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Effects of chronic and early goitrogenic stimulation on thyroidal metabolism in the chicken and in the.. Leung, Peter Chung Kwor 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 of 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 required standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1975  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y I  further  fulfilment  the U n i v e r s i t y of  s h a l l make it  agree  in p a r t i a l  freely  of  the  requirements  B r i t i s h Columbia, I agree  available  for  t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f  of  this  representatives. thesis for  It  financial  this  thesis  The  of  gain shall  Poultry Science  U n i v e r s i t y o f B r i t i s h Columbia  2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5  Date  August 15, 1975  or  i s understood that copying or p u b l i c a t i o n  written permission.  Department  that  reference and study.  f o r s c h o l a r l y purposes may be granted by the Head of my Department by h i s  for  not  be allowed without my  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 or both the growing p e r i o d and the l a y i n g period.  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 .  Control rations  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 . 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 o v e r 90 weeks o f age.  The  were s t u d i e d when the b i r d s were  P a r a m e t e r s measured i n c l u d e d :  weights,  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 the 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 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 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 substances  i n the 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  the 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  hatching.  B i r d s which were b e i n g f e d rapeseed meal a t the t i m e the 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 m a g n i t u d e a c c o r d i n g t o the l e n g t h o f t i m e over which rapeseed 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 rapeseed m e a l - f e d b i r d s 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  increased  time.  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 the t h y r o i d g l a n d  induced  by the 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 the g r o w i n g 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 goitrogen from the d i e t .  Thyroid weights, thyroid iodine  contents  and r a d i o i o d i n e u p t a k e were a l l i n c r e a s e d i n b i r d s w h i c h had g o i t r o g e n d u r i n g the growing p e r i o d .  received  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  -iii-  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 l o w e r in::the t h y r o i d g l a n d s  of  t h e s e b i r d s than 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 .  Since  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 thiocyanates  indicated similar concentrations  i n the eggs from b i r d s  f e d rapeseed meal and from c o n t r o l b i r d s f e d soybean m e a l , the hypot 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 the 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 the amounts r e a c h i n g the d e v e l o p i n g ova were r e d u c e d .  -iv-  TABLE OF CONTENTS Page i i  ABSTRACT TABLE OF CONTENTS  iv  LIST OF TABLES  vi  LIST OF FIGURES  vii  ACKNOWLEDGEMENTS  viii  INTRODUCTION  ix  REVIEW OF LITERATURE BIOSYNTHESIS AND RELEASE OF THYROID HORMONES  1  EFFECTS OF GOITROGENS ON THYROID HORMONE BIOSYNTHESIS  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  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  15  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  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  EXPERIMENTAL  -  V  -  E x p e r i m e n t 5: EitEECTSi,OF GOITROGENIC RSM ON SUSCEPTIBILITY TO HYDROLYSIS OF THYROGLOBULIN  39  E x p e r i m e n t 6A: EFFECTS OF GOITROGENIC RSM ON THE IODINE CONTENT OF EGG YOLK  42  E x p e r i m e n t 6B: EFFECTS OF INCREASED DIETARY IODINE INTAKE ON IODINE DEPOSITION IN EGG YOLKS OF SBMAND RSM-FED BIRDS  46  E x p e r i m e n t 7: OVARIAN UPTAKE OF A SINGLE DOSE OF 1-131 BY DEVELOPING FOLLICLES  50  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  55  THYROIDAL RESPONSE TO CHRONIC GOITROGENIC STIMULATION BY RSM  60  PERSISTENCE OF EFFECTS OF EARLY GOITROGENIC STIMULATION  67  EFFECTS OF ALTERATIONS IN MATERNAL THYROID METABOLISM ON EMBRYONIC THYROID DEVELOPMENT IN THE CHICK  70  DISCUSSION  SUMMARY AND CONCLUSIONS  72  BIBLIOGRAPHY  75  APPENDIX  LIST OF TABLES  Table  Page  1  Percentage composition o f d i e t s fed i n experiment 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-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  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 hydroly^ates a f t e r d i f f e r e n t times o f pronase 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 t i m e 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  - vii -  TABLE OF FIGURES Figure  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  - viii -  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 h e academic  field. 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 extended t o t h e  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 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  Dr. A.M. P e r k s  Department o f Z o o l o g y  Science  Science  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 mention 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.  -IX-  INTRODUCTION  The t h y r o i d g l a n d i s one o f the f i r s t e n d o c r i n e o r g a n s 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 unclear.  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  formation  o f the t h y r o i d hormones, b o t h 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 u n d e r p h y s i o l o g i c a l is s t i l l  conditions  uncertain. The p i c t u r e becomes more c o m p l i c a t e 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 .  when the t h y r o i d Thyroid function  gland 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 the 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 the e f f e c t s o f the a n t i t h y r o i d compounds on t h y r o i d tions of experimental  func-  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 the 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 the t h y r o i d gland.  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  early stimulation?  by g o i t r o g e n s when t h e y a r e l a t e r w i t h d r a w n .  In the 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  chickens  e i t h e r j u s t f o r the 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 the e f f e c t s o f the 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 the most v a l u a b l e o i l s e e d crops i n Canada, both f o r its p o t e n t i a l i n the production  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 m e a l s s u i t a b l e f o r s u p p l e m e n t i n g a n i m a l diets.  I t has been i n d i c a t e d i n the 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 the 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  anti-  -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 ,  the 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 m o n o g e n e s i s , 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 o r t h y o f i n v e s t i g a t i o n from t h e a c a d e m i c 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  standpoint. stimulation  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 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 v  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 t h e 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 (Wilgus 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  alimentary t r a c t , d i e t a r y iodide i s r e a d i l y absorbed i n t o the 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  within the thyroid.  The r o l e o f t h e 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 ext 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 al_, 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 t h e p r e s e n t d i s c u s s i o n , we w i l l review o n l y t h e more  r e c e n t d e v e l o p m e n t s i n t h e 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 agents. 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 h e 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 h y d r o g e n  peroxide,  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  iodothyronines  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 circulation.  - 2 -  I o d i d e t r a p p i n g by the t h y r o i d i s d e p e n d e n t on a d q u a t e p o t a s s i u m concentration  ( 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  agents that uncouple o x i d a t i v e phosphorylation  such as  2,4-dinitrophenol,  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; and I n g b a r , 1955; W o l f f , 1964).  Freinkel  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  process  r e q u i r i n g energy r e l a t e d with the Na-,K -ATPase-dependent t r a n s p o r t +  s y s t e m ( 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 the s i t e  o f t h i s i o d i d e pump mechanism, w h i c h 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 the 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 al_, 1962). been suggested  I t has  t h a t a l e c i t h i n phospholipid i s a c o n s t i t u e n t of the  t o r s i t e ( V i l k k i and J a a k o n m a k i , 1966) and a r o l e o f F e  + +  recep-  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 the t h y r o i d c e l l s et_ al_, 1964; Thomson and G o l d b e r g ,  (Nadler  1968); i t then 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 the 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 L e e d , 1965; Robbins and R a i l , 1960;  R a i l e t a l _ , 1960) and a  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  and  sedimentation  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 the 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 the 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 the 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; V a l e n t a e t a l _ , 1968).  Ermans 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 ; they have r e c e n t l y have d i s c u s s e d by DeGroot e t al_ (1972). require generation,  2I"^I  It i s widely accepted that i n v i v o i o d i n a t i o n s  o f H,,0  2  o x i d i z i n g agents with  thoroughly  which, together with 0  an.jDxidation^reduction  2  a r e the o n l y b i o l o g i c a l  p o t e n t i a l above t h a t o f the  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  2  Rosenberg, 1970).  and  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_ al_, 1966, 1968) a c t i v i t y have been suggested  t o be the o r i g i n o f H 0 , t h e c o f a c t o r NADPH i s l i k e l y t o be 2  2  i n v o l v e d i n t h y r o i d a l H 0 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 NADPH2  cytochrome C reductase  2  (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 , M o r r i s o n , 1967; Mahoney and Igo, 1966;  1959; Hosoya and  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 to Protoporphyrin  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 0 f o r peroxidation of i o d i d e to iodine — 2  2  probably to i t s  f r e e r a d i c a l f o r m , I ( K l e b a n o f f e t al_, 1962; Y i p and H a d l e y , 1966) — 0  pero-  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 iodine to tyrosine present 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 " p i n g - p o n g " mechanism o f o r g a n i f i c a t i o n , w h i c h p r e s u m a b l y 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 H 0 . 2  2  complexed w i t h  U n i o n 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  state.  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 the 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 consonant with a precursor-product s i n e s ( P l a s k e t t e t a l _ , 1963a).  r e l a t i o n s h i p between t h e two  iodotyro-  When the 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 the MIT/DIT r a t i o ( Q u e r i d o e t a l _ , 1957; Ermans e t a l _ , 1 9 6 1 ) , w h i c h , as m e n t i o n e d e a r l i e r , i s d e p e n d e n t 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 pi ling 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 1  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 slower process  ( P i t t - R i v e r s , 1962;  P l a s k e t t e t al_, 1963b).  Although 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 , 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  i s not 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 can be formed i n t h y r o g l o b u l i n (DeGroot e t al_, 1972): rearrangement, i . e . conformational  iodothyronines  1) by  intramolecular  by an o x i d i z i n g a g e n t such as  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 pyruvate  thyronine  changes w i t h i n t h e t h y r o g l o b u l i n w h i c h  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 (produced peroxidase-I")  1952),  (DIHPPA),peroxidation  o f DIT t o form h y d r o x y p h e n o l -  o f the enol 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 al_, 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 al_, 1969a,b), the 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  uncertain.  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 of the t h y r o g l o b u l i n polypeptide.  Recent  electronmicrographical  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 the re-entrance of t h y r o g l o b u l i n i n t o the c e l l f o r r e l e a s e to 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 thelysosomes(Wollman  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 to a f f e c t target 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) the adenohypophysis. t h y r o i d metabolism  from  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 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 feedback c o n t r o l by t h y r o i d hormones and t h e hypothalamic;\ppn,tr p:lcby T S H - r e l e a s i n g hormone (TRF) v i a t h e hypothalamof  hypophyseal  p o r t a l system ( S c h a l l y e t al_, 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 iodine metabolism  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 appears 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).  Solo-  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 animal 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 a c c 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 l o 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 a n d t h e mammalian g l a n d s ( S h e l l a b a r g e r a n d 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 t h e 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 n o 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) h a s 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; Newcomer, 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 t h e 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 t h e 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 e x 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 oithers,  i n o r g a n i c i o n s s u c h a s 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 t h e r a t -- t h e 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 t h e 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; e t al_, 1945).  Astwood  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 -  Disturbances  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 can 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) ^defect 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 presence of elemental  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 iodothyroncines;  5) i n a b i l i t y t o  hydrolyze  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 " substances; tyrosines for reutilization.  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 -  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 .  For 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 i o d i d e w h i l e the p e r o x i d a s e sulfonamides  i s a c t i n g (DeGroot and D a v i s , 1962).  the The  i n t e r f e r e with o r g a n i f i c a t i o n of iodine without 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 the 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 but i n c r e a s e t h y r o i d hypertrophy  i n animals t r e a t e d with sulfonamides  ( M a c K e n z i e 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 the 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 to completely 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 t h i o u r a c i l (TU); p r o p y l t h i o u r a c i l (PTU) and  (MIA);  ()-)-5-vinyl-2-thio-oxazolidone  ( 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  thiourea;  goitrin.  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  goitrin.  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  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 rapeseed  except  in a following  meal.  Many s t u d i e s have been f o c u s e d on the 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 K i r k w o o d , 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.  1958)  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 the 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 - a m i n o b e n z o a t e , and 3-amono1 , 2 , 4 - t r i a z o l e are potent i n h i b i t o r s of i o d i d e peroxidase  (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  also  possess i n h i b i t o r y a c t i v i t y on the v a r i o u s t h y r o i d enzymes such as peroxidase,  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  (Langer 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 the t h y r o g l o b u l i n and of lysosomal i n man  suppression  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  (Beckers 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 the o i l s have been e x p e l l e d o r  are 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  solvent-extracted, feedstuffs  (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 u t k o w s k i , 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 the 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 (VanEtten  1969).  glucosinolates  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 are  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 the 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  gluco-  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 are b i o l o g i c a l l y i n a c t i v e .  Destruction of the  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 the 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 ,  while  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 ; organic isothiocyanates; inorganic thiocyanate  ( 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 buted 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 hydrolysis product  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 .  of  I t i s p r o b a b l y not 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 myros 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 the 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  the  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 the 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 .  This,  however, i s not borne out 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 rapeseed m e a l - t r e a t e 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 mal."  chicken thyroid  and h y p e r p l a s i a o f p a r e n c h y -  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 .  f u r t h e r s u p p o r t e d by the p h o t o m i o r o g r a p h i c  This i s  f i n d i n g s o f Rutkowski  (1971),  i n which d e s q u a m a t i o n 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 the thy-  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 the r a t i o n (Rutkowski,  1971).  A s y n e r g e t i c i n f l u e n c e o f the 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 (the 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)  that  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 the 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 the 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).  Thyroid hypertrophy  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 al_, 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 the known g o i t r o g e n i c compounds o f r a p e s e e d : oxazolidone  (>-)-5-vinyl-2-thio-  (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-vinyl-2-thio-oxazolidone  (goittrim) 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 (Langer e t ^ a l _ , 1971). was f i r s t i d e n t i f i e d i n rapeseed  by Astwood ejt al_ (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 thione.  It  and l a t e r renamed  (-)-5-2-oxazolidine-  Its synthetic analogue,,(+)-5-vinyl-2-oxazolidinethione,  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  was  (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 the 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 u p t a k e i s g r e a t l y reduced 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 . Chick growth r a t e i s d e p r e s s e d . w i t h accompanying h y p e r t r o p h y  Rapid e n l a r g e m e n t o f the g l a n d s and h y p e r p l a s i a .  follows  Iodine uptake 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  reach  physiological e q u i l i b r i u m at increased thyroid-to-body weight r a t i o s . When c o m p a r i n g 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 e t h i m a z o l e ) and 6 - m e t h y l - t h i o u r a c i l  (methiocil) in  g r o w i n g 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 h i c h 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 p t a k e o f 1-131  and the 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 . i n c o r p o r a t i o n o f b l o o d 1-131  They c o n c l u d e d t h a t the  by the t h y r o i d g l a n d i s not i n h i b i t e d and  t h a t , from the 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  secretion  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). g a t o r s showed t h a t w h i l e the PTU c h i c k s do not m a i n t a i n 1-131  These i n v e s t i incorporated  i n the t h y r o i d g l a n d but r e l e a s e i t i n t o the 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 the 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 .  the p e r c e n t a g e  Furthermore,  since  o f iodothyronin.es (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 substances  found i n t h y r o i d homogenates, and t h a t  the 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 the 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 g r o u p , 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 the m o n o i o d i n a t i o n 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.  o f t y r o s i n e , but i t i n h i b i t s The f i n d i n g o f i n c r e a s e d MIT  per-  centage i s i n agreement w i t h r e s u l t s o f l i n o e t al_ (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 o s e s o f  PTU.  Reports on the e f f e c t o f PTU o r t h i o u r a c i l on the 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.  For example, t h i o u r a c i l i n h i b i t s the  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; M a y b e r r y 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 Ingbar, 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 :  oxidation of iodide,  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 1971).  i n both groups ( A k i b a and Matsumoto,  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 the 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 not 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 . Moreover, i n t h e i r study of t h y r o i d f u n c t i o n of chicks a f t e r withdrawal  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 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 .  of t h y r o i d gland  A f t e r withdrawal  of 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 demonstrated. withdrawal  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  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  (D'Angelo e t a l _ , 1951, 1954; L i p n e r e t al_, 1959; S t u d e r and G r e e r ,  pigs 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 the unblocked withdrawal  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 ( D ' A n g e l o , 1961, 1969).  w e i g h t a f t e r g o i t r i n withdrawal PTU w i t h d r a w a l  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  i s i n agreement w i t h t h a t r e p o r t e d f o r  i n c h i c k s ( A k i b a e t al, 1971), r a t s (Shimoda, 1960)  g u i n e a p i g s (D'Angelo e t al,  and  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 p e r f o r m e d 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 ,  -141965; 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). p r o t e i n supplement  In an e x p e r i m e n t where r a p e s e e d meal was used a s a i n t h e 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 t h e 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 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 have normal p h y s i o l o g i c a l function with regard t o the egg-laying process. 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 aggregations o f thyroprotein related t o the observation 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 (1965), 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, m i c e 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 . It appears t h a t g o i t r i n i s not 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^ al_, 1972). newly-hatched  The t h y r o i d g l a n d s o f  progeny of.dams f e d rapes'eed m e a l , however, showed  hypertrophy,  which was g r e a t e r when t h e 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  than 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).  -15E 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 hypertrophy  and h y p e r p l a s i a .  However, s i n c e most o f the h i s t o l o g i c a l  d a t a a r e on g r o w i n g c h i c k s , l i t t l e i s known about the 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 , the e x t e n t t o w h i c h  a d a p t a t i o n o f the 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 the mechanism o f compens a t i o n has not been e s t a b l i s h e d .  When c o m p e n s a t o r y f u n c t i o n s o f 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 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 not 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 the 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 alleviated.  Hence the 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 the 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  exceptions  o f e x p e r i m e n t s 5 and 6B), the b i r d s u s e d were on an e x p e r i m e n t d e s i g n e d 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 meal (RSM)  to rapeseed  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 o w i n g a n d / o r 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 ( S h a v e r 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 g r o w i n g 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 g r o w i n g 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 bronchitis.  Causes o f m o r t a l i t y were  recorded.  -16A 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 and f e d t h e 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.  cages  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 t h e 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 t h e o t h e r h a l f were t r a n s f e r r e d t o t h e 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 g r o w i n g 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 S h a v e r 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 . f o r measurements  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  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 micrometer f i t t e d to the l i g h t microscope. 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 weights only. 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 t h e 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).  -17G 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 W h i t e 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.  Six chicks  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 .  Results:  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  established  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 data o f the t h y r o i d glands o f the 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.  Total percentage thyroid epithelium in  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 . percentage epithelium  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  (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)  a l s o l o w e r than t h a t o f t h e c o n t r o l .  was  -18Consistent with the appearance o f enlarged glands, the average 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 t h a t o f t h e 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 .  over Again,  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 . showed t h e 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 .  The R-R and S-R g l a n d s  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 t h e 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 . 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 .  No  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 t h e c h r o n i c a l l y RSM-stimulated  birds.  -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 experiment  1 and  RSM g o i t r o g e n i c i t y t e s t  Experiment Code L e t t e r  1  Starter-Grower S R  RSM-Goitrogenicity Test S R  Layer S  R  13.1  -  Ingredient Soybean meal Rapeseed meal Ground wheat Ground c o r n Ground o a t s Dehydrated c e r e a l g r a s s Bone meal Limestone Iodized s a l t Feeding t a l l o w DL-Methionine L-Lysine T r i c a l c i u m phosphate Micronutrients  */kg:  11.0 66.5 15.0 2.0 2.0 1.0 0.5 2.0 -  17.5 60.0 15.0 2.0 2.0 1.0 0.5 2.0 -  *  *  60.6 6.0 10.0 2.0 2.5 4.75 0.25 0.5 0.1  **  19.0 53.5 6.0 10.0 2.0 2.5 4.75 0.25 2.0 **  26.3 53.6 10.0 2.0 2.0 1.3 0.5 3.0 0.1 1.2  7.8 30.0 42.6 10.0 2.0 2.0 1.9 0.5 3.0 -  *  *  0.1 0.1  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. 13.0 meg, v i t a m i n A 4400 I.U., v i t a m i n D 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. 2  3  **/kg: 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 , v i t a m i n A 6600 I . I L , v i t a m i n D 660 I.II. 0  2  3.3  meg,  -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  treatment from hatching  Treatments  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  EXPERIMENT 1  Dietary treat.  Ave. t h y r o i d wt. a t 58 weeks o f age* mg/100 g body wt.  Strain A Ave. t h y r o i d wt. a t 91 weeks o f age mg/100 g body wt.  Strain B Ave. t h y r o i d wt. a t 99 weeks o f age mg/100 g body wt.  S-S  11.0 (12)**  10.7 (10) aA  11.2 (10)  R-S  27.1° (12)  23.5 (10) aB  28.4 (10)  R-R  67.8° (12)  112.5° (10)  1 4 9 . 4 (10)  S-R  4 4 . 9 (12)  84.9° (10)  103.4° (10)  a A  C  aA  aB  C  Thiouracil in s t a r t i n g diet until / i o \ 4 wks o f a g e : 1 7 . 7 ^ a  K  D  a D B U  Level o f significance  5%  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 ** number o f d e t e r m i n a t i o n s p e r t r e a t m e n t a D c d  AB  1% laboratory  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 .  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 separate comparison.  -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.  Dietary treatment  Ave.* thyroid wt. g,  S-S  0.19  R-S  0.52  R-R  2.30  S-R  Level o f significance  Ave. epithelial ht./i  Ave. follicle diam. mm  41.l  0.167°  14.8  a  76.5  10.65°  0.227  15.1  a b  334.6°  58  1.39°  10.11°  0.196°  20.1  a b c  277.4°  67  1%  1%  5%  c  0.108  5.72  a  Ave. no. follicles a c r o s s gland  C  a B  a  Wt. epithelium mg  24.4  a A  7.13  % epithelium  a  c  C  5%  a  a  67 51  1%  * f i v e g l a n d s examined p e r 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 ,  -23E 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 the R-R and S-R t h y r o i d s showed the 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 d e g r e e o f a t r o p h y due to c h r o n i c RSM s t i m u l a t i o n , 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 d e t e r m i n e the t o t a l i o d i d e c o n t e n t o f the t h y r o i d t i s s u e as a f f e c t e d by the treatments.  M a t e r i a l s and Methods:  F i v e S h a v e r 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 R-S,  (S-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  Potter-Elvehjem  g l a s s - h o m o g e n i z e r to 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 the measurement o f t o t a l i o d i n e was an a d a p t a t i o n concentration  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  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  the 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 conversion  content  a c i d , which i n v o l v e d  o f the 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 .  o f the 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  The  the  color  twenty m i n u t e s a f t e r the  r e a c t i o n s t a r t e d , and the 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 the o p t i c a l d e n s i t y . 1 ml o f the 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 the 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  -24an oven a t 100°C t o c o m p l e t e 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 t u b e s were then removed and a l l o w e d  to 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 t h e 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 w a t e r 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 w a t e r 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 t h e 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 m i n u t e 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 m i 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 t h e l a p s e d t i m e f o r a l l o f t h e samples were constant. Measurements o f t h e 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 p a p e r .  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 h e 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  -25f 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 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 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  pair of thyroid  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). was weighed i n t o c u l t u r e t u b e s .  0.1 t o 0.2 g o f t h e sample  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 t u b e s 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 mult 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.  Results:  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 iodine content o f the t h y r o i d s of the b i r d s a f t e r c h r o n i c d i e t a r y treatments 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 .  -26The i o d i n e c o n c e n t r a t i o n R-R b i r d s (0.89) compared t o 2.65 mg/g  (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 of the c o n t r o l .  However, b e c a u s e o f  the 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 o f 1.72  content  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 the c o n t r o l was o n l y 0.55  L i k e w i s e , the 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 .  Again, the iodine  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 the S-R t h y r o i d s were between t h o s e o f the R-R and S-S  mg.  intermediate  glands.  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 the 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;  Dietary treatment  Ave. wt. o f thyroid glands* g  Ave. i o d i n e concentration in gland mg/g  Total iodine in gland mg  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  Level o f significance  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 not s i g n i f i c a n t l y d i f f e r e n t .  \  -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.  Analyzed iodine concentrations mg/kg  Rations  In e x p e r i m e n t s 1 and 6B: SBM-supplemented RSM-supplemented  0.85 0.91  In e x p e r i m e n t 6B o n l y : SBM-supplemented + K I 0  3  RSB-supplemented + KIO.,  1.10 1.35  -29E 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 met 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 al_, 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 t h e 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 t h e 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 t h e 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 t h e p e r c e n t a g e u p t a k e o f a dose o f 1-131 by t h e 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 h o u r s 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 for radioisotope studies.  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 t h e l e f t b r a c h i a l v e i n o f each bird.  The,time o f each i n j e c t i o n was n o t e d .  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 .  A t e x a c t l y 6 hours a f t e r e a c h 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 background 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 t h e 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 time o f t h e c o u n t i n g a s s e m b l y .  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 ) time 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 count 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 u p t a k e 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  with  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 c a g e d .  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 e a c h b i r d and t h e time o f i n j e c t i o n was r e c o r d e d . w a t e r ad l i b i t u m .  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  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.  gjass-homogenizer  c o n c e n t r a t i o n 40 mg/ml.  i n Tris-HCl buffer to give a solution o f t i s s u e 100 u l o f t h i s s u s p e n s i o n was taken f o r c o u n t i n g  of r a d i o a c t i v i t y i n a well-type s c i n t i l l a t i o n counter.  Again, the count  r a t e s were c o r r e c t e d f o r b a c k g r o u n d , 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 d o s e  (50yuCi)  of radioiodine. 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 counted f o r r a d i o a c t i v i t y .  -31Resuits:  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 u p t a k e 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 b o t h 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 u p t a k e had dropped from 6 hours t o 22 hours i n t h e S-S and R-S b i r d s , i t r e m a i n e d 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 concentration  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 t h e 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:  Concentrations of radioiodine 6 and 22 hours a f t e r  Dietary treatment  Total thyroid radioactivity % i n j e c t e d dose 6 h  S-S  9.1  R-S  22 h  i n t h e t h y r o i d g l a n d and l i v e r 1  administration.  Thyroid Estimated thy. radioactivity radioactivity % i n j e c t e d dose % i n j e c t e d dose /100 mg t h y r o i d /mg e p i t h e l i u m 6 h  22r.h  6 h  22 h  Liver radioactivity a t 22 h cpm/g  a  7.8  a  4.7  b  4.2  bc  .194  .172  556  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  Level 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 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 .  -33E 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  S i n c e t h e c h r o n i c a l l y R S M - s t i m u l a t e d 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  m i g h t 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 al_, 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 pronase 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 t h e p o p u l a t i o n . 50,uCi o f 1-131 (Atomic E n e r g y , 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 D e E s c o b a r , 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.  -34The 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 a g e n t 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  several times, nitrogen  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 .  During  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 frothing.  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 w a t e r b a t h a t 37 C f o r 21-24 tf  hours.  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 t h e d i g e s t i o n tube and was a p p l i e d on Whatmann 3 MM chromatography p a p e r s t r i p s (4.5;.em w i d e ) .  A p p l i c a t i o n took 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 t h e 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  equilibrated  w i t h s o l v e n t f o r a b o u t 12 hours b e f o r e chromatography was s t a r t e d .  Freshly  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 u s e d .  The a s c e n d i n g chromatogram was d e v e l o p e d f o r a b o u t 16  hours a t room t e m p e r a t u r e i n t h e d a r k (Lamas and D e E s c a r b a r , 1972).  The  chromatogram was t h e n 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 . t o MIT, DIT, T^ and T  3 >  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 g e t h e r w i t h t h e 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 t h e s i t e : ,  -35o f a p p l i c a t i o n , b u t t o a l l 1-131 1-131  r a d i o a c t i v i t y up t o t h e DIT band.  The  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  A r e f e r e n c e chromatogram  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 t h e chromatogram by Agerbaek  was i d e n t i c a l t o t h a t g i v e n  (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  (1972),  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 ( 1 9 7 2 ) .  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  run.  Results:  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 quantitatively reversed  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 t i m u l a t e d t h y r o i d t i s s u e s was e v i d e n t : 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 i n t h e R-R and S-R chromatograms,  In t h e S-S  was f o u n d i n the DIT band, whereas  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  -36MIT 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 t h e  RSM-stimulated  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  birds  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 probably represented degradative loss o r heterogeneity o f labeled iodoprotein 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 appeared c l e a r l y v i s i b l e e x c e p t f o r T g . 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 t h 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 t h e 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 ) . T h 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 ^ (Figure 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 h e I " and t h e DIT l e v e l s . d e s i g n a t e d by ( ? ) i n T a b l e 8.  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  I t might 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 t h e 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 T g + T^/(MIT/DIT), lower f o r t h e RSM-stimulated  was  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 parameters o f t h y r o i d m e t a b o l i s m m e a s u r e d , 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 patteiti 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 order:  R-R>S-R>R-S>S-S.  -37T 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 .into t h y r o i d a l compounds.  Dietary treatment  % of total thyroidal a c t i v i t y Origin* I" MIT DIT ? T & T 3  4  MIT/DIT  T3+T4 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.  -38F 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 treatments. n-butanol-ethanol-ammonia  Chromatographic  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-monoiodotyrosine),  R - R  S - S  R  solvent:  S  S  -  R  -39E x p e r i m e n t 5:  EFFECTS OF GOITROGENIC RSM ON SUSCEPTIBILITY TO HYDROLYSIS OF THYROGLOBULIN  Heterogeneity  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 the l i t e r a t u r e w i t h r e g a r d t o the 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 the 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 recognized  ( 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  suggested  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 the c o l l o i d may perhaps a l s o be h e t e r o g e n o u s 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; and U i , 1969b; Nunez e t al_, 1966; 1971;  Tarutani  Koyayashi and G r e e r , 1971; K o h l e r e t a l _ ,  Rosenberg et_ al_, 1964). S i n c e our 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 c h r o m a t o -  grams o f the 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 indeed 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 the v a r i o u s t r e a t m e n t  groups.  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 RSM-  The f o l l o w i n g e x p e r i -  i n the t h y r o i d t i s s u e s o f  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-  or  RSM-  s u p p l e m e n t e d d i e t s same as t h o s e f o r the 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  1ibitum.  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  vein of each.  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  -40from 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 w a t e r 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 t h e 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 p r o n a s e d i g e s t i o n .  Results:  Percent d i s t r i b u t i o n of r a d i o a c t i v i t y in thyroid tissues of 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 t h e chromatograms.  However, t h e r e was no 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  r a d i o a c t i v i t y remaining a t the o r i g i n .  A f t e r 24 h o u r s 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 control.  T h e r e a l s o 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 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 t h e ' 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 t h e 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).  -41T 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 pronase d i g e s t i o n .  Dietary treatment  % of total thyroidal radioactivity Origin  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  -42E 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_ al_, 1933; Asmundson ejt a l _ , 1936) and t h e 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 d e v e l o p m e n t 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; W i l g u s e t al_, 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 al_, 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_ al_, 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 t h e 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 r e s e m b l a n c e between t h e p r o g e n y 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 t h e a s s u m p t i o n o f an i o d i n e d e f i c i e n c y i n t h e eggs o f t h e 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 t h e eggs o f t h e 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 n o t 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 (experiment 1 ) .  S i n c e t h e egg p r o d u c t i o n a n d 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  -43the 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 :  stimulated birds.  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 .  Quadri-  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  to 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 a d e q u a t e to 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  to 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 particles.  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  -44Romanoff, 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  iodide(PBl)  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 r a m e t e r i n estimating avian iodine  metabolism.  Results:  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. 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 .  The egg y o l k s from t h e c o n t r o l  T h i s v a l u e i s i n agreement w i t h  average v a l u e s g i v e n i n the 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 the 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 the lower l i m i t o f the working range o f the a s s a y under the d e s c r i b e d e x p e r i m e n t a l  conditions.  A l a r g e r y o l k sample from t h i s pool would not be a p p r o p r i a t e b e c a u s e 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  during the i n c i n e r a t i o n process.  A g a i n the i o d i n e c o n c e n t r a t i o n i n the S-R egg y o l k s was 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 .  inter-  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 the 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 treatments i n experiment  Dietary treatment  6A.  Iodine concentration i n egg y o l k ,  S-S  273  a  R-S  286  a  R-R  < 85  S-R  ' 144  ppb  b  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 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 t h e 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 6 A ) , 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 to the hypothesis that the peripheral blood c i r c u l a t i o n o f these 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 p a r a m e t e r i n t h e a v e s , i t was p o s 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 , t h e R S M - s t i m u l a t e d 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 e l e m e n t i n t h e i r eggs.  M a t e r i a l s and  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 .  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 c a g e d .  They were g i v e n one o f t h e  following experimental 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  R-3:  RSM-supplemented  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  iodine/kg);  d i e t as i n e x p e r i m e n t 1; iodine/kg).  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  -47p 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  jfiupther?m6dTfication:  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 the 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 the p r e s u m a b l y low i o d i n e c o n c e n t r a t i o n o f the 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 the y o l k s a r e g i v e n . in t h i s  short-term  "feeding study,  Even  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 the 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 the d i e t a r y i o d i n e l e v e l was locbeasledyby ?iarjdadd4-t tohal :  1  0.3 mg per kg  r a t i o n , i n the form o f potassium iodate, the r e s p e c t i v e i o d i n e o f the SBM-  o r R S M - t r e a t e d egg y o l k s were a l s o e l e v a t e d  concentration  correspondingly.  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 the 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 the R S M - s t i m u l a t e d 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 i n T a b l e 6.  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  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 the 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  -48-  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 . t h a t s m a l l samples (0.1  I t s h o u l d be n o t e d  - 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 treatments i n experiment  Dietary  Iodine concentrations i n  treatment  egg y o l k ,  SBM - d i e t  SBM - d i e t p l u s K I 0  105  3  RSM « d i e t  RSM - d i e t p l u s K I 0  a b  6B.  242  a A  b  41  3  73  ppb  aB  a  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<0.01,  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.  -50E x p e r i m e n t 7:  OVARIAN UPTAKE OF A SINGLE DOSE OF 1-131  BY DEVELOPING FOLLICLES  The growth o f the 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 C o n r a d , 1939;  Romanoff, 1943).  Experi-  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).  Since  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 w e r 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 u p t a k e 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 f i v e l a r g e s t ova were removed.  the  P r e l i m i n a r y t e s t s i n w h i c h 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 , cut up and c o u n t e d p i e c e by p i e c e t h a t most o f the r a d i o a c t i v i t y o f the n e w l y - d e p o s i t e d  1-131  was  revealed  concentrated  t o a r i n g towards the p e r i p h e r y o f the ovum. T h e r e f o r e i n the p r e s e n t 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  ex-  follicles  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 tubes f o r counting i n a well-type s c i n t i l l a t i o n counter. o f the ova were r e c o r d e d , and t o t a l 1-131 o f the c o u n t r a t e o f the o r i g i n a l d o s e . 1-131  Individual weights  u p t a k e was e x p r e s s e d  as a p e r c e n t a g e  M o r e o v e r , p e r m e a b i l i t y o f the ova t o  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 the e q u a t i o n o f Smith (1959) which  expressed  t h e a c t i v i t y o f the 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 the 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-  Results:  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 e r t r e a t m e n t  are presented  i n T a b l e 12.  Since  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 t h e p e r m e a b i l i t y t o 1-131 and t h e s u r f a c e a r e a o f t h e 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. equations  The r e g r e s s i o n  and R v a l u e s were computed a s f o l l o w s :  Treatments  Regression  Equations  R_  S-S  l o g 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 t h e 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 t h e f i v e l a r g e s t o v a i n t h e o v a r y measured a f t e r 22 h o u r s .  Radioiodine  concen-  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 t i m e s those i n t h e SBM-fed b i r d s .  Average r a d i o i o d i n e concentration  four appeared  h i g h e r i n t h e 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 t h e 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 .  Radio-  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 t h e 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  the b i r d s had been f e d RSM d u r i n g t h e g r o w i n g p e r i o d and o v a 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 the ova i n the b i r d s on the d i f f e r e n t treatments  are 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  was maximal i n the 10 t o 12 cm until ovulation.  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  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  linearly  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 the f o l l i c u l a r w a l l , which i s not u n i f o r m but reaches an e a r l y maximum and then d e c l i n e s (Smith, 1959).  The r a t e o f u p t a k e 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 al_ ( 1 9 7 5 ) , which conformed w e l l w i t h our p r e s e n t f i n d i n g .  ,53-  T a b l e 12:  Radioiodine concentrations  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.  Ave. t o t a l r a d i o Dietary treatment  S-S  *  Ave.  radioiodine  in t h y r o i d gland % o f i n j e c t e d dose  7.8  ovarian  follicles  % o f i n j e c t e d dose  3  6.7  a  5.8  a  R-S  10.9  a  R-R  40.6  b  S-R  42.4  b  average o f determinations  iodine i n 5 largest  on f i v e b i r d s o f each  . 2.8  2.6  it  b  b  treatment.  v a l u e s w i t h t h e same s u p e r s c r i p t a r e n o t s i g n i f i c a n t l y d i f f e r e n t , p<0.01.  -54-  4-Or-  F i g u r e 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  1-0  OVUM  5 U R F A  2 0  C  E  AREA  -55E 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 R S M - s t i m u l a t e d b i r d s were f o u n d t o be lower than i n eggs from t h e c o n t r o l b i r d s ( e x p e r i m e n t 6 A ) , 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 e l e m e n t 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 t h e 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 6 B ) , 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.  Thyroid  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.  -56Results:  T h y r o i d w e i g h t s o f t h e 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 a s 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 t h e 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 the control  (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 t h e 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 h e 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 of the r e s p e c t i v e groups.  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 t h e 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 h e growing p e r i o d . F u r t h e r m o r e , t h e 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 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 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 t h e 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 S M - s t i m u l a t e d 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 t h e t h y r o i d g l a n d s o f newlyh 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.  Ave. t h y r o i d wt.  Ave. t h y r o i d wt.  o f progeny when  o f progeny when  dams were 77 wks,  dams were 91 wks,  o f age  o f age ( e x p t . 8)  mg/100 body wt.  mg/100 body wt.  S-S  11.7  a  ((74)  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  14.0  a  (20)  Dietary treatment  c  (73)  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 t h e 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 .  -58T 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. Dietary treatment  %  no.  Ave. h t .  Ave. wt.  follicles  epithelium  epithelium  a c r o s s the  mg  gland  /j  epithelium  AI  Ave.  follicle  diameter  S-S  43.0  b ;  (5)*'  3.86  a  2.55  57  31.7  R-S  49.2  a b  (5)  4.76  ab  3.10  48  35.1  R-R  55.8  a  (5)  4.71  ab  4.03  47  39.6  S-R  55.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 different dietary  treatments.  Dietary  Ave. a b s o l u t e  %  Ave. wt.  Ave. h t .  treatment  t h y r o i d wt  epithelium  epithelium  epithelium  Dam Progeny  Dam Progeny  Dam Progeny  Dam Progeny  g  mg  %  %  mg  mg  p  / i  S-S  0.19  5.94  24.4  43.0  41.1  2.55  7.13  3.86  R-S  0.52  6.30  14.8  49.2  76.5 3.10  5.72  4.76  R-R  2.30  7.21  15.1  55.8  334.6 4.03  10.65  4.71  S-R  1.39  6.19  20.1  55.1  277.4 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 r e s u l t e d i n continuous  e f f e c t s o f continuous  f e e d i n g o f g o i t r o g e n i c RSM  i n c r e a s e i n t h e 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 -  ruption of the e p i t h e l i a l tissues.  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 c o m p e n s a t o r y 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 t h e 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  stimulation the  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 d e s q u a m a t i o n 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 h e 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 c a u s e o f o v e r a c t i v i t y i s usually a deficiency of iodine or the i n a b i l i t y of the thyroid t o t r a p t h e i o d i n e o r t o p r o d u c e 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 study 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 m e n t i o n e d 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  ;  c a u s e d 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  intermediary  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 u p t a k e seems t o be enhanced i n the RSM-fed b i r d s when compared t o S-S c o n t r o l s .  This i s supported  by the i o d i n e  concentrations  i n 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 , measurement o f t h y r o i d a l 1-131  uptake  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 the 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 the 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 the 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 than times that of the c o n t r o l (experiment 2). 1-131  three  S i m i l a r l y , the r e l a t i v e t h y r o i d  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 the t o t a l 1-131  u p t a k e i n the t h y r o i d  glands  o f t h e l a t t e r g r o u p s 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*• • periment 3).  T h i s conforms w i t h the 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 .  According to these 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 the 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 s l o w t h y r o i d a l r e l e a s e o f the i n jected radioiodine. c h r o n i c RSM t r e a t m e n t s  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 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 a t 22 hours was  g r e a t e r than 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 the b i r d s on  treatments  S-S and R - S - i n 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 the 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 i m e 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  -62i 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 the RSM-fed b i r d s than i n the SBM-fed b i r d s . R e l a t e d t o the 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 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 .  o f the  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 hypertrophic 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 than 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 the  f o l l i c u l a r lumen o f t h e s e g l a n d s a p p e a r t o be b r i g h t l y s t a i n e d , w h i c h 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 .  Although the occurrence of 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 than 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  of  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 the 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 al_, 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_ al_, 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 the 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 i o d o a m i n o 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 administration of radioiodine.  Thus the 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 not d i s t u r b e d i n the RSM-fed b i r d s .  -63T 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 indeed i n h i b i t e d i n t h e 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 . 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  The d i s t r i b u t i o n o f 1-131 r a d i o 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 a s 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 s u 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 a s  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 h o r m o n o g e n e s i s .  Coupling e f f i c i e n c y  i . e . Tg + T / ( M I T / D I T ) , as a measure o f t h e combined r a t e s a t w h i c h d i o d o 4  t y r o s i n e and t h y r o i d hormones a r e p r o 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 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  gland.  i n t h e RSM-fed b i r d s  a r e n o t l o w e r 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 not 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 s e 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 synthetic a c t i v i t y o f the iodothyronines  i n g o i t r i n - f e d c h i c k s i s a b o u t two-  t h i r d o f t h e 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 iodothyronines control.  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 t i m e s t h a t o f t h e  In t h e 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  iodothyronines  i n t h e 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  increase i n the weights o f these glands over t h e c o n t r o l , w i l l give  estimated  iodothyronine values l a r g e r than that 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 t h e 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 g r o u p s 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  a p 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  -64s e c r e t i o n s o f 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  axis.  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 the 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 in t h i s respect.  Further i n v e s t i g a t i o n s i n t h i s area are necessary  before  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 the  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 the follicles.  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 the i n t r o -  duction of a goitrogen.  Under the i n f l u e n c e o f the e a r l y r i s e i n TSH,  f r a c t i o n s o f t h y r o g l o b u l i n take p a r t i n the hormonal exchange and t h e  new 1-131  mixes w i t h a d i f f e r e n t pool than i n the " 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 rather complicated  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 the c i r c u l a t i o n , and t h i s i s  supported  by the 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 g r o w i n g 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  the 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 is depressed  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  the 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 the p i t u i t a r y caused by the 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 continuous  the t h y r o i d g l a n d s were c a p a b l e  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 .  of  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 small 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  to birds  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) f o u n d t h a t the 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 enhanced 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  simultaneously  with  the t h i o u r a c i l . In the p r e s e n t s t u d y , the 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  -65p r o n a s e does not a p p e a r 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 than 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 u b s e q u e n t 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 r e m a i n s a p o s s i bility. 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 not 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 the u r i n e , r e sulting in a disturbance i n r e u t i l i z a t i o n of inorganic iodine in iodide organization (Stanbury,  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 the d e i o d i n a t i o n p r o c e s s has not 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 .  Although counting y  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 the d i f f e r e n t d i e t a r y t r e a t -  ments, the 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 o r g a n s 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  process  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 . Unpublished  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 d o s e 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  turn-  o v e r , measured by r a d i o a c t i v i t y o f the 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 not d i f f e r e n t from t h a t o f the c o n t r o l .  Hence the 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 the t h y r o i d c o u l d not 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 .  This indicated  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 the i n t r a t h y r o i d a l iodoamino a c i d s u n d e r 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 , the p o s s i b i l i t y t h a t the 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  -66f o r m a t i o n o f abnormal i o d o p r o t e i n i n the t h y r o i d g l a n d i s u n l i k e l y .  This  abnormal i o d o p r o t e i n o t h e r than t h y r o g l o b u l i n i n the 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 c h r o m a t o g r a p h y w i t h b u t a n o l , i t w i l l not 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 al_, 1962). 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 .  No such p r o t e i n was  The p e r c e n t a g e s  detected  of 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 the chromatograms o f the t h y r o i d h y d r o l y s a t e s s i m i l a r and w i t h i n normal r a n g e s f o r a l l b i r d s on the f o u r d i e t a r y  are  treatments.  It i s therefore hypothesized that chronic feeding of goitrogenic 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  intermediary  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 a n d / o r the r e l e a s e o r s e c r e t i o n o f the t h y r o i d hormones.  One o r more enzyme systems such as t h y r o i d  i n the t h y r o i d g l a n d may be i n v o l v e d .  peroxidase  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 c a u s e 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 p r o v o k e c o m p e n s a t o r y e n l a r g e m e n t o f the 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 e t a l _ , 1967).  (Fukase  T h u s , i n the 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 a n d / o r 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 suggested.  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 the 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 the 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 the enzyme p a t t e r n c h a n g e s , the 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 tend to become f i x e d (Chance ejt a l _ , 1965).  This idea  on the " 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  metabolic  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 through them, and may w e l l e x p l a i n the h y p e r f u n c t i o n o f the t h y r o i d o f t h e  -67R-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 organification.  The e x p e r i e n c e o f a c c o m o d a t i n g 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 the cell.  M o r e o v e r , 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  long-range  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 s u p e r - i m p o s e d 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 feedback 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 a r a m e t e r s o f t h y r o i d a c t i v i t y show t h a t the 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 the t h y r o i d g l a n d remained a p p a r e n t later life.  in  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 w h i c h had been f e d  RSM d u r i n g the 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 ) . weeks a f t e r withdrawal of c o l l o i d g o i t e r .  T h y r o i d g l a n d s examined from b i r d s 67  o f RSM from the 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  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 the e p i t h e l i a l c e l l s lower.  This persistence of e f f e c t of e a r l y  o v e r - s t i m u l a t i o n o f the 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 the r a t .  In the 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  induced  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 pregnant dam and/or t o the neonate r e s u l t e d i n a p e r s i s t e n t l y enlarged t h y r o i d gland with l a r g e r f o l l i c l e s and lower e p i t h e l i a l h e i g h t . hypothyroidism  These i n v e s t i g a t o r s reported a l s o t h a t p e r i n a t a l  caused a d e l a y i n p u b e r t y .  E a r l i e r experiments i n t h i s labo-  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 the i n g e s t i o n o f g o i t r i n by  chickens  up t o 24 weeks o f age a f f e c t e d t h e time o f sexual 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 time (March e t _ a l _ , 1975).  Thyroidal  e n l a r g e m e n t was r e l a t e d t o the l e n g t h o f time o v e r which t h e g o i t r o g e n i c  RSM  -68was 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 than 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 t h e 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 t h e c o n t r o l S-S b i r d s b u t t h e c o n c e n t r a t i o n p e r 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  observation  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 u p t a k e when e x p r e s s e d  r e l a t i v e to the  weight o f gland (experiment 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 h e 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 normal.  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 s p o n d s 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 requirements o f the birds i s not adversely 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 a n d / o r s e c r e t i o n i s w i t h d r a w n , 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 at 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 h e c i r c u l a t i o n stops 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 retarded i n the former.  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  -69was f o l l o w e d by c o l l o i d g o i t e r because the i o d i n e c o n c e n t r a t i o n by the 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  gland.  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 the 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 the  feeding  o f RSM t o b i r d s d u r i n g the g r o w i n g p e r i o d was s t i l l e v i d e n t more than a y e a r l a t e r i n the 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 w h i c h had neven been fed a goitrogenic d i e t .  T h i s a s p e c t o f the 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  heightened  c a p a c i t y f o r u p t a k e 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 the 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  onibiosynthesis  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 a b s e n c e o f l a b e l e d Normally t h i s blocking e f f e c t of large concentrations s y n t h e s i s i s a t r a n s i e n t phenomenon.  iodothyronines.  o f i o d i d e on hormone  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 the high l e v e l o f i o d i d e i n which t h e r e is a rapid loss of t h y r o i d a l capacity to concentrate  iodide.  In the R-S  birds  in the 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 the  Wplff-  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 the 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 the 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 remain a poss 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  may.persist.  -70I t i s therefore concluded  that the e f f e c t s of 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 the 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 the 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  goitrogen.  c o n t a i n s l a r g e r than 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), the h y p o t h e s i s was t e s t e d t h a t , i n the 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 u p t a k e 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 the 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 ova.  To  e l i m i n a t e the 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 the 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 i n the b i r d s f e d the SBM and RSM d i e t s ( e x p e r i m e n t 7 ) .  compared  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 the i n j e c t e d r a d i o i o d i n e than ova i n the SBM-fed b i r d s . F i g u r e 2 t h a t the 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  I t i s a p p a r e n t from f o l l i c l e i s pro-  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 the 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 the 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 the f o l l i c u l a r growth and  the  c o n c e n t r a t i o n o f i o d i n e i n the p l a s m a .  ova,  In c o n t r a s t t o the d e v e l o p i n g  the 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 the 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 .  -71-  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 t h e 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 t h e 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 t h e d i e t a r y i o d i n e i n t a k e  o f t h e 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 c a n 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 e l e m e n t 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 h e 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 t h e growing a n d / o r 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 t h e 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 laying diets.  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 maturity.  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 h e 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 t h e 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 in the 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 the thyroid glands 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 t h e o t h e r hand,  progeny from dams f e d RSM d u r i n g t h e 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 t h e 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 following conclusions concerning the chronic e f f e c t s o f feeding  goitrogenic  RSM t o b i r d s can be drawn. A n a l y s e s . o f Jhe7,thyroid g l a n d s showed t h a t t h e i o d i n e  content  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 control.  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 h e 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 g r o w i n g p e r i o d ) had o v e r 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 t h e c o n t r o l b i r d s . the goitrous s t a t e o f the RSM-stimulated glands.  twice  T h i s was m a i n l y due t o 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  the a c t i v e l y func-  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 a n d / o r SBM treatments.  The t h y r o i d s o f t h e 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 than 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 treatments,  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  diameters i n the t h y r o i d glands o f b i r d s i n these groups.  T h i s gave s u p p o r t  to the hypothetical p i c t u r e o f a stimulated t h y r o i d gland o f the c h r o n i c a l l y RSM-stimulated b i r d s a c t i v e l y accumulating  i o d i n e from t h e 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 t h e 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 f o u n d 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  u p t a k e was measured 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 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).  This four- to f i v e - f o l d  in t h y r o i d a l r a d i o i o d i n e uptake could account f o r the decrease  increase  in iodine  d e p o s i t i o n i n the 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  iodothyronines  was e s t i m a t e d t o be a d e q u a t e 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 the 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 h t  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 the e x t r a n e o u s enzyme p r o n a s e f a i l e d t o r e v e a l d i f f e r e n c e between the SBM-  and RSM-fed b i r d s .  any  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 the 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 further investigation. From t h e above d a t a , i t was h y p o t h e s i z e d  that the 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  accumulating  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 e l e m e n t was  available  f o r d e p o s i t i o n to the 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 indeed f o u n d t o be s i g n i f i c a n t l y l o w e r 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  regarded  as too 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  that  -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. was f u r t h e r s u p p o r t e d t r a t e d a decrease  This  by a r a d i o i o d i n e study ( e x p e r i m e n t 7) w h i c h demons-  i n the d e p o s i t i o n o f 1-131  i n the d e v e l o p i n g  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 .  ovarian  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 the 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 i o d i n e d e p o s i t i o n i n the eggs o f the RSM-  intravenously, this difference and SBM-fed b i r d s c o u l d not  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 contained a s l i g h t l y higher c o n c e n t r a t i o n  of  i o d i n e than 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 also u n l i k e l y , since unpublished  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  to the  SBM-  o r RSM-fed b i r d s . Thyroidal hypertrophy  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 the p r o g e n y o f the RSM-fed dams. t h a t the 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 t i s concluded  therefore  i n the p r o g e n y 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. 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 the eggs i s caused by a d i v e r s i o n o f a h i g h p r o p o r t i o n o f d i e t a r y i o d i n e i n t o the t h y r o i d g l a n d w i t h the r e s u l t t h a t amounts r e a c h i n g the d e v e l o p i n g ova a r e r e d u c e d .  -75-  BIBLIOGRAPHY  Adams, A.E. and J . M. B u s s , 1952. The e f f e c t o f a s i n g l e i n j e c t i o n o f an a n t i t h y r o i d drug on h y p e r p l a s i a i n t h e t h y r o i d o f t h e c h i c k embryo. E n d o c r 50:234. A g e r b a c k , H. 1972. 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E n d o c r 42:468.  homostatic  chemical  -93Wolff, J. and N.S. H a l m i , 1963. T h y r o i d a l i o d i d e t r a n s p o r t : V. Role o f Na -K - a c t i v a t e d , o u a b a i n - s e n s i t i v e a d e n o s i n e t r i p h o s p h a t a s e a c t i v i t y . J B i o l Chem 238:847. +  +  W o l f f , J . and J.R. Maurey, 1958. C a r d i a c g l y c o s i d e s and t h y r o i d a l i o d i d e t r a n s p o r t . N a t u r e 182:957. Wollman, S.H., 1961. E f f e c t s o f f e e d i n g t h i o u r a c i l on t h y r o i d g l a n d s o f r a t s . J Nat C a n c e r I n s t 26:473. Wollman, S.H., S.S. S p i c e r a n d M.S. B u r s t o n e , 1964. L o c a l i z a t i o n o f e s t e r a s e a n d a c i d p h o s p h a t a s e i n g r a n u l e s and c o l l o i d d r o p l e t s i n r a t t h y r o i d e p i t h e l i u m . J C e l l B i o l 21:191. Wyngaarden, J . B . , J.B. S t a n b u r y a n d B. Rapp, 1953. E f f e c t s o f i o d i d e , p e r c h l o r a t e , t h i o c y a n a t e and n i t r a t e a d m i n i s t r a t i o n upon i o d i d e c o n c e n t r a t i n g mechanism o f r a t t h y r o i d . Endocr 52:568. Yamamoto, K. a n d L . J . DeGroot, 1974. P e r o x i d a s e a n d NADPH-cytochrome C r e d u c t a s e a c t i v i t y d u r i n g t h y r o i d h y p e r p l a s i a and i n v o l u t i o n . Endocr 95:606. Y i p , C C . and L.D. H a d l e y , 1966. T h e i o d i n a t i o n o f t y r o s i n e by m y e l o peroxidase and beef t h y r o i d s . The p o s s i b l e involvement o f f r e e r a d i c a l s . B i o c h i m Biophys A c t a 122:406. Y i p , C a n d 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 . E n d o c r 70:931.  -94-  A P P E N D I X  -95Table 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) Analysis of variance: Source o f v a r i a t i o n  df  MS  Among groups  3  23719.7  W i t h i n groups  36  493.9  Total  39  F  P  48.0  <0.0l  i i ) D u n c a n ! s m u l t i p l e range t e s t : Dietary treatment  S-S  R-S  S-R  R-R  10.7  23.5  84.9  112.5  a  a  Ave. t h y r o i d wt. mg/100 body wt. Duncan's c o m p a r i s o n P<0.01!  b  c  i i i ) S e p a r a t e c o m p a r i s o n 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  df  MS  F  Among groups  1  819.2  39.4  W i t h i n groups  18  Total  19  20.8  P <0.0it  -96-  Table 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 pullets (DeKalb-strain)  f e d RSM and/or SBM, 99 weeks  o f age.  i) Analysis of variance: Source o f v a r i a t i o n  df  MS  F  Among groups  3  35005.0  W i t h i n groups  36  1693.0  Total  39  P  20.7  <0.01  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  S-S  R-S  S-R  R-R  11.2  28.4  103.4  149.4  a  a  b  Ave. t h y r o i d wt. mg/100 g body wt. Duncan's c o m p a r i s o n P<0.0l„  c  i i i ) S e p a r a t e c o m p a r i s o n 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  df  MS  F  Among groups  1  1477.5  W i t h i n groups  18  21.2  Total  19  69.7  P *0.0L  -97-  Table 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  a v 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 a g e .  i) Analysis of variance: Source o f v a r i a t i o n  df  MS  F  Among groups  3  4.5067  W i t h i n groups  16  0.1016  Total  19  P  44.4  <0.0J..  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  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 c o m p a r i s o n 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  df  MS  Among groups  1  0.2720  W i t h i n groups  8  0.0241  Total  9  F 11.29  P <0.0-L  -98Table 4(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  average e p i t h e l i a l heights i n t h y r o i d glands o f White Leghorn p u l l e t s a t 91 weeks o f a g e .  i) Analysis of variance: Source o f v a r i a t i o n  df  MS  Among groups  3  28.050  W i t h i n gorups  16^  2.433  Total  19  F  P  11.53  <0.01  i i ) Duncan's m u l t i p l e range t e s t : Di e t a r y treatment  R-S  S-S  S-R  R-R  5.61)  7.13  10.11  10.65  a  a  b  b  Ave. e p i t h e l i a l h e i g h t s , ja Duncan's c o m p a r i s o n P<0.0i:  -99-  Table 5(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 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) Analysis of variance: Source o f v a r i a t i o n  df  MS  Among groups  3  12905  Within groups  16_  1128  Total  19  F 11.44  P <0.0i»!  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment Ave.  S-S  R-S  S-R  R-R  .108  .167  .196  .227  a  b  bc  c  follicle  d i a m e t e r , mm Duncan's c o m p a r i s o n P< 0.05'.".  -100Table 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  percentage e p i t h e l i u m i n t h y r o i d glands o f White Leghorn p u l l e t s a t 91 weeks o f age.  i) Analysis o f variance: Source o f v a r i a t i o n  df  MS  Among groups  3  111.44  Within groups  16_  37.32  Total  19  F 2.99  P ^0.05  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  R-S  R-R  S-R  S-S  Percentage epithelium Duncan's c o m p a r i s o n P < O.OB:  14.2 a  15,1 ab  20.1 abc  24.4 c  -101Table 7(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 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) Analysis of variance: Source o f v a r i a t i o n  df  MS  Among groups  3  105615  W i t h i n groups  li5  Total  19  F 18.92  P <0.0-lI  5580  i i ) Duncan's m u l t i p l e range t e s t : Di e t a r y treatment  S-S  R-S  S-R  R-R  Weight o f e p i t h e l i u m , mg Duncan's c o m p a r i s o n P^O.OII  41.1 a  76.5 a  277.4 b  334.6 b  -102Table 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  a v e r a g e w e i g h t s o f t h y r o i d g l a n d s o f 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) Analysis of variance: Source o f v a r i a t i o n :  df  MS  F  Among groups  3  3.8623  23.09  W i t h i n groups  16_  0.1673  Total  19  P <0.0l  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  S-S  R-S  S-R  R-R  0.2039  0.4733  1.5115  2.9781  Ave. t h y r o i d weights, g Duncan's c o m p a r i s o n P<0.0'5  a  a  b  c  -103Table 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  average i o d i n e concentrations i n t h y r o i d glands o f White Leghorn p u l l e t s .  i)  Analysis of variance:  Source o f v a r i a t i o n  df  MS  F  P  Among groups  3  4.879  29.25  <0.01  W i t h i n groups  16^  Total  19  0.1668  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  R-R  S-R  S-S  R-S  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 c o m p a r i s o n P< 0.0.1':,  a  a  b  Ave. i o d i n e  b  -104Table 10(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  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) Analysis of variance: Source o f v a r i a t i o n  df  MS  F  P  Among groups  3  1.4305  25.8  Within groups  16^  0.0554  Total  19  <0.0l  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  S-S  R-S  S-R  R-R  0.55  1.25  1.65  1.72  a  b  c  c  Total iodine i n g l a n d , mg Duncan's c o m p a r i s o n P^O.OSc  -105Table 11(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 of the radioiodine.  i) Analysis of variance: Source o f v a r i a t i o n  df  MS  F  Among groups  3  851.00  18.4  W i t h i n groups  16  46.26  Total  19  P < 0.01  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  S-S  R-S  S-R  R-R  9.08  13.55  30.14  35.36  a  b  b  Percentage o f i n j e c t e d dose Duncan's c o m p a r i s o n P<0.01'  a  -106Table 12(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 of the radioiodine.  i) Analysis of variance: Source o f v a r i a t i o n  df  Among g r o u p s  3  W i t h i n groups  16.  Total  19  MS 1731.54  F 40.5  P <.0.01  42.72  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  S-S  R-S  R-R  S-R  7.83  10.87  40.57  42.40  Percentage p f i n j e c t e d dose Duncan's c o m p a r i s o n P <0.0i:  a  a  b  b  -107Table 13(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 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 6 hours a f t e r a d m i n i s t r a t i o n  i)  thyroid)  of the radioiodine.  Analysis o f variance:  Source o f v a r i a t i o n  df  MS  Among groups  3  933.54  Within groups  16  89.73  Total  19  F  P  10.40  <0.01  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  R-R  R-S  S-R  S-S  15.41  23.13  26.98  47.18  a  a  a  b  % i n j e c t e d dose per g t h y r o i d Duncan's c o m p a r i s o n P<0.01-'  -108Table 14(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 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)  Analysis of variance:  Source o f v a r i a t i o n  df  MS  Among g r o u p s  3  376.34  Within groups  _16  96.91  Total  19  F  P  3.88  <0.G5  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  R-R  S-R  R-S  S-S  21.61  29.55  35.58  41.96  a  ab  bc  % i n j e c t e d dos>e per g t h y r o i d Duncan's c o m p a r i s o n P<0.05;  bc  -109Table 15(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 different dietary treatments.  i)  Analysis of variance:  Source o f v a r i a t i o n  df  MS  Among groups  2  50473  Within groups  18_  2292  Total  20  F 22.02  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  S-R  S-S  R-S  144  273  286  a  b  b  I o d i n e cone, i n egg y o l k , ppb Duncan's c o m p a r i s o n P<0.0l-  P <0.01...  -110Table 16(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 dietary treatments.  i)  Analysis o f variance:  Source o f v a r i a t i o n  df  MS  Among groups  3  43093  Within groups  20_  9973  Total  23  F  P  4.321  <0.05  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  RSM  RSM+I  SBM  SBM+I  41  73  105  241  a  a  a  b  I o d i n e cone, i n egg y o l k , ppb Duncan's c o m p a r i s o n P<0.01  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 : Source o f v a r i a t i o n  df  MS  Among groups  1  12352  Within groups  lfj  1145  Total  11  F 10.78  P <0.0l  Table 17(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  average 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 ovarian 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)  Analysis of variance:  Source o f v a r i a t i o n  df  MS  Among groups  3  21.5  W i t h i n groups  16_  0.55  Total  19  F  P  39.09  <0.01  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment  S-R  R-R  R-S  S-S  5.796  6.717  % i n j e c t e d dose in 5 largest ovarian f o l l i c l e s Duncan's c o m p a r i s o n P<0.01  2.624 a  2.830 a  b  b  i-3-ttIHDS—PERMCA3IL IT V REGRESSION  A N A L Y S I S ,  A?i|) f-CUl. I C u L A'* 3 1 7 F  T H L OFPE'M.'C'. f  V A ^ I A H I F  IS  L O T , PERM  CONt»rri!|t./fiM STANDARD  I »:r-l'.( LM)i M V A I' I A h L f;  Hr G R F S S I ON CCEhT J C U NT  ;  S.AKTA L (.1 r; A W r. A INTERCEPT STANDARD ERROR OF E S T I M A T E -RESIDUAL "VARIANCE M U L T I P L E CORRELATION" C O E F F I C I E N T R SQUARED -VARrA^CE-RATTO—CF1 THE V A R I A B L E TO 8E 0 * I T T l D IS  REGRESSION  A N A L Y S I S ,  RtGR.  •0.982Sn3F-01 0.2708SSK+01  0. 1 1 1586C-01 0, J.286Q4E + 00  VARIABLE  I S  P i - T Til.  INTERCEPT STANDARD CRROR OF E S T I M A T E "RE S t at)* C VTl'RIA M C E M U L T I P L E CORRELATION COEFFICIENT R SQUARED "VTRTA'NCE RATIO Cr )  77.S36 6 7,9C0 H  "  '  "  " 00  i: G R E e s~nr-F R F F D c * ~  L O G PERM  STANDARD REGRESSION  S.AREA  VARIANCE RATIO (K}  •10.27  CONTRIBUTION  INDEPENDENT -VARlAHLF  REGRfcSSI 0"  cf  = l O G AREA  T H E OFPENUENT  •  Cut f F I C 11 'N r O F DfcTfcRuI'NATIOM  COEFF.  3atP'n«—WITH—2——22  _..  "  T IPLE  03  : -0.119S75E+0 1 = o.i634H<iEi-oo = O.2672.70K-01 = 0.HU278 = 0.77931  "  ERROR  Of- T H L :  TO  ERROR  ~"RF-"' R7""C'0EFr',~  •0. I 0 8 9 l 8 t - 0 l  0.689903L-02  = 0 . 3 9 l 6 l 4 b " + UO = 0.323268E+00 " =—TJTTO"fl STbTTOXT = 0,31269 = ' 0 .09777 -770-9-2  ,  REGRESSION  W X  — T O — X)  PARTIAL COEFFICIENT  OF T H E  "COEFFICIENT"'  TO M U L T I P L E  F  -  DF T E R  V  I N ATIGV  3  VARIANCE  -RATT0~TFT"  3 rt  2.«92  9.78  "W" H n co  Vt TH  I  A~WO  23  -40-  DTTTJRECS OF FRfcEDO fr  O -prPTrvorrrr-vTVRTA-SLE  is  ptnx-  CCNTRI3UTI0N ~S'TAN"D"A'RD r  REGRESSION COEFFICIENT  I NDEPENDF.NT VARIABLE  ARE AAA2  INTERCEPT TANHARD"ERROR OF EST I H A T E RESIDUAL VARIANCE MULTIPLE CORRELATION COEFFICIENT -R—STJARED VARIANCE RATIO CF) THE V A R I A B L E TO BE OMITTED IS  REGR,  0.712H40r. + i)0 •0.388R21E-01 -Q-.- ;6-220-8E-iDT  S.AREA ~A R E"A"3F*3  OF  ERROR"  OF  COE'- f I.LTcNT DETERMINATION •275,2h  0, l«7068E->00 0.923H8E-02  69S.30  -34 r.SQ'  c  _  - 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 AR E A* *3  WITH  AND  21  DEGREES  REGRESSION  PA-RTTAT  THE COEFF,  T O MULT lPi.E  OF  FRKEDOM  VARIANCE RATIO ( F )  23,u94 17.740 ~I 0 , 7 /  i  Pi  ca ca  /  rv3-Bik0S-  REGRESSION  rm ANALYSIS,  THE  DEPENDENT  is  VARIABLE  LOG  PFRM COivT-< I H I I I  INDEPENDENT VARIABLE S . AREA ICG 4RFA INTERCEPT STANDARD ERROR OF E S T I M A T E -RCStOUAt -VA-RrA-WCC M U L T I P L E CORRELATION COEFFICIENT R SQUARED -VSRTA-N C E-R-ATIO tfl THE V A R I A B L E TO BC OMITTED IS  = =  REGRESSION  ANALYSIS,  THE  •0.7B5U29E-01 0.2i«004c+01  = =  DEPENDENT  0, 106521E-01 0.27n9fl,TE + 00  PAKTJ AL COf.Fr IC1FNT O E T E V " I N A T tOl4.11 71 . 2 3  54.366 61.060  _  (t> r-»  S.AREA  IS  2 —AND  2D  SO  DEC R ETS~  LOG PERM CONTRIBUTION  STANDARD ERROR OF THE —REER,--COEFF—"  REGRESSION "COEFFICIENT'  INDEPENDENT — VARIABLE"  INTCRCEPT STANDARD ERROR OF E S T I M A T E -RESIDUAL"VARIANCE" M U L T I P L E CORRELATION C O E F F I C I E N T R SQUARED -VXR17TNCE RATIO ( D :  v«?f i A>.r E RATIO CF)  H  WTT H  •-  VARIABLE  rLOG  VtGRESSICM.  0.66795 0.7^335-  =  =  fiF  --rit M f L t  -0.9731C6E+00 0.1S6303E+UO  ~s—"0"- 2 W S 0 7 E - ' 0 1  -  STANDARD CRWOR ~ CF THE REGR. C O E F F .  RECESSION COEFFICIENT  TO  rnfM  AREA  v  0 . 2 5 3 5 5 5 E + 00'  = -0.801994E-01 = 0.294138E+00 "=0.86517IE-01 = 0.28783 = 0,09284 = r.""897  0.184100E+00  TO  MULTIPLE  PARTIAL COEFFICIENT "OF "OE'TERMINAT I OF! ...  8.26  REGRESSION  VARIANCE RATIC"CF} 1.897  W X -tfa H  S rt -sv-  •~"T  09  n  TITH-  T  3TN13  2T  (D rn CO H-  DTn5Rnrs~DF~FREED'OH—  O 3  5SIOW7rN"ATTSTS7 T^C" DEF»CEDENT vART*BLE""rS—PERM -  -  INDEPENDENT VARIABLE  REGRESSION COEFFICIENT  S'.AREA ARFA* * 2 ~AREA**3"  0.561084E+00 -0.278921E-01 ~~0;3S"2982EiT;r"  INTCRCEPT "STAND A R D~C R R O R - O F ^ E S T I H 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 OU'A R ETT V A R I A N C E RATIO (F) THE V A R I A B L E TO BE OMITTED IS -  :  1  CONTRiauTICN TO MULTIPLE REGRESSION "ST A W A R D ERHTJR CF THE REGR, C O E F F , 0.213811E+00 0, 152627E-01  "0,3T3TJ2r-0T"  -  PTRTTAL" COEFFICIENT OF DETERMINATION -44.06 197.33 --99753~  = -0,S9295SE+00 -=— 0 . 7 3 61 07E+ 0 0= 0,617964E+00 0,73312  "=  0Y53747" 7', 359  =  ARE A* * 3  WITH  AND  19  DEGREES OF FREEDOM  VARIA\CE RATIO CF) 6,886 3.340 1,271  Co 03 CO  f  S-n-Bjkrys PAGE REGRESSION ANALYSIS,  THE DEPENDENT VARIAHI.K i s  L  „  G  PERM CONTRJhuT IPM TO ruLTCPLt" REGRESSION  I NOEPErJOEN T V A R I ArtL F S'.AREA L O G AREA INTCRCEPT STANDARD ERROR OF ESTIMATE -RrstntTAr-VA-RrATgcrr MULTIPLE CORRELATION COEFFICIENT R SQUARED "VAWTflNTC RATIO (h ) THE VARIABLE TO SE OMITTED IS  REGRESSION  ANALYSIS,  REGRESSION COEF (• ICIb NT  'STANDARD E R R O R S OF THE . REGR. COEFF,  -0.H5704'Jfc-01 0.216407E+01  0.141453E-01 0.363090E+00  OF  P'A»TT AC COEFFICIENT DETER"I NATION 37.95 25.20  LOG  T H EDEPENDENT  VARIAHLF.  S .AR = = = =  OF E S T I M A T E -  MULTIPLE CORRELATION R SQUARED VTRTANCE  RAiiu  COEFFICIENT  cn  REGRE^SrOfr-Am-YSIS-TTHC  =  DEPENDENT  36,710 35.523 H Co  -ffTTH  AREA  INDEPENDENT "VTRTA-OCE—  ~ESlDUAL" VATrrffwCE  I S  2 AW  E  A  Trio O  22—TTFGRCEt) Or FRETOTna-  L O G PERM  STANDARD ERROR OF THE RETTR7 COEFT"J—  REGRFSSION '"CTjEFrrcTeMT— -0.823994E-02  -0,117142C+00 0.396438F+00 0.157I63E+00 0.19103 0.03649 0T87T  MIJUUPLF  RA  S'.AREA ARK A A * 2 ARE"A i"*3  _  -STANDARD-ERROR "OF'TSTIMATE  RESIDUAL VARIANCE MULTIPLE CORRELATION COEFFICIENT ~R~S CUT* R ED VARIANCE RATIO CF) THE V A R I A B L E TO B E O M I T T E D I S  i iu u  y  0,871  3,65  -  1  REGRESSION COEFFICIENT 0.216527E+00 •0.114076K-01 "tr7T5'*nr29r-Trr  = -0,625384E-U1 = 0.3n9C37E+00 = 0,l3fa780EtoO = 0.71212 =~r 5071r 7,202 s AREA * *3  3 -»3 rt  09 TTH~  ^A'NO  2 3  DEGREES  UF  a -co—  FREED07T  p.  V A R I A B L E ' S - PERM  INDEPENDENT VARIABLE  X  H  VARIANCE  OF •Ut.ItKMTKJtTTCm-  0.832869E-02  REGRESSION  T3  . PARTIAL COEFF- I C I E N T  CONTRIBUTION TO MULTIPLE  INTERCEPT  (F)  = - 0 , 123046F_tyi = 0.250676EtU0 07628394F-TJT~ = 0.79407 = 0.63150  CONTRIBUTION TO  INTERCEPT S T A N D A R D ERROR  V A R I A N C E R A T I O  "STAND A RT)—ERRCFT OF THE REGR, COEFF, 0.857160C-01 0.598289E-02 '57119723E-TJ3-  OF  PSR7TSL~ COEFFICIENT DETERMINATION -120.55 324.45 ~-T517T9-  f  *ITH  AND  21  DEGREES OF FREEDOM  REGRESSION  VARIANCE RATIO ( F ) 6.3B1 3.636  l.oOO  3 To"" 3 0> CO CO  REGRESSION  A N A L Y S I S , THE DEPENDENT  V A k J ArtL.E  IS  LIT, PERM CONTRIBUTION -T ti  INDEPENDENT V AH I A B L E S.AREA LOG AREA INTERCEPT S T A N D A R D E R R O R OF E S T I M A T E - R E S I D U AL~~V ARI"ANCE~ MULTIPLE CORRELATION C O E F F I C I E N T R SQUARED "-VARIANCE RATTtT^fF) T H E V A R I A B L E TO B E O M I T T E D I S  REGRESSION  REGRESSION COEFFICIENT  STANDARD" ERROR OF THE RFGR. C O E F F .  •O.lCSluE+OC 0.279482E+01  0. 106617E-01 0 281865L+0O  -; i L T J P L E  " ? A R T T A L CUf.r F I C I E N T OF DL T t R'' I (•' AT 101>'  VAKIANCF P AT ] D fF)  C.f-.95  105.123 98.517  15.80  t  RttiKr.SS-lON  - 0 . 144323E+01 0 . 1 5 9 5 7 2 E + 00 0.254633E-01 0.90968 0,82752 \  T27777-  L0G  A N A L Y S I S , T H E DEPENDENT  WITH  AND '  -DEGRFFS"OF"FREEDOM"  AREA  VARIABLE  IS  LOG PERM CONTRITION  INDEPENDENT -—VAR I A X L E -  REGRESSION COEFFICIENT"-  S'. A R E A INTERCEPT' "\. STANDARD ERRORS OF E S T I M A T E - R E S T DO A t V ARTA~NCE M U L T I P L E CORRELATION COEFFICIENT R SQUARED V A R I A N C E R A T I O CF}  STANDARD ERROR Or T H E  PARTIAL COF.FF IC1F.NT "OF "DETER'1 IN ATI ON "  " R E G R ; "coErF'i  •0.926432E-02  TO M U L T I P L E  VARIANCE RATIO"-CF"}  5.67  0,787605E-02  REGRESSION  1.384  = 0.200225E-01 = 0,364969E+00 "="—OT 1 3 a 0,23821 = 0,05674 "T73B"3  "DElTrNDENT  VARTABtTE  TS  -CO-  WITH  T  A"NTT  :3  mrcTfLEs  UF  FREEUDH~  PERWCONTRIBUTION  INUEPENDENT VARIABLE S'.AREA AREA* * 2 -AKEAW3 -  INTERCEPT -STANDARD-ERROR—OF E S T I M A T E " RESIDUAL VARIANCE MULTIPLE CORRELATION C O E F F I C I E N T R SQUARED VARIANCE RATIO ( F } T H E V A R I A B L E TO B E O M I T T E D I S  v  =  REGRESSION COEFFICIENT  WITH  REGRESSION  ""STAND A PTJ—"E R R D R"~ OF T H E REGR, COEFF,  T X R T T"A E COEFFICIENT OF DETERMINATION  V'ARlAr.CE RATIO C F )  0. 1 14627E + 00 0.814305E-02 -0","-ir>72^BE"-0T-  •246.18 64 t . 3 1 •T27TCT-  14.947 10,480 "5.212  0.443172E+00 •0.263608E-01 -0-.4IB8"03t-0"3-  -0.542931E+U0 O",3S3969F + 00 0 . 1 4 7 4 3 2 F + 00 0.30780 -0TG525JI 13.146 ARtA**3  TO M U L T I P L E  ANO  DEGREES  -  OF FREEDOM  -116Table 22(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  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)  Analysis o f variance:  Source o f v a r i a t i o n  df  MS  Among groups  3  .0000175  W i t h i n groups  76_  .0000177  Total  79  F 0.99  P n.s.  -117Table 23(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 glarids o f progeny a t h a t c h i n g .  i)  Analysis of variance:  Source o f v a r i a t i o n  df  MS  F  P  Among groups  3  179.50  3.77  <0.05  W i t h i n groups  16_  47.58  Total  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  43.0  49.2  55.1  55.8  a  ab  b  % thyroid epithelium i n progeny Duncan's c o m p a r i s o n P<0.05  b  -118-  Table 24(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 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)  Analysis of variance:  Source o f v a r i a t i o n  df  MS  Among g r o u p s  3  1.369  Within groups  16  0.473  Total  19  F  P  2.894  <0.10  i i ) Duncan's m u l t i p l e range t e s t : Dietary treatment o f dam;.;  S-S  R-R  R-S  S-R  3.864  4.708  4.761  5.092  a  ab  ab  b  Ave. h e i g h t s o f e p i t h e l i u m , JJ Duncan's c o m p a r i s o n P 4.0.05  R-S  -120-  Photnricrographs: ( I 6 0 x ) Thyroid gland R-R  

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