERROR OF ESTIMATE -RESIDUAL "VARIANCE MULTIPLE CORRELATION" COEFFICIENT R SQUARED -VARrA^CE-RATTO—CF1 " THE VARIABLE TO 8E 0 * I T T l D IS 0. 1 1 1586C-01 0, J.286Q4E + 00 •10.27 : -0.119S75E+0 1 = o.i634H<iEi-oo = O.2672.70K-01 " ' " " = 0.HU278 = 0.77931 _.. 3 a t P ' n « — W I T H — 2 — — 2 2 = lOG AREA i: G R E e s~nr-F R F F D c * ~ 77.S36 6 7,9C0 H 03 cf 00 REGRESSION A N A L Y S I S , T H E O F P E N U E N T V A R I A B L E I S L O G P E R M C O N T R I B U T I O N TO M U L T I P L E R E G R E S S I O N INDEPENDENT - V A R l A H L F S.AREA R E G R E S S I O N " C O E F F I C I E N T " ' •0. I 0 8 9 l 8 t - 0 l S T A N D A R D E R R O R O F T H E ~"RF-"',R7""C'0EFr',~- 0.689903L-02 P A R T I A L C O E F F I C I E N T F D F T E R V I N A T I G V 9.78 V A R I A N C E -RATT0~TFT" 2.«92 INTERCEPT STANDARD CRROR OF ESTIMATE "RE St at)* C VTl'RIA M C E MULTIPLE CORRELATION COEFFICIENT R SQUARED "VTRTA'NCE RATIO Cr ) = 0.39l6l4b" + UO = 0.323268E+00 " =—TJTTO"fl STbTTOXT = 0,31269 = ' 0 .09777 -770-9-2 Vt TH I A~WO 23 DTTTJRECS OF FRfcEDO fr -prPTrvorrrr-vTVRTA-SLE is ptnx- CCNTRI3UTI0N T O MULT lPi.E REGRESSION I N D E P E N D F . N T V A R I A B L E REGRESSION COEFFICIENT ~S'TAN"D"A'RrD E R R O R " O F THE R E G R , COEFF, PA - R T T A T COE'- f I.LTcNT OF DETERMINATION V A R I A N C E R A T I O ( F ) S . A R E A A R E A A A 2 ~A R E"A"3F*3 _ 0.712H40r. + i)0 •0.388R21E-01 -Q-.-c;6-220-8E-iDT 0, l«7068E->00 0.923H8E-02 •275,2h 6 9 S . 3 0 -34 r.SQ' 23,u94 17.740 ~I 0 , 7 / i INTERCEPT TANHARD"ERROR OF EST IHATE RESIDUAL VARIANCE MULTIPLE CORRELATION COEFFICIENT -R—STJARED VARIANCE RATIO CF) THE VARIABLE TO BE OMITTED IS -0 . 7 f i 8 5 6 5 E + 00 "<r.'5 73'13GE"*'0~n" 0,32a«82E+nn 0.rt'.-il83 -QT72562 18.^12 WITH A N D 2 1 D E G R E E S O F F R K E D O M A R E A * * 3 W X X) —TO— 3 3 rt "W" H n co -40- O Pi ca ca / rv3-Bik0S- r m v R E G R E S SION A N A L Y S I S , THE DEPENDENT VARIABLE is LOG PFRM COivT-< I H I I I r n f M TO --rit M f L t V t G R E S S I C M . INDEPENDENT VARIABLE S . AREA ICG 4RFA INTERCEPT STANDARD ERROR OF ESTIMATE -RCStOUAt --VA-RrA-WCC MULTIPLE CORRELATION C O E F F I C I E N T R SQUARED -VSRTA-N C E-R-ATIO tfl THE VARIABLE TO BC OMITTED IS R E C E S S I O N COEFF IC IENT • 0 . 7 B 5 U 2 9 E - 0 1 0 . 2 i « 0 0 4 c + 0 1 STANDARD CRWOR ~ CF THE REGR. C O E F F . 0 , 106521E-01 0.27n9fl,TE + 00 PAKTJ AL COf.Fr IC1FNT fiF OETEV"INAT tOl- 4 .11 71 .23 v«?f i A>.r E RATIO CF) = - 0 . 9 7 3 1 C 6 E + 0 0 = 0.1S6303E+UO ~s—"0"- 2 W S 0 7 E - ' 0 1 _ = 0 . 6 6 7 9 5 = 0 . 7 ^ 3 3 5 - = •- = S . A R E A WTT H 2 —AND 2D DEC R ETS~ 5 4 . 3 6 6 61 .060 H (t> r-» SO REGRESSION A N A L Y S I S , THE DEPENDENT VARIABLE IS LOG PERM C O N T R I B U T I O N TO M U L T I P L E R E G R E S S I O N INDEPENDENT — VARIABLE" r L O G AREA REGRESSION " C O E F F I C I E N T ' 0 .253555E + 00' INTCRCEPT STANDARD ERROR OF ESTIMATE -RESIDUAL"VARIANCE" MULTIPLE CORRELATION C O E F F I C I E N T R SQUARED -VXR17TNCE RATIO ( D : = - 0 . 8 0 1 9 9 4 E - 0 1 •- = 0 .294138E+00 " = 0 . 8 6 5 1 7 I E - 0 1 ~"T = 0 . 2 8 7 8 3 = 0 ,09284 = r.""897 T I T H - 5SIOW-7rN"ATTSTS7 T̂ C" DEF»CEDENT vART*BLE""rS—PERM- INDEPENDENT VARIABLE REGRESSION C O E F F I C I E N T S'.AREA A R F A * * 2 ~AREA**3" 0 .561084E+00 - 0 . 2 7 8 9 2 1 E - 0 1 ~~0;3S"2982EiT;r" STANDARD ERROR OF THE — R E E R , - - C O E F F — " 0 .184100E+00 PARTIAL COEFFIC IENT "OF "OE'TERMINAT I OF! . . . 8 . 2 6 VARIANCE RATIC"CF} 1 . 897 T 3TN13 2T DTn5Rnrs~DF~FREED'OH— CONTRiauTICN TO MULTIPLE REGRESSION "ST AWAR D ERHTJR- CF THE REGR, C O E F F , PTRTTAL" COEFF IC IENT OF DETERMINATION V A R I A \ C E RATIO CF) 0 .213811E+00 0, 152627E-01 "0,3T3TJ2r-0T" - 4 4 . 0 6 197 .33 - -99753~ 6 ,886 3 .340 1,271 W X -tf- a H S rt -sv- 09 n (D rn CO H - O 3 Co 03 CO INTCRCEPT "STAND A R D~C R R O R - O F ^ E S T I H A T E - RESIDUAL VARIANCE MULTIPLE CORRELATION C O E F F I C I E N T ~R~S OU'A R ETT : VARIANCE RATIO (F ) THE VARIABLE TO BE OMITTED IS = - 0 , S 9 2 9 5 S E + 0 0 -=— 0 .73 61 07E+ 0 0- = 0 ,617964E+00 0 , 7 3 3 1 2 "= 0Y53747" = A R E A * * 3 7', 359 WITH AND 19 DEGREES OF FREEDOM 1 f S-n-Bjkrys REGRESSION ANALYSIS, THE DEPENDENT VARIAHI.K i s L „ G PERM CONTRJhuT IPM TO ruLTCPLt" REGRESSION I NOEPErJOEN T V A R I A rt L F S'.AREA L O G AREA REGRESSION COEF (• ICIb NT -0.H5704'Jfc-01 0.216407E+01 'STANDARD E R R O R S OF THE . REGR. COEFF, P'A»TT AC COEFFICIENT OF DETER"I NATION V A R I A N C E R A T I O (F) INTCRCEPT STANDARD ERROR OF ESTIMATE -RrstntTAr-VA-RrATgcrr 0.141453E-01 0.363090E+00 37.95 25.20 MULTIPLE CORRELATION COEFFICIENT R SQUARED "VAWTflNTC RATIO (h ) THE VARIABLE TO SE OMITTED IS = - 0 , 123046F_tyi = 0.250676EtU0 - 07628394F-TJT~ = 0.79407 = 0.63150 36,710 35.523 LOG AREA -ffTTH 2 AW 22—TTFGRCEt) Or FRETOTna- R E G R E S S I O N A N A L Y S I S , T H E D E P E N D E N T V A R I A H L F . I S L O G PERM CONTRIBUTION TO M I J U U P L F REGRESSION INDEPENDENT "VTRTA-OCE— S . A R E A I N T E R C E P T STANDARD ERROR OF ESTIMATE ~ESlDUAL" VATrrffwCE- REGRFSSION '"CTjEFrrcTeMT— -0.823994E-02 STANDARD ERROR OF THE RETTR7 COEFT"J— . PARTIAL COEFF- ICIENT OF •Ut.ItKMTKJtTTCm- VARIANCE RA i iu u y 0.832869E-02 MULTIPLE CORRELATION COEFFICIENT R SQUARED V T R T A N C E R A i i u c n = -0,117142C+00 = 0.396438F+00 = 0.157I63E+00 = 0.19103 0.03649 = 0T87T 3 , 6 5 0,871 T T H ~ 1 ^A'NO 2 3 D E G R E E S UF FREED07T REGRE^SrOfr-Am-YSIS-TTHC D E P E N D E N T V A R I A B L E ' S - P E R M CONTRIBUTION TO MULTIPLE REGRESSION INDEPENDENT VARIABLE REGRESSION C O E F F I C I E N T "STAND A RT)—ERRCFT OF THE REGR, COEFF, S'.AREA A R K A A * 2 _ARE"A i"*3 - PSR7TSL~ COEFFICIENT OF DETERMINATION VARIANCE RATIO ( F ) I N T E R C E P T - S T A N D A R D - E R R O R " O F ' T S T I M A T E R E S I D U A L VARIANCE M U L T I P L E CORRELATION C O E F F I C I E N T ~R~S CUT* R ED - V A R I A N C E RA T I O CF) THE V A R I A B L E TO BE OMITTED I S 0.216527E+00 •0.114076K-01 "tr7T5'*nr29r-Trr 0.857160C-01 0.598289E-02 '57119723E-TJ3- -120.55 324.45 ~-T517T9- = -0,625384E-U1 = 0.3n9C37E+00 = 0,l3fa780EtoO = 0.71212 = ~ r f 5 0 7 1 r 6 . 3 B 1 3 . 6 3 6 l.oOO s AREA * *3 7,202 *ITH AND 21 DEGREES OF FREEDOM PAGE H Co Tr- io O X T3 H 3 -»- 3 rt 09 a -co— p. 3 To"" 3 0> CO CO REGRESSION A N A L Y S I S , THE DEPENDENT VAkJ ArtL.E I S LIT, PERM CONTRIBUTION -T ti v-; i L T J P L E RttiKr.SS-lON I N D E P E N D E N T V AH I A B L E S . A R E A L O G A R E A REGRESSION C O E F F I C I E N T • O . l C S l u E + O C 0 .279482E+01 I N T E R C E P T STANDARD ERROR OF ESTIMATE - R E S IDU AL~~V ARI"ANCE~ M U L T I P L E CORRELATION C O E F F I C I E N T R SQUARED "-VARIANCE RATTtT^fF) - 0 . 144323E+01 0 . 159572E + 00 0 . 2 5 4 6 3 3 E - 0 1 0 . 9 0 9 6 8 0 , 8 2 7 5 2 \ STANDARD" ERROR OF THE RFGR. C O E F F . 0 . 106617E-01 0 t 281865L+0O " ? A R T T A L CUf.r F ICIENT OF DL T t R'' I (•' AT 101>' C.f-.95 15.80 THE V A R I A B L E TO BE OMITTED I S T27777- L0G AREA WITH AND ' - D E G R F F S " O F " F R E E D O M " V A K I A N C F P A T ] D f F ) 105 .123 9 8 . 5 1 7 REGRESSION A N A L Y S I S , THE DEPENDENT V A R I A B L E I S LOG PERM INDEPENDENT - — V A R I A X L E - S'. A R E A R E G R E S S I O N C O E F F I C I E N T " - • 0 . 9 2 6 4 3 2 E - 0 2 INTERCEPT' " \ . STANDARD ERRORS OF ESTIMATE - R E S T DO At V ARTA~NCE MULTIPLE CORRELATION C O E F F I C I E N T R SQUARED VARIANCE RATIO CF} = 0 . 2 0 0 2 2 5 E - 0 1 = 0 , 3 6 4 9 6 9 E + 0 0 "="—OT 1 3 a 0 , 2 3 8 2 1 = 0 , 0 5 6 7 4 C O N T R I T I O N TO M U L T I P L E R E G R E S S I O N "T73B"3 WITH S T A N D A R D E R R O R Or THE " R E G R ; " c o E r F ' i 0 , 7 8 7 6 0 5 E - 0 2 PARTIAL COF.FF IC1F.NT "OF "DETER'1 IN ATI ON " 5 .67 VARIANCE RATIO"-CF"} 1.384 T A"NTT :3 mrcTfLEs UF FREEUDH~ -CO- "DElTrNDENT VARTABtTE TS PERW- I N U E P E N D E N T V A R I A B L E R E G R E S S I O N C O E F F I C I E N T I N T E R C E P T -STANDARD-ERROR—OF E S T I M A T E " R E S I D U A L VARIANCE M U L T I P L E CORRELATION C O E F F I C I E N T R SQUARED VARIANCE RATIO ( F } THE V A R I A B L E TO BE OMITTED I S S'.AREA A R E A * * 2 - A K E A W 3 - 0.443172E+00 • 0 . 2 6 3 6 0 8 E - 0 1 -0-.4IB8"03t-0"3- -0 .542931E+U0 O",3S3969F + 00 0 . 147432F + 00 0 .30780 -0TG525JI C O N T R I B U T I O N TO M U L T I P L E R E G R E S S I O N ""STAND A PTJ—"E RR D R"~ OF T H E R E G R , C O E F F , 0. 1 14627E + 00 0.814305E-02 -0","-ir>72^BE"-0T- T X R T T"A E - COEFFICIENT OF DETERMINATION • 2 4 6 . 1 8 64 t .31 •T27TCT- V'ARlAr.CE RATIO CF) 13 .146 WITH ANO = A R t A**3 DEGREES OF FREEDOM 14.947 10,480 "5 .212 -116- T a b l e 2 2 ( A ) . Experiment 8: S t a t i s t i c a l a n a l y s i s comparing t h e a v e r a g e t h y r o i d w e i g h t s o f progeny when dams were 91 weeks o f age. i ) A n a l y s i s o f v a r i a n c e : S o u r ce o f v a r i a t i o n d f MS F P Among groups 3 .0000175 0.99 n.s. W i t h i n groups 76_ .0000177 T o t a l 79 -117- T a b l e 2 3 ( A ) . E x p e r i m e n t 8: S t a t i s t i c a l a n a l y s i s comparing t h e a v e r a g e p e r c e n t a g e e p i t h e l i u m i n t h y r o i d g l a r i d s o f progeny a t h a t c h i n g . i ) A n a l y s i s o f v a r i a n c e : S o u r ce o f v a r i a t i o n d f MS F P Among groups 3 179.50 3.77 <0.05 W i t h i n groups 16_ 47.58 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : Di e t a r y t r e a t m e n t o f dam S-S R-S S-R R-R % t h y r o i d e p i t h e l i u m i n progeny 43.0 49.2 55.1 55.8 Duncan's c o m p a r i s o n P<0.05 a ab b b -118- T a b l e 2 4 ( A ) . E x p e r i m e n t 8: S t a t i s t i c a l a n a l y s i s comparing t he av e r a g e h e i g h t s o f t h y r o i d e p i t h e l i u m o f progeny o f b r i d s f e d RSM o r SBM f o r d i f f e r e n t p e r i o d s . i ) A n a l y s i s o f v a r i a n c e : S o u r c e o f v a r i a t i o n d f MS F P Among groups 3 1.369 2.894 <0.10 W i t h i n groups 16 0.473 T o t a l 19 i i ) Duncan's m u l t i p l e range t e s t : D i e t a r y t r e a t m e n t o f dam;.; S-S R-R R-S S-R Ave. h e i g h t s o f e p i t h e l i u m , JJ 3.864 4.708 4.761 5.092 Duncan's comparison P 4.0.05 a ab ab b R-S -120- Photnricrographs: (I60x) Thyroid gland R-R

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