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Kinetics of selenium metabolism in the ewe and fetal lamb Shariff, Mohammed Azamatulla 1987

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KINETICS OF SELENIUM METABOLISM IN THE EWE AND FETAL LAMB by MOHAMMED AZAMATULLA SHAJRIFF B.V.Sc, A.P. A g r i c u l t u r a l University, India, 1979 M.Sc (Dairying)., Kurukshetra University, India, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1987 ©Mohammed Azamatulla S h a r i f f , 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Animal Science The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Apr i l 30, 1987 i i ABSTRACT To i n v e s t i g a t e the whole body metabolism of selenium (Se), i s o t o p e k i n e t i c s t u d i e s were undertaken i n which i n d w e l l i n g c a t h e t e r s were implanted i n the j u g u l a r v e i n s of pregnant and nonpregnant ewes as w e l l as the i n f e r i o r vena cava and saphenous v e i n s of 110-120 day (d) o l d ovine 75 f e t u s e s . A s i n g l e i n j e c t i o n of Se-sodium s e l e n i t e was admin i s t e r e d and blood samples were obtained at v a r i o u s times. A 5 d Se balance t r i a l , which commenced on the day of t r a c e r experiment, was a l s o undertaken. The plasma 75 Se s p e c i f i c a c t i v i t y data were used f o r determining the k i n e t i c parameters of Se metabolism and the p l a c e n t a l t r a n s f e r r a t e s . Using the Se balance and t r a c e r data the net a b s o r p t i o n and the f e c a l l o s s e s of Se were c a l c u l a t e d . The f r a c t i o n a l rate constants and °f S e u t i l i z a t i o n i n i n d i v i d u a l t i s s u e s were estimated by s a c r i f i c i n g the ewes and the f e t u s e s at d i f f e r e n t times a f t e r the t r a c e r 75 i n j e c t i o n and determining the t i s s u e Se s p e c i f i c a c t i v i t i e s . The e f f e c t of Se d e f i c i e n c y on the k i n e t i c s , p l a c e n t a l t r a n s f e r , net a b s o r p t i o n and t i s s u e metabolism was s t u d i e d i n a s i m i l a r manner by f e e d i n g the ewes with Se d e f i c i e n t r a t i o n s . The mean plasma Se c o n c e n t r a t i o n s i n the p o s i t i v e nonpregnant and pregnant ewes were 142 187 ng/ml and were s i g n i f i c a n t l y higher than Se and the i i i corresponding values (82 and 69 ng/ml) i n the Se d e f i c i e n t ones r e s p e c t i v e l y . The plasma Se l e v e l s i n the Se p o s i t i v e f e t u s e s (46 ng/ml) were not s i g n i f i c a n t l y d i f f e r e n t from the d e f i c i e n t ones (53 ng/ml). The i r r e v e r s i b l e d i s p o s a l (ID) rates of Se were 7 ug/d/kg body weight (B.W.) i n both nonpregnant and pregnant ewes, whereas, these values d e c l i n e d to 2 ug/d/kg B.W. under d e f i c i e n c y c o n d i t i o n s . The ID values i n the Se p o s i t i v e and d e f i c i e n t f e t u s e s were 20 and 11 ug/d/kg B.W. r e s p e c t i v e l y . These r e s u l t s i n d i c a t e d that the plasma Se turnover was lower i n Se d e f i c i e n t animals than i n the p o s i t i v e ones and that the f e t u s e s had a higher turnover than a d u l t ewes i r r e s p e c t i v e of the maternal Se s t a t u s . Values f o r net Se a b s o r p t i o n (% Se intake) and f e c a l Se l o s s e s (ug/d) i n the Se p o s i t i v e nonpregnant ewes were 51% and 272 ug/d versus 97% and 25 ug/d i n the Se d e f i c i e n t ones. The corresponding values f o r the Se p o s i t i v e pregnant ewes were 56% and 241 ug/d as a g a i n s t 84% and 27 ug/d i n the d e f i c i e n t ones. These r e s u l t s suggested t h a t the e f f i c i e n c y of net Se a b s o r p t i o n expressed as the percent of Se intake decreased with i n c r e a s i n g Se i n t a k e s and that the f e c a l route exerted a homeostatic c o n t r o l on Se metabolism i n ewes. The t i s s u e Se c o n c e n t r a t i o n s were s i g n i f i c a n t l y higher i n the Se p o s i t i v e animals than i n the d e f i c i e n t ones, whereas, the t i s s u e f r a c t i o n a l rate constant values were higher i n the Se d e f i c i e n t animals than i n the p o s i t i v e ones. i v These r e s u l t s i m p l i e d that the t i s s u e Se c o n c e n t r a t i o n s were s i g n i f i c a n t l y decreased under Se d e f i c i e n c y c o n d i t i o n s and t h a t the Se turnover was higher i n t i s s u e s when the d i e t a r y Se intake was minimal. The p l a c e n t a l t r a n s f e r s t u d i e s showed that there was a b i d i r e c t i o n a l exchange of Se across the p l a c e n t a . The p l a c e n t a l Se t r a n s f e r rates from ewe to f e t u s d e c l i n e d from 53 ug/d i n the p o s i t i v e ewes to 29 ug/d i n the d e f i c i e n t ones. The rate of Se t r a n s f e r from f e t u s to ewe a l s o decreased from 24 ug/d i n the p o s i t i v e ewes to 12 ug/d i n the d e f i c i e n t ones. The net p l a c e n t a l Se t r a n s f e r was found to be reduced under c o n d i t i o n s of decreased maternal d i e t a r y supply. The n u t r i t i o n a l i m p l i c a t i o n s of Se s t a t u s of the pregnant ewes on the a v a i l a b i l i t y of the t r a c e m i n e r a l to the f e t u s have been d i s c u s s e d . V TABLE OF CONTENTS INTRODUCTION 1 CHAPTER I. REVIEW OF THE LITERATURE 4 (A) Selenium responsive c o n d i t i o n s i n sheep and other 4 animals (B) Methods of selenium supplementation 9 (C) Methodology to study selenium metabolism i n animals 14 (D) M e t a b o l i c f a t e of selenium i n animals 20 (E) Methodology to study p l a c e n t a l t r a n s f e r of 36 t r a c e minerals (F) Selenium metabolism i n the mother and the f e t u s 40 CONCLUSIONS FROM THE LITERATURE REVIEW 47 CHAPTER I I . SELENIUM METABOLISM IN NONPREGNANT EWES 49 Experiment I: K i n e t i c s of selenium metabolism i n 49 nonpregnant ewes CHAPTER I I I . SELENIUM METABOLISM IN PREGNANT EWES 87 Experiment II A: K i n e t i c s of selenium metabolism 87 i n pregnant ewes Experiment II B: K i n e t i c s of selenium metabolism 125 i n f e t a l lambs Experiment II C: In utero p l a c e n t a l t r a n s f e r r a t e s 150 of selenium i n pregnant ewes GENERAL SUMMARY AND CONCLUSIONS 172 BIBLIOGRAPHY 178 APPENDIX 202 vi L i s t of Figures Figure Page 1. Effe c t of Se intake on plasma Se concentration i n 61 nonpregnant ewes. 75 2. Changes in plasma Se s p e c i f i c a c t i v i t y of Se 62 positi v e nonpregnant ewes. 75 3. Changes in plasma Se s p e c i f i c a c t i v i t y of Se 63 de f i c i e n t nonpregnant ewes. 75 4. B e s t - f i t plasma Se s p e c i f i c activity-time 64 curves of Se positive nonpregnant ewes. 75 5. B e s t - f i t plasma Se s p e c i f i c activity-time 65 curves of Se d e f i c i e n t nonpregnant ewes. 6. Effe c t of Se intake on net Se absorption i n 71 nonpregnant ewes. 75 7. Mean d a i l y excretion of Se i n urine and feces 72 of nonpregnant ewes. 8. Ef f e c t of Se intake on tissue Se pool sizes i n 75 nonpregnant ewes. 9. Effe c t of Se intake on plasma Se concentration in 95 pregnant ewes. 75 10. Changes i n plasma Se s p e c i f i c a c t i v i t y of Se 96 positi v e pregnant ewes. 75 11. Changes in plasma Se s p e c i f i c a c t i v i t y of Se 97 de f i c i e n t pregnant ewes. 75 12. B e s t - f i t plasma Se s p e c i f i c activity-time 99 curves i n Se positive pregnant ewes. 75 13. B e s t - f i t plasma Se s p e c i f i c activity-time 100 curves in Se de f i c i e n t pregnant ewes. 75 14. Mean d a i l y excretion of Se in urine and feces 104 of pregnant ewes. 15. Eff e c t of Se intake on net Se absorption i n 107 pregnant ewes. 16. Eff e c t of Se intake on tissue Se pool size in 109 pregnant ewes. 75 17. Changes i n plasma Se s p e c i f i c a c t i v i t y of Se 134 v n s p e c i f i c a c t i v i t y of Se p o s i t i v e f e t u s e s . 75 18. Changes i n plasma Se d e f i c i e n t f e t u s e s . 75 19. B e s t - f i t plasma Se s p e c i f i c a c t i v i t y - t i m e c u r v e s o f Se p o s i t i v e f e t u s e s . 75, a c t i v i t y - t i m e 20. B e s t - f i t plasma ' JSe s p e c i f i c c u r v e s of Se d e f i c i e n t f e t u s e s . 75 21. Changes i n plasma Se s p e c i f i c a c t i v i t y o f Se p o s i t i v e ewes and t h e i r f e t u s e s f o l l o w i n g t r a c e r i n j e c t i o n i n t o ewes. 75 22. Changes i n plasma Se s p e c i f i c a c t i v i t y o f Se p o s i t i v e ewes and t h e i r f e t u s e s f o l l o w i n g f e t a l t r a c e r i n j e c t i o n . 75 23. Changes i n plasma Se s p e c i f i c a c t i v i t y o f Se d e f i c i e n t ewes and t h e i r f e t u s e s f o l l o w i n g t r a c e r i n j e c t i o n i n t o ewes. 75 24. Changes i n plasma Se a c t i v i t y of Se d e f i c i e n t ewes and t h e i r f e t u s e s f o l l o w i n g f e t a l t r a c e r i n j e c t i o n . 25. P l a c e n t a l ewes. Se t r a n s f e r e f f i c i e n c i e s i n pregnant 135 136 137 160 161 162 163 165 V I 1 1 L i s t of Tables Table Page 7 5 1. B e s t - f i t e x p o n e n t i a l equations of plasma Se 66 s p e c i f i c a c t i v i t y - t i m e curves i n nonpregnant ewes. 75 2. Comparison of areas under the plasma Se 68 s p e c i f i c a c t i v i t y - t i m e curves, plasma Se concentra-t i o n s and k i n e t i c parameters i n nonpregnant ewes. 3. A b s o r p t i o n and r e t e n t i o n of Se i n nonpregnant ewes. 69 4. S t a b l e Se c o n c e n t r a t i o n s and pool s i z e s i n the 73 t i s s u e s of nonpregnant ewes. 5. K i n e t i c parameters of t i s s u e Se metabolism i n 76 nonpregnant ewes. 6. P h y s i o l o g i c a l parameters of pregnant ewes f o l l o w i n g 94 i n t r a u t e r i n e surgery. 75 7. B e s t - f i t e x p o n e n t i a l equations of plasma Se 101 s p e c i f i c a c t i v i t y - t i m e curves i n pregnant ewes. 75 8. Comparison of areas under the plasma Se s p e c i f i c 102 a c t i v i t y - t i m e curves and k i n e t i c parameters i n pregnant ewes. 9. A b s o r p t i o n and r e t e n t i o n of Se i n pregnant ewes. 105 10. S t a b l e Se c o n c e n t r a t i o n and pool s i z e i n the 108 t i s s u e s of pregnant ewes. 1 1 . K i n e t i c parameters of t i s s u e Se metabolism i n 110 pregnant ewes. 12. P o s t - s u r g i c a l p h y s i o l o g i c a l parameters of f e t u s e s . 133 75 1 3 . B e s t - f i t e x p o n e n t i a l equations of plasma Se 138 s p e c i f i c a c t i v i t y - t i m e curves i n f e t u s e s 75 14. Comparison of areas under the plasma Se 139 s p e c i f i c a c t i v i t y - t i m e curves and i n v i v o k i n e t i c parameters i n f e t u s e s . 15. S t a b l e Se c o n c e n t r a t i o n and pool s i z e i n the 141 t i s s u e s of f e t u s e s . 16. Comparison of the t i s s u e Se c o n c e n t r a t i o n s of the 143 f e t u s and the ewe. ix 17. K i n e t i c parameters of t i s s u e Se metabolism i n 144 f e t u s e s . 18. Body w e i g h t s , g e s t a t i o n a l age, plasma Se 152 c o n c e n t r a t i o n s , i n j e c t i o n and sa m p l i n g s i t e s o f Se p o s i t i v e ewes and t h e i r f e t u s e s . 19. Body w e i g h t s , g e s t a t i o n a l age, plasma Se 153 c o n c e n t r a t i o n s , i n j e c t i o n and samp l i n g s i t e s o f Se d e f i c i e n t ewes and t h e i r f e t u s e s . 75 20. B e s t - f i t e x p o n e n t i a l e q u a t i o n s of plasma Se 154 s p e c i f i c a c t i v i t y - t i m e c u r v e s i n Se p o s i t i v e ewes and f e t u s e s . 75 21. B e s t - f i t e x p o n e n t i a l e q u a t i o n s o f plasma Se 155 s p e c i f i c a c t i v i t y - t i m e curve i n Se d e f i c i e n t ewes and f e t u s e s . 75 22. A r e a s under the plasma Se s p e c i f i c 156 a c t i v i t y - t i m e c u r v e s of Se p o s i t i v e and Se d e f i c i e n t ewes and t h e i r r e s p e c t i v e f e t u s e s . 23. P l a c e n t a l t r a n s f e r e f f i c i e n c i e s and r a t e s of Se 164 t r a n s f e r i n pregnant ewes. X ACKNOWLEDGEMENTS I w i s h t o e x p r e s s my h e a r t f e l t g r a t i t u d e and s i n c e r e t h a n k s t o my r e s e a r c h s u p e r v i s o r D r . C R . K r i s h n a m u r t i f o r h i s c o n s t a n t e n c o u r a g e m e n t , c o n s t r u c t i v e c r i t i c i s m , p a t i e n c e and g u i d a n c e d u r i n g t h e c o u r s e o f t h i s s t u d y . I w o u l d l i k e t o t h a n k D r . R . I . McKay f o r s e r v i n g a s my a c t i n g s u p e r v i s o r when D r . C R . K r i s h n a m u r t i was away on h i s s a b b a t i c a l l e a v e and f o r h i s a d v i c e on s t a t i s t i c a l a n a l y s i s o f t h e d a t a . I w i s h t o t h a n k D r . R. B l a i r , Head, D e p a r t m e n t o f A n i m a l S c i e n c e f o r l e t t i n g me u s e t h e d e p a r t m e n t a l f a c i l i t i e s . I w o u l d l i k e t o e x p r e s s my t h a n k s t o D r s . W.T. B u c k l e y , B.D. Mason and J.A. S h e l f o r d , members o f my s u p e r v i s o r y c o m m i t t e e , f o r t h e i r a d v i c e i n t h e d r a f t i n g o f t h i s t h e s i s . I w o u l d l i k e t o e x t e n d my g r a t i t u d e t o my c o l l e a g u e s D r . A l S c h a e f e r and D r . R. G o p i n a t h f o r t h e i r v a l u a b l e and t i m e l y h e l p . S i n c e r e a p p r e c i a t i o n i s e x t e n d e d t o B a r r y Wong, S h e n t o n T a n , D r . P a r e s h P a t t a n i f o r s h a r i n g t h e i r k n o w l e d g e i n t h e u s e o f c o m p u t e r s . I w o u l d l i k e t o a c k n o w l e d g e t h e t e c h n i c a l a s s i s t a n c e o f Sandy J a n s s e n s , K l a r a Shekhtman and C a r o l D yck. S p e c i a l a p p r e c i a t i o n i s e x t e n d e d t o D r . A l S c h a e f e r and Sandy J a n s s e n s f o r t h e i r d e d i c a t e d and s e l f l e s s a s s i s t a n c e . I w i s h t o t h a n k P a u l W i l l i n g , Ed M i r e h o u s e and E l i z a b e t h B a z l e y f o r t h e i r c a r e and m a i n t e n a n c e o f a n i m a l s and t o Ted C a t h c a r t and G i l l e s G a l z y f o r t h e i r t e c h n i c a l h e l p . I w o u l d l i k e t o e x t e n d my t h a n k s t o numerous f r i e n d s i n V a n c o u v e r who h e l p e d me and my f a m i l y i n many ways and made o u r s t a y a memorable one. L a s t b u t n o t t h e l e a s t I t h a n k my f a m i l y members b a c k i n I n d i a f o r t h e i r c o n t i n u e d s u p p o r t and e n c o u r a g e m e n t i n p u r s u i n g my o b j e c t i v e s . xi DEDICATION I wish to d e d i c a t e t h i s t h e s i s daughter Rahima f o r t h e i r moral s a c r i f i c e without which t h i s work c a r r i e d out. to my wife Hamida and support and p e r s o n a l would not have been 1 INTRODUCTION Selenium (Se) i s a t r a c e element d i s c o v e r e d by B e r z e l i u s e a r l y i n the nineteenth century. But the n u t r i t i o n a l e s s e n t i a l i t y of t h i s element was not e s t a b l i s h e d u n t i l 1957 when Schwarz and F o l t z reported f o r the f i r s t time t h a t t r a c e q u a n t i t i e s of Se could a l l e v i a t e the symptoms of l i v e r n e c r o s i s i n r a t s . U n t i l then Se was con s i d e r e d only as a t o x i c a n t because of i t s involvement i n v a r i o u s t o x i c i t y c o n d i t i o n s i n l i v e s t o c k (Franke, 1934; Franke and P o t t e r , 1935). The r o l e of Se i n the p r e v e n t i o n of white muscle disease i n lambs was e s t a b l i s h e d ( O l d f i e l d et a l . I960; Muth, 1963). The mystery behind the b i o c h e m i c a l mode of Se a c t i o n was u n r a v e l l e d when Rotruck et a l . (1973) reported f o r the f i r s t time t h a t Se a c t s as an i n t e g r a l component of g l u t a t h i o n e peroxidase (E.C. 1.11.1.9), an enzyme that removes the o r g a n i c hydroperoxides and hydrogen peroxide and thereby p r o t e c t s the c e l l from the o x i d a t i v e damage due to oxygen f r e e r a d i c a l s . The d i e t a r y e s s e n t i a l i t y of Se has been e s t a b l i s h e d f o r p o u l t r y (Noguchi et a l . 1973), f o r sheep ( O l d f i e l d et a l . 1960, Jenkins and H i d i r o g l o u , 1972a), and f o r c a t t l e ( U l l r e y et a l . 1977). The supplementation of selenium i n the r a t i o n s f o r l i v e s t o c k has been approved by the U.S. Department of Food and Drug a d m i n i s t r a t i o n s i n c e 1974 at a rate of 0.1 ppm f o r c a t t l e , sheep, swine (0.3 ppm f o r p r e s t a r t e r s and s t a r t e r s ) , p o u l t r y 2 and ducks and 0.2 ppm f o r turkeys (NRC, 1983) . Keeping pace with the n u t r i t i o n a l research, metabolic s t u d i e s were a l s o c a r r i e d out e x t e n s i v e l y to understand the f a t e of the inge s t e d or p a r e n t e r a l l y a d m i n i s t e r e d selenium i n l a b o r a t o r y animals as w e l l as i n domestic l i v e s t o c k . E x c e l l e n t reviews have appeared on t h i s s u b j e c t from time to time (Ammerman and M i l l e r , 1974; Bopp et a l . 1982; Burk , 1983; NRC, 1983; Shamberger, 1983; and Combs and Combs, 1984) . R a d i o a c t i v e t r a c e r s of Se, e i t h e r 75 75 Se-selenomethionine or S e - s e l e n i t e , are most commonly employed to study the ab s o r p t i o n , t i s s u e d i s t r i b u t i o n and e x c r e t i o n of selenium i n the b i o l o g i c a l systems. Of l a t e s t a b l e selenium i s o t o p e s have a l s o been employed f o r studying Se metabolism i n humans as w e l l as i n experimental animals (Janghorbani et a l . 1982) . Though these metabolic s t u d i e s were i n d i c a t i v e of the r o l e of v a r i o u s t i s s u e s i n Se metabolism no i n f o r m a t i o n i s obtained with regard to the whole body k i n e t i c s of Se metabolism i n t e g r a t i n g a l l the p h y s i o l o g i c a l p r ocesses, namely, a b s o r p t i o n , plasma and t i s s u e Se turnover o c c u r r i n g i n the animal under d i f f e r e n t n u t r i t i o n a l c o n d i t i o n s . T herefore, the present s t u d i e s were undertaken i n nonpregnant and pregnant ewes employing i s o t o p i c t r a c e r methodology to i n v e s t i g a t e whole body Se metabolism at two d i f f e r e n t l e v e l s of Se i n t a k e . In u t e r o p l a c e n t a l t r a n s f e r and f e t a l Se metabolism were a l s o s t u d i e d 3 under Se p o s i t i v e and d e f i c i e n c y c o n d i t i o n s . The n u t r i t i o n a l i m p l i c a t i o n s of Se s u p p l e m e n t a t i o n t o the pregnant ewe f o r the p r e v e n t i o n o f w h i t e muscle d i s e a s e i n f e t a l lamb have been d i s c u s s e d . 4 CHAPTER I; REVIEW OF LITERATURE A)Selenium responsive conditions in sheep and other animals; As a result of selenium (Se) deficiency in plants, domestic animals consuming such selenium d e f i c i e n t diets were found to develop muscular degeneration or weakness. One of the most common a f f l i c t i o n s i s n u t r i t i o n a l muscular dystrophy (NMD) or white muscle disease (WMD) which occurs mostly i n sheep and to some extent i n c a t t l e (Muth, 1963) . In addition to NMD, reproductive problems and poor growth were also reported in sheep and ca t t l e (Segerson et a l . 1977; Andrews et a l . 1968) . a ) N u t r i t i o n a l Muscular Dystrophy(NMD): N u t r i t i o n a l muscular dystrophy and other related disorders have been reported to occur i n domestic animals, when the feeds consumed are de f i c i e n t i n Se (20-30 ppb or less Se) (Hartley, 1967; de Toledo and Perry, 1985) . Young lambs and calves are usually most severely affected. These lambs develop raised arch and s t i f f hind regions which a f f e c t t h e i r g a i t . They become weak over a period of time and die from starvation. Secondary infections l i k e pneumonia also accompany myopathy. When the cardiac muscle i s involved, animals die suddenly showing degenerative 5 l e s i o n s i n the endocardium and myocardium at autopsy. The 'I' band (myosin) of the sarcomere i s most commonly a f f e c t e d e x h i b i t i n g o x i d a t i v e damage. H i g h l y a c t i v e s k e l e t a l muscles (diaphragm, i n t e r c o s t a l , p e l v i c and hind l e g muscles) are a l s o a f f e c t e d . M i c r o s c o p i c a l l y the l e s i o n s range from h y a l i n e degeneration to a c o a g u l a t i v e n e c r o s i s of muscle f i b e r s . Fibrous t i s s u e tends to re p l a c e the degenerating muscle f i b e r s accompanied by the appearance of macrophages and lymphocytes. Calcium s a l t s may a l s o be dep o s i t e d i n the a f f e c t e d muscles ( H a r t l e y and Grant, 1961; Andrews et a l . 1968) . In experimental animals NMD has been produced a r t i f i c i a l l y and shown to respond to e i t h e r Se or v i t a m i n E a d m i n i s t r a t i o n . Measurements of blood and t i s s u e Se l e v e l s a i d i n the d i a g n o s i s of NMD. Se dependent g l u t a t h i o n e peroxidase (GSH-Px) a c t i v i t y i n the e r y t h r o c y t e s i s measured i n a s s e s s i n g the Se st a t u s of animals (Thompson et a l . 1976; Whanger et a l . 1977) . Abnormally e l e v a t e d l e v e l s of serum glutamate ox a l o a c e t a t e and pyruvate transaminases, l a c t a t e dehydrogenase and c r e a t i n i n e kinase have a l s o been observed with NMD i n animals (Buchanan-Smith et a l . 1969) . b)Reproductive problems: Andrews et a l . (1968) reported that i n f e r t i l i t y i n New Zealand ewes was due to Se d e f i c i e n c y r e s u l t i n g i n embryonic m o r t a l i t y . A d m i n i s t r a t i o n of Se to the ewe before 6 mating was shown to reduce i n f e r t i l i t y and increase lambing percentages. The resulting increase in lambing percentages might have been due to increased ova f e r t i l i z a t i o n or decreased embryonic mortality or a combination of both these factors. Segerson and Ganapathy (1980) suggested that the improvement in ova f e r t i l i t y in Se and vitamin E treated ewes was due to an increase i n the frequency of uterine contractions migrating towards the oviduct at mating. Segerson et a l . (1981) also observed that Se was more important than vitamin E in influencing uterine m o t i l i t y and the contraction v e l o c i t y . Se has been shown to be of importance i n male reproduction. According to Wu et a l . (1979) Se deficiency was shown to produce sperm with impaired m o t i l i t y and a c h a r a c t e r i s t i c mid piece breakage in more than 50% of the experimental rats fed Se d e f i c i e n t (<0.02 ppm) diets over a period of one year. Neither vitamin E nor other antioxidants were ef f e c t i v e in counteracting these effects (Wu et a l . 1973). Se was shown to be associated with a cysteine r i c h structural protein of mitochondrial capsules in rat sperm (Calvin et a l . 1981). Smith et a l . (1979) have reported that i n the b u l l , 75 Se retention in the epididymis was highly correlated (r=0.92) with sperm concentration. They have also demonstrated that when ejaculated bovine spermatozoa were 7 s u b j e c t e d to repeated f r e e z i n g and thawing i n d i s t i l l e d water, Se remained with the spermatozoa suggesting t h a t Se was t i g h t l y bound to the s t r u c t u r a l components of sperm c e l l . On the other hand no c o r r e l a t i o n between the plasma Se and the seminal Se c o n c e n t r a t i o n s was observed i n the study of Heimann et a l . ( 1 9 8 4 ) . Furthermore they a l s o found t h a t the spermatozoal a b n o r m a l i t i e s were not s i g n i f i c a n t l y c o r r e l a t e d with the Se c o n c e n t r a t i o n s i n the r e p r o d u c t i v e t i s s u e s as w e l l as i n the seminal and the blood plasma (Heimann et a l . 1 9 8 4 ) . B e n e f i c i a l e f f e c t s of Se with or without v i t a m i n E i n reducing the i n c i d e n c e of r e t a i n e d placentae i n d a i r y cows have been reported by s e v e r a l workers ( T r i n d e r et a l . 1 9 6 9 ; J u l i e n et a l . 1 9 7 6 ; Ishak et a l . 1 9 8 3 ; D'aleo, 1 9 8 4 and Eger et a l . 1 9 8 5 ) . c)Poor growth; New Zealand workers (Andrews et a l . 1 9 6 8 ) have re p o r t e d t hat poor growth was a common problem i n sheep and c a t t l e s u f f e r i n g from Se d e f i c i e n c y . The Se d e f i c i e n t lambs were shown to l o s e body weight with or without d i a r r h e a . In advanced stages, lambs were repo r t e d to d i e e x h i b i t i n g o s t e o p o r o s i s and emaciation. P r o g r e s s i v e l o s s of c o n d i t i o n , profuse d i a r r h e a and high m o r t a l i t y were re p o r t e d i n c a t t l e (Andrews et a l . 1 9 6 8 ) . T h i s Se-8 r e s p o n s i v e syndrome i n r u m i n a n t s i s o f t e n r e f e r r e d t o as u n t h r i f t i n e s s ( Andrews e t a l . 1968) . S u p p l e m e n t a l d i e t a r y Se was shown t o i m p r o v e w e i g h t g a i n s i n s h e e p a n d c a t t l e b y s e v e r a l w o r k e r s (McLean e t a l . 1959; O l d f i e l d e t a l . 1960; A n d r e w s e t a l . 1968; P a u l s o n e t a l . 1968 and R o t r u c k e t a l . 1969). I t h a s been p r o p o s e d t h a t Se c o u n t e r a c t e d t h i s p o o r g r o w t h syndrome by p r o t e c t i n g t h e immune s y s t e m . Se was shown t o enha n c e t h e p h a g o c y t i c a b i l i t y o f n e u t r o p h i l s t o k i l l b a c t e r i a ( B o y n e a n d A r t h u r , 1979; A z i z e t a l . 1984). Many f o r m s o f Se d e f i c i e n c y d i s e a s e s have b e e n i d e n t i f i e d i n o t h e r d o m e s t i c a s w e l l a s e x p e r i m e n t a l a n i m a l s . T hese i n c l u d e l i v e r n e c r o s i s i n r a t s ( S c h w a r z a n d F o l t z , 1957) , h e p a t o s i s d i e t e t i c a , NMD and m u l b e r r y h e a r t d i s e a s e i n p i g s ( T r a p p e t a l . 1970; v a n V l e e t e t a l . 1970, 1976) , NMD, e x u d a t i v e d i a t h e s i s , g i z z a r d m y o p a t h y and p a n c r e a t i c d e g e n e r a t i o n i n c h i c k e n s a n d t u r k e y s ( S c o t t e t a l . 1967 and Thompson and S c o t t , 1970) , NMD, p o o r s u r v i v a l a n d p o o r g r o w t h r a t e s i n f i s h ( P o s t o n e t a l . 1976 and P o s t o n a n d Combs, 1979), and NMD i n w i l d d e e r ( B r a d y e t a l . 1978) . A d e s c r i p t i o n o f t h e s e d e f i c i e n c y d i s e a s e s i n f a r m a n i m a l s h a s b een r e v i e w e d a t l e n g t h by s e v e r a l a u t h o r s ( H a r t l e y and G r a n t , 1961; M c D o n a l d e t a l . 1976; Underwood, 1977, 1981; v a n V l e e t , 1980; NRC, 1983 and Sh a m b e r g e r , 1983; R o l l e r and E x o n , 1986). 9 B)METHODS OF SELENIUM SUPPLEMENTATION; Pr e v e n t i o n of Se d e f i c i e n c y syndromes i s achieved by supplementing Se to the animals. I f Se i s present i n s u f f i c i e n t c o n c e n t r a t i o n s i n n a t u r a l f e e d s t u f f s there i s no n e c e s s i t y f o r supplemental Se. I f s o i l s are demonstrably d e f i c i e n t , use of f e e d s t u f f s known to be from Se adequate areas or a d m i n i s t r a t i o n of Se supplements i s r e q u i r e d . The p o i n t s to be considered concerning Se a d m i n i s t r a t i o n are d e s c r i b e d below; a) Chemical form of supplement: Two types of Se s a l t s have been used f o r supplementation, sodium s e l e n i t e and sodium s e l e n a t e . Sodium s e l e n i t e i s the most commonly used chemical form of Se. T h i s may be added to the complete d i e t or blended at higher c o n c e n t r a t i o n s with f r e e choice supplements l i k e mineral mixtures or s a l t l i c k s (Rotruck et a l . 1969; U l l r e y et a l . 1977, 1978; Whanger et a l . 1978 and Overnes et a l . 1985b). Both s e l e n i t e and s e l e n a t e have a l s o been used i n p a r e n t e r a l methods of Se supplementation (Jenkins and H i d i r o g l o u , 1972a; Cawley and McPhee, 1984; M a l l i n s o n et a l . 1985 and Overnes et a l . 1985a). b) Route of a d m i n i s t r a t i o n : Se i s administered g e n e r a l l y by e i t h e r o r a l or p a r e n t e r a l routes. O r a l l y Se may be supplemented as a drench or as a f r e e choice supplement through feed or s a l t b l o c k . Se may a l s o be p l a c e d as a 10 heavy p e l l e t i n the reticulum of the ruminants (Hunter et a l . 1981 and Hudson et a l . 1981). The parenteral methods of supplementation include injections or slow-release implants (Jenkins and Hidiroglou, 1972a; Horton et a l . 1978; MacPherson and Chalmers, 1984). Wisconsin workers fed varying levels of Se up to 264 ppm in the s a l t to sheep and found that none of the Se l e v e l s altered s a l t intake nor caused Se t o x i c i t y (Paulson et a l . 1968; Rotruck et a l . 1969). Whanger et a l . (1978), in their studies with sheep, evaluated d i f f e r e n t methods of Se administration on the occurrence of white muscle disease (WMD) and glutathione peroxidase (GSH-Px) a c t i v i t y . They reported that sheep given 10 mg Se as a sodium selenite drench had s i g n i f i c a n t l y higher (P<0.05) blood GSH-Px levels with mild incidence of WMD i n their lambs compared to the control ewes that received unsupplemented low Se oats and hay. They also found that none of the lambs from ewes given Se by i n j e c t i o n or p e l l e t s developed WMD. Whanger et a l . (1978) also observed that either iodized or trace mineralized s a l t containing Se was equally e f f e c t i v e in preventing white muscle disease i n lambs when fed to their dams. Australian workers (Kuchel and Buckley, 1969; Kuchel and Godwin, 1976) developed and investigated the use of intra-ruminal heavy Se p e l l e t s in grazing sheep. They observed that lambs fed a Se d e f i c i e n t ration recovered from the white muscle disease 11 soon a f t e r the a d m i n i s t r a t i o n of ruminal Se p e l l e t s . J enkins and H i d i r o g l o u (1972a) have i n v e s t i g a t e d the use of slow r e l e a s e Se p e l l e t s i n pregnant ewes f o r the c o n t r o l of WMD. P e l l e t s c o n t a i n i n g e i t h e r 20 or 10 mg Se as sodium s e l e n i t e with d i f f e r e n t o i l y v e h i c l e s were implanted i n the loose connective t i s s u e behind the .shoulder of ewes i n mid and l a t e g e s t a t i o n r e s p e c t i v e l y . Both of the i m p l a n t a t i o n treatments were found to i n c r e a s e the blood Se l e v e l s i n the ewes two to four times t h a t of u n t r e a t e d c o n t r o l s . They reported that the a b n o r m a l i t i e s of muscle pathology of the lambs were decreased with the Se i m p l a n t a t i o n of the ewes. S i m i l a r l y , i n another study, white muscle disease was prevented i n neonatal c a l v e s born to beef cows fed Se d e f i c i e n t r a t i o n s by the i m p l a n t a t i o n of slow r e l e a s e Se p e l l e t s ( H i d i r o g l o u et a l . 1972). Horton et a l . (1978) evaluated four d i f f e r e n t methods of Se supplementation based upon he m a t o l o g i c a l responses i n pregnant sheep. They r e p o r t e d t h a t blood GSH-Px enzyme a c t i v i t i e s were 2.3 and 2.8 times higher f o r drench and i n j e c t i o n groups than f o r c o n t r o l animals. Blood Se l e v e l s i n animals fed s a l t mix and Se p e l l e t were only 1.4 times higher than the c o n t r o l s . Based on t h e i r s t u d i e s they concluded that i n t r a m u s c u l a r i n j e c t i o n was the most e f f e c t i v e f o l l o w e d , 12 i n o r d e r , by the o r a l d r e n c h , p e l l e t and f o r t i f i e d s a l t m i x t u r e . R e c e n t l y Overnes e t a l . (1985b) from Norway i n v e s t i g a t e d the e f f e c t of d i f f e r e n t l e v e l s of Se i n m i n e r a l m i x t u r e s and s a l t l i c k s on the Se s t a t u s i n sheep. They m o n i t o r e d the Se s t a t u s of the ewes by measuring b l o o d and m i l k Se c o n c e n t r a t i o n s a f t e r f e e d i n g t h r e e l e v e l s (10, 2 5 and 40 mg Se/kg d i e t ) of sodium s e l e n i t e i n m i n e r a l m i x t u r e s and s a l t l i c k s . They found t h a t 10 mg Se/kg s u p p l e m e n t a t i o n i n m i n e r a l m i x t u r e s and s a l t l i c k s was i n a d e q u a t e i n a r e a s w i t h extreme Se d e f i c i e n c y w h i l e 2 5 mg Se/kg f o r t i f i c a t i o n p r o v i d e d a d a i l y i n t a k e of about 0.4 mg Se and r e s u l t e d i n e l e v a t e d Se l e v e l s i n ewe's b l o o d and m i l k . Se l e v e l s i n lamb's b l o o d were a l s o e l e v a t e d and were c a p a b l e of p r e v e n t i n g w h i t e muscle d i s e a s e w i t h o u t c a u s i n g any t o x i c e f f e c t s . MacPherson and Chalmers (1984) t e s t e d d i f f e r e n t methods of Se s u p p l e m e n t a t i o n i n sheep and c a t t l e . They found t h a t a l l f o u r methods of Se s u p p l e m e n t a t i o n p e l l e t s , subcutaneous i n j e c t i o n s , a d d i t i o n t o water and o r a l s u p p l e m e n t a t i o n of sodium s e l e n i t e s o l u t i o n were e f f e c t i v e i n r a i s i n g b l o o d g l u t a t h i o n e p e r o x i d a s e c o n c e n t r a t i o n s i n c a t t l e and sheep. They c o n c l u d e d t h a t the c h o i c e of Se s u p p l e m e n t a t i o n would depend on c o s t , husbandry system and ease of a d m i n i s t r a t i o n . 13 R e c e n t l y a l o n g a c t i n g c o m m e r c i a l p r e p a r a t i o n o f b a r i u m s e l e n a t e ( D e p o s e l , R y c o v e t L t d . , U.K.) c o n t a i n i n g 50 mg Se/ml was i n t r o d u c e d i n t h e U n i t e d K ingdom. S e v e r a l w o r k e r s have r e p o r t e d i n c r e a s e d b l o o d , p l a s m a a n d t i s s u e Se c o n c e n t r a t i o n s i n s h e e p and c a t t l e w i t h i n two t o e i g h t weeks o f s u b c u t a n e o u s i n j e c t i o n o f b a r i u m s e l e n a t e a t a r a t e o f 1 mg S e / k g w e i g h t ( A l l e n and M a l l i n s o n , 1984; C a w l e y and McPhee, 1984; M a l l i n s o n e t a l . 1985 and O v e r n e s e t a l . 1985b). E u r o p e a n w o r k e r s a l s o f o u n d t h a t b a r i u m s e l e n a t e i n j e c t i o n m a i n t a i n e d an a d e q u a t e Se s t a t u s f o r a t l e a s t s i x months i n s h e e p and a l s o a f f o r d e d p r o t e c t i o n i n f e t a l l a m b s a g a i n s t w h i t e m u s c l e d i s e a s e i f i n j e c t e d i n t o ewes i n autumn. However, t h e r e was c o n c e r n t h a t t h e l a r g e r e s i d u e s (90%) o f i n j e c t e d s e l e n a t e i n t h e m u s c l e a r o u n d t h e s i t e o f i n j e c t i o n c o u l d be a h e a l t h h a z a r d f o r p e o p l e c o n s u m i n g s u c h meat ( A l l e n and M a l l i n s o n , 1984; a n d M a l l i n s o n e t a l . 1985) . The u s e o f C o n t r o l l e d R e l e a s e G l a s s e s (CRG) c o n t a i n i n g Se (43.7% Se W/W) i m p l a n t e d e i t h e r s u b c u t a n e o u s l y o r i n t r a m u s c u l a r l y , was s t u d i e d by A l l e n e t a l . (1981) i n s h e e p . The w h o l e b l o o d Se c o n c e n t r a t i o n s i n c r e a s e d by a p p r o x i m a t e l y 2 -2 .5 t i m e s i n t r e a t e d a n i m a l s c o m p a r e d t o t h e c o n t r o l s . The a p p a r e n t r e t e n t i o n o f Se by t h e t r e a t e d a n i m a l s was h i g h e r t h a n t h e u n t r e a t e d c o n t r o l s . More r e c e n t l y , T r e n g o v e and J u d s o n (1985) s t u d i e d t h e 14 e f f e c t s o f a d m i n i s t e r i n g o r a l l y , s o l u b l e g l a s s b u l l e t s c o n t a i n i n g Se i n s h e e p and f o u n d e l e v a t e d b u t v a r i a b l e l e v e l s o f Se i n b l o o d . Though t h e i r s t u d y i n d i c a t e d t h a t an a d d i t i o n a l b u t n o t t o x i c s u p p l y o f Se t h r o u g h t h e g l a s s b u l l e t was p o s s i b l e , i t f a i l e d t o d e m o n s t r a t e c l e a r l y t h e s u i t a b i l i t y o f g l a s s b u l l e t s f o r c o r r e c t i n g o r p r e v e n t i n g Se d e f i c i e n c y i n s h e e p . C)METHODOLOGY TO STUDY SELENIUM METABOLISM I N ANIMALS: The m e t a b o l i s m o f Se i n a n i m a l s and man h a s b e e n s t u d i e d u s i n g e i t h e r r a d i o a c t i v e l y l a b e l l e d o r s t a b l e Se compounds. C e n t r a l t o t h e s t u d y o f Se m e t a b o l i s m i s a c l e a r u n d e r s t a n d i n g o f t h e n u t r i t i o n a l Se s t a t u s o f t h e a n i m a l . The Se s t a t u s i n a n i m a l s i s a s s e s s e d u s i n g s e v e r a l m e t h o d s w h i c h i n c l u d e a) measurement o f Se c o n c e n t r a t i o n s i n b l o o d , p l a s m a , serum, e r y t h r o c y t e s , h a i r a n d t i s s u e s , b) measurement o f Se d e p e n d e n t g l u t a t h i o n e p e r o x i d a s e a c t i v i t y i n w h o l e b l o o d , p l a s m a , e r y t h r o c y t e s a n d t i s s u e s u s i n g s t a n d a r d l a b o r a t o r y m e t h o d s , c ) p e r f o r m i n g r a p i d ( s p o t ) t e s t s f o r g l u t a t h i o n e p e r o x i d a s e a c t i v i t y i n 75 w h o l e b l o o d o r p l a s m a and d) m e a s u r i n g t h e u p t a k e o f Se by t h e r e d b l o o d c e l l s i n v i t r o . a M e a s u r e m e n t o f s e l e n i u m c o n c e n t r a t i o n s i n b i o l o g i c a l  f l u i d s a n d t i s s u e s ; The Se c o n c e n t r a t i o n s i n p l a s m a o f c a t t l e h a v e b e e n m e a s u r e d b y many w o r k e r s and l e v e l s l e s s t h a n 20 ng/ml 15 a r e c o n s i d e r e d t o be i n d i c a t i v e o f Se d e f i c i e n c y i n c a t t l e ( P e r r y e t a l . 1976; Lane e t a l . 1982; F e n i m o r e e t a l . 1983 and H i d i r o g l o u e t a l . 1985) . R o b i n s o n a n d Thomson (1983) h a v e i n d i c a t e d t h a t p l a s m a Se c o u l d o n l y be u s e d t o i n d i c a t e t h e s h o r t t e r m Se s t a t u s o f an a n i m a l b e c a u s e i t t e n d e d t o f l u c t u a t e o f t e n i n r e s p o n s e t o t h e d i e t a r y Se s u p p l y . Whole b l o o d Se l e v e l s have b e e n r e p o r t e d t o be o f l i t t l e v a l u e i n d i a g n o s i n g w h i t e m u s c l e d i s e a s e i n s h e e p as s i m i l a r b l o o d Se c o n c e n t r a t i o n s were f o u n d i n l a m b s w i t h o r w i t h o u t w h i t e m u s c l e d i s e a s e (Whanger e t a l . 1978) . However, b l o o d Se l e v e l s c o u l d be u s e d t o m o n i t o r t h e e f f i c a c y o f Se s u p p l e m e n t a t i o n u n t i l t h e y r e a c h e d a p l a t e a u , w h i c h o c c u r r e d a f t e r f o u r weeks o f Se i n j e c t i o n . H i d i r o g l o u e t a l . (1965) m e a s u r e d t h e Se c o n c e n t r a t i o n o f h a i r o f p r e g n a n t b e e f cows t o p r e d i c t t h e i n c i d e n c e o f n u t r i t i o n a l m u s c u l a r d y s t r o p h y (NMD) i n c a l v e s . T h e y f o u n d a 40% i n c i d e n c e o f NMD i n c a l v e s f r o m dams h a v i n g Se l e v e l s o f 110 ng/g o f h a i r o r l e s s , 15% i n c i d e n c e i n c a l v e s f r o m dams h a v i n g Se c o n c e n t r a t i o n i n h a i r r a n g i n g b e t w e e n 120 t o 230 ng/g and 0% i n c i d e n c e i n c a l v e s f r o m dams h a v i n g 240 ng Se/g h a i r o r more. The Se c o n t e n t o f h a i r h a s bee n shown t o v a r y d i r e c t l y w i t h Se s u p p l e m e n t a t i o n ( H i d i r o g l o u and S p u r r , 1975; P e r r y e t a l . 1976; and de T o l e d o and P e r r y , 1985) . 16 The l e v e l s of Se i n blood serum have been monitored by de Toledo and Perry (1985) i n an attempt to evaluate the v a r i o u s Se supplements. The serum Se l e v e l was found to reach a p l a t e a u w i t h i n a range of 45 to 60 ng/ml. The serum Se e s t i m a t i o n s would s u f f e r from the same l i m i t a t i o n as plasma Se and t h e r e f o r e would only i n d i c a t e s h o r t term Se st a t u s i n animals. New Zealand workers (Andrews et a l . 1968) have i n d i c a t e d t h a t Se c o n c e n t r a t i o n s of 1.0 ppm i n the kidney c o r t e x and 0.1 ppm or more i n the l i v e r on f r e s h b a s i s were i n d i c a t i v e of Se adequacy i n sheep. They have a l s o suggested t h a t 0.05 ppm and 0.02 ppm of Se i n the l i v e r ( f r e s h b a s i s ) were the r e s p e c t i v e i n d i c a t o r s of border l i n e and severe Se d e f i c i e n c y i n sheep. b)Measurement of g l u t a t h i o n e peroxidase a c t i v i t y i n blood  and t i s s u e s ; Whole blood and e r y t h r o c y t e g l u t a t h i o n e peroxidase (Se dependent) enzyme a c t i v i t i e s have been measured by many workers to assess the Se s t a t u s i n c a t t l e (Wilson and Judson, 1976; Thompson et a l . 1976, Thompson et a l . 1981, R o l l e r et a l . 1983, H i d i r o g l o u et a l . 1985) and i n sheep (Oh et a l . 1976, Whanger et a l . 1977, Anderson et a l . 1979, Peter et a l . 1980, Sheppard and M i l l e r , 1981) . The 17 advantage with GSH-Px e s t i m a t i o n i s that i t i s easy to perform compared to the Se a n a l y s i s and provi d e s a b e t t e r estimate of long term Se s t a t u s i n animals. The l i m i t a t i o n of GSH-Px compared to s t a b l e Se i s th a t the enzyme a c t i v i t y d e t e r i o r a t e s i n a r e l a t i v e l y short time and needs a l a g time before i t gets i n c o r p o r a t e d i n t o the e r y t h r o c y t e s ( R o l l e r et a l . 1984a) . The storage c o n d i t i o n s have a l s o been shown to a f f e c t the plasma enzyme a c t i v i t y (Zhang et a l . 1986). I t has been r e p o r t e d t h a t the l a g p e r i o d f o r GSH-Px a c t i v i t y to reach comparative peak Se c o n c e n t r a t i o n s i n sheep was approximately 30 days (Sheppard and M i l l e r , 1981) and i n c a t t l e n e a r l y 35 days (Scholz and Hutchinson, 1979) . D e f i c i e n t , m a r g i n a l l y d e f i c i e n t and adequate whole blood g l u t a t h i o n e peroxidase a c t i v i t i e s i n beef c a t t l e have been repo r t e d to be < 15, 16-35, and > 35 mU/mg Hb r e s p e c t i v e l y ( R o l l e r et a l . , 1983 and Rice and McMurray, 1986) . H i d i r o g l o u et a l . (1985) suggested that l e s s than 35 IU GSH-Px/g Hb would i n d i c a t e Se d e f i c i e n c y i n d a i r y c a t t l e . B r i t i s h workers (Anderson et a l . 1978; and Anderson et a l . 1979) considered c a t t l e with l e s s than 15 GSH-Px u n i t s per ml RBC or sheep with l e s s than 28 u n i t s per ml RBC to be Se d e f i c i e n t . The maximum reported serum Se c o n c e n t r a t i o n t h a t c o r r e l a t e d with GSH-Px a c t i v i t y i n c a t t l e was 100-120 18 ng/ml, which was equivalent to 320-350 ng Se/ml of whole blood (Backall and Scholz, 1979; Scholz and Hutchinson, 1979). More recently Stevens et a l . (1985) reported that RBC glutathione peroxidase a c t i v i t y was useful in determining the diagnosis of chronic Se t o x i c i t y . In their studies serum Se concentrations upto 0.789 ug/ml, which were approximately 6.2 times higher than the previously reported values for dairy c a t t l e , correlated well with the glutathione peroxidase enzyme a c t i v i t y . Though the a c t i v i t y of glutathione peroxidase i n whole blood i s generally considered to be a better indicator of Se status i n animals than the plasma Se l e v e l s , recent reports by Lane et a l . (1982) and Hidiroglou et a l . (1985) on the contrary suggest that this point needs further c l a r i f i c a t i o n . c)Assay of blood glutathione peroxidase a c t i v i t y by rapid  tests; A rapid spot test u t i l i z i n g the levels of glutathione peroxidase has been introduced with results indicating either adequate, marginal or d e f i c i e n t levels of Se i n the blood of ruminants (Board and Peter, 1976 and Norman et a l . 1981). A k i t for measuring the blood GSH-Px concentration has been devised (Ransel, Randox Labs, U.K.) and evaluated (Rice and Blanchflower, 1986). Radio Immuno Assay (RIA) 19 ( B a r e t e t a l . 1983) a n d E L I S A (Enzyme L i n k e d Immuno S o r b e n t A s s a y ) ( M c M u r r a y e t a l . 1986) methods h a v e b e e n d e v e l o p e d f o r a s s a y i n g t h e r e d b l o o d c e l l G S H - P x . R o b i n s o n a n d Thomson (1983) h a v e r e m a r k e d t h a t a l t h o u g h more r a p i d a s s a y s o f G S H - P x a c t i v i t y were u s e f u l a n d c o m p l e m e n t a r y , t h e y C a n n o t t o t a l l y r e p l a c e t h e m e a s u r e m e n t s o f Se i n t i s s u e s a n d b i o l o g i c a l f l u i d s f o r a more p r e c i s e a s s e s s m e n t o f Se s t a t u s . 75 d ) E r y t h r o c y t e Se U p t a k e s t u d i e s i n v i t r o : 75 I n v i t r o u p t a k e o f Se b y e r y t h r o c y t e s h a s b e e n s t u d i e d t o a s s e s s t h e Se s t a t u s i n s h e e p . A m e r i c a n w o r k e r s ( L o p e z e t a l . 1968) r e p o r t e d t h a t t h e i n i t i a l 7 5 f l u x a n d r a t e o f i n v i t r o u p t a k e o f Se by o v i n e e r y t h r o c y t e s d e c r e a s e d w i t h i n c r e a s i n g Se l e v e l i n t h e d i e t . T h e y c o n c l u d e d t h a t t h i s t e c h n i q u e was p r o m i s i n g a n d u s e f u l t o a s s e s s t h e n u t r i t i o n a l Se s t a t u s i n t h e i m m a t u r e o v i n e . I n c o n t r a s t A u s t r a l i a n w o r k e r s ( J e l i n e k e t a l . 1985) f o u n d a s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n b e t w e e n t h e 75 e r y t h r o c y t e Se u p t a k e v a l u e s a n d e r y t h r o c y t e G S H - P x a c t i v i t i e s . F u r t h e r m o r e t h e y c o u l d n o t d e m o n s t r a t e a n y 75 c o r r e l a t i o n b e t w e e n t h e Se u p t a k e a n d t h e d e v e l o p m e n t o f n u t r i t i o n a l m y o p a t h y i n w e a n e r s h e e p . R e c e n t l y A t r o s h i e t 75 a l . (1985) r e p o r t e d t h a t t h e u p t a k e o f Se by e r y t h r o c y t e s was r e l a t e d t o t h e i r g l u t a t h i o n e (GSH) c o n t e n t . I n t h e i r s t u d i e s w i t h l o w a n d h i g h GSH F i n n s h e e p , t h e f o r m e r g r o u p 20 e x h i b i t e d a higher e r y t h r o c y t e Se uptake and higher GSH-Px a c t i v i t y compared to the l a t t e r . T h i s suggested the presence of more a c t i v e d e t o x i f i c a t i o n mechanisms i n low-GSH animals. D)METABOLIC FATE OF SELENIUM IN ANIMALS: Ingested Se undergoes b i o c h e m i c a l t r a n s f o r m a t i o n s before i t i s completely metabolized i n animals. An understanding of these metabolic changes was made p o s s i b l e by the use of i s o t o p e s , e i t h e r r a d i o a c t i v e or s t a b l e . D i f f e r e n t aspects of the metabolic f a t e of Se are d i s c u s s e d . a ) D i e t a r y i n t a k e of selenium: Se i s in g e s t e d i n many o r g a n i c forms by animals and man through feeds and food. Se occurs mainly as s e l e n o c y s t i n e , s e l e n o c y s t e i n e , selenomethionine, and Se-methylselenomethionine i n p l a n t s and p l a n t products (Peterson and B u t l e r , 1962; S h r i f t , 1969; Olson et a l . 1970). The amount of Se present i n p l a n t s may vary and i s known to depend on many f a c t o r s i n c l u d i n g s o i l type, a p p l i c a t i o n of f e r t i l i z e r s , p l a n t s p e c i e s e t c . This s u b j e c t has been r e c e n t l y reviewed by G i s s e l - N i e l s e n e t a l . (1984). Although organic Se i s the major form f o r animals consuming p l a n t s and p l a n t products, i n o r g a n i c Se s a l t s (sodium s e l e n i t e or s e l e n a t e ) are o f t e n used to supplement feeds d e f i c i e n t i n Se. 21 b ) A b s o r p t i o n ; The extent of a b s o r p t i o n of Se i n animals i s known to vary with the species and the form and amounts of 75 element i n g e s t e d . Studies with Se i n d i c a t e d t h a t the duodenum was the major s i t e of a b s o r p t i o n of Se and there was no a b s o r p t i o n e i t h e r from rumen or abomasum of sheep or from the stomach of pigs (Wright and B e l l , 1966) . When 75 r a t i o n s c o n t a i n i n g 0.35 and 0.50 ppm Se were fed as Se-s e l e n i t e to sheep and swine r e s p e c t i v e l y , about 29% of 75 i n g e s t e d Se was absorbed i n sheep and 77% i n p i g s . McConnell and Cho (1965) showed t h a t selenomethionine was a c t i v e l y t r a n s p o r t e d across the ever t e d duodenal sacs i n v i t r o , whereas s e l e n i t e was absorbed mainly by d i f f u s i o n . Recently Arduser et a l . (1985) i n an i n v i t r o study have shown that s e l e n a t e was tr a n s p o r t e d across the ileum of the r a t a g a i n s t a c o n c e n t r a t i o n g r a d i e n t s i m i l a r to selenomethionine. However, the same group of workers (Wolffram et a l . 1985) f a i l e d to demonstrate such a mechanism f o r s e l e n i t e under e i t h e r i n v i t r o or i n v i v o c o n d i t i o n s . Instead they found t h a t s e l e n i t e was absorbed only by d i f f u s i o n . Se was g e n e r a l l y found to be b e t t e r absorbed and u t i l i z e d i n monogastrics than i n ruminants and t h i s was ex p l a i n e d by the f a c t t h at d i e t a r y Se was reduced to i n s o l u b l e forms by microbes i n the rumen (Cousins and 22 C a i r n e y , 1961; Peterson and Spedding, 1963) . Whanger et a l . (1968) reported that a grea t e r percentage of i n o r g a n i c than o r g a n i c Se was converted i n t o these i n s o l u b l e forms and the convers i o n was higher with s t a r c h based d i e t s than with roughage d i e t s . H i d i r o g l o u et a l . (1968b) have observed that rumen b a c t e r i a were capable of 75 m e t a b o l i z i n g i n o r g a n i c Se and i n c o r p o r a t i n g i t i n t o 75 b a c t e r i a l p r o t e i n i n the form of Se-selenomethionine. E h l i g et a l . (1967) have observed a g r e a t e r Se r e t e n t i o n i n lambs with selenomethionine than w i t h s e l e n i t e and reasoned that t h i s was due to a higher r a t e of u r i n a r y e x c r e t i o n of Se from s e l e n i t e than from selenomethionine. Ashmead and C h r i s t y (1985) have r e c e n t l y reviewed the su b j e c t of i n t e s t i n a l a b s o r p t i o n of t r a c e m i n e r a l s . A c c o r d i n g to them, s e v e r a l f a c t o r s i n c l u d i n g pH changes and c o n c e n t r a t i o n of the i n o r g a n i c s a l t s a f f e c t the a b s o r p t i o n of minerals i n i n o r g a n i c forms. Hence, they recbmmended that amino a c i d c h e l a t e s of min e r a l s were the best forms to be supplemented s i n c e they enhanced the e n t e r i c a b s o r p t i o n . Barbezat et a l . (1984) i n t h e i r review have d e s c r i b e d the methods employed to measure a b s o r p t i o n of Se i n animals and man. They have a l s o d i s c u s s e d the f a c t o r s , both i n t r i n s i c and e x t r i n s i c , t h a t a f f e c t the i n t e s t i n a l a b s o r p t i o n of Se. C o r n e l l workers (Combs, 1976; Combs and P e s t i , 1976) have shown that the ab s o r p t i o n of s e l e n i t e i n chic k was 23 a f f e c t e d by the d i e t a r y c o n c e n t r a t i o n s of vit a m i n s A, C and E which are capable of promoting Se a b s o r p t i o n when added to the d i e t at higher than the r e q u i r e d l e v e l s . R e c e n t l y , Mutanen and Mykkanen (1984) from F i n l a n d 75 r e p o r t e d t h a t S e - s e l e n i t e a b s o r p t i o n from duodenum was lower i n chickens fed vegetable f a t (corn o i l or sunflower o i l ) than i n those fed animal f a t ( b u t t e r ) . Cantor et a l . (1975) have s t u d i e d the b i o l o g i c a l a v a i l a b i l i t y of Se i n f e e d s t u f f s and found that 60-90% of Se from p l a n t sources compared to l e s s than 25% of Se from animal sources was b i o l o g i c a l l y a v a i l a b l e f o r the pr e v e n t i o n of exudative d i a t h e s i s i n c h i c k s . O'Dell (1984) d e f i n e d b i o a v a i l a b i l i t y of Se as the f r a c t i o n of element i n a f e e d s t u f f t h a t was absorbed, t r a n s p o r t e d and converted to a b i o l o g i c a l l y a c t i v e form, Se dependent g l u t a t h i o n e peroxidase. c ) B l o o d T r a n s p o r t ; Wisconsin workers (Oh et a l . 1974) r e p o r t e d t h a t 75-85% of the Se i n ovine red blood c e l l s was a s s o c i a t e d with the selenoenzyme, g l u t a t h i o n e peroxidase (GSH-Px). Whereas almost a l l of the Se i n e r y t h r o c y t e s of r a t s was a s s o c i a t e d with GSH-Px, o n l y 10% of selenium was a s s o c i a t e d with t h i s enzyme i n human red blood c e l l s (Behne and Wolters, 1979) and 20% with GSH-Px of RBC i n the p i g (Xia et a l . 1985). The metabolism of s e l e n i t e by blood i n - v i t r o i s s i m i l a r i n both the primates and other animals though there 24 are d i f f e r e n c e s i n the amount of Se a s s o c i a t e d with GSH-Px enzyme. The uptake and r e l e a s e of s e l e n i t e by human (Lee et a l . 1969) , bovine (Jenkins and H i d i r o g l o u , 1972b) or ovine (McMurray and Davidson, 1979) e r y t h r o c y t e s i n v o l v e s u l f h y d r y l groups, and the b i n d i n g of s e l e n i t e to plasma p r o t e i n was shown to be dependent upon red blood c e l l s (Lee et a l . 1969; Sandholm, 1974, 1975; McMurray and Davidson, 1979) . Symonds et a l . (1981) showed a s i m i l a r 75 i n i t i a l uptake and l a t e r r e l e a s e of Se by the l i v e r i n 75 v i v o w i t h i n 30-60 minutes a f t e r Na2 S e 0 3 i n j e c t i o n i n t o the d a i r y cow. The uptake and subsequent metabolism of s e l e n i t e were found to be dependent upon reduced g l u t a t h i o n e (GSH) c o n c e n t r a t i o n s i n the red blood c e l l s (Gasiewicz and Smith, 1978a). The e r y t h r o c y t e s were reported to take 75 up the S e - s e l e n i t e i n i t i a l l y a f t e r the t r a c e r i n j e c t i o n and r e l e a s e i t back i n t o the blood i n an a l t e r e d form, e i t h e r as l ^Se or s i m i l a r l y reduced s e l e n i d e (Gasiewicz and Smith, 1978b). The compound r e l e a s e d from the e r y t h r o c y t e s was observed to get bound to the plasma p r o t e i n s (Lee et a l . 1969; Jenkins and H i d i r o g l o u , 1972b). The plasma p r o t e i n s that c a r r y selenium have been shown to vary i n d i f f e r e n t s p e c i e s . Albumin i n mice (Sandholm, 1974), gamma g l o b u l i n s i n c a t t l e (Symonds et a l . 1981) and alpha l i p o p r o t e i n s i n man (Sandholm, 1975) were found to be the primary c a r r i e r s of selenium. Burk and Gregory (1982) i s o l a t e d and i d e n t i f i e d f r a c t i o n s of g l u t a t h i o n e 75 peroxidase and another s e l e n o p r o t e i n c a l l e d Se-P i n plasma 25 a n d l i v e r o f r a t s . They a l s o r e p o r t e d t h a t Se 75 d e f i c i e n c y d e c r e a s e d Se i n c o r p o r a t i o n by GSH-Px a t 3 75 and 72 h o u r s a f t e r S e - s e l e n i t e i n j e c t i o n b u t i n c r e a s e d 75 75 Se i n c o r p o r a t i o n by Se-P. C o n t r a r y t o t h i s M o t s e n b o c k e r a n d T a p p e l (1984) have r e p o r t e d t h a t t h e 75 75 i n c o r p o r a t i o n o f Se i n t o p l a s m a s e l e n o p r o t e i n - P ( Se-P) was a f f e c t e d by t h e d i e t a r y Se i n r a t s . They h a v e a l s o s u g g e s t e d t h a t t h e p r o p o r t i o n o f Se i n p l a s m a s e l e n o p r o t e i n -P r e f l e c t e d t h e l o n g t e r m Se s t a t u s o f r a t s . d ) D i s t r i b u t i o n o f s e l e n i u m i n t i s s u e s ; The t i s s u e d i s t r i b u t i o n o f Se i n v a r i o u s a n i m a l s h a s be e n s t u d i e d u s i n g 75 e i t h e r Se o r s t a b l e Se as t r a c e r s . The t i s s u e Se c o n t e n t was shown t o be d e p e n d e n t upon b o t h t h e l e v e l a n d t h e c h e m i c a l f o r m o f t h e e l e m e n t i n t h e d i e t . The k i d n e y , e s p e c i a l l y t h e c o r t e x , h a d t h e h i g h e s t Se c o n c e n t r a t i o n f o l l o w e d b y l i v e r and o t h e r g l a n d u l a r t i s s u e s l i k e s p l e e n , p a n c r e a s a n d p i t u i t a r y . M u s c l e s , b o n e s and b l o o d c o n t a i n e d r e l a t i v e l y l e s s Se. C a r d i a c m u s c l e c o n t a i n e d more Se t h a n t h e s k e l e t a l m u s c l e ( W r i g h t , 1 9 6 5 ; K i n c a i d e t a l . 1 9 7 7 ; Mahan a nd Moxon, 1 9 7 8 ) . The k i d n e y and l i v e r a r e t h e most s e n s i t i v e i n d i c a t o r s o f Se s t a t u s o f an a n i m a l a n d t h e b i o p s y o f t h e s e o r g a n s c a n p r o v i d e v a l u a b l e d i a g n o s t i c i n f o r m a t i o n ( A n d r e w s e t a l . 1 9 6 8 ) . A t t o x i c (>5 ppm) l e v e l s o f Se i n t a k e , Se c o n c e n t r a t i o n s were r e p o r t e d t o r e a c h a s h i g h a s 5-10 ppm i n l i v e r and k i d n e y s a n d 1-2 ppm i n m u s c l e s o f f a r m a n i m a l s (Maag and G l e n n , 1 9 6 7 ; C a s t e e l 26 et a l . 1985; and Hopper et a l . 1985) . 75 Se d e f i c i e n t animals r e t a i n e d Se more e f f i c i e n t l y than Se supplemented animals. T h i s p a t t e r n has been observed i n c h i c k s (Jensen et a l . 1963), r a t s (Burk et a l . 1968) , and sheep (Muth et a l . 1967; Lopez et a l . 1969) and i n c a t t l e ( K i n c a i d et a l . 1977) . The i n c r e a s e d r e t e n t i o n r e f l e c t e d g r e a t e r t i s s u e demands f o r Se. I t i s p o s s i b l e t h a t the Se e x c r e t i o n p a t t e r n may be a f f e c t e d by the d i e t a r y Se l e v e l . Lopez et a l . (1969) measured whole body r e t e n t i o n of Se i n v a r i o u s t i s s u e s along with 75 u r i n a r y and f e c a l Se l o s s e s f o l l o w i n g Se i n j e c t i o n i n t o lambs r e c e i v i n g d i f f e r e n t l e v e l s of Se i n t h e i r d i e t s . The 75 75 whole body l o s s of Se and c o n c e n t r a t i o n of Se i n v a r i o u s t i s s u e s were i n v e r s e l y r e l a t e d to the d i e t a r y Se i n t a k e . The f e e d i n g of f i s h meal to p o u l t r y and swine r e s u l t e d i n poor Se r e t e n t i o n . When Se was fed as selenomethionine i t r e s u l t e d i n gr e a t e r Se c o n c e n t r a t i o n i n muscle (Osman and Latshaw, 1976) and i n egg (Latshaw and B i g g e r t , 1981) than when fed as s e l e n i t e or s e l e n o c y s t i n e or f i s h meal. Mahan and Moxon (1978) observed low serum Se l e v e l s i n four week o l d weanling p i g l e t s f e d f i s h meal and they reasoned t h a t i t was due to the b i n d i n g of Se by mercury present i n f i s h meal. More r e c e n t l y Bern et a l . (1985) s t u d i e d the e f f e c t of mercuric compounds on the i n h i b i t i o n of g l u t a t h i o n e 27 p e r o x i d a s e i n r a t l i v e r . I t was s u g g e s t e d t h a t t h e i n h i b i t i o n o f G S H - P x a n d c o n s e q u e n t u n a v a i l a b i l i t y o f Se was n o t due t o s i m p l e b i n d i n g o f t h e a c t i v e s i t e Se m o i e t y o f g l u t a t h i o n e p e r o x i d a s e t o m e r c u r y b u t due t o a n a l t e r a t i o n i n t h e t e r t i a r y o r q u a r t e r n a r y s t r u c t u r e o f t h e enzyme (Bern e t a l . 1 9 8 5 ) . e ) T i s s u e m e t a b o l i s m ; D e p e n d i n g u p o n i t s c h e m i c a l f o r m , Se i n g e s t e d i n t h e d i e t i s known t o u n d e r g o some m e t a b o l i c p r o c e s s e s b e f o r e i t i s u t i l i z e d b y t h e a n i m a l . I n o r g a n i c Se h a s b e e n shown t o be c o n v e r t e d t o o r g a n i c f o r m s i n t h e t i s s u e s . T h i s was d e m o n s t r a t e d by t h e i n c o r p o r a t i o n o f 75 S e - s e l e n i t e i n t o G S H - P x (Oh e t a l . 1 9 7 4 ) , i n t o d i m e t h y l s e l e n i d e ( H s i e h a n d G a n t h e r , 1975) a n d i n t o s e l e n o a m i n o a c i d s ( O l s o n a n d P a l m e r , 1 9 7 6 ) . When Se was c o n s u m e d a s s e l e n o m e t h i o n i n e o r i n o t h e r o r g a n i c f o r m s p r e s e n t i n n a t u r a l s o u r c e s , i t was shown t o be r e l e a s e d as s e l e n i t e a f t e r u n d e r g o i n g p o s t a b s o r p t i v e c a t a b o l i c c h a n g e s ( O l s o n e t a l . 1 9 7 0 ) . G i v e n t h i s , a n d t h e f a c t t h a t Se s u p p l e m e n t s t o a n i m a l d i e t s c o n s i s t l a r g e l y o f i n o r g a n i c s e l e n i t e , i t i s e v i d e n t t h a t Se m e t a b o l i s m i n a n i m a l s i s i n t i m a t e l y r e l a t e d t o t h e m e t a b o l i s m o f s e l e n i t e . The f o r m a t i o n o f d i m e t h y l s e l e n i d e a n d h y d r o g e n s e l e n i d e f r o m s e l e n i t e h a s b e e n e x t e n s i v e l y s t u d i e d b y G a n t h e r a n d h i s c o l l e a g u e s a n d was r e v i e w e d by G a n t h e r ( 1 9 7 9 ) . The m e t a b o l i c p a t h was shown t o i n v o l v e a s i x e l e c t r o n r e d u c t i o n f r o m t h e +4 o x i d a t i o n s t a t e o f Se i n s e l e n i t e t o t h e 28 -2 l e v e l , followed by methylation of selenide as shown below: (1) H 2 Se0 3 + 4 GSH > GSSeSG + GSSG + 3 H 20 (2) GSSeSG + GSH > GSSeH + GSSG OR GSH reductase (E.C. 1.6.4.2) GSSeSG — — > GSSeH + GSH NADPH+H+ NADP+ GSH reductase (3) GSSeH > H 2 S e + G S H NADPH+H+ NADP+ Se-methyl transferase (4) H 2 Se + 2 S-Adenosyl > (CH 3) 2 Se + S-Adenosyl-methionine homocysteine in the f i r s t step, selenite reacts nonenzymatically with glutathione (GSH) to form the s e l e n o t r i s u l f i d e derivative (GSSeSG) and an equimolar quantity of d i s u l f i d e derivative of GSH (GSSG). Subsequently, the s e l e n o t r i s u l f i d e derivative i s reduced to a selenopersulfide (GSSeH), either nonenzymatically in the presence of excess GSH or by means of NADPH and the enzyme, glutathione reductase. The selenopersulfide i s quite unstable and can decompose to GSH and elemental Se. A l t e r n a t i v e l y , glutathione reductase can catalyse a NADPH linked reduction of the selenopersulfide to an a c i d - v o l a t i l e Se compound, 29 h y d r o g e n s e l e n i d e . T h i s r e d u c t i o n p a t h w a y h a s b e e n shown t o be o p e r a t i v e i n e r y t h r o c y t e s . S e v e r a l 75 w o r k e r s h a v e d e m o n s t r a t e d t h a t S e - s e l e n i t e was r a p i d l y t a k e n up b y m o u s e , b o v i n e , o r human r e d b l o o d c e l l s a n d t h e n r e l e a s e d i n an a l t e r e d f o r m , p o s s i b l y l ^ S e r a t h e r t h a n GSSeSG ( G a n t h e r , 1979) w h i c h was r e a d i l y b o u n d t o p l a s m a p r o t e i n s ( L e e e t a l . 1969; J e n k i n s a n d H i d i r o g l o u , 1972b). A l t h o u g h p a t h w a y s f o r t h e c o n v e r s i o n o f s e l e n i t e t o s e l e n i d e h a v e b e e n f u l l y e s t a b l i s h e d , t h e r e seems t o be some u n c e r t a i n t y c o n c e r n i n g t h e s y n t h e s i s o f s e l e n o c y s t e i n e f r o m s e l e n i t e . I n mammal ian t i s s u e s , s y n t h e s i s o f s e l e n o c y s t e i n e h a s b e e n as sumed t o be s i m i l a r t o t h a t o f c y s t e i n e . J a p a n e s e w o r k e r s h a v e e x t e n s i v e l y s t u d i e d s e l e n o c y s t e i n e m e t a b o l i s m i n a n i m a l s a n d d e s c r i b e d t h e e n z y m o l o g i c a l a s p e c t s o f s y n t h e s i s a n d d e g r a d a t i o n o f s e l e n o c y s t e i n e i n mammal ian t i s s u e s i n a r e c e n t r e v i e w ( T a n a k a e t a l . 1985) . T h o u g h t h e d e g r a d a t i o n o f s e l e n o c y s t e i n e t o s e l e n i t e i s p o s s i b l e , t h e h i g h r e d u c t i o n p o t e n t i a l o f Se f a v o u r s t h e r e l e a s e o f h y d r o g e n s e l e n i d e i n s t e a d ( S u n d e , 1984) . S e l e n o c y s t e i n e h a s b e e n shown t o be t h e a c t i v e s i t e o f g l u t a t h i o n e p e r o x i d a s e i n mammals ( F o r s t r o m e t a l . 1978; L a d e n s t e i n e t a l . 1979) a n d i n b a c t e r i a (Cone e t a l . 1976; J o n e s e t a l . 1979) . S e l e n o c y s t e i n e h a s a l s o b e e n shown t o be p r e s e n t i n o v i n e t i s s u e o r g a n e l l e s ( B i e l s t e i n e t a l . 1981). E v i d e n c e h a s 30 b e e n p r e s e n t e d b y Tappe ' l a n d h i s c o w o r k e r s f o r a s p e c i f i c t r a n s f e r RNA i n r a t l i v e r f o r s e l e n o c y s t e i n e . By t h i s m e c h a n i s m t h e s e l e n o c y s t e i n e was f o u n d t o be i n c o r p o r a t e d i n t o p r o t e i n s d u r i n g t r a n s l a t i o n v i a t h e a c t i o n o f t h i s s p e c i f i c tRNA (Hawkes e t a l . 1982) . O l s o n a n d P a l m e r (1976) h a v e p r o p o s e d an a l t e r n a t i v e h y p o t h e s i s w h e r e b y i n a p r e f o r m e d c y s t e i n e , i f t h e s u l f i d e c o u l d be r e p l a c e d b y s e l e n i d e o r i t s e q u i v a l e n t , t h i s w o u l d r e s u l t i n t h e s y n t h e s i s o f s e l e n o c y s t e i n e . The l a t t e r p o s s i b i l i t y was s u p p o r t e d by Sunde a n d H o e k s t r a (1980) , who 75 d e m o n s t r a t e d t h a t t h e i n c o r p o r a t i o n o f S e - s e l e n o c y s t e i n e i n t o G S H - P x by t h e i s o l a t e d p e r f u s e d r a t l i v e r was d e c r e a s e d by a d d i t i o n o f a 9 - f o l d e x c e s s o f n o n l a b e l l e d s e l e n i t e o r s e l e n i d e t o t h e p e r f u s i o n m e d i u m , w h i l e a d d i t i o n o f a 1 0 0 - f o l d e x c e s s o f u n l a b e l l e d s e l e n o c y s t e i n e was n o t e f f e c t i v e . On t h e o t h e r h a n d , c o n v e r s i o n o f s e l e n i t e t o s e l e n o m e t h i o n i n e a n d p r e s e n c e o f s e l e n o m e t h i o n i n e i n a n i m a l t i s s u e s h a v e n o t b e e n d e m o n s t r a t e d so f a r , a l t h o u g h s e l e n o m e t h i o n i n e was shown t o be p r e s e n t i n t h i o l a s e ( H a r t m a n i s a n d S t a d t m a n , 1982) a n d i n 3 - h y d r o x y b u t y r y l CoA d e h y d r o g e n a s e ( H a r t m a n i s a n d S l i w k o w s k i , 1985) o f C l o s t r i d i u m k l u y v e r i . f ) M e t a b o l i c f u n c t i o n s o f s e l e n i u m : Se as a c o m p o n e n t o f g l u t a t h i o n e p e r o x i d a s e ( G S H - P x ) f u n c t i o n s a s a p a r t o f a m u l t i c o m p o n e n t a n t i o x i d a n t d e f e n c e s y s t e m w i t h i n t h e c e l l . R o t r u c k e t a l . (1973) have d e m o n s t r a t e d t h a t G S H - P x 31 r e d u c e s t o x i c h y d r o p e r o x i d e s t o h y d r o x y a c i d s a n d h y d r o g e n p e r o x i d e t o w a t e r a n d p r o t e c t s t h e b i o m e m b r a n e s f r o m o x i d a n t d a m a g e . The p r o o x i d a n t s t r e s s o r s t h a t i n d u c e o x i d a t i o n i n membranes h a v e b e e n i d e n t i f i e d . Many c e l l u l a r o x i d a t i o n s , b o t h e n z y m a t i c a n d n o n - e n z y m a t i c , h a v e b e e n shown t o g e n e r a t e r e a c t i v e r a d i c a l s , f o r e x a m p l e , s u p e r o x i d e a n i o n ^ ° 2 ^ t n e u n i v a l e n t r e d u c t i o n o f o x y g e n . S u p e r o x i d e d i s m u t a s e s ( b o t h c y t o s o l i c a n d m i t o c h o n d r i a l ) r e d u c e s u p e r o x i d e a n i o n t o h y d r o g e n p e r o x i d e a n d p r e v e n t t h e i n i t i a t i o n o f membrane o x i d a t i o n ( F r i d o v i c h , 1975) . The h y d r o g e n p e r o x i d e f o r m e d i s r e d u c e d b y g l u t a t h i o n e p e r o x i d a s e i n t h e c y t o s o l a n d m i t o c h o n d r i a t o w a t e r , w h i l e c a t a l a s e r e d u c e s t h e h y d r o g e n p e r o x i d e t o w a t e r i n p e r o x i s o m e s . The c o m p e t i t i o n o f b o t h t h e s e enzymes f o r t h e common s u b s t r a t e h y d r o g e n p e r o x i d e was r e p o r t e d t o be v e r y l i t t l e ( F l o h e , 1982) . I n t h e a b s e n c e o f g l u t a t h i o n e p e r o x i d a s e , h y d r o g e n p e r o x i d e may f u r t h e r r e a c t w i t h e x c e s s s u p e r o x i d e a n i o n s t o p r o d u c e a more p o w e r f u l o x i d a n t , h y d r o x y r a d i c a l (OH) w h i c h c a n c a u s e s e v e r e o x i d a t i v e d e g r a d a t i o n o f c e l l u l a r c o m p o n e n t s . G l u t a t h i o n e p e r o x i d a s e i s t h e o n l y i s o l a t e d s e l e n o e n z y m e i n mammals a t t h e p r e s e n t t i m e ( R o t r u c k e t a l . 1973; F l o h e , 1973) . The p r o p e r t i e s a n d b i o c h e m i c a l f u n c t i o n s o f t h i s enzyme h a v e b e e n e x t e n s i v e l y s t u d i e d a n d w e r e r e c e n t l y r e v i e w e d ( T a p p e l , 1984; F l o h e , 1985) . G S H - P x f r o m b o v i n e a n d o v i n e e r y t h r o c y t e s h a s b e e n shown t o c o n t a i n 32 4 g atoms o f Se p e r mole o f enzyme ( F l o h e e t a l . 1973; Oh e t a l . 1974) . T h i s enzyme ha s b e e n p u r i f i e d f r o m t i s s u e s o f c a t t l e , s h e e p , s w i n e , r a t s and humans and shown t o have a m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 80,000 d a l t o n s w i t h f o u r i d e n t i c a l s u b - u n i t s ( G a n t h e r e t a l . 1976) . The r a t l i v e r GSH-Px was f o u n d t o c o n t a i n a t o t a l o f 153 amino a c i d s w i t h two c y s t e i n e and t h r e e m e t h i o n i n e r e s i d u e s i n e a c h s u b - u n i t (Nakamura e t a l . 1974), w h i l e t h a t o f b o v i n e r e d b l o o d c e l l s was f o u n d t o have 178 amino a c i d s ( L a d e n s t e i n e t a l . 1979) . The m e c h a n i s m o f a c t i o n o f GSH-Px has b e e n s t u d i e d e x t e n s i v e l y b y German w o r k e r s and h a s bee n r e c e n t l y r e v i e w e d ( F l o h e , 1982; 1985). F i r s t l y Se p r e s e n t i n t h e enzyme a s s e l e n o c y s t e i n e s e l e n o l (E-SeH) i s o x i d i z e d b y t h e p e r o x i d e s u b s t r a t e and c a u s e s t h e r e l e a s e o f c o r r e s p o n d i n g a l c o h o l a l o n g w i t h t h e f o r m a t i o n o f s e l e n e n i c a c i d ( E - S eOH). S u b s e q u e n t l y a mole o f g l u t a t h i o n e i s ad d e d t o t h e enzyme r e s u l t i n g i n t h e f o r m a t i o n o f a n i n t e r m e d i a t e , s e l e n e n y l s u l f i d e ( E - S e S G ) . F i n a l l y a n o t h e r m o le o f g l u t a t h i o n e r e a c t s w i t h t h e enzyme r e s u l t i n g i n t h e f o r m a t i o n o f o x i d i z e d g l u t a t h i o n e (GSSG) and s e l e n o l ( E -S e H ) . The s e l e n o l r e l e a s e d a f t e r t h e a d d i t i o n o f s e c o n d g l u t a t h i o n e m o l e c u l e r e s t a r t s t h e n e x t c a t a l y t i c c y c l e o f t h e enzyme. T h i s m echanism h e l p s remove t h e h y d r o g e n p e r o x i d e and h y d r o p e r o x i d e s f r o m t h e bod y s y s t e m a n d a l s o a i d s i n p r e v e n t i n g t h e f o r m a t i o n o f s u b s e q u e n t 33 h y d r o p e r o x i d e s due t o l i p i d p e r o x i d a t i o n . A d i r e c t r e l a t i o n s h i p b e t w e e n t h e t i s s u e G S H - P x l e v e l s a n d t h e d i e t a r y Se i n t a k e was f i r s t r e p o r t e d b y H a f e m a n e t a l . (1974) . I t was o b s e r v e d t h a t t h e RBC enzyme a c t i v i t y d e c r e a s e d by 18% i n r a t s f r o m t h a t o f c o n t r o l s w i t h t h e l o w e s t Se i n t a k e (0 .05 p p m ) , w h e r e a s , t h e h i g h e s t Se i n t a k e r a i s e d t h e e r y t h r o c y t e G S H - P x a c t i v i t y by a b o u t 60% (Hafeman e t a l . 1974) . The Se s u p p l e m e n t a t i o n h a s a l s o b e e n shown t o be a s s o c i a t e d w i t h i n c r e a s e d G S H - P x a c t i v i t y i n c h i c k s ( N o g u c h i e t a l . 1973, Omaye a n d T a p p e l , 1974; C a n t o r e t a l . 1975), i n s h e e p ( G o d w i n e t a l . 1975, Oh e t a l . 1976; Whanger e t a l . 1977, 1978; A n d e r s o n e t a l . 1979; J e l i n e k e t a l . 1985) , a n d i n c a t t l e ( A n d e r s o n e t a l . 1978, S t e v e n s e t a l . 1985, H i d i r o g l o u e t a l . 1985) . T h e r e f o r e , a s s a y o f g l u t a t h i o n e p e r o x i d a s e a c t i v i t y i n b l o o d i s o f t e n u s e d a s a t o o l i n d i a g n o s i n g Se d e f i c i e n c y i n a n i m a l s . The u n d e r s t a n d i n g o f Se m e t a b o l i s m was f u r t h e r c o m p l i c a t e d b y t h e d i s c o v e r y o f a n o n - S e d e p e n d e n t g l u t a t h i o n e p e r o x i d a s e enzyme ( E . C . 2 . 5 . 1 . 1 8 ) ( L a w r e n c e a n d B u r k , 1976) . N o n - S e - G S H - P x was shown t o be l o c a l i z e d i n m i t o c h o n d r i a , c y t o s o l a n d m i c r o s o m e s o f r a t l i v e r . A l t h o u g h t h e Se d e p e n d e n t a n d n o n - S e d e p e n d e n t g l u t a t h i o n e p e r o x i d a s e s c a r r y o u t t h e same r e a c t i o n , many o f t h e i r c h a r a c t e r i s t i c s a s enzymes were shown t o be d i f f e r e n t . Se 3 4 d e p e n d e n t GSH-Px d e s t r o y s b o t h t h e s u b s t r a t e s H 2 ° 2 a n c * o r g a n i c h y d r o p e r o x i d e s , w h i l e non-Se-GSH-Px d o e s n o t m e t a b o l i z e H~0 o. Non-Se-GSH-Px a l s o h a s a h i g h e r k t o w a r d s 2 2 3 m o r g a n i c h y d r o p e r o x i d e s u b s t r a t e s t h a n Se-GSH-Px a nd t h e f o r m e r i s f o u n d i n f e w e r c e l l t y p e s t h a n t h e Se d e p e n d e n t enzyme. B u r k (1983) i n a r e c e n t r e v i e w h a s s u g g e s t e d t h a t t h e l a t t e r two c h a r a c t e r i s t i c s make t h e Se d e p e n d e n t GSH-Px t o be o f g r e a t e r i m p o r t a n c e c o m p a r e d t o t h e non-Se d e p e n d e n t GSH-Px i n a n i m a l s . A d d i t i o n a l s e l e n o p r o t e i n s , w i t h unknown f u n c t i o n s a t p r e s e n t t i m e , have been d e m o n s t r a t e d i n a n i m a l t i s s u e s . A 1 0 , 0 0 0 d a l t o n m o l e c u l a r w e i g h t s e l e n o p r o t e i n h a s been i s o l a t e d f r o m t h e s e m i t e n d i n o s u s m u s c l e a n d t h e h e a r t o f n o r m a l l y f e d l a m b s , b u t n o t f r o m Se d e f i c i e n t a n i m a l s ( P e d e r s e n e t a l . 1 9 7 2 ) . Se b i n d i n g p r o t e i n s o f 1 5 , 0 0 0 t o 2 0 , 0 0 0 d a l t o n s have b e e n o b s e r v e d i n s p e r m a t o z o a o f c a t t l e ( P a l l i n i and B a c c i , 1979) and r a t ( C a l v i n , 1978) and i n t h e r a t t e s t i s c y t o s o l ( M c C o n n e l l e t a l . 1979) s u g g e s t i n g some p o s s i b l e f u n c t i o n o f Se i n male r e p r o d u c t i o n and f e r t i l i t y . g ) E x c r e t i o n : Se i s known t o be e x c r e t e d p r i m a r i l y t h r o u g h u r i n e a n d f e c e s a n d t o some e x t e n t i n e x p i r e d a i r . The amounts a n d p r o p o r t i o n s t h a t a p p e a r i n t h e f e c e s , u r i n e o r a i r d e p e n d on t h e l e v e l and f o r m o f i n t a k e , t h e n a t u r e o f t h e r e s t o f t h e d i e t , age and s p e c i e s . 3 5 I n m o n o g a s t r i c a n i m a l s u r i n a r y e x c r e t i o n h a s b e e n r e p o r t e d t o be t h e m a j o r r o u t e i r r e s p e c t i v e o f t h e r o u t e o f Se a d m i n i s t r a t i o n ( B u r k e t a l . 1972 and Bopp e t a l . 1 9 8 2 ) . I n r u m i n a n t s , u r i n a r y e x c r e t i o n seems t o d e p e n d u pon t h e method o f a d m i n i s t r a t i o n . F e c a l e x c r e t i o n o f i n g e s t e d Se was shown t o be g r e a t e r t h a n u r i n a r y e x c r e t i o n i n r u m i n a n t s ( C o u s i n s and C a i r n e y , 1 9 6 1 ; P e t e r s o n a n d S p e d d i n g , 1 9 6 3 , and P a u l s o n e t a l . 1 9 6 6 ) . When Se was i n j e c t e d i n t o s h e e p o r o t h e r r u m i n a n t s p e c i e s t h e u r i n e was t h e m a j o r p a t h w a y o f Se e x c r e t i o n ( W r i g h t and B e l l , 1 9 6 6 ; and L o p e z e t a l . 1 9 6 9 ) . The l a t t e r w o r k e r s s t u d i e d t h e e f f e c t o f r o u t e o f a d m i n i s t r a t i o n and o f h i g h e r d i e t a r y 75 Se l e v e l s on t h e p a t t e r n o f Se e x c r e t i o n i n l a m b s . I n c r e a s e d Se l e v e l s i n t h e d i e t d i d n o t s u b s t a n t i a l l y a f f e c t 75 v o l a t i l e , u r i n a r y , o r f e c a l e x c r e t i o n o f Se a d m i n i s t e r e d o r a l l y . On t h e o t h e r h and t h e y f o u n d t h a t t h e h i g h e r l e v e l s o f Se i n t h e d i e t s i g n i f i c a n t l y i n c r e a s e d t h e 75 e x c r e t i o n o f p a r e n t e r a l l y a d m i n i s t e r e d Se t h r o u g h u r i n e o r f e c e s . Ewan e t a l . (1968) r e p o r t e d t h a t i n y o u n g l a m b s o f 75 8-10 weeks o f a g e , Se f e d o r a l l y a l o n g w i t h s y n t h e t i c l i q u i d d i e t was e x c r e t e d p r e d o m i n a n t l y i n u r i n e t h a n i n f e c e s u n l i k e i n t h e a d u l t s u g g e s t i n g t h e r o l e o f rumen m i c r o b e s on Se e x c r e t i o n . Maximum u r i n a r y e x c r e t i o n o f Se was shown t o be 2 t o 3 d a y s a f t e r a d m i n i s t r a t i o n ( M u t h e t a l . 1 9 6 7 ; B u r k e t a l . 1 9 7 2 ) . The s e l e n i u m s t a t u s o f t h e a n i m a l was shown t o a f f e c t Se e x c r e t i o n p a t t e r n s . 36 I n c a l v e s ( K i n c a i d e t a l . 1 9 7 7 ) , l a m b s ( L o p e z e t a l . 1969) a n d r a t s ( B u r k e t a l . 1972) u r i n a r y Se e x c r e t i o n was f o u n d t o be d i r e c t l y p r o p o r t i o n a l t o t h e Se s t a t u s o f t h e a n i m a l . A t h i g h e r i n t a k e s , Se l o s s t h r o u g h t h e e x p i r e d a i r seems t o be t h e m a j o r r o u t e o f e x c r e t i o n . E ) M e t h o d o l o g y t o s t u d y p l a c e n t a l t r a n s f e r o f t r a c e m i n e r a l s ; I n v i t r o a n d i n v i v o methods a r e commonly e m p l o y e d t o s t u d y t h e p l a c e n t a l t r a n s f e r a n d f e t a l m e t a b o l i s m o f t r a c e m i n e r a l s . T h i s s u b j e c t h a s b e e n r e c e n t l y r e v i e w e d by M o r r i s s a n d C a p r i o l i ( 1 9 8 2 ) . The a d v a n t a g e s , d r a w b a c k s a n d u s e o f t h e s e m e t h o d s i n d i f f e r e n t s p e c i e s w i t h v a r i o u s t r a c e e l e m e n t s a r e d i s c u s s e d b e l o w . a ) I n v i t r o m e t h o d ; The p l a c e n t a l a n d f e t a l u p t a k e o f t r a c e m i n e r a l s h a s b e e n s t u d i e d i n v i t r o by i n c u b a t i n g s l i c e s o f p l a c e n t a l o r f e t a l t i s s u e s i n a medium c o n t a i n i n g r a d i o l a b e l l e d t r a c e m i n e r a l c o m p o u n d s . T h i s a p p r o a c h h a s b e e n u s e d t o s t u d y t h e k i n e t i c s o f t r a n s f e r a n d b i n d i n g o f i r o n t o r a t p l a c e n t a ( L a u r e l l a n d M o r g a n , 1 9 6 4 ) . T h o u g h t h i s m e t h o d i s v e r y g o o d f o r s t u d y i n g t h e e f f e c t s o f enzyme i n h i b i t o r s a n d m e t a b o l i c p o i s o n s on b i n d i n g o f t r a c e m i n e r a l s t o t h e p l a c e n t a o r f e t a l t i s s u e s , i t s u f f e r s f r o m t h e m a j o r d r a w b a c k t h a t i t d o e s n o t m i m i c o r s i m u l a t e 37 t h e p h y s i o l o g i c a l e n v i r o n m e n t p r e s e n t u n d e r i n v i v o c o n d i t i o n s . P r o b a b l y t h i s c o u l d be t h e r e a s o n why t h e r e a r e n o t many r e p o r t s i n t h e l i t e r a t u r e w i t h r e g a r d t o t h e p l a c e n t a l u p t a k e a n d t r a n s f e r o f t r a c e m i n e r a l s u s i n g t h i s m e t h o d . b ) I n v i v o m e t h o d : I n t h i s m e t h o d r a d i o l a b e l l e d t r a c e m i n e r a l s a r e i n j e c t e d i n t o t h e p r e g n a n t dam a n d t h e r a t e o f t r a c e r a p p e a r a n c e i n t h e f e t a l a n d p l a c e n t a l t i s s u e s i s d e t e r m i n e d a t d i f f e r e n t t i m e s f o l l o w i n g s e r i a l s l a u g h t e r . T h i s a p p r o a c h h a s b e e n u s e d by s e v e r a l w o r k e r s t o m e a s u r e t h e p l a c e n t a l t r a n s f e r o f i r o n i n t h e r a t ( G l a s s e r e t a l . 1 9 6 8 , L a u r e l l a n d M o r g a n , 1 9 6 4 , M a n s o u r e t a l . 1972) a n d i n t h e r a b b i t ( D o u g l a s e t a l . 1 9 7 1 , L a r k i n e t a l . 1 9 7 0 ) . S t u d i e s done i n v i v o i n s e v e r a l s p e c i e s h a v e shown t h a t i r o n s u p p l i e d t o t h e f e t u s i n mammals w i t h h e m o c h o r i a l p l a c e n t a i s f r o m t h e i r o n - t r a n s f e r r i n p r o t e i n , w h i l e i n a n i m a l s w i t h e p i t h e l i o c h o r i a l o r e n d o t h e l i o c h o r i a l p l a c e n t a t i o n i s f r o m m a t e r n a l e r y t h r o c y t e s ( H o s k i n s a n d H a n s a r d , 1964a a n d 1 9 6 4 b , S e a l e t a l . 1 9 7 2 , Wong a n d M o r g a n , 1 9 7 4 , V a n D i j k , 1 9 8 1 ) . 59 Wong a n d M o r g a n (1974) i n j e c t e d Fe l a b e l l e d c a t e r y t h r o c y t e s i n t o t h e p r e g n a n t c a t a n d c a l c u l a t e d t h e i r o n u p t a k e b y t h e f e t u s (mg/d) u s i n g t h e f o r m u l a : 38 F e t a l u p t a k e o f I r o n ( m g / d ) % d o s e - m a t e r n a l ( f e t u s ) H c t . X X 100 X d 40 1 0 . 5 B l o o d X 3 . 4 X v o l u m e 100 ( m l ) w h e r e 1 0 . 5 g h e m o g l o b i n (Hb) was a s s u m e d t o be p r e s e n t i n 100 ml b l o o d c o n t a i n i n g 3 . 4 mg F e / g Hb a t a s t a n d a r d h e m a t o c r i t ( H c t ) o f 40%. M o r e r e c e n t l y , t h e s t u d i e s o f B a z e r a n d h i s c o w o r k e r s h a v e shown t h a t a s p e c i f i c p r o t e i n , u t e r o f e r r i n , s y n t h e s i z e d b y t h e u t e r i n e g l a n d s o f p r e g n a n t sow was i n v o l v e d i n t h e t r a n s f e r o f i r o n a c r o s s t h e p l a c e n t a o f t h e p i g ( R e n e g a r e t a l . 1 9 8 2 , R o b e r t s e t a l . 1 9 8 6 ) . F u r t h e r m o r e , t h e f e t a l p i g was shown t o i n c o r p o r a t e t h e 59 F e - l a b e l l e d u t e r o f e r r i n i n t o i t s t i s s u e s when i n j e c t e d v i a t h e u m b i l i c a l v e i n o f t h e p r e g n a n t sow ( B u h i e t a l . 1 9 8 2 ) . The i s o l a t e d , p e r f u s e d p l a c e n t a was a l s o u s e d t o s t u d y t h e p l a c e n t a l t r a n s f e r o f t r a c e m i n e r a l s . T h i s a p p r o a c h h a s b e e n u s e d by s e v e r a l w o r k e r s t o s t u d y t h e p l a c e n t a l t r a n s f e r o f i r o n i n v i v o ( B a k e r a n d M o r g a n , 1 9 7 0 , v a n D i j k e t a l . 1985) a n d o t h e r n u t r i e n t s i n v i t r o ( Y u d i l e v i c h , 1 9 7 9 ) . H o w e v e r , t h e m a j o r c r i t i c i s m a g a i n s t s u c h s t u d i e s i s t h e l a c k o f p r o o f o f v i a b i l i t y o f 39 t h e p r e p a r a t i o n . I n d o m e s t i c a n i m a l s t h e p l a c e n t a l t r a n s f e r a n d f e t a l m e t a b o l i s m o f t r a c e e l e m e n t s a r e commonly s t u d i e d b y i n j e c t i n g t h e r a d i o l a b e l l e d compounds i n t o t h e p r e g n a n t dam a t d i f f e r e n t s t a g e s o f g e s t a t i o n a n d m e a s u r i n g t h e f e t a l a n d p l a c e n t a l u p t a k e o f r a d i o a c t i v i t y a f t e r s a c r i f i c i n g t h e a n i m a l s . T h i s a p p r o a c h h a s b e e n u s e d f o r s t u d y i n g t h e p l a c e n t a l t r a n s f e r o f s e l e n i u m i n s h e e p ( W r i g h t a n d B e l l , 1 9 6 4 , H i d i r o g l o u e t a l . 1 9 6 9 , J a c o b s s o n a n d O k s a n e n , 1 9 6 6 ) , i r o n i n s h e e p ( H o s k i n s and H a n s a r d , 1964a) a n d i n s w i n e ( H o s k i n s a n d H a n s a r d , 1 9 6 4 b ) , manganese i n c a t t l e , s h e e p a n d s w i n e ( H a n s a r d , 1 9 7 2 ) , z i n c i n c a t t l e ( H a n s a r d a n d Mohammed, 1968b) a n d i n s h e e p ( H a n s a r d a n d Mohammed, 1968c ) a n d s u l f u r i n s h e e p ( H a n s a r d a n d Mohammed, 1968a) a n d i n c a t t l e ( H a n s a r d a n d Mohammed, 1 9 6 9 ) . A l t h o u g h some i n f o r m a t i o n i s o b t a i n e d w i t h r e g a r d t o p l a c e n t a l t r a n s f e r a n d f e t a l d i s t r i b u t i o n o f t r a c e m i n e r a l s by t h e s e i n v i v o s l a u g h t e r s t u d i e s , t h e y s u f f e r f r o m t h e d i s a d v a n t a g e t h a t t h e k i n e t i c s o f p l a c e n t a l t r a n s f e r a n d p o s s i b l e b i d i r e c t i o n a l f l u x e s o f c e r t a i n t r a c e m i n e r a l s c a n n o t be d e t e r m i n e d p r e c i s e l y w i t h t h i s a p p r o a c h . T h i s d r a w b a c k h a s b e e n o v e r c o m e p a r t i c u l a r l y by t h e c h r o n i c c a t h e t e r i z a t i o n o f f e t a l a n d u m b i l i c a l b l o o d v e s s e l s ( M e s c h i a e t a l . 1965) a n d m e a s u r i n g t h e v e n o - a r t e r i a l c o n c e n t r a t i o n d i f f e r e n c e s o f t h e n u t r i e n t s a n d t h e b l o o d f l o w . The r a t e s o f t r a n s p l a c e n t a l e x c h a n g e o f n u t r i e n t s as w e l l a s t h e 40 c o n t r i b u t i o n o f t h e p l a c e n t a t o f e t a l n u t r i t i o n c a n be a s w e l l a s t h e c o n t r i b u t i o n o f t h e p l a c e n t a t o f e t a l n u t r i t i o n c a n be d e t e r m i n e d i n c o n s c i o u s a n i m a l s f r o m t h e m e a s u r e d p a r a m e t e r s . However t h i s m e t h o d may n o t be s u i t a b l e f o r s t u d i e s on t r a c e m i n e r a l s whose u m b i l i c a l v e n o -a r t e r i a l c o n c e n t r a t i o n d i f f e r e n c e s a r e b e l o w t h e d e t e c t i o n l i m i t o f t h e a n a l y t i c a l t e c h n i q u e s e m p l o y e d . F u r t h e r m o r e , t h e p h y s i o l o g i c a l n o r m a l c y o f t h e ewe a n d f e t u s f o l l o w i n g s u r g e r y h a v e t o be e s t a b l i s h e d b e f o r e t h e m e t a b o l i c e x p e r i m e n t s a r e c o n d u c t e d . 75 F) Se m e t a b o l i s m i n t h e p r e g n a n t m o t h e r a n d i n t h e f e t u s ; a ) P l a c e n t a l t r a n s f e r ; 75 The u s e o f r a d i o l a b e l l e d Se as s e l e n i t e h a s f a c i l i t a t e d t h e s t u d y o f Se m e t a b o l i s m i n t h e p r e g n a n t 75 m o t h e r a n d i n t h e f e t u s . The p l a c e n t a l t r a n s f e r o f Se was f i r s t o b s e r v e d by M c C o n n e l l a n d R o t h (1964) i n d o g s . 75 T h e y f o u n d t h a t Se a d m i n i s t e r e d s u b c u t a n e o u s l y a s S e -s e l e n i t e t o t h e l a c t a t i n g b i t c h was p r e s e n t i n m i l k s u g g e s t i n g m i l k t o be a s o u r c e o f Se f o r t h e n e o n a t e . I t was a l s o r e p o r t e d t h a t Se was r e t a i n e d i n t h e t i s s u e s o f b i t c h f o r a p e r i o d o f 236 d a f t e r t r a c e r i n j e c t i o n a n d was i n c o r p o r a t e d i n t o t h e t i s s u e s o f t h e pup b o r n i n s u b s e q u e n t p r e g n a n c y ( M c C o n n e l l a n d R o t h , 1 9 6 4 ) . W r i g h t a n d B e l l (1964) i n t h e i r s t u d i e s w i t h ewes f e d Se d e f i c i e n t h a y 41 w i t h o r w i t h o u t a Se s u p p l e m e n t d e m o n s t r a t e d t h e t r a n s f e r o f 75 Se a c r o s s t h e p l a c e n t a . T h e y o b s e r v e d t h a t t h e t o t a l 75 k i d n e y Se c o n t e n t i n t h e f e t u s e s f r o m ewes c o n s u m i n g a Se d e f i c i e n t r a t i o n was h i g h e r t h a n i n t h o s e f r o m ewes r e c e i v i n g t h e Se s u p p l e m e n t , t h o u g h t h e l i v e r c o n t e n t was l o w e r . T i s s u e s f r o m t w i n f e t u s e s were f o u n d t o c o n t a i n 75 a p p r o x i m a t e l y 50% as much Se as t i s s u e s f r o m t h e 75 s i n g l e t o n s . The r a t i o o f Se a c t i v i t y (% o f d o s e / g ) i n t h e m a t e r n a l t o f e t a l p l a s m a was 1 2 : 1 f o r t h e s i n g l e f e t u s e s a n d 2 2 : 1 f o r t h e t w i n f e t u s e s . T h e y a l s o d e m o n s t r a t e d t h a t a d d i t i o n o f a l p h a t o c o p h e r o l t o t h e 75 e w e ' s r a t i o n d i d n o t a f f e c t t h e p a s s a g e o f Se a c r o s s t h e p l a c e n t a l m e m b r a n e s . S e v e r a l w o r k e r s h a v e r e p o r t e d t h a t m a t e r n a l t i s s u e s o f t h e ewe i n g e n e r a l were f o u n d t o 75 c o n t a i n a g r e a t e r amount o f Se t h a n f e t a l t i s s u e s ( M c C o n n e l l a n d R o t h , 1964; W r i g h t a n d B e l l , 1 9 6 4 ; J a c o b s s o n a n d O k s a n e n , 1966 ; H i d i r o g l o u e t a l . 1 9 6 9 ; a n d B u c k e t a l . 1 9 8 1 ) . T h e s e o b s e r v a t i o n s s u g g e s t e d t h e p r e s e n c e o f a p l a c e n t a l b a r r i e r f o r t h e p a s s a g e o f Se i n t h e s h e e p . I n c o n t r a s t t o t h e s e f i n d i n g s , 75 H a n s s o n a n d J a c o b s s o n (1966) s t u d i e d S e - s e l e n o m e t h i o n i n e u p t a k e i n t h e p r e g n a n t mouse u s i n g a u t o r a d i o g r a p h y a n d f o u n d e q u a l d i s t r i b u t i o n o f t h e t r a c e r i n t h e m a t e r n a l a n d f e t a l t i s s u e s d e m o n s t r a t i n g t h e l a c k o f a p l a c e n t a l b a r r i e r i n t h e m o u s e . The c o n f l i c t i n g o b s e r v a t i o n s may be due t o s p e c i e s d i f f e r e n c e s w i t h r e g a r d t o p l a c e n t a t i o n , d i f f e r e n t Se i n t a k e s o r d i f f e r e n t i a l p l a c e n t a l t r a n s f e r 42 b e t w e e n o r g a n i c a n d i n o r g a n i c Se s o u r c e s . I n 75 a n o t h e r s t u d y S e - s e l e n o m e t h i o n i n e was r e p o r t e d t o be a c t i v e l y t r a n s p o r t e d a g a i n s t a c o n c e n t r a t i o n g r a d i e n t t o t h e human f e t u s f r o m t h e m o t h e r ' s b l o o d . The i n f l u x o f t r a c e r f r o m m o t h e r t o f e t u s was u s e d as a n i n d e x o f a n i n s t a n t a n e o u s m e a s u r e o f f e t a l g r o w t h r a t e a n d p l a c e n t a l f u n c t i o n ( G a r r o w , 1 9 7 1 ) . B u c k e t a l . (1981) d e m o n s t r a t e d a p r e f e r e n t i a l 75 a c c u m u l a t i o n o f Se i n s u c h r e p r o d u c t i v e t i s s u e s a s c o r p u s l u t e u m , n o n - l u t e a l o v a r y a n d p l a c e n t o m e o f t h e p r e g n a n t ewe a n d i n t h e a d r e n a l a n d p i t u i t a r y g l a n d s o f b o t h ewe a n d f e t u s s u g g e s t i n g a n o r m a l r e q u i r e m e n t o f t h e s e t i s s u e s f o r S e . R e c e n t l y S h a r i f f e t a l . (1984) r e p o r t e d t h a t t h e 75 s h e e p p l a c e n t a i s p e r m e a b l e t o S e - s e l e n i t e i n e i t h e r d i r e c t i o n . I n an i n u t e r o s t u d y u s i n g c h r o n i c a l l y c a t h e t e r i z e d f e t a l l a m b s t h e y d e m o n s t r a t e d t h a t 75 i n t r a v e n o u s l y a d m i n i s t e r e d S e - s e l e n i t e e i t h e r t o t h e ewe o r t h e f e t u s r a p i d l y e n t e r e d t h e r e s p e c t i v e f e t a l o r m a t e r n a l c i r c u l a t i o n s . b ) C o n c e n t r a t i o n s o f s e l e n i u m i n m a t e r n a l a n d f e t a l t i s s u e s ; T he n e e d f o r Se i n p r e g n a n t a n i m a l s f o r t h e p r e v e n t i o n o f w h i t e m u s c l e d i s e a s e i n t h e i r o f f s p r i n g h a s b e e n w e l l e s t a b l i s h e d . I n s t u d i e s w i t h c a t t l e , g o o d p r o t e c t i o n a g a i n s t t h e c o n g e n i t a l f o r m o f w h i t e m u s c l e d i s e a s e was a c h i e v e d 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 Se t o p r e g n a n t cows 43 i n l a t e g e s t a t i o n (Mace e t a l . 1 9 6 3 ; N e l s o n e t a l . 1 9 6 4 ; a n d H i d i r o g l o u e t a l . 1 9 6 5 ) . S u b c u t a n e o u s i n j e c t i o n o f e i t h e r s e l e n a t e o r s e l e n i t e t o p r e g n a n t ewes was s u c c e s s f u l i n p r e v e n t i n g t h e c o n g e n i t a l o r j u v e n i l e w h i t e m u s c l e d i s e a s e i n l a m b s ( Y o u n g e t a l . 1 9 6 1 ; B u r t o n e t a l . 1962 a n d Hamdy e t a l . 1 9 6 3 ) . H i d i r o g l o u e t a l . ( 1969) r e p o r t e d i n c r e a s e d Se c o n c e n t r a t i o n s i n b o t h t h e m a t e r n a l a n d f e t a l t i s s u e s f o l l o w i n g a n i n t r a m u s c u l a r i n j e c t i o n o f 6 mg o f Se t o p r e g n a n t ewes f e d Se d e f i c i e n t h a y . T h e y f o u n d t h a t m a t e r n a l k i d n e y , l i v e r a n d m u s c l e c o n t a i n e d more Se ( 0 . 6 5 0 , 0 . 3 6 0 a n d 0 . 0 5 0 ppm) t h a n t h e c o r r e s p o n d i n g f e t a l t i s s u e s ( 0 . 2 1 4 , 0 . 1 7 4 a n d 0 . 0 3 6 ppm) r e s p e c t i v e l y . New Z e a l a n d w o r k e r s ( H a r t l e y a n d G r a n t , 1961) h a v e o b s e r v e d t h a t w h i t e m u s c l e d i s e a s e i n s h e e p o c c u r r e d as a c o n g e n i t a l d i s e a s e a n d o f t e n l a m b s were e i t h e r s t i l l b o r n o r d i e d s h o r t l y a f t e r b i r t h . H a r t l e y (1967) a l s o r e p o r t e d t h a t Se c o n c e n t r a t i o n i n t h e m a t e r n a l , f e t a l a n d j u v e n i l e t i s s u e s o f a n i m a l s e x h i b i t i n g w h i t e m u s c l e d i s e a s e was s i g n i f i c a n t l y l o w e r t h a n t h a t o f u n a f f e c t e d o n e s . A c o m p a r i s o n b e t w e e n s h e e p f e d n o r m a l v e r s u s d y s t r o p h o g e n i c h a y showed t h a t t h e t i s s u e Se l e v e l s o f ewes a n d t h e i r o f f s p r i n g were a b o u t t h r e e f o l d h i g h e r i n t h e f o r m e r g r o u p t h a n i n t h e l a t t e r ( B u r t o n e t a l . 1 9 6 2 ) . 44 F o l l o w i n g s u p p l e m e n t a t i o n o f Se t o p r e g n a n t ewes on d y s t r o p h o g e n i c h a y , t h e t i s s u e Se l e v e l s were a l m o s t s i m i l a r t o t h o s e c o n s u m i n g n o n d y s t r o p h o g e n i c h a y a n d o c c u r r e n c e o f c o n g e n i t a l NMD i n t h e o f f s p r i n g was p r e v e n t e d . B u r t o n e t a l . (1962) a l s o o b s e r v e d t h a t Se c o n c e n t r a t i o n i n t i s s u e s o f f e t u s e s f r o m Se i n j e c t e d ewes a p p r o a c h e d t h e c o n c e n t r a t i o n i n t h o s e o f ewes g i v e n a d i e t o f n o r m a l h a y . H i d i r o g l o u e t a l . (1969) h a v e r e p o r t e d t h a t Se i s n o t r e t a i n e d i n f e t a l t i s s u e s f o r f u t u r e u s e when p r e g n a n t ewes f e d w h i t e m u s c l e d i s e a s e - p r o d u c i n g h a y were i n j e c t e d w i t h Se a t t h e t i m e o f b r e e d i n g . F u r t h e r m o r e , t h e y s u g g e s t e d t h a t t h e l i m i t e d s t o r a g e c a p a c i t y o f t h e f e t u s w o u l d n e c e s s i t a t e a c o n t i n u i n g a d e q u a t e s u p p l y o f Se b y t h e d e v e l o p i n g f e t u s . R e c e n t l y H i d i r o g l o u e t a l . (1985) h a v e r e p o r t e d t h a t i n t r a r u m i n a l Se p e l l e t a d m i n i s t r a t i o n t o p r e g n a n t d a i r y cows r e s u l t e d i n a n i n c r e a s e d p l a s m a Se l e v e l s a n d a d e c r e a s e d i n c i d e n c e o f n u t r i t i o n a l m u s c u l a r d y s t r o p h y i n c a l v e s a t b i r t h . W e i s s e t a l . (1982) r e p o r t e d t h a t Se s u p p l e m e n t a t i o n o f p r e g n a n t d a i r y cows w i t h 1 mg o r 5 mg o f s o d i u m s e l e n i t e d a i l y i n c r e a s e d t h e Se c o n c e n t r a t i o n s s i g n i f i c a n t l y i n t h e b l o o d s e r u m o f h e i f e r c a l v e s a t b i r t h c o m p a r e d t o t h o s e f r o m u n s u p p l e m e n t e d c o w s . R o l l e r e t a l . ( 1984b) o b s e r v e d a s i m i l a r r e s p o n s e i n b e e f c a l v e s b o r n t o cows 45 t h a t r e c e i v e d Se. A t b i r t h t h e c a l v e s f r o m t r e a t e d cows h a d 0 . 2 4 2 mg/kg o f Se i n t h e i r w h o l e b l o o d c o m p a r e d t o t h o s e f r o m u n t r e a t e d cows t h a t h a d o n l y 0 . 0 8 1 mg S e / k g i n t h e i r w h o l e b l o o d . I t a n o e t a l . (1984) e x a m i n e d t h e Se l e v e l s i n t h e s t r i p e d d o l p h i n a t f e t a l and s u c k l i n g s t a g e s . T hey f o u n d t h a t t h e Se l e v e l i n t h e f e t u s e s was h i g h e r i n l i v e r c o m p a r e d t o o t h e r t i s s u e s , w h e r e a s i n t h e s u c k l i n g s t h e b l u b b e r c o n t a i n e d h i g h e r Se c o n c e n t r a t i o n s . de T o l e d o and P e r r y (1985) i n v e s t i g a t e d t h e e f f e c t s o f d i f f e r e n t m ethods and amounts o f Se s u p p l e m e n t a i o n on d i s t r i b u t i o n o f Se i n p e r i p a r t u r i e n t d a i r y cows. They o b s e r v e d t h a t b o t h o r a l and i n j e c t i o n t r e a t m e n t s r a i s e d t h e se r u m Se c o n c e n t r a t i o n s o f cows. Serum Se l e v e l s a t b i r t h w e r e g r e a t e r o n l y f o r c a l v e s whose dams r e c e i v e d Se b y i n j e c t i o n b u t n o t by o r a l s u p p l e m e n t a t i o n . c ) S e l e n i u m d e p e n d e n t g l u t a t h i o n e p e r o x i d a s e a c t i v i t y i n  m a t e r n a l a n d f e t a l t i s s u e s ; Se h a s be e n shown t o be an i n t e g r a l component o f g l u t a t h i o n e p e r o x i d a s e (GSH-Px) ( R o t r u c k e t a l . 1 9 7 3 , F l o h e e t a l . 1 9 7 3 ) . H i g h l y s i g n i f i c a n t c o r r e l a t i o n s b e t w e e n t i s s u e Se d e p e n d e n t GSH-Px a c t i v i t y a n d b l o o d Se c o n c e n t r a t i o n s i n c a t t l e and s h e e p were e s t a b l i s h e d (Whanger e t a l . 1 9 7 7 ; A n d e r s o n e t a l . 1978 ; P a y n t e r , 1 9 7 9 ) . M e a s u r e m e n t o f GSH-Px a c t i v i t y h a s t h e r e f o r e b e e n 46 recommended f o r a s s e s s i n g t h e Se s t a t u s i n a n i m a l s (Whanger e t a l . 1978) . R o l l e r e t a l . (1984b) m e a s u r e d t h e b l o o d GSH-Px l e v e l s i n p r e g n a n t b e e f cows and t h e i r o f f s p r i n g a t b i r t h . T h ey r e p o r t e d t h a t Se t r e a t e d cows a nd t h e i r c a l v e s h a d h i g h e r b l o o d GSH-Px a c t i v i t i e s (144 and 154 mll/mg o f Hb) co m p a r e d t o t h e u n t r e a t e d cows and t h e i r c a l v e s (30 and 50 mll/mg o f Hb) r e s p e c t i v e l y . H i d i r o g l o u e t a l . (1985) r e c e n t l y r e p o r t e d t h a t t h e b l o o d GSH-Px a c t i v i t y a t b i r t h was h i g h e r i n c a l v e s b o r n f r o m t r e a t e d cows t h a n t h o s e f r o m t h e u n t r e a t e d (134 I U / g Hb v s 29 I U / g H b ) . R u d o l p h and Wong (1978) f o u n d t h a t Se d e p e n d e n t g l u t a t h i o n e p e r o x i d a s e a c t i v i t y i n r e d c e l l s and p l a s m a o f human f e t u s was s i g n i f i c a n t l y l o w e r t h a n i n t h e a d u l t . A c c o r d i n g t o t h e s e w o r k e r s , t h e l o w a c t i v i t y o f t h i s enzyme i n t h e f e t u s was p r o b a b l y an a d a p t a t i o n t o a l o w r e q u i r e m e n t f o r p r o t e c t i o n a g a i n s t t h e a n t i o x i d a n t s t r e s s . E r y t h r o c y t e g l u t a t h i o n e p e r o x i d a s e a c t i v i t y i n new b o r n i n f a n t s was f o u n d t o be l o w i n c o m p a r i s o n t o a d u l t s ( G r o s s e t a l . 1 9 6 7 ; B r a c c i e t a l . 1 9 6 9 ; Emerson e t a l . 1 9 7 2 ) . Low GSH-Px a c t i v i t y i n c r e a s i n g w i t h age h a s been r e p o r t e d i n t h e n e o n a t a l l i v e r s u g g e s t i n g an age r e l a t e d d e v e l o p m e n t a l m e c h a n i s m o f t h i s enzyme ( P i n t o and B a r t l e y , 1 9 7 7 ) . The i s o l a t i o n , p u r i f i c a t i o n and k i n e t i c p r o p e r t i e s 47 o f GSH-Px f r o m human p l a c e n t a have b e e n r e p o r t e d by A w a s t h i e t a l . ( 1 9 7 9 ) . I n a l a t e r s t u d y A w a s t h i and Dao (1981) r e p o r t e d t h a t t h e d e c o m p o s i t i o n o f h y d r o p e r o x i d e s o f l i p i d s was c a r r i e d o u t i n t h e p l a c e n t a by Se d e p e n d e n t GSH-Px a l o n e and n o t by GSH S - t r a n s f e r a s e s , a n o t h e r g r o u p o f enzymes w h i c h e x h i b i t e d GSH-Px l i k e a c t i v i t y . U n d e r n o r m a l c o n d i t i o n s t h e p r o t e c t i o n a g a i n s t t h e a n t i o x i d a n t i n j u r y t o t h e f e t u s f r o m h y d r o p e r o x i d e s , p e r o x i d e s , e l e c t r o p h i l i c d r u g s and x e n o b i o t i c s was a f f o r d e d b y b o t h GSH-Px as w e l l as GSH S - t r a n s f e r a s e s . S e v e r a l w o r k e r s h a v e shown t h a t t h e l e v e l s o f GSH S - t r a n s f e r a s e s w e re v e r y l o w i n t h e f e t a l l u n g and l i v e r ( M u k h t a r and B r e s n i c k , 1 9 7 6 ; K l a s s a n , 1975 and Pegg and Hook, 1 9 7 7 ) . T h i s w o u l d s u g g e s t t h a t t h e GSH-Px a c t i v i t y o f GSH S - t r a n s f e r a s e w i l l p r o b a b l y n o t be a v a i l a b l e t o p r o v i d e a d d e d p r o t e c t i o n t o t h e f e t u s a g a i n s t t o x i c h y d r o - p e r o x i d e s . Thus i t h a s b e e n s u g g e s t e d t h a t Se d e f i c i e n c y c a n c a u s e more o x i d a n t damage t o t h e f e t u s t h a n t o t h e n e o n a t e . CONCLUSIONS FROM THE LITERATURE REVIEW: From t h e f o r e g o i n g s u r v e y o f t h e l i t e r a t u r e i t c o u l d be s e e n t h a t t h e r e i s a p a u c i t y o f i n f o r m a t i o n on t h e q u a n t i t a t i v e a s p e c t s o f Se m e t a b o l i s m i n t h e p r e g n a n t a n d n o n p r e g n a n t s h e e p . To be more s p e c i f i c t h e r e i s no a v a i l a b l e d a t a w i t h r e g a r d t o t h e amounts o f Se t r a n s p o r t e d t r a n s p l a c e n t a l l y f r o m t h e p r e g n a n t ewe t o t h e f e t u s o r 48 v i c e v e r s a u n d e r c o n d i t i o n s o f a d e q u a t e o r d e f i c i t Se s u p p l y . B e s i d e s t h e r e i s no i n f o r m a t i o n a v a i l a b l e i n t h e l i t e r a t u r e on t h e k i n e t i c a s p e c t s o f Se m e t a b o l i s m e i t h e r i n t h e p r e g n a n t ewe o r t h e g r o w i n g f e t u s s h o w i n g 75 q u a n t i t a t i v e Se u p t a k e a n d t u r n o v e r i n t h e e x c h a n g e a b l e c o m p a r t m e n t s . S i m i l a r l y no s u c h i n f o r m a t i o n i s a v a i l a b l e f o r t h e t h e n o n p r e g n a n t e w e s . A l t h o u g h some d a t a a r e a v a i l a b l e m o s t l y f r o m s l a u g h t e r s t u d i e s on t h e d i s t r i b u t i o n o f Se i n t h e p r e g n a n t ewe a n d t h e f e t a l l a m b s t h e y a r e n o t a d e q u a t e t o e x p l a i n how e i t h e r t h e p r e g n a n t m o t h e r o r h e r f e t u s a d a p t s t o t h e c h a n g i n g Se a v a i l a b i l i t y s i t u a t i o n s . F u r t h e r m o r e t h e s e r e p o r t s a r e n o t a i m e d a t e l u c i d a t i n g t h e p o s s i b l e r o l e o f t h e p l a c e n t a u n d e r Se a d e q u a t e o r d e f i c i e n t c o n d i t i o n s . T h e s e o b s e r v a t i o n s p r o v i d e d t h e b a c k g r o u n d f o r u n d e r t a k i n g t h e e x p e r i m e n t s r e p o r t e d i n t h i s t h e s i s on t h e m e t a b o l i s m o f Se i n t h e n o n p r e g n a n t a n d p r e g n a n t ewes f e d Se p o s i t i v e o r d e f i c i e n t r a t i o n s . 49 CHAPTER I I ; SELENIUM METABOLISM IN NONPREGNANT EWES.  E X P E R I M E N T I ; K i n e t i c s o f s e l e n i u m m e t a b o l i s m i n n o n p r e g n a n t  ewes f e d Se p o s i t i v e o r d e f i c i e n t r a t i o n s . INTRODUCTION S e l e n i u m m e t a b o l i s m h a s b e e n i n v e s t i g a t e d i n s h e e p o f v a r y i n g p h y s i o l o g i c a l s t a t e s s u c h as p r e g n a n c y ( W r i g h t a n d B e l l , 1964; H i d i r o g l o u e t a l . 1 9 6 9 ) , l a c t a t i o n ( P a u l s o n e t a l . 1966) a n d g r o w t h ( W r i g h t , 1 9 6 5 ; E h l i g e t a l . 1 9 6 7 ; M u t h e t a l . 1967 ; Pope e t a l . 1 9 7 9 ; L a n g l a n d s e t a l . 1 9 8 6 ) . The e a r l y f i n d i n g o f M u t h e t a l . (1967) i n d i c a t e d t h a t s e v e r a l h o m e o s t a t i c m e c h a n i s m s a r e i n v o l v e d i n r e g u l a t i n g Se m e t a b o l i s m i n n o n p r e g n a n t ewes f e d Se a d e q u a t e o r d e f i c i e n t r a t i o n s . A k n o w l e d g e o f t h e s e r e g u l a t o r y m e c h a n i s m s i s n e c e s s a r y t o i m p r o v e Se u t i l i z a t i o n by t h e a n i m a l . T h e r e f o r e , t h e p r e s e n t s t u d y was u n d e r t a k e n t o i n v e s t i g a t e t h e w h o l e b o d y a n d t i s s u e m e t a b o l i s m o f Se a t two d i f f e r e n t l e v e l s o f Se i n t a k e w i t h a v i e w t o a d d r e s s t h e f o l l o w i n g q u e s t i o n s : (a ) A r e t h e r e a n y d i f f e r e n c e s i n t h e k i n e t i c p a r a m e t e r s o f Se m e t a b o l i s m i n t h e w h o l e b o d y a n d i n i n d i v i d u a l t i s s u e s when ewes a r e f e d Se p o s i t i v e o r d e f i c i e n t r a t i o n s ? a n d (b) I s t h e r e a d i f f e r e n c e i n t h e a b s o r p t i o n , e x c r e t i o n a n d 50 r e t e n t i o n o f Se i n ewes a t t h e s e two l e v e l s o f Se i n t a k e ? M A T E R I A L S AND METHODS ( a ) A n i m a l s a n d Management ; E i g h t n o n p r e g n a n t ewes ( D o r s e t X S u f f o l k c r o s s b r e d ) w e i g h i n g 59 + 6 kg (mean + SEM) were u s e d i n t h i s s t u d y . T h e y were r a n d o m l y a l l o t t e d t o two g r o u p s w i t h f o u r a n i m a l s i n e a c h a n d were f e d a Se p o s i t i v e o r a d e f i c i e n t r a t i o n The Se p o s i t i v e ewes ( G r o u p I ) were f e d a l f a l f a h a y c u b e s , c o n t a i n i n g 0 . 1 5 mg S e / k g , t o p r o v i d e e n e r g y a n d p r o t e i n as p e r NRC r e c o m m e n d a t i o n s ( 1 9 7 8 ) . The Se d e f i c i e n t ewes ( G r o u p I I ) were f e d h a y d e f i c i e n t i n Se (<0 .01 mg S e / k g ) . The Se d e f i c i e n t h a y (95% c a n a r y g r a s s a n d 5% t i m o t h y ) was p u r c h a s e d a t t h e s u g g e s t i o n o f B . C . M i n i s t r y o f A g r i c u l t u r e a n d F o o d ' s (BCMAF) n u t r i t i o n a l a n a l y s i s l a b o r a t o r y , K e l o w n a f r o m a l o c a l f a r m e r whose l i v e s t o c k were r e p o r t e d t o be e x p e r i e n c i n g p r o b l e m s o f Se d e f i c i e n c y . The f a r m e r h a d b e e n s u p p l e m e n t i n g Se ( 0 . 1 ppm) as s o d i u m s e l e n i t e i n t h e f e e d a t t h e a d v i c e o f BCMAF o f f i c i a l s t o h i s l i v e s t o c k . Random s a m p l e s o f h a y were c o l l e c t e d on f i v e d i f f e r e n t o c c a s i o n s d u r i n g t h e e x p e r i m e n t , m i x e d t h o r o u g h l y a n d a l i q u o t s were t a k e n f o r Se a n a l y s i s . F e e d s a m p l e s were g r o u n d t o p a s s t h r o u g h 1 mm mesh s c r e e n . F e e d a n a l y s i s showed t h a t a l f a l f a a n d t h e Se d e f i c i e n t h a y s c o n t a i n e d 15.8% a n d 9.7% o f c r u d e p r o t e i n 51 r e s p e c t i v e l y w i t h a s i m i l a r g r o s s e n e r g y c o n c e n t r a t i o n ( 1 7 . 6 K J / g ) . Ewes i n b o t h g r o u p s were p r o v i d e d w i t h w a t e r (30 ng S e / m l ) a n d i o d i z e d b l o c k s a l t a d l i b . A f t e r b e i n g f e d a p p r o x i m a t e l y f o r s i x t o e i g h t weeks on t h e s e r a t i o n s t h e ewes w e r e s h o r n a n d moved t o s e p a r a t e m e t a b o l i s m c a g e s f o r i n d i v i d u a l f e e d i n g a n d d i g e s t i b i l i t y s t u d i e s . ( b ) I s o t o p i c t r a c e r e x p e r i m e n t s a n d Se b a l a n c e s t u d i e s ; One d a y p r i o r t o t h e t r a c e r e x p e r i m e n t , two p o l y e t h y l e n e c a t h e t e r s ( I n t r a m e d i c " P E - 9 0 " , C l a y A d a m s , P a r s i p p a n y , N . J . ) were i m p l a n t e d i n b o t h j u g u l a r v e i n s t o f a c i l i t a t e t r a c e r i n j e c t i o n a n d w i t h d r a w a l o f b l o o d . A b o l u s d o s e o f a p p r o x i m a t e l y 200 u C i ( s p e c i f i c 75 a c t i v i t y ; 1 8 7 - 9 1 9 m C i / m g Se) o f s o d i u m S e - s e l e n i t e ( I C N c h e m i c a l s , I r v i n e , C a l i f o r n i a ) was i n j e c t e d i n t o t h e l e f t j u g u l a r v e i n . B l o o d s a m p l e s were w i t h d r a w n f r o m t h e r i g h t j u g u l a r v e i n a t 2 , 5 , 1 0 , 1 5 , 3 0 , 4 5 , 6 0 , 1 2 0 , 1 8 0 , 2 4 0 , 3 0 0 , 360 m i n p o s t i n j e c t i o n a n d o n c e d a i l y t h e r e a f t e r . B l o o d s a m p l e s were p l a c e d i n c h i l l e d p y r e x t u b e s c o n t a i n i n g h e p a r i n a n d c e n t r i f u g e d i m m e d i a t e l y . , The o p l a s m a was s t o r e d a t - 2 0 C u n t i l Se a n a l y s e s were p e r f o r m e d . A f i v e d a y Se b a l a n c e t r i a l was c o n d u c t e d on t h r e e ewes f r o m e a c h g r o u p ( G r o u p I ; ewe n o s . 6 5 , 99 a n d 1 0 0 ; G r o u p I I : ewes 9 9 8 , 123 a n d 325) i n c o n j u n c t i o n w i t h t h e t r a c e r 52 e x p e r i m e n t . The b a l a n c e s t u d i e s were commenced on t h e d a y o f t r a c e r i n j e c t i o n . F e e d a n d w a t e r i n t a k e s were r e c o r d e d e v e r y d a y . D a i l y f e c a l a n d u r i n a r y v o i d s w e r e m e a s u r e d a n d r e p r e s e n t a t i v e a l i q u o t s o f t h e same were o c o l l e c t e d a n d s t o r e d a t - 2 0 C . One ewe f r o m e a c h g r o u p was s a c r i f i c e d on d a y s 1 (622 a n d 9 9 9 ) , 5 (65 a n d 9 9 8 ) , 9 (99 a n d 123) a n d 14 (100 a n d 325) p o s t i n j e c t i o n by a d m i n i s t e r - i n g 20 ml o f T - 6 1 ( H o e c h s t P h a r m a c e u t i c a l s L t d ) , an e u t h a n a s i a s o l u t i o n . The ewes were d i s s e c t e d a n d i n d i v i d u a l t i s s u e s were c o l l e c t e d , t o w e l d r i e d , w e i g h e d a n d s t o r e d i n d o u b l e s e a l e d o p l a s t i c c o n t a i n e r s a t - 2 0 C u n t i l t h e Se a n a l y s e s w e r e c a r r i e d o u t . ( c ) A n a l y t i c a l m e t h o d s : 75 The p l a s m a Se r a d i o a c t i v i t y was c o u n t e d i n a n a u t o m a t i c gamma c o u n t e r ( P a c k a r d m o d e l 500) w i t h a c o u n t i n g e f f i c i e n c y o f 86%. P l a s m a Se c o n c e n t r a t i o n was d e t e r m i n e d i n d u p l i c a t e u s i n g h y d r i d e g e n e r a t i o n a t o m i c a b s o r p t i o n s p e c t r o m e t r y as p e r t h e m e t h o d o f Tam a n d L a c r o i x ( 1 9 8 2 ) . B r i e f l y , 10 ml o f a s h i n g a i d (8 g MgO a n d 80 g M g f N O j ^ - 6^2° d i s s o l v e d i n 200 ml o f d e i o n i z e d w a t e r ) w e r e a d d e d t o 2 ml o f p l a s m a s a m p l e s ( i n d u p l i c a t e ) i n a n 53 a c i d w a s h e d g l a s s b e a k e r a n d t h o r o u g h l y m i x e d . The b e a k e r n II a n d t h e c o n t e n t s were c o v e r e d w i t h a 3 X 3 p i e c e o f c a l u m i n u m f o i l ( w i t h h o l e s on i t ) a n d d r i e d o v e r n i g h t a t 110 C i n a h o t a i r o v e n . The b e a k e r s were t h e n t r a n s f e r r e d t o a c o l d m u f f l e f u r n a c e a n d t h e c o n t e n t s were a s h e d o v e r n i g h t o b y s l o w l y r a i s i n g t h e t e m p e r a t u r e t o 500 C . The a s h e d s a m p l e was c o o l e d t o room t e m p e r a t u r e . D e i o n i z e d w a t e r (5 m l ) was a d d e d t o wet t h e a s h a n d t h e n t h e c o n t e n t s w e r e d i s s o l v e d i n 35 ml o f 6 N H C l . The s o l u t i o n was h e a t e d t o b o i l i n g on a h o t p l a t e f o r 2 0 - 3 0 m i n u t e s t o r e d u c e t h e s e l e n a t e t o s e l e n i t e . T h e n t h e c o n t e n t s were t r a n s f e r r e d t o a n a c i d washed 50 ml v o l u m e t r i c f l a s k a n d d i l u t e d t o v o l u m e w i t h 6 N H C l . S i m u l t a n e o u s l y , d u p l i c a t e b l a n k s ( w i t h o n l y 10 ml o f a s h i n g a i d s o l u t i o n ) a n d Se s t a n d a r d s i n d u p l i c a t e ( c o n t a i n i n g 0 . 1 , 1, 1 0 , 100 ppm o f Se) w e r e c a r r i e d t h r o u g h t h e a b o v e p r o c e d u r e s a l o n g w i t h t h e s a m p l e s . One ml a l i q u o t o f d i g e s t e d s a m p l e was t r e a t e d w i t h 3 ml o f 1% NaBH^ s o l u t i o n (3 g o f NaBH^ i n 300 ml o f 1% NaOH) t o g e n e r a t e g a s e o u s h y d r o g e n s e l e n i d e , w h i c h was a n a l y z e d b y a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y ( P e r k i n E l m e r , m o d e l 5 6 0 ) . B l a n k a n d s t a n d a r d s o l u t i o n s were t r e a t e d i n t h e same m a n n e r . The m e t h o d was s t a n d a r d i z e d i n o u r l a b o r a t o r y a g a i n s t t h e N a t i o n a l B u r e a u o f S t a n d a r d s r e f e r e n c e m a t e r i a l 1577 f o r t h e b o v i n e l i v e r . The d e t e c t i o n l i m i t (3 X s t a n d a r d d e v i a t i o n o f t h e b l a n k s a m p l e ) b a s e d on a 10 g s a m p l e was 5 p p b . The i n t e r - a n d i n t r a - a s s a y c o e f f i c i e n t s o f v a r i a t i o n f o r 54 Se d e t e r m i n a t i o n were 13% ( r a n g e 10-15%) a n d 10% ( r a n g e 8 -12%) r e s p e c t i v e l y . The f e c a l p e l l e t s were g r o u n d t o p a s s t h r o u g h a 1 mm mesh s c r e e n . A r e p r e s e n t a t i v e f e c a l s a m p l e o f 1 g was 75 75 u s e d f o r d e t e r m i n i n g S e . The Se r a d i o a c t i v i t y i n u r i n e (1 m l ) was a l s o m e a s u r e d . A p p r o x i m a t e l y 1 g o f t i s s u e s a m p l e s was t a k e n a n d r i n s e d t h o r o u g h l y i n p h y s i o l o g i c a l s a l i n e (0.9% N a C l ) , t o remove a n y p o s s i b l e e x t e r n a l c o n t a m i n a t i o n f r o m b l o o d , b l o t t e d d r y a n d t h e n c o u n t e d . Se c o n c e n t r a t i o n s i n u r i n e (2 m l ) , f e c e s (2 g ) , t i s s u e s (1 g) a n d f e e d s a m p l e s (2 g) were d e t e r m i n e d i n d u p l i c a t e a s p e r t h e m e t h o d o f Tarn a n d L a c r o i x (1982) w i t h m i n o r m o d i f i c a t i o n s w h i c h i n c l u d e d i ) p r e - w e t t i n g o f f e e d a n d f e c a l s a m p l e s w i t h d e i o n i z e d w a t e r a n d i i ) h e a t i n g t h e m t o 1 5 0 ° C , u n t i l d r y a n d f l u f f y , t o p r e v e n t l o s s o f t h e s e s a m p l e s d u r i n g a s h i n g . Se c o n c e n t r a t i o n s were e x p r e s s e d on a wet b a s i s . (d ) D a t a a n a l y s i s a n d c a l c u l a t i o n s ; 7 5 The p l a s m a Se s p e c i f i c a c t i v i t y d a t a a t e a c h t i m e p o i n t were e x p r e s s e d as a f r a c t i o n o f i n i t i a l 75 d o s e i n j e c t e d p e r m i c r o g r a m S e . The p l a s m a Se s p e c i f i c a c t i v i t y - t i m e d a t a were f i t t e d t o e x p o n e n t i a l e q u a t i o n s u s i n g t h e c u r v e p e e l i n g c o m p u t e r p r o g r a m , AUTOAN (Sedman a n d W a g n e r , 1 9 7 6 ) . The g o o d n e s s o f f i t o f t h e e x p o n e n t i a l 55 75 e q u a t i o n s t o p l a s m a Se s p e c i f i c a c t i v i t y d a t a , was e v a l u a t e d b a s e d on ( i ) t h e s q u a r e d c o r r e l a t i o n c o e f f i c i e n t 2 (R ) v a l u e s a n d ( i i ) " F " v a l u e s , f o r t h e o v e r a l l f i t , c a l c u l a t e d as p e r t h e f o l l o w i n g f o r m u l a . F - ( S A o b s " S A e s t > 2 75 w h e r e S A 0 D S " O b s e r v e d p l a s m a Se s p e c i f i c a c t i v i t y 75 S A e s t ™ E s t i m a t e d p l a s m a Se s p e c i f i c a c t i v i t y T he e x p o n e n t i a l e q u a t i o n s w i t h m i n i m a l " F " v a l u e s a n d 2 w i t h t h e R v a l u e s a p p r o a c h i n g 1 were c h o s e n a s t h e b e s t f i t e q u a t i o n s . The a r e a s u n d e r t h e e x p o n e n t i a l c u r v e s were c a l c u l a t e d b y i n t e g r a t i o n f r o m z e r o t i m e t o i n f i n i t y . The f o l l o w i n g e q u a t i o n s were u s e d f o r t h e e s t i m a t i o n o f k i n e t i c p a r a m e t e r s o f Se m e t a b o l i s m . i ) I r r e v e r s i b l e r a t e 1 o f d i s p o s a l (I.D.) «= ( S h i p l e y a n d ( u g S e / d / k g B . W . ) J " S . A . ( d t ) C l a r k , 1 9 7 2 ) /o> 75 S . A . ( d t ) - A r e a u n d e r t h e p l a s m a Se s p e c i f i c a c t i v i t y - t i m e c u r v e 1 - N o r m a l i z e d d o s e B . W . - B o d y w e i g h t ( n o r m a l i z e d t o 70 k g ) . The I.D. i s t h e r a t e o f l o s s o f m a t e r i a l f r o m a p r i m a r y l a b e l l e d p o o l w h i c h d o e s n o t r e t u r n t o i t d u r i n g 56 t h e c o u r s e o f t h e t r a c e r e x p e r i m e n t ( S h i p l e y a n d C l a r k , 1 9 7 2 ) . I n t h e p r e s e n t s t u d y t h e m a t e r i a l h a s b e e n a s s u m e d t o be t h e t r a c e e i e . s t a b l e S e , a n d t h e p r i m a r y p o o l a s b l o o d p l a s m a w h i c h h a s b e e n l a b e l l e d w i t h ^ S e -s e l e n i t e . 1 i i ) P o o l s i z e ( P . S ) - ( W h i t e e t a l . 1969) ( u g Se) 2 ( A . ) i=i 1 75 w h e r e A - Z e r o t i m e i n t e r c e p t o f Se s p e c i f i c a c t i v i t y i - e x p o n e n t i a l c o m p o n e n t number n = number o f e x p o n e n t i a l c o m p o n e n t s The q u a n t i t y o f u n l a b e l l e d m a t e r i a l ( t r a c e e ) p r e s e n t i n a p o o l i s known as t h e p o o l s i z e ( W h i t e e t a l . 1 9 6 9 ) . I n t h e c u r r e n t s t u d y t h e p o o l s i z e r e f e r s t o t h e t o t a l amount o f s t a b l e Se p r e s e n t i n t h e b l o o d p l a s m a . i i i ) P l a s m a c l e a r a n c e I . D . r a t e ( P . C . R ) = ( S h i p l e y a n d ( m l / d / k g B . W . ) P l a s m a Se C o n e . C l a r k , 1972) The p l a s m a c l e a r a n c e r a t e c a n be d e f i n e d as t h e v o l u m e o f p l a s m a n o m i n a l l y c l e a r e d p e r u n i t t i m e . A l t h o u g h no v o l u m e i s l i t e r a l l y c l e a r e d , a m a t h e m a t i c a l e q u i v a l e n t r e l a t e s t h e v o l u m e w h i c h , a t t h e 57 e x i s t i n g c o n c e n t r a t i o n o f s o l u t e , c o n t a i n s a n amount o f s o l u t e c o r r e s p o n d i n g t o t h e q u a n t i t y i r r e v e r s i b l y l o s t f r o m t h e s p e c i f i e d v o l u m e ( e . g . , b l o o d p l a s m a ) p e r u n i t t i m e ( S h i p l e y a n d C l a r k , 1 9 7 2 ) . i v ) V o l u m e o f d i s t r i - P o o l s i z e 1 b u t i o n ( V . D . ) = X ( W h i t e e t ( m l / k g B . W . ) P l a s m a Se c o n e . B o d y w t . a l . 1969) The v o l u m e o f d i s t r i b u t i o n r e f e r s t o t h e t o t a l v o l u m e o f t h e p o o l i n w h i c h t h e e n t i r e m a t e r i a l ( t r a c e e o r t r a c e r ) i s p r e s e n t ( W h i t e e t a l . 1 9 6 9 ) . I n t h e p r e s e n t s t u d y t h e t o t a l p l a s m a v o l u m e c o n t a i n i n g a l l t h e s t a b l e Se c o n s t i t u t e s t h e v o l u m e o f d i s t r i b u t i o n . 7 5 The u r i n a r y a n d f e c a l Se l o s s e s were c a l c u l a t e d 75 as mean d a i l y e x c r e t i o n o f Se p e r d a y a n d e x p r e s s e d as f r a c t i o n o f t h e i n i t i a l d o s e i n j e c t e d . S t a b l e Se b a l a n c e was c a l c u l a t e d as p e r t h e s t a n d a r d n u t r i t i o n a l m e t h o d s ( M a y n a r d a n d L o o s l i , 1 9 6 9 ) . The e n d o g e n o u s f e c a l Se l o s s e s were c a l c u l a t e d u s i n g t h e f o l l o w i n g e q u a t i o n o f H a n s a r d e t a l . (1957) w i t h s l i g h t m o d i f i c a t i o n w h i c h i n c l u d e d u s e o f t h e a r e a s 75 u n d e r t h e f e c a l a n d p l a s m a Se s p e c i f i c a c t i v i t y - t i m e c u r v e s (5 d) a s n u m e r a t o r a n d d e n o m i n a t o r o f t h e e q u a t i o n r e s p e c t i v e l y i n s t e a d o f mean s p e c i f i c 58 a c t i v i t i e s o f t h e l a s t two d a y s o f t h e b a l a n c e t r i a l . E n d o g e n o u s A U C - F e c e s S p . A c t - t i m e c u r v e (5d) A v e r a g e f e c a l Se = X d a i l y f e c a l l o s s ( u g / d ) A U C - P l a s m a S p . A c t - t i m e c u r v e (5d) Se l o s s w h e r e t h e s p e c i f i c a c t i v i t y o f f e c e s a n d p l a s m a a r e 75 f r a c t i o n s o f t h e Se d o s e p e r m i c r o g r a m S e , a n d AUC r e p r e s e n t s t h e a r e a u n d e r t h e c u r v e . The f r a c t i o n a l r a t e c o n s t a n t s o f Se u t i l i z a t i o n i n i n d i v i d u a l t i s s u e s were o b t a i n e d by s a c r i f i c i n g t h e ewes on d a y 1 , 5 , 9 a n d 14 a f t e r t r a c e r a d m i n i s t r a t i o n a n d d e t e r m i n i n g t h e s p e c i f i c a c t i v i t y i n t h e t o t a l t i s s u e . The e x p o n e n t i a l e q u a t i o n s were f i t t e d t o t h e s p e c i f i c a c t i v i t y -t i m e d a t a o f t h e i n d i v i d u a l t i s s u e s a n d t h e f r a c t i o n a l r a t e c o n s t a n t s a s w e l l a s t h e v a l u e s were d e r i v e d . T h o u g h one a n i m a l was s a c r i f i c e d a t e a c h t i m e p o i n t f o r r e a s o n s o f economy i t was a s s u m e d t h a t t h e s l o p e o f t h e c u r v e w o u l d p r o v i d e a n a p p r o x i m a t e t u r n o v e r o f Se i n t h e t i s s u e s . The t o t a l p o o l o f Se i n t h e i n d i v i d u a l t i s s u e s was d e r i v e d by m u l t i p l y i n g t h e Se c o n c e n t r a t i o n w i t h t h e w e i g h t o f t h e w h o l e o r g a n / t i s s u e . The t o t a l w e i g h t o f t h e s k e l e t a l m u s c l e t i s s u e was a s sumed t o be 40% o f t h e b o d y w e i g h t a s p e r A r n a l ( 1 9 7 7 ) . 59 S t a t i s t i c a l a n a l y s i s : A l l r e s u l t s were e x p r e s s e d as means + S E M . The d a t a were s u b j e c t e d t o one way a n a l y s i s o f v a r i a n c e a n d t h e s i g n i f i c a n c e o f d i f f e r e n c e b e t w e e n t h e means was a s s e s s e d b y S t u d e n t ' s ' t ' t e s t ( S A S , 1 9 8 5 ) . The r e g r e s s i o n l i n e s a n d t h e c o r r e l a t i o n c o e f f i c i e n t s b e t w e e n s e l e c t e d p a r a m e t e r s were c a l c u l a t e d b y t h e method o f l e a s t s q u a r e s . 60 RESULTS The plasma Se concentration in the Se positive ewes was 142 + 6 ng/ml, which was s i g n i f i c a n t l y higher (P < 0.01) than i n the d e f i c i e n t ones (82 + 10 ng/ml). A posit i v e l i n e a r relationship (r=0.90) between the Se intake and the plasma Se concentration was observed (Fig. 1 ) . 75 The plasma Se s p e c i f i c a c t i v i t y changes during the f i r s t 24 hr after tracer i n j e c t i o n into Se positive and de f i c i e n t ewes are depicted in Figs. 2 and 3. In the Se positi v e ewes (Fig. 2) the tracer disappeared i n i t i a l l y from the plasma in an exponential fashion u n t i l approximately 1 hr after i n j e c t i o n . This was followed by an increase in the plasma r a d i o a c t i v i t y which reached a peak at approximately 4 hr postinjection before f i n a l l y d eclining gradually over several days. At the peak of reappearance the r a d i o a c t i v i t y was 44% of that present at 2 min at which time the f i r s t sample of blood was co l l e c t e d . A similar pattern was observed i n Se d e f i c i e n t ewes (Fig. 3) except that the maximum reappearance of the r a d i o a c t i v i t y occurred at 3 hr and was 49% of the a c t i v i t y present i n the 2 min plasma sample. The best f i t plasma s p e c i f i c activity-time curves of Se positive and de f i c i e n t ewes are shown i n Figs. 4 and 5 respectively. The exponential equations describing 61 160-1 C 140-120-O C o i n IOO ^ 80 -60 -0 200 400 Se intake (pg/d) 600 F i g . 1 . E f f e c t o f Se i n t a k e on p l a s m a Se c o n c e n t r a t i o n i n n o n p r e g n a n t ewes f e d Se p o s i t i v e (A) o r d e f i c i e n t (A) r a t i o n s . The r e g r e s s i o n e q u a t i o n i s Y * 7 9 . 8 4 + 0 . 1 3 x a n d r - 0 . 9 0 . F i g . 2 . C h a n g e s i n p l a s m a Se s p e c i f i c a c t i v i t y d u r i n g f i r s t 24 h r a f t e r t r a c e r i n j e c t i o n i n t o Se p o s i t i v e n o n p r e g n a n t e w e s . • , O i • » • r e f e r t o ewe n o s . 6 2 2 , 6 5 , 100 a n d 99 r e s p e c t i v e l y . Fra. = Fraction i n 6 CD o . o o o i - l 1 1 0 10 20 30 Time (Hours) F i g . 3 . C h a n g e s i n p l a s m a Se s p e c i f i c a c t i v i t y d u r i n g f i r s t 24 h r a f t e r t r a c e r i n j e c t i o n i n t o Se d e f i c i e n t n o n p r e g n a n t ewes . 9, O, u, • r e f e r t o ewe n o s . 9 9 9 , 9 9 8 , 325 a n d 123 r e s p e c t i v e l y . 64 0.01 0) m CO CU 0.0001-f 1 1 1 — 0 20 40 60 80 Time (Hours) F i g . 4. B e s t - f i t p l a s m a 7 5 S e s p e c i f i c a c t i v i t y - t i m e c u r v e s o f Se p o s i t i v e n o n p r e g n a n t e w e s . # , O, •» • r e f e r t o ewe n o s . 6 2 2 , 6 5 , 100 a n d 99 r e s p e c t i v e l y . 65 O.OOOH 0 20 40 60 Time (Hours) F i g . 5 . B e s t - f i t p l a s m a 7 5 S e s p e c i f i c a c t i v i t y - t i m e c u r v e s o f Se d e f i c i e n t n o n p r e g n a n t e w e s . • , O, • , • r e f e r t o ewe n o s . 9 9 9 , 9 9 8 , 325 a n d 123 r e s p e c t i v e l y . T a b l e 1 . B e s t - f i t e x p o n e n t i a l e q u a t i o n s o f p l a s m a Se s p e c i f i c a c t i v i t y - t i m e c u r v e s o f Se p o s i t i v e a n d d e f i c i e n t n o n p r e g n a n t e w e s . A n i m a l # A l A 2 A 3 a l a 2 a 3 Se p o s i t i v e ewes : 622 .000770 .000413 8 . 5672 .0126 65 .004078 .000743 22 .8084 . 0096 99 .000487 .000343 6 . 2382 . 0071 100 .001064 .000408 6 . 6 9 3 0 .0052 Se d e f i c i e n t ewes • • 999 .005010 .002867 7 .0774 . 0202 998 .003329 .002562 13 . 2316 . 0 1 0 0 123 .005357 .002187 8 . 6 3 3 5 . 0 0 4 7 325 .000956 .000431 .000281 9 . 3 2 0 5 . 0372 .0012 * T h e e q u a t i o n i s : A t " A l e a fc + A e 1 + A 2 e - a t 2 + A 3 e - a t 3 ' w h e r e A - p l a s m a Se s p e c i f i c a c t i v i t y ( f r a c t i o n o f i n j e c t e d d o s e / u g S e ) , a - r a t e c o n s t a n t , e - l o g 1 n and t - t i m e ( h r ) . 67 these curves are summarized in Table 1. Two exponentials were Required to f i t the plasma s p e c i f i c a c t i v i t y data i n a l l ewes except in one, (#325) which required three 75 exponentials. The plasma Se clearance was thus biphasic in a l l animals except ewe #325. The two pool k i n e t i c model i s based on the assumption that the tracer injected into the blood (pool a) mixes instantaneously and gets d i s t r i b u t e d into the tissues (pool b) i n i t i a l l y . After a while the tracer leaves the system obeying f i r s t ' order k i n e t i c s . The values of plasma Se i n the Se posit i v e and d e f i c i e n t ewes were 1.95 + 0.44 d and 2.02 + 0.28 d respectively (Table 2 ) . 75 The areas under the plasma Se s p e c i f i c a c t i v i t y -time curves and the kin e t i c parameters of Se metabolism i n the ewes are presented in Table 2. S i g n i f i c a n t l y higher i r r e v e r s i b l e disposal of Se (P _< 0.05), plasma Se clearance (P <^  0.05) and plasma Se pool size (P< 0.10) were observed in Se positive ewes than in the de f i c i e n t ones. On the other hand the area under the plasma 75 Se s p e c i f i c activity-time curves was s i g n i f i c a n t l y higher (P < 0.05) i n the Se de f i c i e n t ewes than in the posit i v e ones. The metabolic parameters of Se intake and excretion are given i n Table 3. The Se intake was s i g n i f i c a n t l y (P < 0.01) higher i n Se positive ewes (459 + 53 ug : mean + SEM) T a b l e 2 . C o m p a r i s o n o f a r e a s u n d e r t h e c u r v e s ( A U C ) , p l a s m a Se c o n c e n t r a t i o n s a n d k i n e t i c p a r a m e t e r s o f Se p o s i t i v e a n d d e f i c i e n t n o n p r e g n a n t e w e s . Se p o s i t i v e ewes ( n » 4 ) Se d e f i c i e n t ewes ( n - 4 ) P a r a m e t e r Mean + S . E . M Mean + S . E . M AUC ( F r a c t i o n o f d o s e / u g S e ) . h r 0 . 0 6 a + 0 .01 0 . 2 8 b + 0 . 0 7 P l a s m a Se C o n e , ( n g / m l ) 1 4 2 ° + 6 8 2 d + 10 I r r e v e r s i b l e D i s p o s a l o f Se ( u g S e / d / k g B . W . ) 7 a + 2 2 b + 0 . 3 P l a s m a C l e a r a n c e R a t e ( m l / d / k g B . W . ) 4 7 a + 10 1 9 b + 5 P l a s m a Se P o o l S i z e ( u g Se) 7 3 4 e + 207 2 5 8 f + 229 V o l u m e o f d i s t r i b u t i o n ( m l / k g B . W . ) 72 + 18 40 + 12 T l / 2 ( d ) 1 . 9 5 + 0 .44 2 . 0 2 + 0 . 2 8 Means w i t h i n t h e rows d i f f e r s i g n i f i c a n t l y ( a , b P < 0 . 0 5 ; c , d P < 0 . 0 1 ; e ' r P < 0 . 1 0 ) . 69 T a b l e 3 . A b s o r p t i o n a n d r e t e n t i o n o f Se i n n o n p r e g n a n t ewes f e d Se p o s i t i v e o r d e f i c i e n t r a t i o n s . Se p o s i t i v e ewes Se d e f i c i e n t ewes P a r a m e t e r Mean (n-+ =3) S . E . M ( n - 3 ) Mean + S . E . M Se i n t a k e ( u g / d ) 4 5 9 a + 53 4 4 b + 5 F e c a l Se l o s s ( u g / d ) 2 7 2 a + 48 2 5 b + 6 E n d o g e n o u s f e c a l Se 46 + 6 24 + 9 l o s s ( u g / d ) U r i n a r y Se l o s s ( u g / d ) 3 5 c + 7 6 d + 1 * A p p a r e n t Se a b s o r p t i o n ( a ) e s t i m a t e d ( u g / d ) 1 8 6 a + 36 2 0 b + 11 (b)% o f Se i n t a k e 41 + 7 40 + 20 N e t Se a b s o r p t i o n ( a ) e s t i m a t e d ( u g / d ) 2 3 2 a + 41 4 4 b + 11 (b)% o f Se i n t a k e 5 1 e + 8 9 7 f + 16 N e t Se b a l a n c e ^ 1 5 2 ° + 35 1 3 d + 13 ( u g / d ) ' ' Means w i t h i n t h e rows d i f f e r s i g n i f i c a n t l y ( a ' b P < 0 . 0 1 ; c , d P < 0 . 0 5 ; e , f P < 0 . 1 0 ) . * A p p a r e n t Se a b s o r p t i o n = Se i n t a k e - F e c a l Se l o s s . * N e t a b s o r p t i o n = S e i n t a k e - ( F e c a l l o s s - E n d o g e n o u s Se l o s s ) . ® S e b a l a n c e = Se i n t a k e - F e c a l Se l o s s - U r i n a r y Se l o s s . 70 than i n the d e f i c i e n t ones (44 + 5 ug : mean + SEM). Measurement of Se intake i n Se p o s i t i v e ewes showed t h a t approximately 70% of Se was from hay and 30% from water, while i n Se d e f i c i e n t ewes only 10% of Se in t a k e was from hay and 90% was from water. F e c a l and u r i n a r y Se l o s s e s were s i g n i f i c a n t l y higher i n Se p o s i t i v e ewes than i n the d e f i c i e n t ones. The combined s t a b l e Se l o s s e s through feces and u r i n e of Se p o s i t i v e ewes were 59% and 8% of Se i n t a k e r e s p e c t i v e l y , whereas i n Se d e f i c i e n t ewes the • corresponding values were 57% and 14% r e s p e c t i v e l y . Though the endogenous f e c a l Se l o s s i n Se p o s i t i v e ewes was almost two f o l d higher than i n d e f i c i e n t ones i t was not s t a t i s t i c a l l y s i g n i f i c a n t . Although the estimated apparent Se a b s o r p t i o n values (ug/d) were s i g n i f i c a n t l y (P _< 0.01) higher i n the Se p o s i t i v e ewes than i n the d e f i c i e n t ones, the d i f f e r e n c e s proved to be n o n s i g n i f i c a n t when expressed as a per cent of Se i n t a k e . The net Se a b s o r p t i o n expressed as ug/d was found to be s i g n i f i c a n t l y higher (P £ 0.01) i n the Se p o s i t i v e ewes than i n the d e f i c i e n t ones, whereas, on a per cent Se intake b a s i s the Se d e f i c i e n t ewes had a s i g n i f i c a n t l y higher (P <^  0.10) net Se a b s o r p t i o n than the p o s i t i v e ones. At higher Se i n t a k e s the net Se a b s o r p t i o n was low i n Se p o s i t i v e ewes, whereas i n the Se d e f i c i e n t ewes i t was high i n d i c a t i n g an i n v e r s e l i n e a r r e l a t i o n s h i p between the Se intake and the net Se a b s o r p t i o n ( F i g . 6 ) . 71 F i g . 6. E f f e c t o f Se i n t a k e on net Se a b s o r p t i o n i n nonpregnant ewes. The r e g r e s s i o n e q u a t i o n s are' Y -2 4 . 3 3 + 0 . 4 5 X ( r - 0 . 9 4 ) and Y - 1 0 0 . 8 0 - 0 . 1 1 X ( r — 0 . 7 6 ) f o r t he net Se a b s o r p t i o n , e x p r e s s e d as ug/d and as % Se i n t a k e r e s p e c t i v e l y . 72 3-1 1 2 3 4 5 DAYS AFTER TRACER INJECTION F i g . 7 . Mean d a i l y e x c r e t i o n o f Se i n u r i n e a n d f e c e s o f n o n p r e g n a n t ewes f e d Se p o s i t i v e o r Se d e f i c i e n t r a t i o n s . E E B , r e f e r t o f e c e s a n d u r i n e o f Se p o s i t i v e e w e s , andE&3, Z D r e f e r t o f e c e s a n d u r i n e o f Se d e f i c i e n t ewes r e s p e c t i v e l y . T a b l e 4. S t a b l e Se c o n c e n t r a t i o n s and p o o l s i z e s i n t h e t i s s u e s o f Se p o s i t i v e a n d d e f i c i e n t n o n p r e g n a n t ewes . T i s s u e Se c o n c e n t r a t i o n ( u g / g t i s s u e ) P o o l s i z e ( u g Se ) Se p o s i t i v e Se d e f i c i e n t Se p o s i t i v e Se d e f i c i L i v e r 0. 1 8 a + 0. ,01 0. l l b + 0. ,01 1 9 9 e + 15 8 1 £ + 12 K i d n e y 1 .00 + 0. ,06 0 .84 + 0. .12 1 8 7 g + 9 1 3 1 h + 22 S k . M u s c l e 0 .07 + 0. ,01 0 .06 + 0. ,01 2066 + 166 1623 + 134 H e a r t 0 .21 + 0. .01 0 .17 + 0. .03 63 + 7 51 + 8 L u n g 0. 2 1 c + 0. .01 0. 1 2 d + 0. .01 1 7 4 e + 7 9 4 f + 11 S p l e e n 0. 3 9 e + 0. .01 0. 2 2 f + 0. ,02 1 2 5 a + 14 4 0 b + 6 P a n c r e a s 0. 3 0 a + 0. .02 0. 2 0 b + 0. .02 26* + 4 1 4 h + 3 Mammary 0 .10 + 0. .01 0 .06 + 0. .01 2 9 g + 4 1 3 h + 4 g l a n d a , b t Means w i t h d i f f e r e n t s u p e r s c r i p t s w i t h i n t h e row d i f f e r s i g n i f i c a n t l y ( ' P _< 0.01; c ' a P < 0.0001; e , t P < 0.001; g ' n P < 0.05). 74 The maximum e x c r e t i o n of Se through feces and u r i n e was observed on day 1 a f t e r the t r a c e r i n j e c t i o n i n Se p o s i t i v e ewes and t h i s d e c l i n e d g r a d u a l l y over a 5 d p e r i o d ( F i g . 7 ) . On the other hand i n Se d e f i c i e n t ewes the 75 h i g h e s t Se a c t i v i t y was found i n u r i n e on day 1, whereas 7 5 the d a i l y f e c a l Se l o s s was hi g h e s t 3 d a f t e r the 75 t r a c e r i n j e c t i o n . A gradual d e c l i n e of Se was observed i n u r i n e but was not the case with feces ( F i g . 7 ) . The c o n c e n t r a t i o n s and pool s i z e s of Se i n the e n t i r e t i s s u e s are presented i n Table 4 . The Se c o n c e n t r a t i o n was the hi g h e s t i n the kidney of the Se p o s i t i v e ( 1 . 0 0 ug/g) as w e l l as i n the Se d e f i c i e n t ( 0 . 8 4 ug/g) ewes fo l l o w e d by spleen and pancreas. The c a r d i a c muscle had approximately three times as much Se c o n c e n t r a t i o n as the s k e l e t a l muscle of ewes i n both groups. The Se c o n c e n t r a t i o n i n the l i v e r and pancreas of Se p o s i t i v e ewes was s i g n i f i c a n t l y (P _< 0 . 0 5 ) higher than i n the d e f i c i e n t ones. S i g n i f i c a n t l y h igher c o n c e n t r a t i o n s of Se were n o t i c e d i n the spleen (P < 0 . 0 0 0 1 ) and i n the lung (P <^  0 . 0 0 1 ) of the Se p o s i t i v e ewes than i n the d e f i c i e n t ones. The c o n c e n t r a t i o n s of Se i n the s k e l e t a l and c a r d i a c muscle were s i m i l a r i n the ewes of both Se p o s i t i v e and d e f i c i e n t groups (P > 0 . 1 0 ) . The Se pool s i z e was s i g n i f i c a n t l y lower i n t i s s u e s such as l i v e r and lung (P < 0 . 0 0 1 ) , kidney and pancreas (P _< 0 . 0 5 ) and spleen (P <^  0 . 0 1 ) of Se d e f i c i e n t ewes than i n the Se intake (ug/d) F i g . 8 . E f f e c t o f Se i n t a k e o n t i s s u e Se p o o l s i z e i n n o n p r e g n a n t ewes f e d Se p o s i t i v e o r d e f i c i e n t r a t i o n s . The r e g r e s s i o n e q u a t i o n s a r e Y - 6 4 . 6 5 + 0 . 2 7 X , r « 0 . 8 6 , l i v e r (A); Y - 1 0 6 . 2 7 + 0 . 1 6 X , r - 0 . 8 9 , k i d n e y (ffi); a n d Y - 1 4 3 9 . 5 4 + 1 . 5 5 X , r - 0 . 8 9 , s k e l e t a l m u s c l e ( • ) r e s p e c t i v e l y . T a b l e 5 . K i n e t i c p a r a m e t e r s o f t i s s u e Se m e t a b o l i s m i n n o n p r e g n a n t e w e s * . T i s s u e . * * F r a c t i o n a l r a t e c o n s t a n t { F r a c t i o n / d ) Se p o s i t i v e Se d e f i c i e n t Se p o s i t i v e Se d e f i c i e n t L i v e r 0 . 0 4 9 5 0 .1201 14 6 K i d n e y 0 . 1062 0 .1013 7 7 S k . M u s c l e 0 . 0 3 4 7 0 .0462 20 15 H e a r t 0 . 0 3 6 3 0 .0557 19 12 L u n g 0 .0588 0 .0768 12 9 S p l e e n 0 .0927 0 .1084 7 6 P a n c r e a s 0 . 0 5 9 1 0 .0801 12 9 Mammary g l a n d 0 . 0 5 4 0 0 .0540 13 13 n -4 ewes i n e a c h g r o u p . * * o f Se u t i l i z a t i o n i n i n d i v i d u a l t i s s u e s . 77 p o s i t i v e o n e s . The c a r d i a c and s k e l e t a l m u s c l e o f Se d e f i c i e n t ewes t e n d e d t o h a v e l o w e r Se p o o l s i z e s t h a n t h e p o s i t i v e o n e s a n d t h e s e d i f f e r e n c e s were 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 . H o w e v e r , t h e s k e l e t a l m u s c l e Se p o o l s i z e was f o u n d t o r e s p o n d t o t h e c h a n g e s i n t h e d i e t a r y Se i n t a k e i n a l i n e a r f a s h i o n ( F i g . 8). The f r a c t i o n a l r a t e c o n s t a n t s a n d t h e T i / 2 v a l u e s i n d i c a t i n g t h e u t i l i z a t i o n o f Se i n t h e t i s s u e s o f ewes a r e p r e s e n t e d i n T a b l e 5. The r a t e c o n s t a n t s o f Se u t i l i z a t i o n t e n d e d t o be h i g h e r i n t h e Se d e f i c i e n t ewes t h a n i n t h e p o s i t i v e o n e s . T h u s , t h e t i s s u e Se v a l u e s were g e n e r a l l y h i g h e r i n t h e Se p o s i t i v e ewes t h a n i n t h e d e f i c i e n t o n e s . D I S C U S S I O N T h o u g h t h e p l a s m a Se c o n c e n t r a t i o n s o f ewes i n g r o u p I I w e r e l o w e r t h a n i n g r o u p I ( T a b l e 2 ) , t h e d u r a t i o n o f t h e f e e d i n g o f t h e Se d e f i c i e n t r a t i o n was p r e s u m a b l y n o t l o n g e n o u g h t o r e s u l t i n a n a b s o l u t e d e f i c i e n t s t a t u s i n g r o u p I I ewes b a s e d on t h i s c r i t e r i o n . C o u p l e d w i t h t h e t i s s u e Se l e v e l s ( T a b l e 4) t h e ewes i n g r o u p I I may be c o n s i d e r e d t o be m a r g i n a l l y Se d e f i c i e n t u n d e r t h e c o n d i t i o n s o f t h i s s t u d y as c o m p a r e d t o t h o s e i n g r o u p I . The i n i t i a l d i s a p p e a r a n c e f o l l o w e d b y r e a p p e a r a n c e o f 78 75 Se a f t e r 3-4 h r o f i n j e c t i o n i n t h e p l a s m a o f t h e ewes i s s i m i l a r t o o b s e r v a t i o n s i n d a i r y cows (Symonds e t a l . 1981) a n d i n g o a t s ( A l l e n a n d M i l l e r , 1 9 8 1 ) . Symonds e t a l . ( 1 9 8 1 ) h a v e r e p o r t e d t h a t t h e l i v e r p l a y e d a r o l e i n t h e p l a s m a Se c l e a r a n c e o f d a i r y cows a n d a c c o u n t e d f o r 75 a b o u t 40% o f t h e Se d o s e t h a t i s c l e a r e d i n i t i a l l y 75 f r o m t h e p l a s m a . The maximum r e a p p e a r a n c e o f Se i n t o t h e p l a s m a t o o k p l a c e a f t e r 1 h r i n d a i r y cows (Symonds e t a l . 1981) a n d 3-6 h r i n g o a t s ( A l l e n a n d M i l l e r , 1981) a f t e r t r a c e r i n j e c t i o n . I n t h e c u r r e n t s t u d y t h e h i g h e s t 75 Se a c t i v i t y was o b s e r v e d i n i t i a l l y i n t h e 2 m i n p l a s m a s a m p l e i n a l l t h e ewes . The r e s u l t s o f t h e p r e s e n t s t u d y a r e more i n a g r e e m e n t w i t h t h o s e o f Symonds e t a l . ( 1 9 8 1 ) 75 w i t h r e g a r d t o t h e amount o f Se t h a t r e a p p e a r e d i n t o t h e p l a s m a a n d w i t h t h o s e o f A l l e n a n d M i l l e r ( 1 9 8 1 ) w i t h r e g a r d 75 t o t h e t i m i n g a t w h i c h Se r e a p p e a r a n c e o c c u r r e d . The 7 5 d i f f e r e n c e s i n t h e amounts a n d t i m i n g s o f p l a s m a Se r e a p p e a r a n c e c o u l d be due t o t h e v a r i a t i o n s i n t h e s p e c i e s a n d p h y s i o l o g i c a l s t a t u s . The a v i d u p t a k e o f Se b y t h e e r y t h r o c y t e s as shown by S a n d h o l m ( 1 9 7 3 ) may h a v e a l s o c o n t r i b u t e d t o t h e i n i t i a l r a p i d d i s a p p e a r a n c e o f 75 Se f r o m t h e p l a s m a . F o r t h e p u r p o s e o f c a l c u l a t i n g t h e k i n e t i c p a r a m e t e r s a n a s s u m p t i o n was made t h a t t h e ewes i n b o t h g r o u p s w e r e i n a s t e a d y s t a t e c o n d i t i o n . The a n i m a l s i n g r o u p I I were a t d i f f e r e n t s t a g e s o f a t t a i n i n g m a r g i n a l Se 79 d e f i c i e n c y a n d f o r t h e s h o r t d u r a t i o n o f t h e t r a c e r s t u d i e s t h e b o d y p o o l s were a s s u m e d t o r e m a i n c o n s t a n t i n s i z e ( S h i p l e y a n d C l a r k , 1 9 7 2 ) . 75 The a r e a s u n d e r t h e p l a s m a Se s p e c i f i c a c t i v i t y -t i m e c u r v e s o f Se d e f i c i e n t ewes were s i g n i f i c a n t l y h i g h e r (P _< 0 . 0 5 ) t h a n t h o s e o f Se p o s i t i v e ewes i n d i c a t i n g 75 g r e a t e r u p t a k e a n d r e t e n t i o n o f Se i n t h e t i s s u e s o f t h e f o r m e r . T h i s may be due t o t h e f a c t t h a t t h e p o o l o f s t a b l e ( c o l d ) Se i n p l a s m a was s m a l l e r i n Se d e f i c i e n t 75 ewes t h a n i n t h e p o s i t i v e o n e s . A g r e a t e r t i s s u e Se u p t a k e a n d r e t e n t i o n were r e p o r t e d u n d e r Se d e f i c i e n c y c o n d i t i o n s i n s h e e p ( M u t h e t a l . 1967) a n d i n c a t t l e 75 ( K i n c a i d e t a l . 1 9 7 7 ) . The h i g h e r AUC o f p l a s m a Se s p e c i f i c a c t i v i t y - t i m e c u r v e s o f Se d e f i c i e n t ewes c o u p l e d w i t h t h e i r l o w e r p l a s m a Se l e v e l s (82 + 10 n g / m l ) i n d i c a t e d t h a t t h e d u r a t i o n a n d t h e l e v e l o f t h e n u t r i t i o n a l r e g i m e i m p o s e d were a d e q u a t e t o make t h e ewes m a r g i n a l l y Se d e f i c i e n t d u r i n g t h e p e r i o d o f s t u d y . 75 The p l a s m a Se s p e c i f i c a c t i v i t y - t i m e d a t a o f ewe #325 were d i f f e r e n t f r o m t h o s e o f o t h e r ewes i n t h e same g r o u p ( F i g s . 3 a n d 5 ) . T h o u g h Se i n t a k e ( 3 8 + 6 u g / d : mean + SEM) a n d Se b a l a n c e ( - 6 . 5 3 + 8 . 5 1 u g / d : mean + SEM) i n ewe #325 were l o w e r t h a n t h o s e o f i t s g r o u p m a t e s , t h e 75 p l a s m a Se c o n c e n t r a t i o n (111 n g / m l ) a n d t h e p l a s m a Se s p e c i f i c a c t i v i t y - t i m e d a t a ( F i g s . 3 a n d 5) i n d i c a t e d a 80 h i g h e r p l a s m a Se s t a t u s i n ewe #325. T h i s d i s c r e p a n c y may be due t o t h e l a r g e r body s i z e (73 kg) o f ewe #325 t h a n h e r g r o u p m a t e s . A n i m a l v a r i a t i o n a n d Se s t a t u s p r i o r t o e x p e r i m e n t a t i o n may have a l s o c o n t r i b u t e d t o t h e d i f f e r e n c e s o b s e r v e d . The i r r e v e r s i b l e d i s p o s a l ( I D ) and p l a s m a c l e a r a n c e r a t e s o f Se ( T a b l e 2) were s i g n i f i c a n t l y h i g h e r (P _< 0 . 0 5 ) i n Se p o s i t i v e ewes t h a n i n t h e d e f i c i e n t o n e s . A s one w o u l d e x p e c t a h i g h e r (P < 0 . 1 0 ) p l a s m a Se p o o l s i z e was a l s o o b s e r v e d i n Se p o s i t i v e ewes, r e f l e c t i n g t h e h i g h e r Se i n t a k e , t h a n i n t h e d e f i c i e n t o n e s . The s i g n i f i c a n t l y l o w e r ID and p l a s m a c l e a r a n c e r a t e s w o u l d s u g g e s t a d e c r e a s e d t u r n o v e r o f Se u n d e r c o n d i t i o n s o f e i t h e r Se d e f i c i e n c y o r l o w e r e d Se i n t a k e . T h i s c o u l d be e x p l a i n e d b y t h e f a c t t h a t t h e e n t r y o f Se i n t o p l a s m a p o o l t h r o u g h t h e d i e t a r y s o u r c e s was l i m i t e d a n d i f t h i s s i t u a t i o n was n o t r e v e r s e d t h e p l a s m a p o o l w o u l d e v e n t u a l l y be d e p l e t e d o f e n t i r e Se c o n t e n t . However, t h e Se d e f i c i e n t ewes w o u l d a t t e m p t t o m a i n t a i n Se h o m e o s t a s i s n o t o n l y b y d e c r e a s i n g t h e p l a s m a Se t u r n o v e r b u t a l s o b y i n c r e a s i n g t h e t u r n o v e r o f Se i n t h e t i s s u e s ( T a b l e 5 ) . I n a d d i t i o n , t h e e f f i c i e n c y o f n e t a b s o r p t i o n (% Se i n t a k e ) was i n c r e a s e d c o u p l e d w i t h a r e d u c t i o n i n t h e t o t a l b o dy l o s s e s t h r o u g h f e c e s and u r i n e t o m a i n t a i n t h e p l a s m a Se l e v e l s . T h e r e f o r e t h e p l a s m a ID o f Se, w h i c h r e f l e c t s t h e m e t a b o l i c a d a p t a t i o n s o f t h e e n t i r e b o d y , w o u l d 81 be a.good i n d i c a t o r o f t h e t u r n o v e r o f Se u n d e r b o t h Se a d e q u a t e a n d d e f i c i e n t c o n d i t i o n s . The v a l u e s o f p l a s m a Se i n t h e Se p o s i t i v e a n d d e f i c i e n t ewes were s i m i l a r . The v a l u e s r e p o r t e d i n s h e e p (20 d: L o p e z e t a l . 1969) and d a i r y c a t t l e (64 d : Symonds e t a l . 1981) a r e d i f f e r e n t f r o m t h o s e o b t a i n e d i n t h e p r e s e n t s t u d y (2 d ) . T hese d i f f e r e n c e s i n t h e ^1/2 v a l u e s c o u l d be due t o v a r i a t i o n s i n ( i ) S e s t a t u s o f a n i m a l s , ( i i ) d u r a t i o n o f e x p e r i m e n t a l p e r i o d , ( i i i ) t h e a n a l y t i c a l ( w h o l e body c o u n t i n g v s . p l a s m a c o u n t i n g ) a n d ( i v ) c u r v e p e e l i n g methods ( m a n u a l v s . c o m p u t e r ) u s e d i n f i t t i n g e q u a t i o n s t o t h e o b s e r v e d s p e c i f i c a c t i v i t y - t i m e c u r v e s . 75 I n g e n e r a l t h e d a i l y e x c r e t i o n o f Se i n f e c e s t e n d e d t o be s l i g h t l y h i g h e r t h a n t h e u r i n a r y l o s s e s b o t h i n t h e Se p o s i t i v e a n d d e f i c i e n t ewes ( F i g . 7). T h i s i s c o n t r a r y t o t h e f i n d i n g o f L o p e z e t a l . (1969) who 7 5 r e p o r t e d a h i g h e r e x c r e t i o n o f Se i n t h e u r i n e t h a n i n t h e f e c e s . R e c e n t l y L a n g l a n d s e t a l . (1986) h a v e c o n c l u d e d t h a t t h e f e c a l e x c r e t i o n o f Se w o u l d v a r y d e p e n d i n g on t h e o r g a n i c m a t t e r i n t a k e o f t h e ewes. I t i s n o t c l e a r w h e t h e r t h e d i f f e r e n c e s o b s e r v e d a r e r e a l o r due t o p o s s i b l e e x p e r i m e n t a l e r r o r s . The Se i n t a k e was s i g n i f i c a n t l y l o w e r i n t h e Se 82 d e f i c i e n t ewes t h a n i n t h e p o s i t i v e o nes ( T a b l e 3 ) . The r e l a t i v e c o n t r i b u t i o n o f h a y and w a t e r t o t h e t o t a l Se i n t a k e o f ewes was d i f f e r e n t b e t w e e n t h e g r o u p s . I n Se p o s i t i v e ewes h a y p r o v i d e d a m a j o r (70%) p o r t i o n o f t h e Se i n t a k e , w h e r e a s , w a t e r was t h e p r i m a r y s o u r c e (90%) o f Se i n t h e d e f i c i e n t o n e s . The e x c r e t i o n o f s t a b l e Se i n t h e f e c e s o f Se d e f i c i e n t ewes (25 + 6 ug Se/d) was s i g n i f i c a n t l y (P <^  0 . 0 1 ) l o w e r t h a n t h a t o b s e r v e d i n t h e Se p o s i t i v e ewes (272 + 48 ug S e / d ) ( T a b l e 3 ) . When e x p r e s s e d a s a p e r c e n t o f Se i n t a k e t h e r e a p p e a r e d t o be no d i f f e r e n c e i n t h e amount e x c r e t e d t h r o u g h t h e f e c e s w h i c h was 59 and 57% i n t h e Se p o s i t i v e and d e f i c i e n t ewes r e s p e c t i v e l y . When, h o w e v e r , t h e c o r r e c t i o n s were made f o r t h e e n d o g e n o u s l o s s t h e f e c a l Se l o s s e s r e p r e s e n t e d 49 and 2% o f t h e i n t a k e s i n t h e Se p o s i t i v e and d e f i c i e n t ewes r e s p e c t i v e l y . B a s e d on t h e s e r e s u l t s i t may be c o n c l u d e d t h a t t h e f e c a l e x c r e t o r y r o u t e d o e s e x e r t a h o m e o s t a t i c c o n t r o l on Se m e t a b o l i s m i n t h e body . From t h e Se b a l a n c e d a t a i t c a n be s e e n t h a t t h e a p p a r e n t a b s o r p t i o n o f Se r a n g e d f r o m 40-41% o f Se i n t a k e i n n o n p r e g n a n t ewes. However t h e s e v a l u e s a r e much l o w e r t h a n t h o s e r e p o r t e d i n r a t s (80% : C a r y e t a l . 1973) and humans (72% : G r e g e r and M a r c u s , 1 9 8 1 ) . T h i s may be e x p l a i n e d by t h e f a c t t h a t o r g a n i c Se f r o m t h e f e e d i s c o n v e r t e d t o i n s o l u b l e Se compounds ( P e t e r s o n and B u t l e r , 1 9 6 2 ; P e t e r s o n and S p e d d i n g , 1963) i n t h e rumen a nd h e n c e 8 3 t h e a b s o r p t i o n i s l i k e l y t o be l o w e r i n r u m i n a n t s . V e r y r e c e n t l y L a n g l a n d s e t a l . (1986) h a v e r e p o r t e d a n e n d o g e n o u s f e c a l l o s s o f a p p r o x i m a t e l y 14 ug S e / d (29% o f Se i n t a k e ) i n w e t h e r s c o n s u m i n g 48 ug S e / d b a s e d on a 1 7 - 4 5 d s t u d y . T h i s i s l o w e r t h a n t h e e n d o g e n o u s f e c a l l o s s o f 24 u g S e / d o b s e r v e d i n Se d e f i c i e n t ewes c o n s u m i n g 44 ug S e / d . T h i s v a r i a t i o n c o u l d be e i t h e r due t o t h e d i f f e r e n c e s i n f e c a l Se l o s s e s a n d Se r e t e n t i o n o r c o u l d be due t o t h e d i f f e r e n c e s i n t h e d u r a t i o n o f t h e s t u d y . The o r i g i n o f t h e e n d o g e n o u s f e c a l Se l o s s e s c o u l d be e i t h e r f r o m l i v e r o r f r o m p a n c r e a s . U n t i l now i t was t h o u g h t t h a t l i v e r c o n t r i b u t e d v e r y l i t t l e t o t h e e n d o g e n o u s Se l o s s e s i n r u m i n a n t s (Symonds e t a l . 1981) a n d i n n o n r u m i n a n t s ( L e v a n d e r a n d Baumann, 1 9 6 6 ) . However L a n g l a n d s e t a l . (1986) h a v e r e p o r t e d t h a t l i v e r i s t h e m a j o r s o u r c e o f t h e e n d o g e n o u s s e c r e t i o n o f Se i n t h e d i g e s t i v e t r a c t o f r u m i n a n t s a n d t h e c o n t r i b u t i o n o f p a n c r e a s i s v e r y m i n i m a l . The l a c k o f d i f f e r e n c e b e t w e e n t h e a p p a r e n t a n d n e t Se a b s o r p t i o n (on a p e r c e n t Se i n t a k e b a s i s ) i n Se p o s i t i v e ewes ( T a b l e 3) c o u l d be due t o t h e r e l a t i v e l y h i g h Se i n t a k e c o u p l e d w i t h a l o w e r p e r c e n t o f i n t a k e (10%) b e i n g l o s t a s e n d o g e n o u s f e c a l l o s s i n t h e s e a n i m a l s . T h i s may be s u p p o r t e d b y t h e d a t a f r o m t h e human Se s t u d i e s o f J a n g h o r b a n i e t a l . (1982) a n d Thomson e t a l . (1978) w h i c h i n d i c a t e no d i f f e r e n c e b e t w e e n t h e a p p a r e n t a n d n e t Se 84 a b s o r p t i o n s i n human s u b j e c t s f e d more t h a n 1 mg S e / d . On t h e o t h e r h a n d i n Se d e f i c i e n t ewes t h e n e t Se a b s o r p t i o n was two f o l d h i g h e r t h a n t h e a p p a r e n t Se a b s o r p t i o n a n d t h i s c o u l d be a s a r e s u l t o f r e d u c e d d i e t a r y Se i n t a k e a s s o c i a t e d w i t h a h i g h p e r c e n t o f Se i n t a k e b e i n g l o s t e n d o g e n o u s l y (58%). T h e r e a r e no a v a i l a b l e d a t a i n t h e l i t e r a t u r e on n e t Se a b s o r p t i o n i n d o m e s t i c a n i m a l s t o d i s c u s s t h e r e s u l t s f r o m t h e p r e s e n t s t u d y . However t h e n e t Se a b s o r p t i o n e x p r e s s e d as p e r c e n t o f Se i n t a k e was 75 r e p o r t e d t o r a n g e f r o m 90-97% i n t r a c e r ( Se) a n d d o s i n g ( s e l e n i t e / s e l e n o m e t h i o n i n e ) s t u d i e s i n humans (Thomson e t a l . 1 9 7 8 ; G r i f f i t h s e t a l . 1 9 7 6 ) . R e s u l t s f r o m t h e c u r r e n t s t u d y on Se d e f i c i e n t ewes (97%) a r e i n a g r e e m e n t w i t h t h o s e o f a b o v e w o r k e r s a n d a t v a r i a n c e w i t h t h o s e o f J a n g h o r b a n i e t a l . (1982) who f o u n d a n e t a b s o r p t i o n o f 6 8 -74 76% i n man u s i n g Se a s t h e t r a c e r . The d i s c r e p a n c y b e t w e e n t h e p r e s e n t s t u d y a n d t h a t o f J a n g h o r b a n i e t a l . (1982) may be due t o t h e d i f f e r e n c e s i n t h e t r a c e r s e m p l o y e d a n d t h a t 74 t h e s t a b l e Se i s o t o p e ( Se) u s e d was c o n s t i t u t i n g more t h a n 50% o f t h e t o t a l d o s e a d m i n i s t e r e d i n t h e s t u d y o f l a t t e r w o r k e r s . The h e t e r o g e n i t y o f t h e s o u r c e s f r o m w h i c h t h e e x p e r i m e n t a l a n i m a l s were p u r c h a s e d a n d t h e i r unknown Se s t a t u s m i g h t h a v e i n t r o d u c e d c o n s i d e r a b l e e r r o r s i n t h e p r e s e n t b a l a n c e s t u d y . I n a d d i t i o n , i t i s a l s o l i k e l y t h a t o v e r e s t i m a t i o n o f Se i n t a k e , i n c o m p l e t e f e c a l c o l l e c t i o n , n e g l e c t o f o t h e r b o d y l o s s e s t h r o u g h w o o l a n d e x p i r e d g a s e s may h a v e a d d e d t o t h e d i f f e r e n c e s . 85 The Se c o n c e n t r a t i o n s i n most of the t i s s u e s of Se p o s i t i v e ewes were s i g n i f i c a n t l y higher than i n the d e f i c i e n t ones (Table 4). Because of the f a c t t h a t the Se in t a k e was s i g n i f i c a n t l y higher i n the Se p o s i t i v e ewes than i n the d e f i c i e n t ones i t can be suggested that the Se in t a k e had a d i r e c t p o s i t i v e e f f e c t on the t i s s u e Se c o n c e n t r a t i o n of the former ewes. Although the t i s s u e s of the Se d e f i c i e n t ewes responded to the r e d u c t i o n i n the d i e t a r y Se supply by de c r e a s i n g t h e i r Se c o n c e n t r a t i o n s , no c l i n i c a l or p a t h o l o g i c a l changes a s s o c i a t e d with Se d e f i c i e n c y were observed e i t h e r i n the animals or i n the t i s s u e s . T h i s could be due to the short d u r a t i o n of the n u t r i t i o n a l regime imposed i n the present study. The t i s s u e Se data of the ewes a l s o i n d i c a t e d t h a t the pool s i z e of Se i n v a r i o u s t i s s u e s was d i r e c t l y i n f l u e n c e d by the Se intake (Table 4). The s i g n i f i c a n t l y h igher t i s s u e Se pool s i z e s observed i n the Se p o s i t i v e ewes demonstrated the r f a c t t h a t at higher Se i n t a k e s there was indeed a gre a t e r i n c o r p o r a t i o n of Se i n t o the t i s s u e metabolic pools c o n t r a r y to the o b s e r v a t i o n s i n man (Janghorbani et a l . 1982; Thomson et al.1978). The d e p l e t i o n of Se observed i n the t i s s u e s of Se d e f i c i e n t ewes i n d i c a t e d that Se was m o b i l i z e d from the t i s s u e depots to meet requirements f o r the mineral (Table 5). Though the c o n c e n t r a t i o n of Se i n s k e l e t a l muscle was lower 86 t h a n i n o t h e r t i s s u e s , t h e l a r g e s k e l e t a l m u s c l e mass i n t h e b o d y ( a p p r o x i m a t e l y 40% o f t h e BW - A r n a l , 1977) w o u l d r e n d e r t h e p o o l s i z e i n t h i s t i s s u e v u l n e r a b l e t o c h a n g e s i n Se i n t a k e ( F i g 8 ; r = 0 . 8 9 ) . I t i s t h e r e f o r e l o g i c a l t o e x p e c t t h a t Se f r o m t h i s p o o l w o u l d be d e p l e t e d t o a g r e a t e r e x t e n t t o meet t h e demands o f t h e b o d y u n d e r c o n d i t i o n s o f Se d e f i c i e n c y w h i c h i n f a c t was o b s e r v e d t o be t h e c a s e ( T a b l e 5 ) . The t i s s u e Se m e t a b o l i c p a r a m e t e r s a l s o i n d i c a t e d t h a t t h e u t i l i z a t i o n o f Se a p p e a r e d t o be g e n e r a l l y h i g h e r i n t h e t i s s u e s o f Se d e f i c i e n t ewes t h a n i n t h e p o s i t i v e o n e s ( T a b l e 5 ) . The t i s s u e s o f t h e d e f i c i e n t ewes t e n d e d t o h a v e l o w e r ^1/2 v a l u e s t h a n t h o s e i n t h e p o s i t i v e o n e s s u g g e s t i n g a l o w e r e d Se r e t e n t i o n . T h e s e c a l c u l a t e d T ^ 2 v a l u e s w o u l d r e f l e c t b o t h t h e e n t r y o f Se f r o m p l a s m a i n t o t h e t i s s u e s a n d i t s d i s a p p e a r a n c e f r o m t h e t i s s u e s . H o w e v e r , t h e e x p e r i m e n t a l p r o t o c o l e m p l o y e d i n t h e c u r r e n t s t u d y w o u l d n o t p e r m i t t h e r e s o l u t i o n o f t i s s u e k i n e t i c s a c c u r a t e l y . I n a d d i t i o n t o t h i s t h e f a c t t h a t t h e t i s s u e Se p o o l s i z e s i n t h e d e f i c i e n t ewes were s i g n i f i c a n l y l o w e r ( T a b l e 4) t h a n i n t h e p o s i t i v e o n e s w o u l d c a u s e t h e t i s s u e s t o e x h a u s t t h e i r Se d e p o t s a t a f a s t e r r a t e a n d t h i s w o u l d a c c o u n t f o r t h e l o w e r e d v a l u e s o b s e r v e d u n d e r d e f i c i e n c y c o n d i t i o n s . 87 CHAPTER I I I ; SELENIUM METABOLISM IN PREGNANT EWES. EXPERIMENT IIA: K i n e t i c s of selenium metabolism i n pregnant  ewes fed Se p o s i t i v e or d e f i c i e n t r a t i o n s . INTRODUCTION Studies of Se metabolism i n sheep have concentrated mainly on t i s s u e d i s t r i b u t i o n and whole body e x c r e t i o n and r e t e n t i o n (Wright, 1965 ; Muth et a l . 1 9 6 7 ; Wright and B e l l , 1 9 6 4 ; Lopez et a l . 1 9 6 9 ; H i d i r o g l o u et a l . 1 9 6 9 ) . Only two of these s t u d i e s were conducted i n pregnant ewes (Wright and B e l l , 1964; H i d i r o g l o u et a l . 1 9 6 9 ) . Though these s t u d i e s were h e l p f u l i n e l u c i d a t i n g the r o l e of v a r i o u s t i s s u e s e i t h e r i n Se storage or metabolism, they were not aimed at determining ( i ) the k i n e t i c s of Se metabolism a n d , ( i i ) the f a t e of Se i n the pregnant animal as a whole with due emphasis on a b s o r p t i o n and t i s s u e metabolism. For t h i s reason the present i n v e s t i g a t i o n was undertaken to study the k i n e t i c s of whole body Se metabolism i n pregnant ewes fed Se p o s i t i v e or d e f i c i e n t r a t i o n s . MATERIALS AND METHODS (a)Animals and management: Two groups of Dorset X S u f f o l k c r o s s b r e d ewes weighing 88 6 0 - 8 0 kg were used i n t h i s study. The Se p o s i t i v e ewes (group I, n=4) were fed a r a t i o n c o n s i s t i n g of a l f a l f a hay cubes and b a r l e y to provide n u t r i e n t s as per NRC recommendations ( 1 9 7 8 ) . The Se d e f i c i e n t ewes (group I I , n=3) were fed hay and b a r l e y d e f i c i e n t i n Se. The Se d e f i c i e n t hay used and d e t a i l s of i t s procurement were the same as d e s c r i b e d i n Expt. I (p 5 0 ) . The Se d e f i c i e n t b a r l e y was purchased l o c a l l y . The f e e d i n g of Se d e f i c i e n t r a t i o n commenced at the time of breeding and was continued through g e s t a t i o n f o r approximately 4 months u n t i l the experiments were completed. The d u r a t i o n of the t r a c e r and balance s t u d i e s was 5 d. Group I and group II ewes were e a t i n g on an average 1 . 2 5 kg of hay cubes (16.1% CP) and 0 . 9 6 kg of Se d e f i c i e n t hay (9.7% CP) r e s p e c t i v e l y per day which contained s i m i l a r gross energy c o n c e n t r a t i o n of 1 7 . 6 KJ/g. Ewes i n both groups had f r e e access to water ( c o n t a i n i n g 30 ng Se/ml) and c o b a l t - i o d i z e d s a l t b l o c k s at a l l times. The estrous c y c l e s i n a l l the ewes were synchronized by p l a c i n g progestagen impregnated sponges i n the v a g i n a . The sponges were removed manually from the vagina f o u r t e e n days a f t e r t h e i r placement. The ewes were bred n a t u r a l l y and the breeding dates were recorded. Pregnancy was confirmed by l i s t e n i n g to the f e t a l heart beat with a Doppler u l t r a s o u n d machine. Approximately four to s i x weeks p r i o r to the experiment the ewes were shorn and moved to separate metabolic cages f o r i n d i v i d u a l f e e d i n g . I n t r a u t e r i n e surgery was performed on these 89 ewes t o c a t h e t e r i z e t h e f e t a l b l o o d v e s s e l s a n d t h e d e t a i l s o f t h e s u r g e r y a r e g i v e n i n E x p t . I I B (p 126). D e t a i l s o f t h e p h y s i o l o g i c a l s t a t u s o f t h e s e a n i m a l s a l o n g w i t h t h e i r f e t u s e s a r e g i v e n i n E x p t . I I C . ( b ) E x p e r i m e n t a l P r o c e d u r e : A f i v e d a y p o s t s u r g i c a l r e c o v e r y p e r i o d was g i v e n b e f o r e t h e s t a r t o f t h e t r a c e r e x p e r i m e n t s . The p h y s i o l o g i c a l n o r m a l c y o f t h e ewe was v e r i f i e d b y r e c o r d i n g ( d e t a i l s i n A n a l y t i c a l methods s e c t i o n ) b l o o d g a s t e n s i o n s ( P C C ^ a n d P C ^ ) , b l o o d p H , h e m a t o c r i t , h e m o g l o b i n a n d b o d y t e m p e r a t u r e b e f o r e e a c h e x p e r i m e n t a s d e s c r i b e d . J u g u l a r v e i n c a t h e t e r s were i n t r o d u c e d on b o t h s i d e s o f t h e n e c k one d a y p r i o r t o t h e e x p e r i m e n t s t o f a c i l i t a t e t r a c e r i n j e c t i o n a n d w i t h d r a w a l o f b l o o d . A b o l u s d o s e o f a p p r o x i m a t e l y 300 u C i ( s p e c i f i c a c t i v i t y : 187-919 75 m C i / m g Se) o f S e - s o d i u m s e l e n i t e ( I C N C h e m i c a l s , I r v i n e , C a l i f o r n i a ) was i n j e c t e d i n t o j u g u l a r v e i n o f t h e Se p o s i t i v e ewes ( g r o u p I ewes : 9 7 7 , 982, 818 a n d 840) a n d Se d e f i c i e n t ewes ( g r o u p I I ewes: 58, 707Y a n d 724) r e s p e c t i v e l y . B l o o d s a m p l e s were c o l l e c t e d 2 , 5, 10, 1 5 , 3 0 , 4 5 , 6 0 , 1 2 0 , 1 8 0 , 2 4 0 , 300 a n d 360 m i n p o s t i n j e c t i o n a n d o n c e d a i l y f o r 3 d t h e r e a f t e r . The t o t a l b l o o d c o l l e c t e d f r o m e a c h ewe on t h e d a y o f t r a c e r e x p e r i m e n t d i d n o t e x c e e d 36 m l . S i m u l t a n e o u s b l o o d s a m p l e s f r o m t h e c o r r e s p o n d i n g f e t u s were a l s o c o l l e c t e d t o 90 d e t e r m i n e t h e i n u t e r o p l a c e n t a l t r a n s f e r r a t e o f Se f r o m ewe t o f e t u s ( E x p t . I I C ) . A l l b l o o d s a m p l e s were p l a c e d i n c h i l l e d p y r e x t u b e s c o n t a i n i n g h e p a r i n a n d c e n t r i f u g e d i m m e d i a t e l y . The p l a s m a was k e p t a t - 2 0 ° C u n t i l Se a n a l y s e s were c a r r i e d o u t . A f i v e d a y Se b a l a n c e t r i a l commenc ing on t h e d a y o f t r a c e r i n j e c t i o n was c o n d u c t e d on t h e e w e s . F e c a l p e l l e t s were c o l l e c t e d s e p a r a t e l y i n a meshed t r a y w h i l e t h e u r i n e was c o l l e c t e d i n t o a p a i l t h r o u g h a f u n n e l p l a c e d b e n e a t h t h e m e t a b o l i c c a g e s . T h i s p r e v e n t e d t h e Se c o n t a m i n a t i o n b e t w e e n t h e f e c e s a n d u r i n e . F e e d a n d w a t e r i n t a k e s a l o n g w i t h t h e u r i n a r y a n d f e c a l o u t p u t s were r e c o r d e d e v e r y d a y . A l i q u o t s o f u r i n e , a s a n d when v o i d e d , were c o l l e c t e d on t h e d a y o f t r a c e r e x p e r i m e n t a n d t h e v o l u m e s were n o t e d . D a i l y u r i n a r y a n d f e c a l s u b s a m p l e s were a l s o c o l l e c t e d . Se p o s i t i v e ewes , n o s . 9 8 2 , 8 4 0 , 977 a n d 818 were s a c r i f i c e d on d a y s 6 , 1 4 , 18 a n d 41 r e s p e c t i v e l y a f t e r t r a c e r i n j e c t i o n b y a d m i n i s t e r i n g 20 ml o f T - 6 1 ( H o e c h s t P h a r m a c e u t i c a l s L t d . ) , a n e u t h a n a s i a s o l u t i o n , a n d t h e f e t u s e s were d e l i v e r e d b y l a p a r o t o m y . Se d e f i c i e n t e w e s , n o s . 58 , 707Y a n d 725 were s a c r i f i c e d on d a y s 4 , 6 , a n d 12 r e s p e c t i v e l y . The ewes were d i s s e c t e d a n d l i v e r , k i d n e y , h e a r t , s p l e e n , l u n g a n d s k e l e t a l m u s c l e ( g a s t r o c n e m i u s ) were c o l l e c t e d , t o w e l d r i e d a n d w e i g h e d . S u b s a m p l e s o f a b o u t 5-10 g were o b t a i n e d f r o m e a c h o f t h e 7 5 t i s s u e s f o r Se r a d i o a c t i v i t y m e a s u r e m e n t s a n d r e s t w e r e 91 s t o r e d i n d o u b l e s e a l e d p l a s t i c c o n t a i n e r s a t - 2 0 C u n t i l t h e Se a n a l y s e s were p e r f o r m e d . ( d ) A n a l y t i c a l M e t h o d s : H e m o g l o b i n was d e t e r m i n e d a c c o r d i n g t o t h e c y a n o m e t h e m o g l o b i n m e t h o d ( H y c e l I n c . , H o u s t o n ) . H e m a t o c r i t was d e t e r m i n e d by t h e m i c r o h e m a t o c r i t p r o c e d u r e . W h o l e b l o o d ( v e n o u s ) g a s t e n s i o n s (PCC<2 a n d PC^) a n d pH w e r e d e t e r m i n e d a n e r o b i c a l l y u s i n g a R a d i o m e t e r d i g i t a l a c i d -b a s e a n a l y z e r ( R a d i o m e t e r , m o d e l pHM 7 2 , C o p e n h a g e n ) e q u i p p e d w i t h i n d i v i d u a l m o d u l e s a n d e l e c t r o d e s f o r r e c o r d i n g b l o o d P C C ^ , P O j a n d pH r e s p e c t i v e l y . The P C O ^ , PC>2 a n d pH e l e c t r o d e s were t h e r m o s t a t i c a l l y m a i n t a i n e d a t a t e m p e r a t u r e o f 3 7 . 5 ° C . The b l o o d g a s e l e c t r o d e s w e r e c a l i b r a t e d b y b u b b l i n g a g a s m i x t u r e ( 1 2 - 1 5 b u b b l e s / m i n ) w i t h known p a r t i a l p r e s s u r e s o f C O j a n d C ^ , c o r r e c t e d f o r d a i l y b a r o m e t r i c p r e s s u r e t h r o u g h t h e e l e c t r o d e s . T h e pH e l e c t r o d e was c a l i b r a t e d u s i n g l o w a n d h i g h pH s t a n d a r d s ( R a d i o m e t e r , C o p e n h a g e n ) . The P C C ^ , PC»2 a n d pH v a l u e s w e r e r e c o r d e d a s d i s p l a y e d o n t h e m a c h i n e f o l l o w i n g i n j e c t i o n o f v e n o u s w h o l e b l o o d s a m p l e s o n c e t h e r e s p e c t i v e e l e c t r o d e s w e r e c o m p l e t e l y c a l i b r a t e d . S t a b l e a n d r a d i o a c t i v e Se a n a l y t i c a l m e t h o d s a n d t h e d a t a a n a l y s i s were t h e same as d e s c r i b e d u n d e r E x p t . I . (p 52) . 92 S t a t i s t i c a l a n a l y s i s : A l l r e s u l t s were expressed as means + SEM. The plasma and t i s s u e Se c o n c e n t r a t i o n data were analysed by a two way a n a l y s i s of va r i a n c e u s i n g SAS ( 1 9 8 5 ) . The r a t i o n and the sheep were assumed to be the main e f f e c t s . The remaining data were subjected to one way a n a l y s i s of v a r i a n c e (SAS, 1 9 8 5 ) . The s t a t i s t i c a l s i g n i f i c a n c e of d i f f e r e n c e s between treatment means was determined by Students 't' t e s t (SAS, 1 9 8 5 ) . A l e a s t squares method was employed f o r the c a l c u l a t i o n of the r e g r e s s i o n equations and the c o r r e l a t i o n c o e f f i c i e n t s when comparing the r e l a t i o n s h i p between parameters. 93 RESULTS The Se c o n c e n t r a t i o n s i n a l f a l f a c u b e s and b a r l e y f e d t o g r o u p I ewes were 0 . 2 3 mg/kg a nd 0 . 2 2 mg/kg r e s p e c t i v e l y . Se d e f i c i e n t h a y and b a r l e y h a d Se c o n c e n t r a t i o n s o f 0 . 0 1 mg/kg and 0 .04 mg/kg r e s p e c t i v e l y . The mean p h y s i o l o g i c a l p a r a m e t e r v a l u e s o f t h e p r e g n a n t ewes f e d Se p o s i t i v e o r d e f i c i e n t r a t i o n s a r e p r e s e n t e d i n T a b l e 6 . These v a l u e s were r e c o r d e d d u r i n g t h e e x p e r i m e n t a l p e r i o d w h i c h commenced 5 d a y s a f t e r s u r g e r y . The p l a s m a Se c o n c e n t r a t i o n i n t h e Se p o s i t i v e ewes (187 + 20 ng/ml) was s i g n i f i c a n t l y (P <^  0 . 0 0 0 1 ) h i g h e r t h a n i n t h e d e f i c i e n t o nes (69 + 14 n g / m l ) . A p o s i t i v e c o r r e l a t i o n b e t w e e n t h e Se i n t a k e and t h e p l a s m a Se c o n c e n t r a t i o n was o b s e r v e d ( F i g . 9 , r = 0 . 5 3 ) . 75 F o l l o w i n g t h e s i n g l e i n j e c t i o n o f S e - s o d i u m s e l e n i t e 75 i n t o t h e Se p o s i t i v e p r e g n a n t ewes t h e p l a s m a Se r a d i o a c t i v i t y showed a c h a r a c t e r i s t i c p a t t e r n o f c l e a r a n c e . D u r i n g t h e i n i t i a l h a l f an h o u r a f t e r t h e t r a c e r 75 i n j e c t i o n t h e p l a s m a Se r a d i o a c t i v i t y d e c l i n e d e x p o n e n t i a l l y f o l l o w e d by an i n c r e a s e d u r i n g t h e n e x t 3-4 h. 7 5 The h i g h e s t p l a s m a Se a c t i v i t y was o b s e r v e d i n t h e i n i t i a l 75 2 m i n s a m p l e , w h e r e a s t h e p l a s m a Se t h a t r e e n t e r e d s y s t e m i c c i r c u l a t i o n was maximum a r o u n d 4 h ( F i g . 1 0 ) . The peak 7 ^ S e r a d i o a c t i v i t y o b s e r v e d a t 3-4 h was T a b l e 6 . P h y s i o l o g i c a l p a r a m e t e r s o f Se p o s i t i v e a n d d e f i c i e n t p r e g n a n t e w e s . Se p o s i t i v e ( n - 9 ) Se d e f i c i e n t ( n - 6 ) P a r a m e t e r Mean + S . E . M Mean + S . E . M B l o o d pH - 7 . 5 0 + 0 .01 7 . 4 4 + 0 .04 B l o o d P C 0 2 (mm Hg) 2 8 . 3 + 1.2 2 6 . 6 + 0 . 8 B l o o d PC»2 (mm Hg) 2 5 . 4 + 1 .9 2 5 . 1 + 1 .3 H e m o g l o b i n (g %) 1 3 . 0 + 0 . 7 1 3 . 0 + 0 . 4 H e m a t o c r i t (%) 3 3 . 0 + 1.2 3 4 . 9 + 1 .1 B l o o d s a m p l e s were o b t a i n e d f r o m t h e j u g u l a r v e i n . 95 F i g . 9 . E f f e c t o f Se i n t a k e on p l a s m a Se c o n c e n t r a t i o n i n p r e g n a n t ewes f e d Se p o s i t i v e ( A ) o r d e f i c i e n t ( A ) r a t i o n s . The r e g r e s s i o n e q u a t i o n i s Y - 7 4 . 4 8 + 0 . 2 2 X r - 0 . 5 3 . F i g . 1 0 . C h a n g e s d u r i n g f i r s t 24 p o s i t i v e p r e g n a n t 8 1 8 , 8 4 0 , 977 a n d i n p l a s m a Se h r a f t e r t r a c e r e w e s . • , o» • / 982 r e s p e c t i v e l y . s p e c i f i c a c t i v i t y i n j e c t i o n i n t o Se • r e f e r t o ewe n o s . 97 O . O l - i 0.0001-0 10 20 Time (Hours) F i g . 1 1 . C h a n g e s i n p l a s m a Se s p e c i f i c a c t i v i t y d u r i n g f i r s t 24 h r a f t e r t r a c e r i n j e c t i o n i n t o Se d e f i c i e n t p r e g n a n t e w e s . • , o , o r e f e r t o ewe n o s . 5 8 , 707Y a n d 725 r e s p e c t i v e l y . 98 a p p r o x i m a t e l y 61% o f t h e a c t i v i t y p r e s e n t a t 2 m i n p o s t i n j e c t i o n . A s i m i l a r phenomenon was a l s o o b s e r v e d i n t h e Se d e f i c i e n t ewes i n w h i c h t h e peak o f r e a p p e a r a n c e l a s t e d a p p r o x i m a t e l y f o r a b o u t 3-4 h and a c c o u n t e d f o r 55% o f t h e r a d i o a c t i v i t y p r e s e n t i n t h e i n i t i a l 2 m i n s a m p l e 75 ( F i g . 1 1 ) . The p l a s m a Se r a d i o a c t i v i t y d e c l i n e d t h e r e a f t e r g r a d u a l l y o v e r s e v e r a l d a y s i n b o t h g r o u p s . 75 The b e s t f i t p l a s m a Se s p e c i f i c a c t i v i t y - t i m e c u r v e s i n t h e Se p o s i t i v e a n d d e f i c i e n t ewes a r e g i v e n i n F i g . 12 and 13 r e s p e c t i v e l y . The e x p o n e n t i a l e q u a t i o n s d e s c r i b i n g t h e s e c u r v e s a r e g i v e n i n T a b l e 7 . A sum o f two e x p o n e n t i a l s was r e q u i r e d t o o b t a i n t h e b e s t f i t e q u a t i o n s i n t h e ewes o f b o t h g r o u p s . T h i s i n d i c a t e d t h a t t h e c l e a r a n c e o f Se f r o m t h e p l a s m a was b i p h a s i c i n a l l t h e ewes and t h a t a p p r o x i m a t e l y e q u a l amounts o f Se were l o s t i n e a c h o f t h e s e p h a s e s . The T^^ 2 °f S e w a s s i m i l a r (4 d j i n b o t h g r o u p s ( T a b l e 8 ) . 75 The a r e a s u n d e r t h e p l a s m a Se s p e c i f i c a c t i v i t y - t i m e c u r v e s (AUC) and t h e k i n e t i c p a r a m e t e r s o f Se m e t a b o l i s m i n t h e ewes a r e p r e s e n t e d i n T a b l e 8 . The AUC i n t h e Se d e f i c i e n t ewes was s i g n i f i c a n t l y h i g h e r (P < 0 . 0 0 1 ) t h a n i n t h e Se p o s i t i v e o nes i n d i c a t i n g a g r e a t e r p l a s m a r e t e n t i o n 7 5 o f Se. T h i s c o u l d be due t o t h e s m a l l e r p o o l s i z e o f t h e c o l d Se i n t h e p l a s m a o f t h e d e f i c i e n t ewes. I n f a c t , t h e t o t a l p l a s m a Se p o o l o b s e r v e d i n t h e p r e s e n t s t u d y 99 F i g . 1 2 . B e s t - f i t p l a s m a 7 5 S e s p e c i f i c a c t i v i t y - t i m e c u r v e s i n Se p o s i t i v e p r e g n a n t e w e s . • , O/ • » • r e f e r t o ewe n o s . 8 1 8 , 8 4 0 , 977 a n d 982 r e s p e c t i v e l y . 100 O . O l - i GO fciC a 0) o cc o <: d CO CC 6 o.ooH 0.000H 0 20 40 60 Time (Hours) 75, F i g . 1 3 . B e s t - f i t p l a s m a '"Se s p e c i f i c c u r v e s i n Se d e f i c i e n t p r e g n a n t e w e s . • t o ewe n o s . 5 8 , 707Y a n d 725 r e s p e c t i v e l y . a c t i v i t y - t i m e o, • r e f e r T a b l e 7. B e s t - f i t e x p o n e n t i a l e q u a t i o n s o f p l a s m a Se s p e c i f i c a c t i v i t y - t i m e c u r v e s o f p r e g n a n t ewes . Ewe # A 2 a^ &2 Se p o s i t i v e ewes: 818 .000825 .000701 5 .9871 . 0 1 2 7 840 .000909 . 001107 9 . 0 3 5 5 . 0 1 4 9 977 .000505 . 000671 8 .2206 . 0 1 6 9 982 . 000595 .000519 1 4 . 8 4 8 2 . 0131 Se d e f i c i e n t ewes : 58 .002218 . 0 0 2 3 7 5 8 .4231 . 0133 707Y .002038 . 001586 10.7979 . 0 1 2 5 725 .001741 .001187 5 . 3 7 9 1 .0086 * T h e e q u a t i o n i s : A^. - A e " a fc  A l e 1 + A 2 e "V' 75 w h e r e A - p l a s m a Se s p e c i f i c a c t i v i t y ( f r a c t i o n o f d o s e / u g S e ) , a - r a t e c o n s t a n t , e - l o g 1 n a n d t « t i m e ( h r ) . T a b l e 8 . C o m p a r i s o n o f a r e a s u n d e r t h e c u r v e s (AUC) a n d k i n e t i c p a r a m e t e r s o f Se p o s i t i v e and d e f i c i e n t p r e g n a n t ewes . Se p o s i t i v e ewes (n=4) Se d e f i c i e n t ewes ( n - 3 ) P a r a m e t e r Mean + S . E . M Mean + S . E . M AUC ( f r a c t i o n o f d o s e / u g S e ) . h r 0 . 0 5 a + 0 . 0 1 0 . 1 5 b + 0 . 0 2 I r r e v e r s i b l e D i s p o s a l o f Se ( u g S e / d / k g B . W . ) 7 C + 1 2 d + 0 . 2 P l a s m a C l e a r a n c e R a t e ( m l / d / k g B . W . ) 39 + 2 26 + 3 P l a s m a Se P o o l S i z e ( u g Se) 7 2 5 c + 93 2 7 9 d + 36 P l a s m a Se C o n e , ( n g / m l ) 1 8 7 e + 20 6 9 f + 14 V o l u m e o f d i s t r i b u t i o n ( m l / k g B . W . ) 56 + 4 45 + 7 T l / 2 ( d ) 4 . 4 2 + 0 . 4 9 4 . 4 0 + 0 . 9 2 Means w i t h . d i f f e r e n t s u p e r s c r i p t s w i t h i n t h e row d i f f e r s i g n i f i c a n t l y ( P < 0 . 0 0 1 ; c a P < 0 . 0 1 ; e i P < 0 . 0 0 0 1 ) . 103 was s i g n i f i c a n t l y (P < 0 . 0 1 ) l o w e r i n t h e Se d e f i c i e n t ewes t h a n i n t h e p o s i t i v e o n e s . A s i g n i f i c a n t d i f f e r e n c e i n i r r e v e r s i b l e Se d i s p o s a l ( ID) was o b s e r v e d b e t w e e n t h e Se p o s i t i v e a n d d e f i c i e n t ewes w i t h t h e h i g h e r v a l u e b e i n g p r e s e n t i n t h e f o r m e r g r o u p . The p l a s m a Se c l e a r a n c e t e n d e d t o be s l i g h t l y h i g h e r i n t h e g r o u p I t h a n i n t h e g r o u p I I e w e s . H o w e v e r , t h i s d i f f e r e n c e was 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 (P > 0 . 1 0 ) . 75 The e x c r e t o r y p a t t e r n s o f Se i n t o f e c e s a n d u r i n e o f p r e g n a n t ewes f e d Se p o s i t i v e a n d d e f i c i e n t r a t i o n s a r e g i v e n i n F i g . 1 4 . When t h e o v e r a l l (5 d) Se e x c r e t i o n p a t t e r n s were e x a m i n e d i t was f o u n d t h a t t h e d a i l y u r i n a r y 75 Se l o s s t e n d e d t o be h i g h e r t h a n t h e f e c a l l o s s on a l l 5 d o f t h e t r i a l i n Se p o s i t i v e e w e s . On t h e o t h e r h a n d i n 75 Se d e f i c i e n t ewes t h e d a i l y f e c a l Se l o s s was g e n e r a l l y h i g h e r t h a n t h e u r i n a r y l o s s e x c e p t o n d a y 1 a t w h i c h t i m e t h e u r i n a r y Se e x c r e t i o n was s l i g h t l y g r e a t e r t h a n t h e f e c a l l o s s . I f t h e d a i l y Se e x c r e t i o n p a t t e r n s w e r e 75 e x a m i n e d i t was o b s e r v e d t h a t t h e Se l o s s e s i n t o f e c e s a n d u r i n e o f Se p o s i t i v e ewes were maximum on d a y 1 a s c o m p a r e d t o t h e o t h e r d a y s . On t h e o t h e r h a n d i n Se 75 d e f i c i e n t ewes t h e d a i l y u r i n a r y Se l o s s was maximum on 75 d a y 1 , w h i l e t h e f e c a l Se l o s s p e a k e d on d a y 2 o f t h e b a l a n c e t r i a l . The b a l a n c e d a t a s h o w i n g t h e d e t a i l s o f t h e Se 6 F i g . 1 4 . Mean d a i l y e x c r e t i o n o f 7 5 S e i n u r i n e a n d f e c e s o f p r e g n a n t ewes f e d Se p o s i t i v e o r d e f i c i e n t r a t i o n s .LZ3, E H r e f e r t o f e c e s a n d u r i n e o f Se p o s i t i v e ewes a n d E S , [ZD r e f e r t o f e c e s a n d u r i n e o f Se d e f i c i e n t ewes r e s p e c t i v e l y . 105 T a b l e 9 . A b s o r p t i o n a n d r e t e n t i o n o f Se i n p r e g n a n t ewes f e d Se p o s i t i v e and d e f i c i e n t r a t i o n s . Se p o s i t i v e ewes Se d e f i c i e n t ewes P a r a m e t e r Mean ( n -+ =8) S . E . M Mean (n= + =6) S . Se i n t a k e ( u g / d ) 4 3 0 a + 40 9 1 b + 11 F e c a l Se l o s s ( u g / d ) 2 4 1 a + 28 2 7 b + 2 E n d o g e n o u s f e c a l Se l o s s + (ug S e / d ) 5 2 c + 10 1 4 d + 4 U r i n a r y Se l o s s ( u g / d ) 4 5 e + 8 8 f + 2 A p p a r e n t Se a b s o r p t i o n ^ ( a ) e s t i m a t e d ( u g / d ) 1 8 9 e + 36 6 4 f + 11 (b)% o f Se i n t a k e 4 3 e + 5 6 7 f + 5 N e t Se a b s o r p t i o n 7 " ( a ) e s t i m a t e d ( u g / d ) 2 4 1 e + 36 7 8 f + 11 (b)% o f Se i n t a k e 56^ + 5 8 4 h + 3 A p p a r e n t Se b a l a n c e ( u g / d ) 145^ + 35 5 6 u + 10 a b , c d , e f , g h M e a n g w i t h i n t h e rows d i f f e r s i g n i f i c a n t l y ( a ' b P < 0 . 0 0 0 1 ; C ' d P < 0 . 0 5 ; e , f P < 0 . 0 0 1 ; g ' h P < 0 . 0 1 ) . it Se b a l a n c e f o r ewe #58 was f r o m a 4 d c o l l e c t i o n o n l y . """Endogenous f e c a l Se l o s s was c a l c u l a t e d f r o m d a t a o f t r a c e r i n j e c t e d (n=4 f o r Se p o s i t i v e a n d n=3 f o r Se d e f i c i e n t ) ewes The t o t a l number i n c l u d e s f e t a l t r a c e r i n j e c t e d ewes a l s o . $ ""Apparent Se a b s o r p t i o n = Se i n t a k e - F e c a l Se l o s s . # N e t a b s o r p t i o n = S e i n t a k e - ( F e c a l l o s s - E n d o g e n o u s Se l o s s ) " A p p a r e n t b a l a n c e = Se i n t a k e - F e c a l l o s s - U r i n a r y Se l o s s . 106 metabolism t r i a l are given i n Table 9 . The Se in t a k e was s i g n i f i c a n t l y (P _< 0 . 0 0 0 1 ) higher i n the Se p o s i t i v e ewes than i n the d e f i c i e n t ones. A l f a l f a hay, b a r l e y and water were found to provide 67%, 11% and 22% of Se in t a k e r e s p e c t i v e l y to the Se p o s i t i v e ewes. I n t e r e s t i n g l y , i n Se d e f i c i e n t ewes the primary source of Se intake was water (75%) whereas, b a r l e y and Se d e f i c i e n t hay accounted f o r 18 and 7% of the Se intake r e s p e c t i v e l y . A s i g n i f i c a n t l y h igher endogenous f e c a l Se l o s s (P < 0 . 0 5 ) and u r i n a r y Se l o s s (P < 0 . 0 0 1 ) were a l s o observed i n the Se p o s i t i v e ewes than i n the d e f i c i e n t ones. The apparent Se a b s o r p t i o n r a t e s were 189 and 64 ug/d i n the group I and group II ewes r e s p e c t i v e l y . When these were expressed as a percent of Se intake the values were 43 and 67% r e s p e c t i v e l y i n the ewes fed Se p o s i t i v e and d e f i c i e n t r a t i o n s . When the endogenous f e c a l Se l o s s e s were added to the apparent a b s o r p t i o n r a t e s the r e s u l t i n g net Se a b s o r p t i o n r a t e s were 241 and 78 ug/d r e s p e c t i v e l y i n the group I and group II ewes. A p o s i t i v e l i n e a r r e l a t i o n s h i p (r= 0 . 8 9 ) was observed between the Se intake and net Se a b s o r p t i o n (ug/d), whereas, a negative r e l a t i o n s h i p ( r = - 0 . 6 6 ) e x i s t e d between i n t a k e and net Se a b s o r p t i o n expressed as per cent of i n t a k e ( F i g . 1 5 ) . The apparent as w e l l as the net Se a b s o r p t i o n r a t e s were s i g n i f i c a n t l y higher (P _< 0 . 0 0 1 ) i n the Se p o s i t i v e ewes than i n the d e f i c i e n t ones. The apparent Se balance values were 145 and 56 ug/d r e s p e c t i v e l y i n the ewes fed Se p o s i t i v e and d e f i c i e n t 107 Se intake (pg/d) F i g . 1 5 . E f f e c t o f Se i n t a k e on n e t Se a b s o r p t i o n i n p r e g n a n t e w e s . The r e g r e s s i o n e q u a t i o n s a r e Y - 2 3 . 7 5 + 0 . 5 2 X ( r - 0 . 8 9 ) a n d Y = 8 5 . 9 1 - 0 . 0 6 X ( r — 0 . 6 6 ) f o r t h e n e t Se a b s o r p t i o n , e x p r e s s e d as p g / d a n d a s % Se i n t a k e r e s p e c t i v e l y . T a b l e 1 0 . S t a b l e Se c o n c e n t r a t i o n a n d ^ p o o l s i z e i n t h e t i s s u e s o f Se p o s i t i v e and d e f i c i e n t p r e g n a n t ewes . Se C o n c e n t r a t i o n P o o l s i z e ( u g / g ) ( u g S e ) T i s s u e Se p o s i t i v e Se d e f i c i e n t Se p o s i t i v e Se d e f i c i e n t L i v e r 0 . 3 7 a + 0 .02 0 . 1 1 b + 0 .02 3 4 7 a + 29 1 0 2 b + 11 K i d n e y 1 . 0 9 c + 0 .06 0 . 8 7 d + 0 .06 1 8 4 e + 13 1 3 7 f + 14 S k . M u s c l e 0 . 1 3 a + 0 .01 0 . 0 6 b + 0 . 0 1 3 6 6 4 9 + 332 1 7 0 9 h + 145 H e a r t 0 . 3 1 a + 0 .02 0 . 1 7 b + 0 .02 7 6 ° + 8 5 0 d + 4 L u n g 0 . 3 1 a + 0 .02 0 . 1 4 b + 0 .02 2 0 0 C + 24 1 0 3 d + 12 S p l e e n 0 . 5 7 a + 0 . 0 3 0 . 2 6 b + 0 .03 l l l e + 24 5 0 f + 9 a b Means w i t h . d i f f e r e n t s u p e r s c r i p t s w i t h i n t h e row d i f f e r s i g n i f i c a n t l y ( ' P < 0 . 0 0 0 1 ; C ' ° P < 0 . 0 1 ; 6 , t P < 0 . 0 5 ; g , n < 0 . 0 0 1 ) . * n - 6 ewes i n e a c h g r o u p . 5000 • • Se intake (ug/d) F i g . 1 6 . E f f e c t o f Se i n t a k e on t i s s u e Se p o o l s i z e i n p r e g n a n t ewes f e d Se p o s i t i v e o r d e f i c i e n t r a t i o n s . The r e g r e s s i o n e q u a t i o n s a r e Y - 7 5 . 4 7 + 0 . 5 3 X , r - 0 . 7 6 , l i v e r ( A ) ; Y - 1 4 7 7 . 3 5 + 4 . 5 5 X , r - 0 . 7 1 , s k e l e t a l m u s c l e ( • ) ; Y - 4 3 . 3 7 + 0 . 0 8 X , r - 0 . 7 1 , h e a r t (a ) a n d Y - 2 8 . 3 9 + 0 . 2 3 X , r - 0 . 7 6 , s p l e e n ( $ ) . T a b l e 1 1 . K i n e t i c p a r a m e t e r s o f t i s s u e Se m e t a b o l i s m i n Se p o s i t i v e a n d d e f i c i e n t p r e g n a n t ewes. F r a c t i o n a l r a t e c o n s t a n t ( F r a c t i o n / d ) T i s s u e Se p o s i t i v e (n=4) Se d e f i c i e n t ( n - 3 ) Se p o s i t i v e ( n - 4 ) Se d e f i c i e n t ( n - 3 ) L i v e r 0 . 0 4 3 9 0 .1245 16 6 K i d n e y 0 .0618 0 .1488 11 5 Sk. M u s c l e 0 .0122 0 .0320 57 22 H e a r t 0 .0273 0 .0760 25 9 Lung 0 .0534 0 .1143 13 6 S p l e e n 0 . 0 1 3 9 0 .1313 50 5 * o f Se u t i l i z a t i o n i n t h e i n d i v i d u a l t i s s u e s . I l l r a t i o n s and were s i g n i f i c a n t l y d i f f e r e n t ( P <^  0 . 0 5 ) . The t i s s u e Se c o n c e n t r a t i o n s and pool s i z e s of the Se p o s i t i v e and d e f i c i e n t ewes are presented i n Table 1 0 . The Se c o n c e n t r a t i o n s were s i g n i f i c a n t l y (P < 0 . 0 0 0 1 ) higher i n l i v e r , s k e l e t a l muscle, heart, lung and spleen of Se p o s i t i v e ewes than i n d e f i c i e n t ones. A s i g n i f i c a n t l y higher (P <^  0 . 0 1 ) t o t a l s t a b l e Se pool was observed i n the l i v e r of Se p o s i t i v e ewes than i n the d e f i c i e n t ones. S i m i l a r l y the s k e l e t a l muscle, heart and lung of group I ewes had s i g n i f i c a n t l y (P _< 0 . 0 5 ) higher s t a b l e Se pools than i n the t i s s u e s of the group II ewes. There was a p o s i t i v e l i n e a r r e l a t i o n s h i p between the Se int a k e and the t i s s u e Se pool s i z e s of l i v e r , s k e l e t a l muscle, heart and spleen ( F i g . 1 6 ) .The k i n e t i c parameters of t i s s u e Se metabolism are given i n Table 1 1 . The Se d e f i c i e n t ewes had g e n e r a l l y higher u t i l i z a t i o n r a t e constant values than the p o s i t i v e ones i n d i c a t i n g a higher turnover of Se. On the other hand the Se p o s i t i v e ewes tended to have s l i g h t l y higher values i n the t i s s u e s than the d e f i c i e n t ones. DISCUSSION The values of v a r i o u s p o s t - o p e r a t i v e p h y s i o l o g i c a l parameters observed i n the ewe were i n agreement with those reported by other workers (Anand et a l . 1 9 7 9 ; Hodgson 112 et a l . 1 9 8 0 ; K i t t s and Krishnamurti, 1 9 8 2 ; Schaefer and K r i s h n a m u r t i , 1984) f o r ewes be a r i n g f e t u s e s t h a t had c h r o n i c a l l y implanted v a s c u l a r c a t h e t e r s . T h i s coupled with the f a c t t h a t the ewes s t a r t e d to eat normal amounts of feed w i t h i n 2 d a f t e r the surgery i n d i c a t e d t h a t the ewe had recovered from the s t r e s s of surgery at the time of t r a c e r experiment. 75 The plasma Se s p e c i f i c a c t i v i t y d e c l i n e d i n i t i a l l y f o r about h a l f an hour i n the Se p o s i t i v e as w e l l as i n the Se d e f i c i e n t ewes. This was followed by an i n c r e a s e d reappearance which l a s t e d f o r about 6 hr before d e c l i n i n g g r a d u a l l y over s e v e r a l days. This type of c h a r a c t e r i s t i c 75 plasma Se clearance was a l s o observed i n the nonpregnant ewes fed Se p o s i t i v e and d e f i c i e n t r a t i o n s (Expt I ) . A s i m i l a r phenomenon was a l s o noted by other workers i n mice (Sandholm, 1 9 7 3 ) , i n d a i r y c a t t l e (Symonds et a l . 1981) and i n goats ( A l l e n and M i l l e r , 1 9 8 1 ) . Sandholm ( 1973) r e p o r t e d that i n mice w i t h i n 1 min a f t e r the t r a c e r 75 i n j e c t i o n 50% of i n t r a v e n o u s l y administered Se dose was taken up by the e r y t h r o c y t e s (RBC). Se was found to be r e l e a s e d from e r y t h r o c y t e s and taken up by the l i v e r w i t h i n 15 min p o s t t r a c e r i n j e c t i o n at which time the 75 l i v e r had the h i g h e s t Se a c t i v i t y . Sandholm (1973) a l s o 75 suggested that there was an e x p u l s i o n of Se t a k i n g p l a c e from the l i v e r i n t o the plasma around 1 hr a f t e r the t r a c e r 75 i n j e c t i o n at which time the plasma Se was found to be 113 g r a d u a l l y i n c r e a s i n g . I t was s p e c u l a t e d t h a t t h e t r a n s p o r t o f Se f r o m e r y t h r o c y t e s t o l i v e r c e l l s c o u l d be due t o an a l b u m i n - g l u t a t h i o n e - S e c o m p l e x ( S a n d h o l m , 1 9 7 3 ) . I n c o n t r a s t , Symonds e t a l . (1981) s u g g e s t e d t h a t t h e c l e a r a n c e o f Se f r o m t h e b l o o d p l a s m a was m a i n l y due t o t h e a c t i o n o f l i v e r i n c a t t l e . I t was r e p o r t e d 75 t h a t t h e Se r a d i o a c t i v i t y i n t h e p o r t a l v e n o u s p l a s m a was g r e a t e r t h a n i n t h e h e p a t i c v e n o u s p l a s m a d u r i n g t h e r a p i d d i s t r i b u t i o n p h a s e a n d t h a t t h e l i v e r was 75 a c c o u n t i n g f o r 40% o f t h e r e m o v a l o f i n j e c t e d S e 0 3 f r o m t h e p l a s m a . The s t u d y o f Symonds e t a l . (1981) a l s o 75 showed t h a t t h e Se r e - e n t e r e d t h e p l a s m a w i t h i n 1 h r a f t e r i n j e c t i o n b o u n d t o a p l a s m a component p r o b a b l y a r -75 g l o b u l i n . The peak r e a p p e a r a n c e o f Se i n t o t h e p l a s m a was f o u n d t o o c c u r a t 1 h r p o s t i n j e c t i o n w i t h t h e a c t i v i t y b e i n g 50% o f t h e r a d i o a c t i v i t y p r e s e n t i n t h e i n i t i a l 2 m i n s a m p l e 75 (Symonds e t a l . 1 9 8 1 ) . On t h e o t h e r h a n d t h e peak Se r e a p p e a r a n c e c o n c e n t r a t i o n s i n b l o o d a n d p l a s m a o f g o a t s were r e p o r t e d t o o c c u r a r o u n d 3-6 h r f o l l o w i n g t r a c e r i n j e c t i o n a n d t h e s e were e v e n h i g h e r t h a n t h o s e o b s e r v e d i n t h e i n i t i a l s a m p l e s t a k e n i m m e d i a t e l y a f t e r t h e t r a c e r i n j e c t i o n ( A l l e n a n d M i l l e r , 1 9 8 1 ) . O u r s t u d y i s more i n a g r e e m e n t w i t h t h a t o f Symonds e t a l . ( 1981) 7 5 w i t h r e g a r d t o t h e amount o f Se r a d i o a c t i v i t y t h a t r e a p p e a r e d i n t h e p l a s m a a n d w i t h t h a t o f A l l e n a n d M i l l e r ( 1 9 8 1 ) w i t h r e g a r d t o t h e t i m e a t w h i c h maximum r e a p p e a r a n c e i n t o p l a s m a t o o k p l a c e . D i f f e r e n c e s i n t h e 114 plasma Se reappearance a c t i v i t i e s and timings c o u l d be due to ( i ) the d i f f e r e n c e s i n the s y n t h e s i s of plasma p r o t e i n components that bind to Se and ( i i ) v a r i a t i o n s i n the rate of plasma Se clearance by the l i v e r and e r y t h r o c y t e s i n d i f f e r e n t s p e c i e s . An i n i t i a l d e c l i n e followed by an i n c r e a s e d reappearance of the t r a c e r i n t o the systemic 64 c i r c u l a t i o n was a l s o observed with Cu i n j e c t e d i n t o the pregnant ewes i n the p l a c e n t a l t r a n s f e r s t u d i e s of copper 64 (Sansom et a l . 1979). Since the p e r i o d of Cu r e e n t r y i n t o the plasma was much longer (10-24 hr) a f t e r i n j e c t i o n ( i n f a c t d u r i n g the e n t i r e d u r a t i o n of the t r a c e r 64 experiment) and the f a c t that the h a l f l i f e of Cu was much s h o r t e r (12 h r ) , these workers a p p l i e d a c o r r e c t i o n f a c t o r to account f o r the over-estimates i n the mean s p e c i f i c r a d i o a c t i v i t y . In the present study the longer c o l l e c t i o n of plasma samples (3 d) and the curve p e e l i n g of the plasma 75 Se ' r a d i o a c t i v i t y by n o n - l i n e a r r e g r e s s i o n methods (AUTOAN, Sedman and Wagner, 1976) f a c i l i t a t e d the 75 c o r r e c t i o n f o r over-estimates i n the plasma Se s p e c i f i c a c t i v i t y a u t o m a t i c a l l y and r e s u l t e d i n smooth curves and e x p o n e n t i a l equations with best f i t s . However du r i n g the course of f i t t i n g the c h a r a c t e r i s t i c t r a c e r disappearance followed by reappearance (hump) i s l o s t . Thus the b i o l o g i c a l s i g n i f i c a n c e of Se t r a n s p o r t d u r i n g the very e a r l y phase f o l l o w i n g t r a c e r i n j e c t i o n may 115 also be missed. In order to obtain p h y s i o l o g i c a l l y best simulated curves more powerful kinetic programs l i k e SAAM (Simulation, Analysis And Modeling) were developed by Berman and Weiss ( 1 9 7 8 ) . Ramberg et a l . (1973) used SAAM along with the deconvolution analysis to study the i n utero placental transfer and the in vivo metabolism of calcium in pregnant sheep. The SAAM program has also been used l a t e l y for the development of kin e t i c models describing the metabolism of various nutrients such as glucose (Wastney et a l . 1 9 8 3 ) , copper (Weber et a l . 1983) and calcium and phosphorus (Schneider et a l . 1985) in sheep and zinc (Foster et a l . 1984) in humans. Symonds et a l . (1981) reported a T^^2 °f approximately 64 d in dairy cows that were injected intravenously with 75 Se-selenite. The discrepancy between our study and that of Symonds et a l . (1981) could be the result of the shorter duration of the present study (3 d). The T.^ 2 values were smaller as compared to those reported i n sheep by Lopez et a l . (1969) which were approximately 20 d for lambs fed a ration containing 0 . 26 ppm Se and approximately 69 d for lambs fed a Se def i c i e n t ration containing 0 . 0 1 ppm Se. The values in the study of Lopez et a l . (1969) were calculated based on the whole body counting of lambs at various times for 14 d after the tracer i n j e c t i o n and thi s might have contributed to the discrepancy to some extent. As pointed out by Hansen and Kristensen ( 1 9 7 9 ) , i n 116 prolonged s t u d i e s of Se a grea t e r p r o p o r t i o n of whole body-counting would be due to the c o n c e n t r a t i o n of Se i n the h a i r or wool although i t was e l i m i n a t e d from the body f o r a l l p r a c t i c a l purposes. The e s t i m a t i o n of b i o l o g i c a l T l / 2 * s D a s e a - o n *-he assumption that the e l i m i n a t i o n process can be d e s c r i b e d by a f i r s t order r e a c t i o n with an e q u i l i b r i u m being a t t a i n e d between the c e n t r a l pool (blood or plasma) and a l l the organs. Under such circumstances where an element i s i n c o r p o r a t e d i n t o h a i r or wool the assumption f o r c a l c u l a t i n g the b i o l o g i c a l h a l f l i f e may not be v a l i d as the decrease i n whole body counting would depend on the growth rate of h a i r or wool r a t h e r than on the metabolic processes o c c u r r i n g i n the animal. Moreover there i s a cont r o v e r s y i n the l i t e r a t u r e w ith regard to the values of b i o l o g i c a l °f S e observed i n v a r i o u s s p e c i e s . The reported T ^ 2 values are: i n r a t s -1.5 to 8.0 d (Burk et a l . 1972), 13 d ( B l i n c o e , 1960), 43 d (Ewan et a l . 1968) and 50-59 d (Thomson and Stewart, 1973); i n mice - 14 d (Hansen and K r i s t e n s e n , 1979) and 17-18 d (Furchner et a l . 1975); i n sheep - 20-69d (Lopez et a l . 1969); i n d a i r y c a t t l e - 64 d (Symonds et a l . 1981); i n monkeys - 65 d ( B i e l s t e i n et a l . 1984) and 46-53 d (Furchner et a l . 1975); and i n dogs - 46 to 60 d (Furchner et a l . 1975) . The v a r i a t i o n s i n the T ^ 2 could be due to many f a c t o r s such as s p e c i e s , d i e t a r y Se l e v e l s , Se s t a t u s of animals, amount of t r a c e r dose administered, route of 117 a d m i n i s t r a t i o n , d u r a t i o n of experimental p e r i o d , e r r o r s i n a n a l y t i c a l methods (whole body counting) and the d i f f e r e n c e s i n the curve p e e l i n g (manual vs. computer) methods. A s i g n i f i c a n t l y higher i r r e v e r s i b l e d i s p o s a l of Se was observed i n the Se p o s i t i v e ewes than i n the d e f i c i e n t ones suggesting a g r e a t e r plasma turnover of Se i n the former ewes. T h i s could be e x p l a i n e d by the g r e a t e r e n t r y of Se from the d i e t a r y sources i n t o the plasma pool of the Se p o s i t i v e ewes u n l i k e i n the d e f i c i e n t ones. Though the Se i n t a k e was s i g n i f i c a n t l y (P < 0 . 0 0 0 1 ) lower i n Se d e f i c i e n t ewes than i n the p o s i t i v e ones, the d e f i c i e n t ewes were found to maintain t h e i r Se homeostasis by d i f f e r e n t metabolic adaptations which i n c l u d e d i ) decreased plasma Se turnover (Table 8 ) , i i ) reduced metabolic l o s s e s through feces and u r i n e (Table 9 ) , i i i ) i n c r e a s e d net Se a b s o r p t i o n (% Se intake b a s i s ) , i v ) i n c r e a s e d t i s s u e Se turnover (Table 11) and v) decreased net p l a c e n t a l Se t r a n s f e r (Expt. II C). I t may be suggested t h a t the u l t i m a t e aim of a l l these metabolic adjustments i n the d e f i c i e n t ewes was to maintain the plasma Se l e v e l at an optimum l e v e l . Therefore the plasma ID of Se would r e f l e c t the turnover of Se i n the body of pregnant ewes. The e x c r e t i o n of Se through u r i n e was g e n e r a l l y higher than i n the feces i n Se p o s i t i v e ewes on a l l 5 d of 75 the t r i a l . The combined u r i n a r y Se l o s s e s over a 5 d 118 p e r i o d a c c o u n t e d f o r a p p r o x i m a t e l y 14% o f t h e d o s e i n j e c t e d i n Se p o s i t i v e ewes , w h e r e a s t h e f e c a l l o s s e s were a b o u t 9% o f t h e d o s e a d m i n i s t e r e d . L o p e z e t a l . (1969) r e p o r t e d 75 t h a t t h e u r i n a r y a n d f e c a l Se l o s s e s were 31% a n d 5% 75 o f t h e i n t r a v e n o u s l y i n j e c t e d d o s e o f S e - s e l e n i t e r e s p e c t i v e l y i n l a m b s . The r e s u l t s f r o m t h e p r e s e n t s t u d y were a l m o s t i n a g r e e m e n t w i t h t h o s e o f L o p e z e t a l . ( 1 9 6 9 ) . The l o w e r u r i n a r y Se l o s s e s o b s e r v e d i n t h e c u r r e n t s t u d y (14%) c o u l d be due t o t h e s h o r t e r c o l l e c t i o n p e r i o d e m p l o y e d (5 d) a s a g a i n s t 12 d o f L o p e z e t a l . ( 1 9 6 9 ) . 75 On t h e o t h e r h a n d i n Se d e f i c i e n t ewes t h e f e c a l Se l o s s e s w e r e g e n e r a l l y h i g h e r t h a n t h e u r i n a r y l o s s e s . The 75 c u m u l a t i v e f e c a l a n d u r i n a r y Se l o s s e s w e r e a p p r o x i m a t e l y 7 a n d 3% o f t h e d o s e i n j e c t e d . T h e s e r e s u l t s w e r e i n c o n t r a s t w i t h t h o s e r e p o r t e d by L o p e z e t a l . (1969) who o b s e r v e d a l m o s t e q u a l amounts o f t r a c e r t o be l o s t i n t h e f e c e s a n d u r i n e o f Se d e f i c i e n t s h e e p . The Se i n t a k e was s i g n i f i c a n t l y l o w e r i n t h e Se d e f i c i e n t ewes t h a n i n t h e p o s i t i v e o n e s ( T a b l e 9 ) . The s o u r c e o f d i e t a r y Se was f o u n d t o v a r y b e t w e e n t h e Se p o s i t i v e a n d d e f i c i e n t ewes . I n Se p o s i t i v e ewes t h e d i e t a r y Se s o u r c e s were i n t h e o r d e r o f h a y (67%) > w a t e r (22%) > b a r l e y (11%), w h e r e a s , i n Se d e f i c i e n t o n e s w a t e r (75%) was t h e m a j o r s o u r c e o f Se f o l l o w e d by b a r l e y (18%) a n d h a y (7%). The e s t i m a t e d f e c a l a n d u r i n a r y Se l o s s e s were s i g n i f i c a n t l y r e d u c e d i n Se d e f i c i e n c y ( T a b l e 9 ) . When 119 the f e c a l Se l o s s e s were expressed on a percent Se i n t a k e b a s i s the v a l u e s were 58 and 33% i n Se p o s i t i v e and d e f i c i e n t ewes r e s p e c t i v e l y . T h i s i n d i c a t e d t h a t the g a s t r o i n t e s t i n a l t r a c t exerted a homeostatic c o n t r o l on Se metabolism under c o n d i t i o n s of Se d e f i c i e n c y . However when the u r i n a r y Se l o s s e s were expressed on a percent Se intake b a s i s the values were 11 and 9% i n ewes fed Se p o s i t i v e and d e f i c i e n t r a t i o n s r e s p e c t i v e l y . T h i s suggested t h a t the u r i n a r y Se route d i d not e x e r t a c o n t r o l on the Se r e t e n t i o n . C o n t r a r i l y Swanson et a l . (1983) r e c e n t l y reported a r e n a l r e g u l a t i o n of Se homeostasis i n pregnant women. The d i f f e r e n c e s between these s t u d i e s c o u l d be due to the f a c t t h at the Se e x c r e t i o n i s p r i m a r i l y through the feces i n ruminants (Cousins and Cairney, 1961; Peterson and Spedding, 1963) whereas i t i s through the u r i n e i n monogastrics (Bopp et a l . 1982, Levander et a l . 1981) i n d i c a t i n g a s p e c i e s v a r i a t i o n i n the Se metabolism. The endogenous f e c a l Se l o s s e s were s i g n i f i c a n t l y reduced i n Se d e f i c i e n t ewes (14 ug/d) as compared to the p o s i t i v e ones (52 ug/d) (Table 9) . The o r i g i n of endogenous f e c a l Se would be e i t h e r through the b i l i a r y or p a n c r e a t i c s e c r e t i o n s e n t e r i n g the duodenum. The b i l i a r y s e c r e t i o n of Se had been shown to be very l i t t l e (1-2% of dose i n j e c t e d ) and v a r i a b l e i n r a t s (Levander and Baumann, 1966) and very minimal (0.6% of dose i n j e c t e d ) i n 120 r u m i n a n t s (Symonds e t a l . 1 9 8 1 ) . However, Se e x c r e t i o n i n t h e b i l e h a d b e e n shown t o be g r e a t l y e n h a n c e d o n l y when a r s e n i c compounds were a d m i n i s t e r e d ( L e v a n d e r a n d Baumann, 1 9 6 6 ) . Se was a l s o shown t o be s e c r e t e d i n t o t h e duodenum by t h e p a n c r e a s i n t h e f o r m o f p r o t e i n enzymes a n d was f o u n d t o be r e a b s o r b e d and r e c i r c u l a t e d i n man ( L a k e - B a k a a r e t a l . 1 9 8 0 ) . C o n t r a r i l y L a n g l a n d s e t a l . (1986) u s i n g w e t h e r s w i t h o r w i t h o u t l i g a t e d p a n c r e a t i c d u c t s r e p o r t e d t h a t t h e c o n t r i b u t i o n o f p a n c r e a s was minimum i n r u m i n a n t s and t h e m a j o r s o u r c e o f t h e endo g e n o u s f e c a l Se was o n l y f r o m t h e l i v e r t h r o u g h t h e b i l i a r y s e c r e t i o n s . An e s t i m a t e d e n d o g e n o u s f e c a l l o s s o f 14 ug Se/d was o b s e r v e d i n Se d e f i c i e n t w e t h e r s whose Se i n t a k e was 48 ug S e / d ( L a n g l a n d s e t a l . 1 9 8 6 ) . The en d o g e n o u s f e c a l Se v a l u e o f 14 u g / d o b s e r v e d i n t h e Se d e f i c i e n t ewes i s a p p r o x i m a t e l y 50% o f t h a t r e p o r t e d by L a n g l a n d s e t a l . (1986) on t h e p e r c e n t Se i n t a k e b a s i s . As d i s c u s s e d e a r l i e r t h i s c o u l d be due t o a h o m e o s t a t i c m e c h a n i s m o p e r a t i n g i n t h e ewes t o i n c r e a s e t h e e f f i c i e n c y o f Se u t i l i z a t i o n u n d e r c o n d i t i o n s o f d e f i c i e n c y . The a p p a r e n t Se a b s o r p t i o n r a t e s were s i g n i f i c a n t l y h i g h e r i n t h e Se p o s i t i v e ewes t h a n i n t h e d e f i c i e n t o n e s ( T a b l e 9 ) . T h i s i n d i c a t e d t h a t t h e r a t e o f a p p a r e n t Se a b s o r p t i o n ( u g / d ) was d i r e c t l y p r o p o r t i o n a l t o t h e amount o f Se consumed by t h e ewes. When t h e a p p a r e n t Se a b s o r p t i o n r a t e s were e x p r e s s e d a s p e r c e n t o f Se i n t a k e t h e 121 v a l u e s w e re 43 and 6 7 % r e s p e c t i v e l y i n t h e ewes f e d Se p o s i t i v e a n d d e f i c i e n t r a t i o n s . T h ese v a l u e s f o r a p p a r e n t a b s o r p t i o n o f Se i n t h e p r e s e n t s t u d y were l o w e r t h a n i n m o n o g a s t r i c s p e c i e s w h i c h were 9 1 - 9 7 % i n r a t s (Thomson and S t e w a r t , 1 9 7 3 ) and 6 6 - 7 5 % i n humans ( R o b i n s o n e t a l . 1 9 7 8 ) . T h i s c o u l d be a s a r e s u l t o f c o n v e r s i o n o f o r g a n i c Se p r e s e n t i n t h e f e e d ( S h r i f t , 1 9 6 9 ) i n t o i n s o l u b l e compounds by t h e b a c t e r i a i n t h e rumen ( P e t e r s o n a n d S p e d d i n g ( 1 9 6 3 ) r e s u l t i n g i n l o w e r e d Se a b s o r p t i o n . On t h e o t h e r h a n d i n m o n o g a s t r i c a n i m a l s t h e o r g a n i c Se was f o u n d t o be a b s o r b e d a g a i n s t a c o n c e n t r a t i o n g r a d i e n t b y a n a c t i v e t r a n s p o r t p r o c e s s ( M c C o n n e l l and Cho, 1 9 6 5 , R e a s b e c k e t a l . 1 9 8 1 ) a t a f a s t e r r a t e . When t h e e n d o g e n o u s f e c a l Se l o s s e s were a d d e d t o t h e a p p a r e n t Se a b s o r p t i o n v a l u e s t h e r e s u l t i n g n e t Se a b s o r p t i o n r a t e s were 2 4 1 and 78 u g / d r e s p e c t i v e l y i n t h e Se p o s i t i v e a n d d e f i c i e n t ewes. T h e s e v a l u e s w e r e s i g n i f i c a n t l y h i g h e r i n t h e Se p o s i t i v e ewes t h a n i n t h e d e f i c i e n t o n e s . A l i n e a r r e l a t i o n s h i p was o b s e r v e d b e t w e e n t h e Se i n t a k e and t h e n e t Se a b s o r p t i o n ( F i g 1 5 ; r = 0 . 8 9 ) . When t h e n e t Se a b s o r p t i o n r a t e s were e x p r e s s e d a s a p e r c e n t o f Se i n t a k e t h e v a l u e s were 56 and 8 4 % r e s p e c t i v e l y i n t h e ewes f e d Se p o s i t i v e a n d d e f i c i e n t r a t i o n s . On a p e r c e n t a g e i n t a k e b a s i s t h e n e t Se a b s o r p t i o n v a l u e s were s i g n i f i c a n t l y h i g h e r i n t h e Se d e f i c i e n t ewes t h a n i n t h e p o s i t i v e o n e s . T h i s h i g h e r n e t Se 122 a b s o r p t i o n (%) i n Se d e f i c i e n t ewes m i g h t be a m e t a b o l i c a d a p t a t i o n t o i n c r e a s e t h e Se a v a i l a b i l i t y t o t h e t i s s u e s o f t h e a n i m a l u n d e r c o n d i t i o n s o f r e d u c e d d i e t a r y Se s u p p l y . The a p p a r e n t Se b a l a n c e o r r e t e n t i o n was s i g n i f i c a n t l y (P £ 0 . 0 5 ) h i g h e r i n Se p o s i t i v e ewes (145 ug/d) t h a n i n d e f i c i e n t o n e s (56 u g / d ) . However when t h e s e w e re e x p r e s s e d on a p e r c e n t Se i n t a k e b a s i s t h e v a l u e s w e re 32 and 58% r e s p e c t i v e l y i n t h e ewes f e d Se p o s i t i v e a n d d e f i c i e n t r a t i o n s . These d i f f e r e n c e s were s t a t i s t i c a l l y s i g n i f i c a n t (P _< 0 . 0 1 ) i n d i c a t i n g t h a t t h e Se d e f i c i e n t ewes were r e t a i n i n g more Se i n s p i t e o f t h e i r l o w e r e d Se i n t a k e . The t i s s u e Se c o n c e n t r a t i o n s were s i g n i f i c a n t l y h i g h e r i n Se p o s i t i v e ewes t h a n i n t h e d e f i c i e n t o nes ( T a b l e 1 0 ) . T h i s i n d i c a t e d t h a t t h e c o n c e n t r a t i o n o f Se i n t h e t i s s u e s was d i r e c t l y i n f l u e n c e d by t h e amount o f Se p r e s e n t i n t h e r a t i o n consumed by t h e ewes. The h i g h e s t Se c o n c e n t r a t i o n was o b s e r v e d i n t h e k i d n e y f o l l o w e d b y t h e s p l e e n a n d t h e l i v e r i n t h e Se p o s i t i v e ewes. On t h e o t h e r h a n d i n Se d e f i c i e n t ewes t h e t i s s u e Se c o n c e n t r a t i o n s were i n t h e o r d e r o f k i d n e y > s p l e e n > h e a r t > l u n g > l i v e r . T h ese v a l u e s were i n a g r e e m e n t w i t h t h o s e r e p o r t e d b y Maag a nd G l e n n (1967) i n ewes and P a u l s o n e t a l . (1968a ) i n l a m b s . The t o t a l t i s s u e Se p o o l s i z e s w e r e d e t e r m i n e d a f t e r o b t a i n i n g t h e i n d i v i d u a l t i s s u e s f o l l o w i n g t h e s e q u e n t i a l s l a u g h t e r o f t h e ewes a t d i f f e r e n t t i m e s . 123 The t i s s u e Se p o o l s i z e s were s i g n i f i c a n t l y h i g h e r i n t h e Se p o s i t i v e ewes t h a n i n t h e d e f i c i e n t o n e s . T h i s c o u l d be e x p l a i n e d by t h a t t h e Se i n t a k e s were s i g n i f i c a n t l y h i g h e r i n t h e Se p o s i t i v e ewes t h a n i n t h e d e f i c i e n t o n e s ( T a b l e 9 ) . I n d e e d , a l i n e a r p o s i t i v e r e l a t i o n s h i p was o b s e r v e d b e t w e e n t h e Se i n t a k e and t h e t o t a l Se p o o l s i z e i n t h e s k e l e t a l m u s c l e , l i v e r , h e a r t and s p l e e n ( F i g . 1 6 ) . Though t h e t i s s u e Se c o n c e n t r a t i o n v a l u e s w e re i n d i c a t i v e o f t h e Se s t a t u s o f t h e ewes ( H a r t l e y a n d G r a n t , 1 9 6 1 ; Underwood, 1981) t h e y do n o t p r o v i d e i n f o r m a t i o n on t h e k i n e t i c s o f Se m e t a b o l i s m i n t h e t i s s u e s . F o r t h i s r e a s o n t h e f r a c t i o n a l r a t e c o n s t a n t s o f Se u t i l i z a t i o n and T i / 2 w e r e d e t e r m i n e d a f t e r f i t t i n g e x p o n e n t i a l e q u a t i o n s t o t h e i n d i v i d u a l ( w h o l e ) t i s s u e 75 Se s p e c i f i c a c t i v i t y d a t a ( T a b l e 1 1 ) . T h e s e d a t a i n d i c a t e d t h a t t h e t u r n o v e r o f Se was g e n e r a l l y h i g h e r i n d e f i c i e n c y c o n d i t i o n s . However t h e s e T^^ 2 v a l u e s , a s p o i n t e d o u t i n t h e p r e v i o u s e x p e r i m e n t w i t h n o n p r e g n a n t ewes ( E x p t . I ) , w o u l d r e p r e s e n t b o t h r a t e s o f e n t r y and d i s a p p e a r a n c e o f Se i n t o / f r o m t h e t i s s u e s and t h e r e f o r e a r e o n l y an a p p r o x i m a t i o n o f t h e a c t u a l t i s s u e t u r n o v e r . T h i s i n c r e a s e d t u r n o v e r c o u p l e d w i t h t h e s i g n i f i c a n t l y l o w e r t i s s u e Se p o o l s i n t h e d e f i c i e n t ewes ( T a b l e 10) w o u l d r e s u l t i n a f a s t e r d e p l e t i o n o f Se f r o m t h e t i s s u e s and m i g h t p r e d i s p o s e t h e ewe and h e r f e t u s t o Se d e f i c i e n c y s y n d r o m e s . 124 A d i r e c t c o m p a r i s o n o f Se m e t a b o l i s m i n p r e g n a n t v s n o n p r e g n a n t ewes i s c o n f o u n d e d w i t h s e v e r a l v a r i b l e s s u c h a s t h e t i m e a n d d u r a t i o n o f e x p e r i m e n t a t i o n , p r i o r Se s t a t u s a n d p h y s i o l o g i c a l c o n d i t i o n o f t h e a n i m a l s . The Se i n t a k e o f p r e g n a n t a n d n o n p r e g n a n t ewes i n t h e Se p o s i t i v e g r o u p was s i m i l a r . On t h e o t h e r h a n d Se c o n s u m p t i o n o f p r e g n a n t ewes f e d Se d e f i c i e n t r a t i o n s (91 u g / d ) was s i g n i f i c a n t l y (P _< 0 . 0 5 ) h i g h e r t h a n t h e c o r r e s p o n d i n g n o n p r e g n a n t ewes (44 u g / d ) . T h i s i s a l s o r e f l e c t e d i n h i g h e r n e t Se a b s o r p t i o n a n d a p p a r e n t Se b a l a n c e i n Se d e f i c i e n t p r e g n a n t ewes t h a n i n n o n p r e g n a n t ewes ( T a b l e s 3 a n d 9 ) . When t h e f i n a l s l o p e s 75 o f p l a s m a Se c l e a r a n c e c u r v e s were e x a m i n e d , t h e y were f o u n d t o be h i g h e r i n Se p o s i t i v e p r e g n a n t ewes t h a n t h e c o r r e s p o n d i n g n o n p r e g n a n t ewes . T h i s may be a t t r i b u t e d t o i n c r e a s i n g demands o f Se by t h e p r e g n a n t ewes t o meet t h e t i s s u e r e q u i r e m e n t s o f t h e g r o w i n g c o n c e p t u s . 125 EXPERIMENT I I B: K i n e t i c s o f s e l e n i u m m e t a b o l i s m i n Se  p o s i t i v e o r d e f i c i e n t f e t a l l a mbs i n u t e r o . r INTRODUCTION E a r l y s t u d i e s on f e t a l n u t r i e n t m e t a b o l i s m i n v o l v e d t h e u s e o f e i t h e r a c u t e s u r g i c a l p r e p a r a t i o n s o r c o m p a r a t i v e s l a u g h t e r t e c h n i q u e s . R e s e a r c h e r s i n C o l o r a d o d e v e l o p e d s u r g i c a l p r o c e d u r e s f o r t h e p l a c e m e n t o f c h r o n i c f e t a l v a s c u l a r c a t h e t e r s ( M e s c h i a e t a l . 1965) and t h i s p a v e d t h e way f o r t h e f e t a l m e t a b o l i c s t u d i e s i n u t e r o i n c o n s c i o u s ewes. U s i n g s u c h c h r o n i c p r e p a r a t i o n s t h e m e t a b o l i s m o f v a r i o u s s u b s t r a t e s s u c h as g l u c o s e (Hay e t a l . 1 9 8 1 ; K i t t s and K r i s h n a m u r t i , 1982) and a m i n o a c i d s (Lemons and S c h r i e n e r , 1 9 8 3 ; S c h a e f e r a n d K r i s h n a m u r t i , 1984) was s t u d i e d i n t h e s h e e p f e t u s . A l t h o u g h t h e r e c o v e r y 75 o f Se i n t h e f e t u s f o l l o w i n g m a t e r n a l t r a c e r i n j e c t i o n a c c o m p a n i e d by s l a u g h t e r a t d i f f e r e n t t i m e s d e m o n s t r a t e d t h e p l a c e n t a l Se t r a n s f e r ( W r i g h t a n d B e l l , 1 9 6 4 ; J a c o b s s o n and O k s a n e n , 1966 ; H i d i r o g l o u e t a l . 1 9 6 9 ) , t h e r e i s no i n f o r m a t i o n a v a i l a b l e on t h e k i n e t i c s o f Se u t i l i z a t i o n i n t h e f e t u s u n d e r i n u t e r o c o n d i t i o n s . T h e r e f o r e t h e p r e s e n t s t u d y was d e s i g n e d t o i n v e s t i g a t e t h e Se m e t a b o l i s m i n Se p o s i t i v e o r d e f i c i e n t s h e e p f e t u s i n u t e r o . 1 2 6 MATERIALS AND METHODS A n i m a l s , f e e d i n g and management d e t a i l s were t h e same as d e s c r i b e d i n E x p t . I I A. ( a ) S u r g i c a l P r o c e d u r e s ; A r o u n d 1 1 0 - 1 2 0 d o f g e s t a t i o n p o l y e t h y l e n e c a t h e t e r s ( I n t r a m e d i c "PE - 9 0 and P E - 5 0 " , C l a y Adams, P a r s i p p a n y , N.J.) w i t h s i l a s t i c t i p s were i n t r o d u c e d s u r g i c a l l y i n t o t h e b l o o d v e s s e l s o f t h e ewe and t h e f e t u s a c c o r d i n g t o t h e p r o c e d u r e r e p o r t e d b y K i t t s e t a l . ( 1 9 7 9 ) . The d e t a i l s o f t h e s u r g e r y and p o s t - o p e r a t i v e c a r e o f t h e a n i m a l s a r e d e s c r i b e d b e l o w : The ewes were d e p r i v e d o f f o o d , b u t n o t w a t e r f o r 2 4 -36 h r p r i o r t o s u r g e r y t o r e d u c e t h e rumen s i z e . The ewes were g i v e n an i n t r a m u s c u l a r i n j e c t i o n o f a c e p r o m a z i n e m a l e a t e ( A t r a v e t , A y e r s t ) , a t a d o s e o f 0 . 0 1 m l / k g w t , 2 -3 h r b e f o r e t h e s u r g e r y t o p r e v e n t e x c i t e m e n t d u r i n g p r e - o p e r a t i v e p r e p a r a t i v e p r o c e d u r e s . The w o o l o v e r t h e abdomen was c l i p p e d c o a r s e and t h e o p e r a t i v e s i t e was w a s h ed t h o r o u g h l y w i t h i o d i n e s o a p . G e n e r a l a n e s t h e s i a was i n d u c e d by s l o w (1 ml o v e r a p e r i o d o f 1-2 m i n ) i n t r a v e n o u s i n j e c t i o n o f s o d i u m t h i o p e n t a l ( P e n t o t h a l , A b b o t t L a b o r a t o r i e s ) a t a do s e o f 20 mg/kg body w e i g h t . An 127 e f f e c t i v e a n e s t h e s i a was e n s u e d by c h e c k i n g t h e c o r n e a l r e f l e x a n d t h e m a n d i b u l a r movement. The a n i m a l was i n t u b a t e d and a m i x t u r e o f 1 .0 -1 .5% h a l o t h a n e ( F l u o t h a n e , A y e r s t ) i n o x y g e n was u s e d f o r m a i n t a i n i n g a n e s t h e s i a w i t h t h e ewe i n t h e s u p i n e p o s i t i o n . A s t e r i l e d e x t r o s e s a l i n e was a d m i n i s t e r e d t o t h e a n i m a l i n t r a v e n o u s l y by d r i p t o r e p l a c e e l e c t r o l y t e and f l u i d l o s s e s d u r i n g s u r g e r y . A t r o p i n e s u l f a t e (BDH p h a r m a c e u t i c a l s ) was g i v e n s u b c u t a n e o u s l y ( 0 . 0 6 mg/kg) h a l f an h o u r b e f o r e t h e s u r g e r y t o p r e v e n t e x c e s s i v e s a l i v a t i o n d u r i n g s u r g e r y . The h e a r t b e a t a n d t h e r e s p i r a t i o n r a t e were m o n i t o r e d f r e q u e n t l y d u r i n g t h e c o u r s e o f s u r g e r y . The w o o l o v e r t h e o p e r a t i v e s i t e was removed w i t h a f i n e c l i p p e r a n d t h e a r e a was washed a g a i n w i t h i o d i n e s o a p ( S u r g i d i n e , I n g r a m a nd B e l l ) . A 1 0 - 1 5 cm m i d l i n e s k i n i n c i s i o n was made b e l o w t h e u m b i l i c u s and t h e u t e r u s was e x p o s e d by c u t t i n g t h r o u g h t h e p e r i t o n e u m . The g r a v i d u t e r i n e h o r n was p a l p a t e d a n d t h e h i n d l i m b s o f t h e f e t u s were b r o u g h t t o t h e e x t e r i o r t h r o u g h t h e a b d o m i n a l i n c i s i o n . A l e a s t v a s c u l a r a r e a , d e v o i d o f c o t y l e d o n s , e x p o s i n g t h e h o o v e s o f t h e f e t a l h i n d l i m b s was c h o s e n a nd a s m a l l i n c i s i o n was made on t h e u t e r i n e w a l l . A p u r s e s t r i n g s u t u r e w i t h a 2 -0 c h r o m i c c a t g u t ( E t h i c o n ) was made a r o u n d t h e u t e r i n e o p e n i n g i n c l u d i n g t h e f e t a l membranes t o f a c i l i t a t e t h e f e t a l h a n d l i n g and t h e p r e v e n t i o n o f l e a k a g e o f a m n i o t i c f l u i d . The h i n d l i m b s o f t h e f e t u s w e re e x t e r i o r i z e d b y c u t t i n g t h r o u g h t h e f e t a l membranes 128 c a r e f u l l y . The w o o l o v e r t h e h i n d l i m b s was c l i p p e d a b o v e t h e h o c k j o i n t w i t h s c i s s o r s and t h e a r e a was c l e a n e d t o l o c a t e t h e e x t e r n a l s a p h e n o u s v e i n . An i n c i s i o n p a r a l l e l t o t h e s a p h e n o u s v e i n was made and a few d r o p s o f l i d o c a i n e H C l ( D u r a c a i n e , R o g a r / S T B ) , a s t e r i l e l o c a l a n e s t h e t i c w e r e i n s t i l l e d . By b l u n t d i s s e c t i o n t h e e x t e r n a l s a p h e n o u s v e i n was c l e a r e d o f f a s c i a and was i s o l a t e d f o r c a t h e t e r i z a t i o n . A s m a l l i n c i s i o n was made on t h e v e i n a n d a PE-90 c a t h e t e r was i n s e r t e d t o a d i s t a n c e o f 20 cm so t h a t t h e t i p o f t h e c a t h e t e r was l o c a t e d i n t h e i n f e r i o r v e n a c a v a . The e x t e r n a l s a p h e n o u s v e i n and o c c a s i o n a l l y t h e p e d a l a r t e r y on t h e o t h e r h i n d l i m b were a l s o c a t h e t e r i z e d . The f e t a l c a t h e t e r s were s t e r i l i z e d by k e e p i n g them o v e r n i g h t i n c i d e x s o l u t i o n ( S u r g i c o s , C a n l a b ) . The c a t h e t e r s were r i n s e d and f i l l e d w i t h s t e r i l e h e p a r i n i z e d s a l i n e (50 U/ml) b e f o r e t h e i r i n t r o d u c t i o n i n t o t h e f e t a l b l o o d v e s s e l s . The c a t h e t e r s w e re a n c h o r e d t o t h e f e t a l s k i n on t h e h i n d l i m b s w i t h a b r a i d e d 2-0 s i l k a n d t h e f e t a l s k i n was c l o s e d i n a c o n t i n u o u s f a s h i o n w i t h a b r a i d e d 4-0 s i l k s u t u r e . F o l l o w i n g t h e v a s c u l a r c a t h e t e r i z a t i o n and a n c h o r i n g t h e f e t a l h i n d l i m b s w e re p l a c e d c a r e f u l l y b a c k i n t o t h e u t e r u s . The a n t i b i o t i c , a m p i c i l l i n (500 mg, A y e r s t ) was i n s t i l l e d i n t o t h e a m n i o t i c c a v i t y b e f o r e t h e u t e r u s was c l o s e d . An a m n i o t i c f l u i d c a t h e t e r ( T y g o n ) was i n t r o d u c e d i n t o t h e a m n i o t i c c a v i t y t o i n s t i l l a n t i b i o t i c d u r i n g t h e p o s t o p e r a t i v e p e r i o d . The u t e r u s was c l o s e d a l o n g w i t h t h e f e t a l membranes u s i n g 129 2-0 chromic catgut (Ethicon) as a continuous suture which was then b u r i e d i n t o the u t e r i n e musculature without damaging the cotyledons with a second s u t u r e . The c a t h e t e r s were e x t e r i o r i z e d v i a a small tunnel made through the peritoneum, f l a n k muscles and s k i n on the r i g h t s i d e of the ewe. The peritoneum, abdominal muscles and s k i n were sutured s e p a r a t e l y i n a continuous f a s h i o n u s i n g E t h i f l e x - O , P l a i n - 0 and P r o l i n e - 0 ( E t h i c o n ) r e s p e c t i v e l y . The c a t h e t e r s were tucked under the s t e r i l e bandage a p p l i e d over the wound around the abdomen. Both the j u g u l a r v e i n s were c a t h e t e r i z e d i n the ewe u s i n g p o l y e t h y l e n e (PE - 9 0 ) t u b i n g . The patency of the c a t h e t e r s was maintained by f i l l i n g them d a i l y with s t e r i l e h e p a r i n i z e d s a l i n e (100 U/ml). The ewes were moved to i n d i v i d u a l metabolic c r a t e s provided with i n f r a r e d h e a t i n g and were given 5 ml of long a c t i n g P e n i c i l l i n (Penlong-S p l u s , Rogar/STB) i n t r a m u s c u l a r l y . Each ml of Penlong-S p l u s contained 2 0 0 , 0 0 0 i . u . P e n i c i l l i n G and 250 mg d i h y d r o s t r e p t o m y c i n . The a n t i b i o t i c was a d m i n i s t e r e d once d a i l y f o r 3 days. A 2 ml of A m p i c i l l i n s o l u t i o n ( P e n b r i t i n , A y e r s t ) c o n t a i n i n g 100 mg A m p i c i l l i n was i n s t i l l e d i n t o the amniotic c a v i t y through the amniotic f l u i d c a t h e t e r once d a i l y f o r 3 days. The ewes recovered u n e v e n t f u l l y and resumed normal f e e d i n g w i t h i n 2 days a f t e r the surgery. 130 (b)Experimental procedure; F o l l o w i n g the i n t r a v a s c u l a r surgery the f e t u s e s were allowed to recover q u i e t l y f o r f i v e days from the s u r g i c a l s t r e s s . The p o s t s u r g i c a l normalcy of the f e t u s was v e r i f i e d by r e c o r d i n g blood gas tensions ( p C 0 2 a n o - p 0 2 ^ ' P H ' hematocrit and hemoglobin as per the methods d e s c r i b e d i n Expt. IIA p r i o r to each t r a c e r experiment. A bolus dose of approximately 70 uCi ( s p e c i f i c a c t i v i t y : 187-919 75 mCi/mg Se) of sodium S e - s e l e n i t e (ICN Chemicals, I r v i n e , C a l i f o r n i a ) was i n j e c t e d i n t o i n f e r i o r vena cava of Se p o s i t i v e f e t u s e s (group I f e t u s e s : 5 7 2 , 5 8 7 , 6 5 4 , 665 and 707W) and Se d e f i c i e n t f e t u s e s (group II f e t u s e s : 7 2 5 , 909 and 1000) r e s p e c t i v e l y . Blood samples from the f e t u s were c o l l e c t e d at 2 , 5 , 1 0 , 1 5 , 3 0 , 4 5 , 6 0 , 1 2 0 , 1 8 0 , 2 4 0 , 300 and 360 min p o s t i n j e c t i o n and once d a i l y t h e r e a f t e r f o r 3 d. The t o t a l blood withdrawn from the f e t u s over the 6 hr p e r i o d on the day of experiment d i d not exceed 18 ml. Blood samples from the corresponding ewes were a l s o obtained simultaneously f o r determining the p l a c e n t a l t r a n s f e r of Se from the f e t u s to ewe (Expt. I I C ) . Blood samples were c o l l e c t e d i n t o c h i l l e d pyrex tubes c o n t a i n i n g h e p a r i n and c e n t r i f u g e d immediately. Plasma samples were kept f r o z e n a t - 2 0 ° C f o r f u r t h e r Se a n a l y s i s . The Se p o s i t i v e ewes were s a c r i f i c e d on days 7 (# 131 6 5 4 ) , 9 (# 587) and 23 (# 572) and the d e f i c i e n t ewes were k i l l e d on days 5 (# 9 0 9 ) , 7 (# 1000) and 21 (# 724) a f t e r t r a c e r i n j e c t i o n i n t o the f e t u s e s . The f e t u s e s were d e l i v e r e d by laparotomy as d e s c r i b e d under Expt. II A. The f e t u s was towel d r i e d , weighed and d i s s e c t e d f o r t i s s u e c o l l e c t i o n . L i v e r , kidney, heart, spleen and s k e l e t a l muscle (gastrocnemius) were c o l l e c t e d , towel d r i e d , weighed and s t o r e d at - 2 0 ° C. 75 S t a b l e Se and Se a n a l y s i s and the k i n e t i c a n a l y s i s of data were the same as d e s c r i b e d under Expt. I. A l l the r e s u l t s were expressed as means + SEM. The f e t a l plasma and t i s s u e Se ( s t a b l e ) c o n c e n t r a t i o n data were combined with the maternal data and were analysed by a two way a n a l y s i s of v a r i a n c e u s i n g SAS ( 1 9 8 5 ) . The r a t i o n and the sheep were assumed to be the main e f f e c t s . The remaining data were subjected to one way a n a l y s i s of v a r i a n c e (SAS, 1 9 8 5 ) . Student's ' t ' t e s t was used to t e s t the s i g n i f i c a n c e of d i f f e r e n c e between the means of the two groups of f e t u s e s . 132 RESULTS The mean p h y s i o l o g i c a l p a r a m e t e r v a l u e s o f Se p o s i t i v e a n d d e f i c i e n t f e t u s e s a r e p r e s e n t e d i n T a b l e 12. T h e s e v a l u e s were r e c o r d e d d u r i n g t h e e x p e r i m e n t a l p e r i o d w h i c h commenced 5 d a y s a f t e r s u r g e r y . 75 F o l l o w i n g t h e i n j e c t i o n o f S e - s e l e n i t e i n t o t h e i n f e r i o r v e n a c a v a o f t h e f e t u s e s t h e r e was an e x p o n e n t i a l 75 d e c l i n e i n t h e p l a s m a Se r a d i o a c t i v i t y ( F i g 17). I n c o n t r a s t t o t h e a d u l t ewe t h e r e a p p e a r a n c e (hump) o f Se 75 i n t o t h e p l a s m a was m i n i m a l . S i m i l a r l y when Se was i n j e c t e d i n t o t h e Se d e f i c i e n t f e t u s e s t h e t r a c e r d i s a p p e a r e d e x p o n e n t i a l l y f r o m t h e p l a s m a ( F i g . 18) w i t h a r e l a t i v e l y s m a l l e r r e a p p e a r a n c e o f t h e t r a c e r i n t o t h e p l a s m a . 75 The b e s t f i t p l a s m a Se s p e c i f i c a c t i v i t y - t i m e c u r v e s o f t h e Se p o s i t i v e and d e f i c i e n t f e t u s e s a r e p r e s e n t e d i n F i g s . 19 and 20 r e s p e c t i v e l y . The b e s t f i t e x p o n e n t i a l e q u a t i o n s d e s c r i b i n g t h e s e c u r v e s a r e g i v e n i n T a b l e 13. Two t o f o u r e x p o n e n t i a l s were r e q u i r e d t o o b t a i n t h e b e s t 75 f i t c u r v e s o f t h e p l a s m a Se r a d i o a c t i v i t y d a t a . T h r e e o f t h e Se p o s i t i v e f e t u s e s (# 707, 654 and 665) showed a 75 b i e x p o n e n t i a l p a t t e r n o f Se c l e a r a n c e w h i l e t h e o t h e r two (# 572 and 587) e x h i b i t e d a t r i - and t e t r a p h a s i c c l e a r a n c e p a t t e r n s r e s p e c t i v e l y . A b i p h a s i c p a t t e r n o f T a b l e 1 2 . P h y s i o l o g i c a l p a r a m e t e r s o f Se p o s i t i v e a n d d e f i c i e n t f e t u s e s . Se p o s i t i v e (n=9) Se d e f i c i e n t (n=6) P a r a m e t e r Mean + S . E . M Mean + S . E . M B l o o d pH 7 . 3 5 + 0 .02 7 . 3 7 + 0 . 0 2 B l o o d PCC»2 (mm Hg) 3 7 . 1 + 1.0 3 5 . 6 + 1 .2 B l o o d PC<2 (mm Hg) 1 0 . 1 + 0 . 7 1 2 . 3 + 0 . 4 H e m o g l o b i n (g %) 1 1 . 4 + 0 .6 1 3 . 4 + 0 . 4 H e m a t o c r i t (%) 3 4 . 0 + 1.3 3 5 . 1 + 0 . 9 B l o o d s a m p l e s were o b t a i n e d f r o m t h e i n f e r i o r v e n a c a v a . 134 O . l - i CD CO ton B E O . O O l i 1 1 ' 0 10 20 30 Time (Hours) F i g . 1 7 . C h a n g e s i n p l a s m a 7 ^ S e s p e c i f i c a c t i v i t y d u r i n g f i r s t 24 h r a f t e r t r a c e r i n j e c t i o n i n t o Se p o s i t i v e f e t u s e s , o i • > O » • > A r e f e r t o f e t u s n o s . 6 5 4 , 707W, 6 6 5 , 572 a n d 587 r e s p e c t i v e l y . 135 F i g . 18. Changes i n plasma Se s p e c i f i c a c t i v i t y d u r i n g f i r s t 24 hr a f t e r t r a c e r i n j e c t i o n i n t o Se d e f i c i e n t f e t u s e s . O , • , O r e f e r t o f e t u s nos. 909, 724 and 1000 r e s p e c t i v e l y . 136 m 0) o CJ d oo CO 0.01 o.oon 0 20 40 60 Time (Hours) F i g . 19. B e s t - f i t plasma 7 5 S e s p e c i f i c * c t i v i t y - t i m e curves of Se posit i v e fetuses. O » Bfl to feturnoL P654, 707W, 665, 572 and 587 respectively. 137 Time (Hours) 7 ^ S e s p e c i f i c F i g . 2 I . » . W 1 ) J 1 » S^/oS"... ! 0 0 0 r e s p e c t i v e ! * . a c t i v i t y - t i m e D r e f e r t o T a b l e 13. B e s t - f i t e x p o n e n t i a l e q u a t i o n s o f p l a s m a Se s p e c i f i c a c t i v i t y - t i m e c u r v e s o f f e t u s e s . F e t u s # A l A 2 A 3 A 4 a l a 2 a 3 a 4 Se p o s i t i v e f e t u s e s : 707W .019508 .010324 3 . 3 2 7 9 . 0 1 8 7 654 .016472 .015369 1 . 3 2 3 5 . 0 1 2 7 665 . 014707 .008532 3 . 3 6 6 2 . 0 5 2 9 572 .015218 .008985 .006662 6 . 2 3 2 6 . 4 9 0 6 . 0 0 8 2 587 .025661 .006159 .003542 .002687 3 5 . 0 1 4 7 . 0 4 2 1 . 2938 . 0 0 2 7 Se d e f i c i e n t f e t u s e s : 724 .020478 .010593 4 . 6 9 8 7 . 0192 909 . 051408 .021717 6 .2838 . 0 1 5 8 1000 .032005 .018706 5 . 3 9 1 6 . 0 1 9 7 ^"The e q u a t i o n i s : A f c - v "\ t + A 2 e "V + A 3 e ~V + A e ~ a t  A 4 e 4 w h e r e A - p l a s m a Se s p e c i f i c a c t i v i t y ( f r a c t i o n o f d o s e / u g S e ) , a - r a t e c o n s t a n t , e « l o g . n and t - t i m e ( h r ) . T a b l e 1 4 . C o m p a r i s o n o f a r e a s u n d e r t h e c u r v e s (AUC) a n d i n - v i v o k i n e t i c p a r a m e t e r s o f Se p o s i t i v e and d e f i c i e n t f e t u s e s . Se p o s i t i v e ( n » 4 ) Se d e f i c i e n t ( n » 3 ) P a r a m e t e r Mean + S . E . M Mean + S . E . M AUC ( f r a c t i o n o f d o s e / u g S e ) . h r 0 .79 + 0 .20 0 . 9 6 + 0 . 2 4 I r r e v e r s i b l e D i s p o s a l o f Se (ug S e / d / k g B . W . ) 20 + 9 11 + 3 P l a s m a C l e a r a n c e R a t e ( m l / d / k g B . W . ) 444 + 157 301 + 91 P l a s m a Se P o o l S i z e (ug ) 3 0 a + 1 2 2 b + 5 P l a s m a Se C o n e , ( n g / m l ) 46 + 4 53 + 10 V o l u m e o f d i s t r i b u t i o n ( m l / k g B . W . ) 310 + 41 232 + 64 T l / 2 ( d ) 6 . 1 0 + 1.74 3 . 0 8 + 0 . 2 6 ' Means w i t h d i f f e r e n t s u p e r s c r i p t s w i t h i n t h e row d i f f e r s i g n i f i c a n t l y ( P < 0 . 1 0 ) . 140 Se c l e a r a n c e was o b s e r v e d i n a l l t h e t h r e e Se d e f i c i e n t f e t u s e s . The a r e a s u n d e r t h e p l a s m a s p e c i f i c a c t i v i t y - t i m e c u r v e s a n d t h e i n v i v o k i n e t i c p a r a m e t e r s o f Se p o s i t i v e a n d d e f i c i e n t f e t u s e s a r e p r e s e n t e d i n T a b l e 1 4 . A l t h o u g h t h e 75 a r e a u n d e r t h e p l a s m a Se s p e c i f i c a c t i v i t y - t i m e c u r v e o f Se d e f i c i e n t f e t u s e s t e n d e d t o be two f o l d h i g h e r t h a n i n t h e p o s i t i v e o n e s , a s t a t i s t i c a l s i g n i f i c a n c e c o u l d n o t be e s t a b l i s h e d . I r r e v e r s i b l e d i s p o s a l a n d p l a s m a c l e a r a n c e v a l u e s o f Se were l o w e r i n Se d e f i c i e n t f e t u s e s t h a n i n t h e p o s i t i v e o n e s a n d t h e s e d i f f e r e n c e s were s t a t i s t i c a l l y n o t s i g n i f i c a n t . The a p p a r e n t v o l u m e s o f Se d i s t r i b u t i o n i n Se p o s i t i v e a n d d e f i c i e n t f e t u s e s were 310 a n d 232 m l / k g r e s p e c t i v e l y a n d t h e s e i n d i c a t e d t h a t Se was w e l l d i s t r i b u t e d i n t o t h e f e t a l t i s s u e s . The p l a s m a Se p o o l s i z e i n t h e f e t u s e s o f b o t h g r o u p s d i f f e r e d s i g n i f i c a n t l y (P _< 0 . 1 0 ) w i t h h i g h e r v a l u e s b e i n g o b s e r v e d i n Se p o s i t i v e f e t u s e s . The mean (+ SEM) v a l u e s o f Se a r e 6 . 1 0 + 1 .74 d a n d 3 . 0 8 + 0 . 2 6 d r e s p e c t i v e l y i n t h e Se p o s i t i v e a n d d e f i c i e n t f e t u s e s . The t i s s u e Se c o n c e n t r a t i o n a n d p o o l s i z e d a t a o f t h e f e t u s e s a r e p r e s e n t e d i n T a b l e 1 5 . I n Se p o s i t i v e f e t u s e s t h e h i g h e s t Se c o n c e n t r a t i o n was o b s e r v e d i n t h e k i d n e y f o l l o w e d by l i v e r a n d s p l e e n . On t h e o t h e r h a n d i n Se d e f i c i e n t f e t u s e s t h e k i d n e y , l i v e r a n d s p l e e n h a d a l m o s t T a b l e 1 5 . S t a b l e Se c o n c e n t r a t i o n and p o o l s i z e i n t h e t i s s u e s o f Se p o s i t i v e a n d d e f i c i e n t f e t u s e s . Se C o n c e n t r a t i o n P o o l s i z e ( u g / g ) (ug S e ) T i s s u e Se p o s i t i v e Se d e f i c i e n t Se p o s i t i v e Se d e f i c i e n t L i v e r 0 . 2 9 a + 0 .02 0 . 1 5 b + 0 .02 2 9 . 6 C + 4 . 0 1 4 . 0 d + 1 .1 K i d n e y 0 . 4 5 ° + 0 .06 0 . 1 7 d + 0 .06 8 . 1 « + 2 . 0 2 . 7 h + 0 . 4 S k . M u s c l e 0 . 1 0 e + 0 . 0 1 0 . 0 3 f + 0 .01 9 6 . 2 a + 1 2 . 3 3 1 . 6 b + 5 . 1 H e a r t 0 . 2 0 e + 0 .02 0 . 0 7 f + 0 .02 5 . 4 C + 1.2 1 . 5 d + 0 . 2 S p l e e n 0 . 2 9 c + 0 . 0 3 0 . 1 5 d + 0 .03 2 . 1 * + 0 . 3 l . l h + 0 . 3 Means w i t h d i f f e r e n t s u p e r s c r i p t s w i t h i n t h e row d i f f e r s i g n i f i c a n t l y ( ' P < 0 . 0 0 1 ; C ' d P < 0 . 0 1 ; 6 ' £ P < 0 . 0 0 0 1 ; g , n P < 0 . 0 5 ) . n=6 f e t u s e s i n e a c h g r o u p . 142 e q u a l c o n c e n t r a t i o n o f Se. The t i s s u e Se c o n c e n t r a t i o n s i n g e n e r a l were s i g n i f i c a n t l y h i g h e r i n Se p o s i t i v e f e t u s e s t h a n i n t h e d e f i c i e n t o n e s . The c a r d i a c and t h e s k e l e t a l m u s c l e Se c o n c e n t r a t i o n s o f Se p o s i t i v e f e t u s e s w e re s i g n i f i c a n t l y (P < 0 . 0 0 0 1 ) h i g h e r t h a n i n t h e Se d e f i c i e n t o n e s . The l i v e r , k i d n e y and s p l e e n o f t h e Se p o s i t i v e f e t u s e s h a d a l m o s t t w i c e t h e Se c o n c e n t r a t i o n o f Se d e f i c i e n t f e t u s e s and t h e s e d i f f e r e n c e s were s t a t i s t i c a l l y s i g n i f i c a n t . When t h e t o t a l t i s s u e Se p o o l s i z e s w e r e e x a m i n e d , a s i g n i f i c a n t l y l o w e r v a l u e was o b s e r v e d i n t h e k i d n e y (P < 0 . 0 5 ) , h e a r t (P < 0 . 0 5 ) , s k e l e t a l m u s c l e (P < 0 . 1 0 ) and s p l e e n (P < 0 . 0 1 ) o f Se d e f i c i e n t f e t u s e s t h a n i n t h e p o s i t i v e o n e s . The l i v e r Se p o o l s i z e i n Se d e f i c i e n t f e t u s e s t e n d e d t o be l o w e r t h a n i n t h e p o s i t i v e o n e s an d t h i s d i f f e r e n c e , h o w e v e r , was s t a t i s t i c a l l y n o t s i g n i f i c a n t . When t h e f e t a l t i s s u e Se c o n c e n t r a t i o n s w e r e c o m p a r e d w i t h t h e m a t e r n a l d a t a i t was f o u n d t h a t t h e Se c o n c e n t r a t i o n was s i g n i f i c a n t l y h i g h e r i n t h e m a t e r n a l t i s s u e s t h a n i n t h e f e t u s ( T a b l e 1 6 ) . The e x c e p t i o n was l i v e r w h ere s i m i l a r c o n c e n t r a t i o n o f Se was p r e s e n t i n b o t h t h e f e t a l a n d m a t e r n a l (P > 0 . 1 0 ) h e p a t i c t i s s u e s . The k i n e t i c p a r a m e t e r s o f t i s s u e Se m e t a b o l i s m i n t h e f e t u s e s a r e p r e s e n t e d i n T a b l e 1 7 . The f r a c t i o n a l r a t e c o n s t a n t s o f Se u t i l i z a t i o n were a l m o s t s i m i l a r i n b o t h g r o u p s . The e x c e p t i o n was l i v e r where t h e v a l u e t e n d e d t o be l o w e r i n t h e Se d e f i c i e n t f e t u s e s t h a n i n t h e p o s i t i v e Table 1 6 . Comparison of the t i s s u e Se co n c e n t r a t i o n s of the f e t u s and the ewe. Se c o n c e n t r a t i o n (ug/g t i s s u e ) T i s s u e Se p o s i t i v e Se d e f i c i e n t Fetus (n - 6 ) Ewe (n - 6 ) Fetus (n - 6 ) Ewe (n - 6 ) L i v e r 0 . 2 9 a + 0 .02 0 . 3 7 b + 0 .02 0 . 1 5 + 0 . 0 2 0 . 1 1 + 0 . 0 2 Kidney 0 . 4 5 c + 0 . 06 1 . 0 9 d + 0 .06 0 . 1 7 c + 0 . 0 6 0 . 8 7 d + 0 . 0 6 Sk. Muscle 0 . 1 0 a + 0 . 0 1 0 . 1 3 b + 0 .01 0 . 0 3 a + 0 . 0 1 0 . 0 6 b + 0 . 0 1 Heart 0 . 2 0 e + 0 .02 0 . 3 1 f + 0 .02 0 . 0 7 e + 0 . 0 2 0 . 1 7 f + 0 . 0 2 Lung 0 . 2 1 g + 0 .02 0 . 3 1 h + 0 .02 0 . 0 7 a + 0 . 0 2 0 . 1 4 b + 0 . 0 2 Spleen 0 . 2 9 c + 0 . 0 3 0 . 5 7 d + 0 .03 0 . 1 5 a + 0 . 0 3 0 . 2 6 b + 0 . 0 3 ' ' ' y Means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the rows of each group d i f f e r s i g n i f i c a n t l y ( a ' D P < 0 . 0 5 ; c ' d P < 0 . 0 0 0 1 ; e , £ P < 0 . 0 0 1 ; g ' n P < 0 . 0 1 ) . T a b l e 1 7 . K i n e t i c p a r a m e t e r s , o f t i s s u e Se m e t a b o l i s m i n t h e Se p o s i t i v e a n d d e f i c i e n t f e t u s e s . F r a c t i o n a l r a t e c o n s t a n t T T i s s u e ( F r a c t i o n / d ) (< Se p o s i t i v e Se d e f i c i e n t Se p o s i t i v e Se d e f i c i e n t L i v e r 0 . 1000 0 .0595 7 12 K i d n e y 0 .0480 0 .0518 14 13 S k . M u s c l e 0 .0954 0 .0761 7 9 H e a r t 0 . 0612 0 .0793 11 9 * n - 3 f e t u s e s i n e a c h g r o u p . * * o f Se u t i l i z a t i o n i n t h e i n d i v i d u a l t i s s u e s . 145 ones. When the T j y 2 v a * u e s w e r e examined i t was found that these v a l u e s were almost s i m i l a r i n both groups except i n the l i v e r of Se d e f i c i e n t f e t u s e s which tended to be higher than i n the p o s i t i v e ones. DISCUSSION The v a l u e s of v a r i o u s p o s t - o p e r a t i v e p h y s i o l o g i c a l parameters observed i n the f e t u s are i n agreement with those r e p o r t e d by other researchers ( B a t t a g l i a and Meschia, 1 9 7 8 ; K i t t s et a l . 1 9 7 9 ; S i l v e r , 1 9 8 1 ; Krishnamurti and Schaefer, 1984) i n d i c a t i n g t h at the f e t u s has recovered from the s t r e s s of surgery and the a n e s t h e s i a at the time of t r a c e r experiment. The c l e a r a n c e of 7^Se from the plasma of Se p o s i t i v e and d e f i c i e n t f e t u s e s i n d i c a t e s that the p h y s i o l o g i c a l mechanisms i n v o l v e d i n the i n i t i a l uptake and l a t e r r e l e a s e of the Se by the l i v e r and the e r y t h r o c y t e s as observed i n the a d u l t sheep (Expts. I and II A), c a t t l e (Symonds et a l . 1981) and goats ( A l l e n and M i l l e r , 1981) may not have yet developed i n the f e t u s at t h i s stage of g e s t a t i o n . A low GSH-Px a c t i v i t y i n the neonatal l i v e r t h a t i n c r e a s e d with age has been reported by P i n t o and B a r t l e y (1977) suggesting an age r e l a t e d developmental mechanism of t h i s s e l e n o p r o t e i n . Furthermore a lower amount of Se dependent GSH-Px was reported i n the e r y t h r o c y t e s of 146 the human f e t u s (Rudolph and Wong, 1978) and the newborn i n f a n t s (Gross et a l . 1967 ; B r a c c i et a l . 1 9 6 9 ; Emerson et a l . 1972) than i n the a d u l t s . The reduced s y n t h e s i s of f u n c t i o n a l s e l e n o p r o t e i n s such as GSH-Px i n the f e t a l t i s s u e s may thus account f o r the absence of r e g u l a t o r y mechanisms and an almost smooth appearance of the plasma 7^Se decay curves. The s i g n i f i c a n t l y higher plasma Se pool s i z e of the Se p o s i t i v e f e t u s e s (30 ug) than i n the d e f i c i e n t ones (22 ug) coupled with s i m i l a r plasma Se c o n c e n t r a t i o n s would suggest e i t h e r a reduced volume of d i s t r i b u t i o n or a reduced metabolic clearance or a combination of both i n d e f i c i e n t f e t u s e s . The l a t t e r would be an advantage to the f e t u s under c o n d i t i o n s of Se d e f i c i e n c y . Although the ID , plasma clearance and the volume of d i s t r i b u t i o n (Table 14) were lower i n the Se d e f i c i e n t f e t u s , these d i f f e r e n c e s were not s t a t i s t i c a l l y s i g n i f i c a n t probably because of l a r g e animal v a r i a t i o n s i n these parameters. However the reduced p l a c e n t a l t r a n s f e r e f f i c i e n c i e s and the ra t e of Se t r a n s f e r from the fe t u s to the ewe i n Se d e f i c i e n c y c o n d i t i o n s observed i n Expt. IIC (p 81) would support the hypothesis that there i s an i n c r e a s e d r e t e n t i o n of Se i n the f e t u s when the maternal Se supply i s low. The t i s s u e Se data of the f e t u s (Table 15) i n d i c a t e d t h a t the t i s s u e Se pools were s i g n i f i c a n t l y l a r g e r i n 147 Se p o s i t i v e f e t u s e s t h a n i n t h e d e f i c i e n t o n e s . T h i s may be a c c o u n t e d f o r by t h e i n c r e a s e d t r a n s f e r o f Se f r o m t h e ewe t o t h e f e t u s a c r o s s t h e p l a c e n t a i n Se p o s i t i v e ewes as shown l a t e r i n E x p t . I I C . T h i s was f u r t h e r c o n f i r m e d b y t h e o b s e r v a t i o n t h a t t h e t i s s u e s o f t h e Se d e f i c i e n t f e t u s e s c o n t a i n e d s i g n i f i c a n t l y l o w e r c o n c e n t r a t i o n s o f Se t h a n i n t h e p o s i t i v e o n e s . O f i n t e r e s t was t h e o b s e r v a t i o n t h a t t h e m a t e r n a l t i s s u e s c o n t a i n e d s i g n i f i c a n t l y h i g h e r c o n c e n t r a t i o n s o f Se t h a n t h o s e i n t h e f e t u s e s o f e i t h e r g r o u p ( T a b l e 1 6 ) . T h i s i n d i c a t e d t h a t t h e f e t a l t i s s u e s do n o t have as much Se r e t e n t i o n c a p a b i l i t y a s t h e m a t e r n a l o n e s . The l i m i t e d r e t e n t i o n o f t h e Se o b s e r v e d i n t h e f e t a l l a m b s may be due t o t h e a b s e n c e o f t h e m e c h a n i s m s , a s d i s c u s s e d b e f o r e , t o i n c o r p o r a t e Se i n t o t h e f u n c t i o n a l s e l e n o p r o t e i n s t h a t s t o r e t h i s m i n e r a l . T h o u g h i t i s n o t known a t t h e p r e s e n t t i m e t h e f o r m i n w h i c h Se i s t r a n s f e r r e d f r o m t h e ewe t o f e t u s i t c a n be a s s u m e d t h a t Se i s t r a n s f e r r e d p r o b a b l y a s o r g a n i c s e l e n o - a m i n o a c i d s . On t h e o t h e r h a n d i f Se i s t r a n s f e r r e d i n a n i n o r g a n i c f o r m e i t h e r a s s e l e n i t e o r s e l e n i d e t h e f e t u s c o u l d s t i l l u t i l i z e t h e s e l e n i t e S e . T h i s c o u l d be a r g u e d f r o m t h e f a c t t h a t t h e e n z y m e , c y s t a t h i o n i n e B s y n t h a s e , r e q u i r e d f o r c o n v e r s i o n o f s e l e n i d e t o s e l e n o c y s t e i n e h a s b e e n r e p o r t e d t o be p r e s e n t i n t h e f e t a l l i v e r a n d k i d n e y ( S t u r m a n e t a l . 1 9 7 6 ) . The a c t i v i t y o f c y s t a t h i o n i n e 148 s y n t h a s e enzyme was f o u n d t o be h i g h e r i n t h e f e t a l l i v e r a n d k i d n e y t h a n i n t h e a d u l t and was shown t o be d e c r e a s i n g r a p i d l y a f t e r b i r t h a n d r e a c h i n g t h e a d u l t l e v e l s a t t h e age o f 2 weeks p o s t n a t a l l y ( S t u r m a n e t a l . 1 9 7 6 ) . S t u d i e s by T a p p e l (1984) on i n c o r p o r a t i o n o f v a r i o u s Se compounds i n t o GSH-Px have s u g g e s t e d t h a t s e l e n i t e i s m e t a b o l i c a l l y c l o s e r t o t h e s e l e n o c y s t e i n e a c t i v e s i t e o f GSH-Px t h a n e i t h e r o f t h e s e l e n o - a m i n o a c i d s , s e l e n o c y s t e i n e and s e l e n o m e t h i o n i n e . A s i m i l a r i n c o r p o r a t i o n o f r a d i o a c t i v e l y l a b e l l e d s e l e n i d e o r s e l e n i t e i n t o s e l e n o c y s t e i n e m o i e t y o f g l u t a t h i o n e p e r o x i d a s e was d e m o n s t r a t e d b y Sunde and H o e k s t r a (1980) a n d t h i s w o u l d g i v e a d d e d s u p p o r t t o t h e s u g g e s t i o n t h a t t h e l a b e l l e d s e l e n i t e u s e d i n t h e p r e s e n t s t u d y was a c t u a l l y m e t a b o l i z e d and i n c o r p o r a t e d i n t o t h e s e l e n o c y s t e i n e component o f GSH-Px enzyme i n t h e f e t u s . A p a r t f r o m i t s s y n t h e s i s f r o m s e l e n i d e o r s e l e n i t e , s e l e n o c y s t e i n e c o u l d a l s o be p r e s e n t i n t h e f e t u s e i t h e r b y i t s f o r m a t i o n f r o m s e l e n o m e t h i o n i n e , s i m i l a r t o t h e f o r m a t i o n o f c y s t e i n e f r o m m e t h i o n i n e v i a t r a n s s u l f u r a t i o n p a t h w a y , o r by i t s t r a n s f e r f r o m t h e m o t h e r a c r o s s t h e p l a c e n t a . However t h e l a s t enzyme o f t h e t r a n s s u l f u r a t i o n p a t h w a y , c y s t a t h i o n a s e r e q u i r e d f o r t h e c o n v e r s i o n o f c y s t a t h i o n i n e t o c y s t e i n e was f o u n d t o be a b s e n t i n t h e f e t a l t i s s u e s a s w e l l a s i n t h e p l a c e n t a ( S t u r m a n e t a l . 1970) o f humans and t h i s l e d t o t h e 149 s u g g e s t i o n that c y s t ( e ) i n e i s an e s s e n t i a l amino a c i d f o r the f e t u s (Sturman et a l . 1 9 7 0 ; G a u l l et a l . 1 9 7 2 ; G a u l l , 1973) although formation of c y s t e i n e from s u l p h i d e was r e p o r t e d (Braunstein et a l . 1 9 6 9 ) . Even i f c y s t a t h i o n a s e i s assumed to be present i n the sheep f e t u s and p l a c e n t a there may not be adequate s e l e n o c y s t e i n e formation from s e l e n o c y s t a t h i o n i n e because c y s t a t h i o n a s e was found to c a t a l y s e the e l i m i n a t i o n r e a c t i o n of selenohomocysteine, the precursor of s e l e n o c y s t a t h i o n i n e (Tanaka et a l . 1 9 8 5 ) . However, the e s s e n t i a l i t y of s e l e n o c y s t e i n e i n the f e t u s can not be e s t a b l i s h e d u n t i l 1) the c o n t r i b u t i o n of s e l e n i d e or s e l e n i t e to s e l e n o c y s t e i n e s y n t h e s i s i s known q u a n t i t a t i v e l y and 2) the form i n which Se t r a n s p o r t e d from the mother to f e t u s i s determined. The k i n e t i c parameters of t i s s u e Se metabolism i n d i c a t e d t h a t the rate of Se u t i l i z a t i o n was almost s i m i l a r i n the Se p o s i t i v e and d e f i c i e n t f e t u s e s (Table 1 7 ) . However the s i g n i f i c a n t l y reduced t i s s u e Se pool s i z e s noted i n the d e f i c i e n t f e t u s e s (Table 15) would be d e p l e t e d a t a f a s t e r rate under c o n d i t i o n s of decreased Se supply from the ewe. Indeed a lowered p l a c e n t a l t r a n s f e r of Se was observed from the ewe to the f e t u s (Expt. IIC) i n Se d e f i c i e n c y . These metabolic changes d i s c u s s e d above would be a disadvantage to the Se d e f i c i e n t f e t u s and would p r e c i p i t a t e the d e f i c i e n c y symptoms at a f a s t e r r a t e . 150 EXPERIMENT I I C : I n u t e r o p l a c e n t a l t r a n s p o r t r a t e s o f  Se i n p r e g n a n t ewes f e d Se p o s i t i v e o r d e f i c i e n t r a t i o n s . INTRODUCTION Whole body Se m e t a b o l i s m i n p r e g n a n t ewes i s i n f l u e n c e d n o t o n l y by t h e m e t a b o l i c p r o c e s s e s o c c u r r i n g w i t h i n t h e m a t e r n a l c o m p a r t m e n t b u t a l s o by t h e e x t e n t o f p l a c e n t a l t r a n s f e r and f e t a l m e t a b o l i s m o f Se. The q u a n t i t a t i v e d e t e r m i n a t i o n o f t h e l a t t e r two p a r a m e t e r s i n u t e r o i s c o n f r o n t e d w i t h t h e d i f f i c u l t y o f s a m p l i n g t h e p l a c e n t a l and f e t a l c o m p a r t m e n t s s e p a r a t e l y . To overcome t h i s p r o b l e m s e v e r a l w o r k e r s have r e s o r t e d t o t h e s e q u e n t i a l s l a u g h t e r o f t h e p r e g n a n t ewes f o l l o w i n g t h e a d m i n i s t r a t i o n o f r a d i o a c t i v e Se compounds t o t h e ewe and m e a s u r i n g t h e i r r e c o v e r y i n t h e f e t a l a n d p l a c e n t a l t i s s u e s t o i n d i c a t e t h e e f f e c t i v e p l a c e n t a l Se t r a n s f e r ( J a c o b s s o n and O k s a n e n , 1966; W r i g h t a n d B e l l , 1964; H i d i r o g l o u e t a l . 1969; Buck e t a l . 1982) . However, i n a d d i t i o n t o t h e e x p e n s e i n v o l v e d i n t h e s l a u g h t e r o f s e v e r a l e x p e r i m e n t a l a n i m a l s t h i s m e t hod s u f f e r s f r o m t h e d r a w b a c k t h a t t h e b i d i r e c t i o n a l Se e x c h a n g e s a c r o s s t h e p l a c e n t a , i f a n y , a r e d i f f i c u l t t o d e t e r m i n e q u a n t i t a t i v e l y . P r e l i m i n a r y 75 s t u d i e s ( S h a r i f f e t a l . 1984) i n d i c a t e d t h a t S e -s e l e n i t e i n j e c t e d i n t o t h e ewe o r t h e f e t u s c r o s s e d t h e p l a c e n t a i n e i t h e r d i r e c t i o n . W i t h t h e a d v e n t o f t h e 151 c h r o n i c f e t a l c a t h e t e r i z a t i o n t e c h n i q u e ( M e s c h i a e t a l . 1965) i t i s now p o s s i b l e t o measure t h e t r a n s p l a c e n t a l n u t r i e n t t r a n s f e r a s w e l l as t h e p l a c e n t a l and f e t a l s u b s t r a t e u t i l i z a t i o n s e p a r a t e l y u n d e r i n u t e r o c o n d i t i o n s i n u n a n e s t h e t i z e d ewes. U s i n g t h i s a p p r o a c h i n c o n j u n c t i o n w i t h t h e r a d i o i s o t o p e d i l u t i o n t e c h n i q u e t h e p l a c e n t a l Se t r a n s f e r h a s b e e n q u a n t i t a t i v e l y d e t e r m i n e d i n u t e r o i n ewes f e d Se p o s i t i v e o r d e f i c i e n t r a t i o n s i n t h e p r e s e n t s t u d y . MATERIALS AND METHODS A n i m a l s a n d f e e d i n g , s u r g i c a l p r o c e d u r e s , r a d i o t r a c e r i n j e c t i o n s a n d b l o o d s a m p l i n g p r o t o c o l were t h e same a s d e s c r i b e d i n e x p e r i m e n t s I I A and I I B . The p h y s i o l o g i c a l s t a t u s o f t h e ewes and t h e i r f e t u s e s a l o n g w i t h t h e t r a c e r i n j e c t i o n a n d s a m p l i n g d e t a i l s a r e p r e s e n t e d i n T a b l e s 18 and 19 r e s p e c t i v e l y f o r t h e Se p o s i t i v e a n d d e f i c i e n t g r o u p s . ( a ) D a t a a n a l y s i s : 75 The p l a s m a Se s p e c i f i c a c t i v i t y - t i m e d a t a o f t h e ewe and o f t h e f e t u s f o l l o w i n g m a t e r n a l as w e l l a s f e t a l i n j e c t i o n s ( i n b o t h t h e g r o u p s ) were f i t t e d t o e x p o n e n t i a l e q u a t i o n s ( T a b l e s 20 and 21) u s i n g AUTOAN (Sedman and Wagner, 1 9 7 6 ) . The a r e a s u n d e r t h e s e c u r v e s 152 Table 18. Body weights, g e s t a t i o n a l age, plasma Se concentrations, i n j e c t i o n and sampling s i t e s of Se p o s i t i v e ewes and t h e i r fetuses • Animal # Body weight Gest a t i o n a l Plasma Se I n j e c t i o n Sampling (kg) age (d) Cone, (ng/ml) s i t e s i t e 818 Ewe 73.6 116 144 JV(L) JV(F) Fetus (T) 2.13 44 SV 84 0 Ewe 69.1 117 118 JV(L) JV(R) Fetus (T) 2.85 63 SV 977 Ewe 59.1 115 251 JV(L) JV(R) Fetus (T) 1.47 50 SV 982 Ewe 60.5 116 255 JV(L) JV(R) Fetus (T) 1.90 50 SV ,57 2 Ewe 88.2 114 181 JV(R) Fetus (S) 3.39 31 IVC SV 587 Ewe 77.3 115 149 — JV(R) Fetus (S) 3.18 32 IVC SV 654 Ewe 79.6 117 286 — JV(R) Fetus (T) 1.44 56 IVC SV 665 Ewe 72.7 116 175 — JV(R) Fetus (S) 1.90 55 IVC SV 7 07W Ewe 59.1 113 128 — JV(R) Fetus (S) 2.72 36 IVC SV Mean Ewe 71 + 3 115 + 1 187 a + 20 . ' 1  + SEM Fetus 2.3 + 0.2 46 D + 4 — — — — IVC • I n f e r i o r vena cava, SV - Saphenous v e i n , JV » Jugular v e i n . Fetus (S) - Single f e t u s , Fetus (T) - Twin f e t u s . "'^Mean plasma Se concentrations d i f f e r e d s i g n i f i c a n t l y (P < 0.0001). 153 Table 19. Body weights, g e s t a t i o n a l age, plasma Se concentrations, i n j e c t i o n and sampling s i t e s of Se d e f i c i e n t ewes and t h e i r fetuses. Animal # Body weight (kg) Ges t a t i o n a l age (d) Plasma Se Cone, (ng/ml) I n j e c t i o n s i t e Sampling s i t e 58 Ewe 42.7 116 97 JV(L) JV(R) Fetus (S) 3.23 32 SV 707Y Ewe 65.5 117 82 JV(L) JV(R) Fetus (S) 2.95 82 SV 725 Ewe 72.7 113 90 JV(L) JV(R) Fetus (S) 3.29 88 SV 72 4 Ewe 66.2 116 18 JV(R) Fetus (S) 3.63 36 IVC SV 909 Ewe 62.7 116 90 — JV(R) Fetus (T) 2.17 39 IVC SV 1000 Ewe 77.2 115 36 — JV(R) Fetus (T) 2.15 41 IVC SV Mean Ewe + SEM Fetus 65 + 5 2.9 + 0.3 116 + 1 69 + 14 53 + 10 — IVC - I n f e r i o r vena cava, SV - Saphenous vein , JV - Jugular • i v e i n . Fetus (S) - Single f e t u s , Fetus (T) » Twin f e t u s . 154 Table 20. B e s t - f i t exponential equations of plasma Se s p e c i f i c a c t i v i t y - t i m e curves i n Se p o s i t i v e ewes and fetuses. Animal # A l A2 A3 A4 a l a2 a3 a4 818 Ewe (j ) .000825 .000701 5 .9871 .0127 Fetus .000021 -.000053 .000074 20 .1604 .0759 .0066 840 Ewe(,) .000909 .001107 9 .0355 .0149 Fetus .000008 -.000028 .000036 16 .5294 .0561 .0012 977 Ewe ( | ) .000505 .000671 6 .2206 .0169 Fetus .000021 .000034 -.000114 , .000101 19 .9807 .2963 .1391 .0004 982 Ewe(\) .000595 .000519 — 14 .8482 .0131 Fetus .000015 -.000039 .000054 — e .8617 .0424 .0008 707 Fetus(\) .019508 .010324 — 3 .3279 .0187 Ewe - .000120 .000120 — .2172 .0015 587 Fetus(|) .025661 .006159 .003542 , .002687 35 .0147 .0421 .2938 .0027 Ewe .000028 .000028 .1319 .0016 665 F e t u s H ) .014707 .008532 3 .3662 .0529 Ewe - .000013 -.000053 .000066 16 .2693 .0937 .0051 654 Fetus(J) .016472 .015369 1 .3235 .0127 Ewe .000096 .000096 .1678 .0074 572 Fetus(J) .015218 .006985 .006662 6 .2326 .4906 .0082 Ewe .000061 .000061 .1532 .0007 i n d i c a t e s 7 5 S e 0 3 ( t r a c e r ) i n j e c t i o n e i t h e r i n t o ewe or fetus. lThe equation i s : A t - h^e + A 2e ~ 8 2 t + A 3e ~ a 3 t + A 4e - * 4 t r where A - plasma 7 5 S e s p e c i f i c a c t i v i t y ( f r a c t i o n of dose/ug Se), a - rate constant, e - l o g . - and t - time ( h r ) . T a b l e 2 1 . B e s t - f i t e x p o n e n t i a l e q u a t i o n s o f p l a s m a Se s p e c i f i c a c t i v i t y - t i m e c u r v e s i n Se d e f i c i e n t ewes and f e t u s e s . A n i m a l # A l A 2 A 3 a l a 2 a 3 58 Ewe(\) .002218 .002375 8 . 4 2 3 1 . 0133 F e t u s .000033 - . 0 0 0 0 9 5 .000128 1 5 . 2 5 9 3 . 0 5 4 7 . 0003 707Y Ewe ( J ) . 002038 .001586 1 0 . 7 9 7 9 . 0 1 2 5 F e t u s .000006 - . 0 0 0 0 2 6 .000032 1 9 . 2 2 9 3 .0323 .0014 725 Ewe(|) .001741 .001187 5 . 3 7 9 1 .0086 F e t u s .000016 - . 0 0 0 0 3 2 .000048 2 0 . 1 6 8 7 . 0090 . 0 0 0 9 724 F e t u s ( \ ) . 020478 .010593 4 . 6 9 8 7 . 0192 Ewe - . 000320 - . 0 0 0 6 3 3 .000953 9 . 1 0 6 7 . 0482 .0064 909 F e t u s ( \ ) . 051408 .021717 6 . 2 8 3 8 .0158 • Ewe . 000761 .000761 . 0701 . 0 2 1 9 1000 F e t u s ( | ) . 032005 .018706 5 . 3 9 1 6 . 0197 Ewe .000087 - . 0 0 0 7 9 6 .000883 3 2 . 3 6 8 3 . 0 3 6 5 . 0 1 5 0 U> 75 i n d i c a t e s S e 0 3 ( t r a c e r ) i n j e c t i o n e i t h e r i n t o ewe o r f e t u s . 1 T h e e q u a t i o n i s : A f c - A x e ~ a l f c + A 2 e " 3 fc + A 2 + A 3 e _ a fc  e 3 ' where A - p l a s m a Se s p e c i f i c a c t i v i t y ( f r a c t i o n o f d o s e / u g S e ) , a - r a t e c o n s t a n t , e - l°9in a n c * fc " t i m e ( h r ) . Table 2 2 . Areas under the plasma Se s p e c i f i c a c t i v i t y - t i m e curves (AUC) of Se p o s i t i v e and deficient.ewes and t h e i r c orresponding f e t u s e s ( F r a c t i o n of dose, ug Se . h r ) . Se p o s i t i v e group Se d e f i c i e n t group Parameter Mean + S.E.M Mean + S.E.M Maternal I n j e c t i o n : AUC (Ewe) . 0 5 a + .01 . 1 5 b ± . 02 AUC (Fetus) .09 + .06 . 1 7 + . 13 F e t a l I n j e c t i o n : AUC (Fetus) . 79 + .20 . 96 + .24 AUC (Ewe) .04 + .02 . 0 7 + .04 a b ' Means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row d i f f e r s i g n i f i c a n t l y ( P < 0 . 0 0 1 ) . 157 (AUC) (Table 2 2 ) , obtained by i n t e g r a t i n g the e x p o n e n t i a l equations from time zero to time i n f i n i t y , were used f o r determining the p l a c e n t a l Se t r a n s p o r t r a t e s and the e f f i c i e n c y of p l a c e n t a l Se t r a n s f e r . The p l a c e n t a l Se t r a n s f e r e f f i c i e n c y and t r a n s p o r t r a t e s were c a l c u l a t e d as per the formulae r e p o r t e d by Donoghue et a l . ( 1 9 8 2 ; 1 9 8 5 ) : P l a c e n t a l Se t r a n s f e r AUC-Fetus a f t e r mat.lnj e f f i c i e n c y from ewe = X 100 to f e t u s (% of I.D. AUC-Ewe a f t e r f e t . I n j . i n ewe) P l a c e n t a l Se P l a c e n t a l t r a n s f e r I r r e v e r s i b l e d i s p o s a l t r a n s f e r r a t e = e f f i c i e n c y from X rate (I.D.) of Se i n from ewe to ewe to f e t u s the ewe f e t u s ( u g Se/d) (from Expt. IIA) S i m i l a r l y p l a c e n t a l Se t r a n s f e r e f f i c i e n c y and t r a n s p o r t r a t e s from f e t u s to ewe were c a l c u l a t e d : P l a c e n t a l Se t r a n s f e r AUC-Ewe a f t e r f e t . Inj e f f i c i e n c y from f e t u s = X 100 to ewe (% of I.D. AUC-Fetus a f t e r mat. I n j . i n f e t u s ) 158 P l a c e n t a l Se P l a c e n t a l t r a n s f e r I r r e v e r s i b l e d i s p o s a l t r a n s f e r r a t e » e f f i c i e n c y from X rate (I.D.) of Se i n from f e t u s to f e t u s to ewe the f e t u s ewe(ug Se/d) (from Expt. IIB) The areas under the curves (AUC) were expressed as means + SEM. The AUC data were analysed by a one way a n a l y s i s of v a r i a n c e and the s i g n i f i c a n c e of d i f f e r e n c e s between the means was t e s t e d by a Student's ' t ' t e s t (SAS, 1 9 8 5 ) . 159 RESULTS When Se-selenite was injected intravenously into Se positive ewes, the tracer crossed the placenta rea d i l y and entered the f e t a l c i r c u l a t i o n within 2 min i n d i c a t i n g the transplacental transfer of Se from the ewe to the fetus (Fig. 21). This phenomenon was also observed when the maternal injections were made into the Se d e f i c i e n t ewes (Fig. 22). The tracer concentration gradually increased i n the f e t a l blood during the f i r s t 6 hr of the experimental period and then began to decline between 1 and 3 d after tracer i n j e c t i o n . S i m i l a r l y , when the tracer was injected into the i n f e r i o r vena cava of the fetus i t appeared i n the maternal blood c i r c u l a t i o n in less than 2 min indicating that placenta was permeable to Se on the f e t a l side as well (Fig. 23). A similar pattern of Se transfer from the fetus to the ewe was also observed under Se d e f i c i e n t conditions (Fig. 24). The placental transfer rates and e f f i c i e n c i e s of Se are given in Table 23. In Se positive ewes the placental Se transfer rate from the ewe to the fetus was 53 ug/d and from the fetus to the ewe was 24 ug/d resulting in a net transfer of 29 ug Se/d to the fetus. On the other hand in Se d e f i c i e n t group the ewe to fetus and fetus to ewe placental transfer rates of Se were 29 and 12 ug/d respectively with a net transfer of 17 ug/d from the ewe to 8 Time (Hours) P i g . 2 1 . C h a n g e s i n p l a s m a ,DSe s p e c i f i c a c t i v i t y o f Se p o s i t i v e ewes ( c l o s e d s y m b o l s ) a n d t h e i r c o r r e s p o n d -i n g f e t u s e s ( o p e n s y m b o l s ) a f t e r m a t e r n a l t r a c e r i n j e c t i o n . • , • , • , • r e f e r t o ewe n o s . 8 1 8 , 8 4 0 , 977 a n d 982 r e s p e c t i v e l y . 75 , , v Plasma Se Activity in ewe (cpm/ml) ON H-TJ CO n tn 3 O CD r*-vQ » CD Q a r O * O cn r r a 01 N> Cf\ H - v Q ui o r r • * 3 • CD < n cn < fD 3* - J • CD 0» tsJ CO M> 3 CD \Q fl) rr CD C oi a o 01 CD H» Ul CD 0) 3 3 -s>0 O 1— I-l ^ c/> l_> at CD • O 01 w 3 oi 3 T5 - CT fD Al CD O a n * t-< r r w o» m H - n ~ CO < fD 3 fD fD ffc cr fD CU O on •< 1 Ml i—a r r 0) fD r r fD — O O «i 0) Ht r-h 3 o r u a n r r fD C r r r r oi Oi 3* fl* M H O 3 H - r r o 01 < • r r O r r - J n -1 »< o Or if »J O fD 01 O cn -75 Plasma Se Activity in fetus (cpm/ml) 191 75 Plasma Se Activity in fetus (cpm/ml) 1 ftH-» i 3 •o o (» (9 lt» n i n rr • rr H - C < • tn (0 <v CO o * T3 • 3 vQ a • i » Ml to O • <o a 3 3* rr 01 3 f0 03 * l » <D a 01 01 — «— n •< O 3 H 3 H Hi cr o ro O cn T J 1 H (ft H tn Q. QI — 0) rr tn 3 O 0» "< » M» 3 rr Or -J (0 (D O ui < n H W <D tn fo 3 O 01 3 0) ft» 0» rr 3 »o <0 Q. (0 n O ui 3 rr H -00 01 3* rt» - M (D H -H - O O 3 Q> •~J<-»- O O * (9 Q cr o n rt n < (0 H-O ot rr 3 T J K O 3 o a o 0* 3 a to U l 75 Plasma Se Activity in ewe (cpm/ml) 291 75 Plasma Se Activity in ewe (cpm/ml) n rr co i-i o> 01 T3 O CD CD o M rr • < • fD CO rr O co rr c at 3 O at to TJ cn ^  o » H -3 iO Q. Q> • 3 H i (O ifl H - A 0 • CO< i t n fD 3 3* Bl rr » 3 MlvQ *— CD fD O f t » n c co at H -3 fD 3 cn 0> T3 — M 3 O Of O" t— « 0 O 3 H- cn fl» cn fD — a -4 cn cn ca 01 (D MI 3 rr CT fD O 01 n (-"O 01 fD H» ^- r> fD M« rr 01 M i fl> 3 H -Mao vo 3 3" Of O U fD O vo'fl H«- rr O M H -0» rr < 3 O H » a O O rr 3 M *< H * M O fD O O 01 O O M l I M l o o P0 -o C -I .CiO. cw -cn -cn o b P ft-* N * O a "• o o o o 75 Plasma Se Activity in fetus (cpm/ml) £91 Table 2 3 . P l a c e n t a l t r a n s f e r e f f i c i e n c i e s and rat e s of Se t r a n s f e r i n Se p o s i t i v e and d e f i c i e n t ewes. Parameter Se p o s i t i v e ewes Se d e f i c i e n t ewes P l a c e n t a l Se t r a n s f e r  E f f i c i e n c y Ewe to f e t u s (% of maternal i r r e v e r s i -b l e Se d i s p o s a l ) Fetus to ewe (% of f e t a l i r r e v e r s i b l e Se d i s p o s a l ) P l a c e n t a l Se t r a n s f e r r a t e s : 11 80 17 44 Ewe to Fetus (ug Se/d) Fetus to Ewe (ug Se/d) Net Se t r a n s f e r to fe t u s (ug Se/d) . 53 24 29 29 12 17 165 F i g . 2 5 . P l a c e n t a l Se t r a n s f e r e f f i c i e n c i e s i n p r e g n a n t ewes f e d Se p o s i t i v e o r d e f i c i e n t r a t i o n s . LZ2 EWE TO FETUS ES FETUS TO EWE 166 t h e f e t u s . The p l a c e n t a l Se t r a n s f e r e f f i c i e n c i e s o f Se p o s i t i v e a n d d e f i c i e n t ewes a r e g i v e n i n F i g . 2 5 . The e f f i c i e n c i e s o f p l a c e n t a l Se t r a n s f e r f r o m t h e ewe t o t h e f e t u s a n d t h e f e t u s t o t h e ewe were 11 a n d 80% o f m a t e r n a l a n d f e t a l i r r e v e r s i b l e d i s p o s a l r a t e s ( f r o m E x p t s . I I A a n d I I B ) r e s p e c t i v e l y i n t h e ewes f e d Se p o s i t i v e r a t i o n s . On t h e o t h e r h a n d i n ewes f e d Se d e f i c i e n t r a t i o n s , t h e p l a c e n t a l Se t r a n s f e r e f f i c i e n c i e s f r o m t h e ewe t o t h e f e t u s a n d t h e f e t u s t o t h e ewe were 17 a n d 44% o f m a t e r n a l a n d f e t a l i r r e v e r s i b l e d i s p o s a l r a t e s ( f r o m E x p t s . I I A a n d I I B ) r e s p e c t i v e l y . D I S C U S S I O N The h i g h e r p l a c e n t a l t r a n s f e r e f f i c i e n c i e s f r o m t h e f e t u s t o t h e ewe i n b o t h t h e Se p o s i t i v e a n d d e f i c i e n t ewes i n d i c a t e d t h a t t h e t u r n o v e r o f Se i n t h e f e t u s was h i g h and t h a t t h e f e t a l t i s s u e s h a d o n l y a l i m i t e d Se s t o r i n g c a p a b i l i t y . A s i m i l a r o b s e r v a t i o n was made by H i d i r o g l o u e t a l . (1969) on t h e b a s i s o f s l a u g h t e r e x p e r i m e n t s . On t h e o t h e r h a n d t h e h i g h e r p l a c e n t a l t r a n s f e r e f f i c i e n c y f r o m ewe t o f e t u s ( F i g . 25) o b s e r v e d i n Se d e f i c i e n t ewes t h a n i n t h e p o s i t i v e o nes i n d i c a t e d t h e a c t i v e r o l e o f t h e p l a c e n t a i n t r a n s f e r i n g g r e a t e r amount o f Se t o e n s u r e a d e q u a t e Se s u p p l y t o t h e f e t u s u n d e r m a t e r n a l Se d e f i c i e n c y c o n d i t i o n s . 167 The e x a m i n a t i o n o f t h e c o n c e n t r a t i o n s o f s t a b l e Se i n p l a s m a o f Se p o s i t i v e and d e f i c i e n t ewes a nd t h e i r r e s p e c t i v e f e t u s e s r e v e a l e d some i n t e r e s t i n g d i f f e r e n c e s ( T a b l e s 18 and 1 9 ) . I n Se p o s i t i v e ewes, t h e f e t a l p l a s m a Se c o n c e n t r a t i o n i s s i g n i f i c a n t l y l o w e r (P _< 0 . 0 0 0 1 ) t h a n m a t e r n a l c o n c e n t r a t i o n w i t h t h e f e t a l t o m a t e r n a l r a t i o b e i n g l e s s t h a n one ( 0 . 2 7 + 0 . 0 4 ) . T h i s m i g h t i n d i c a t e t h a t t h e t r a n s f e r o f Se f r o m t h e ewe t o t h e f e t u s i s a c c o m p l i s h e d by a s i m p l e d i f f u s i o n a l o n g t h e c o n c e n t r a t i o n g r a d i e n t a c r o s s t h e p l a c e n t a . On t h e o t h e r h a n d i n Se d e f i c i e n t ewes t h e c o n c e n t r a t i o n o f Se i n f e t a l p l a s m a i s e q u a l o r t e n d e d t o be s l i g h t l y g r e a t e r t h a n i n m a t e r n a l p l a s m a w i t h t h e m a t e r n a l t o f e t a l r a t i o b e i n g c l o s e t o one ( 0 . 9 8 + 0 . 2 5 ) . T h i s i s s u g g e s t i v e o f an i n v o l v e m e n t o f a c t i v e Se t r a n s p o r t m e c h a n i s m a c r o s s t h e p l a c e n t a t o t h e f e t u s t o overcome t h e l i m i t e d Se a v a i l a b i l i t y . We a r e n o t aware o f any r e p o r t s a v a i l a b l e i n t h e l i t e r a t u r e i n d i c a t i n g t h e i n u t e r o t r a n s f e r p r o c e s s e s o f any o f t h e s e l e n o a m i n o a c i d s o r o t h e r Se compounds a c r o s s t h e p l a c e n t a . However t h e r e a r e some a v a i l a b l e d a t a on t h e i n u t e r o p l a c e n t a l t r a n s f e r p r o c e s s e s o f s u l p h u r a n a l o g s o f s e l e n o a m i n o a c i d s . M e t h i o n i n e was f o u n d t o be t r a n f e r r e d by an a c t i v e t r a n s p o r t p r o c e s s f r o m m a t e r n a l t o f e t a l p l a s m a i n man a g a i n s t a t h r e e f o l d c o n c e n t r a t i o n g r a d i e n t s i m i l a r t o most o t h e r amino a c i d s ( G a u l l e t a l . 1 9 7 3 ) . C o n t r a r i l y c y s t ( e ) i n e was f o u n d t o be u n i q u e i n t h e s e n s e t h a t i t s c o n c e n t r a t i o n i n t h e m o t h e r 168 was e q u a l o r g r e a t e r t h a n t h a t i n t h e f e t a l p l a s m a . F u r t h e r m o r e , p l a c e n t a l t r a n s f e r o f c y s t ( e ) i n e was f o u n d t o be s t e r e o s p e c i f i c ( L - c y s t ( e ) i n e o n l y ) a n d an i n c r e a s i n g c o n c e n t r a t i o n o f c y s t ( e ) i n e was o b s e r v e d i n t h e f e t a l p l a s m a when t h e m a t e r n a l c o n c e n t r a t i o n o f c y s t ( e ) i n e was d e c r e a s i n g ( G a u l l e t a l . 1 9 7 3 ) . T h e s e a u t h o r s , t h e r e f o r e s u g g e s t e d t h a t t h e p l a c e n t a l t r a n s f e r o f c y s t ( e ) i n e was a c a r r i e r m e d i a t e d a c t i v e p r o c e s s , w h e r e i n t h e f e t a l / m a t e r n a l c o n c e n t r a t i o n r a t i o was n o t g r e a t e r t h a n o r e q u a l t o one. More r e c e n t l y K o r p e l a e t a l . (1984) r e p o r t e d t h a t a l m o s t e q u a l c o n c e n t r a t i o n s o f Se a r e p r e s e n t i n t h e b l o o d o f Se d e f i c i e n t F i n n i s h p r e g n a n t women and t h e i r f e t u s e s (58 + 12 V s . 61 + 14 u g / 1 ; Mean + S D ) . T h i s a l o n g w i t h t h e s t u d i e s o f Thomson and R o b i n s o n ( 1980) who a l s o f o u n d s i m i l a r c o n c e n t r a t i o n s o f Se i n Se d e f i c i e n t p r e g n a n t women and t h e i r f e t u s e s g i v e a d d e d s u p p o r t t o o u r h y p o t h e s i s t h a t Se i s t r a n s f e r r e d by an a c t i v e p r o c e s s f r o m m o t h e r t o f e t u s i n Se d e f i c i e n c y . T h i s c o u l d be e x p l a i n e d by t h e i n i t i a l t r a n s f e r o f Se t o t h e p l a c e n t a f r o m t h e m o t h e r a g a i n s t a c o n c e n t r a t i o n g r a d i e n t . T h i s phenomenon c o u l d be s u b s t a n t i a t e d by t h e f a c t t h a t t h e Se c o n c e n t r a t i o n h a s be e n f o u n d t o r a n g e f r o m 1 . 5 t o 3 f o l d h i g h e r i n t h e p l a c e n t a t h a n i n t h e m a t e r n a l b l o o d ( K o r p e l a e t a l . 1984; H a d j i m a r k o s e t a l . 1 9 5 9 ) . I n t h e p r e s e n t s t u d y t h e c o n c e n t r a t i o n o f Se i n t h e 169 p l a c e n t a was 1 .3 times (90 ng/g) higher than i n the maternal plasma (69 ng/ml). In the second step, Se i s t r a n s f e r r e d from the p l a c e n t a to the f e t u s along the c o n c e n t r a t i o n g r a d i e n t . Since t h i s type of process was found to be o p e r a t i n g i n the e p i t h e l i o c h o r i a l type of p l a c e n t a i n the present study and i n c a t t l e ( R o l l e r et a l . 1984) and a l s o i n the hemochorial type of p l a c e n t a i n humans (Korpela et a l . 1984; Thomson and Robinson, 1980) i t i n d i c a t e s that ( i ) the a c t i v e t r a n s f e r of Se to the f e t u s i n Se d e f i c i e n c y i s not s p e c i e s s p e c i f i c and a l s o not governed by the type of p l a c e n t a t i o n and ( i i ) t h a t p l a c e n t a has a d e f i n i t e r e g u l a t o r y r o l e i n the t r a n s f e r of Se. The whole blood Se c o n c e n t r a t i o n s of the neonatal c a l v e s ( 0 . 0 8 1 mg Se/kg) were reported to be higher than t h e i r dams ( 0 . 0 5 2 mg Se/kg) i n Se d e f i c i e n t beef c a t t l e ( R o l l e r et a l . 1 9 8 4 b ) . Based on the whole blood Se and GSH-Px c o n c e n t r a t i o n s i n the neonatal c a l f and the dam, these workers concluded that Se i s e f f e c t i v e l y t r a n s f e r r e d to the f e t u s from the dam and the f e t u s can sequester higher amounts of Se i n the face of low maternal blood Se c o n c e n t r a t i o n s . Furthermore they suggested an hypothesis that the f e t u s of a Se d e f i c i e n t dam can compensate f o r the low maternal blood Se by c o n c e n t r a t i n g or s t o r i n g Se to meet i t s f u t u r e requirement of t h i s e s s e n t i a l t r a c e element. This hypothesis does 170 n o t a p p e a r t o be t r u e i n t h e l i g h t o f t h e l i m i t e d Se s t o r a g e c a p a b i l i t y o f t h e f e t u s o b s e r v e d i n t h e p r e s e n t s t u d y . F u r t h e r m o r e t h e d e c r e a s e i n t h e n e t p l a c e n t a l Se t r a n s f e r f r o m 29 t o 17 u g / d d u r i n g d e f i c i e n c y (12 ug/d) o b s e r v e d i n t h e c u r r e n t s t u d y f r o m ewe t o f e t u s a t 115 d o f g e s t a t i o n w o u l d q u i t e s i g n i f i c a n t l y a f f e c t t h e Se s t a t u s o f t h e lamb o v e r t h e r e s t o f t h e g e s t a t i o n and c o n s e q u e n t l y a t b i r t h . A d e c r e a s e d p l a c e n t a l t r a n s f e r o f n u t r i e n t s i n g e n e r a l f r o m m o t h e r t o f e t u s h a s be e n r e p o r t e d b y s e v e r a l w o r k e r s t o o c c u r d u r i n g m a t e r n a l s t a r v a t i o n o r m a t e r n a l n u t r i t i o n a l d e f i c i e n c i e s ( Young a n d Wi d d o w s o n , 1 9 7 5 ; M o r r i s s e t a l . 1 9 8 0 ; Munro e t a l . 1 9 8 3 ) . The m a j o r c a u s e s f o r d e c r e a s e d p l a c e n t a l n u t r i e n t t r a n s f e r u n d e r s u c h c o n d i t i o n s were f o u n d t o be (1) d e c r e a s e d n u t r i e n t o r m e t a b o l i t e l e v e l s i n t h e m a t e r n a l c i r c u l a t i o n ( C r a n d e l l e t a l . 1 9 8 2 ) , (2) d e c r e a s e d m a t e r n a l c a r d i a c o u t p u t and u t e r i n e b l o o d f l o w ( W i l k e n i n g e t a l . 1 9 8 2 ) , and (3) d e c r e a s e d p l a c e n t a l i r r i g a t i o n ( R o s e n f e l d , 1 9 8 4 ) . The d e c r e a s e d n e t p l a c e n t a l Se t r a n s f e r t o t h e f e t u s o b s e r v e d i n t h e p r e s e n t s t u d y i n Se d e f i c i e n t ewes c o u l d be due t o a d e c r e a s e d p l a s m a Se c o n c e n t r a t i o n i n t h e ewe (189 v s 69 n g / m l ) . The r o l e o f o t h e r f a c t o r s e n u m e r a t e d above h a s y e t t o be e s t a b l i s h e d i n Se d e f i c i e n c y . Though p l a c e n t a l t r a n s f e r o f Se h a s be e n r e p o r t e d b y 171 s e v e r a l w o r k e r s i n v a r i o u s s p e c i e s i n c l u d i n g s h e e p ( J a c o b s s o n a n d O k s a n e n , 1966; W r i g h t a n d B e l l , 1 9 6 4 ) ; d o g ( M c C o n n e l a n d R o t h , 1 9 6 4 ) ; c a t t l e ( H i d i r o g l o u e t a l . 1972 a n d 1 9 8 5 ; R o l l e r e t a l . 1 9 8 4 b ; de T o l e d o a n d P e r r y , 1 9 8 5 ) , t h i s a p p e a r s t o be t h e f i r s t t i m e t o t h e b e s t o f o u r k n o w l e d g e , t h a t q u a n t i t a t i o n o f t r a n s p l a c e n t a l Se t r a n s f e r i n u t e r o h a s b e e n r e p o r t e d . However d i f f e r e n c e s i n t h e e x p e r i m e n t a l t e c h n i q u e s a n d t h e s p e c i e s e m p l o y e d i n t h e p l a c e n t a l Se t r a n s f e r s t u d i e s by o t h e r w o r k e r s r e n d e r i t d i f f i c u l t t o compare o u r d a t a d i r e c t l y w i t h t h e i r s . I t c o u l d be c o n c l u d e d f r o m t h e p r e s e n t e x p e r i m e n t t h a t b i d i r e c t i o n a l t r a n s f e r o f Se h a s b e e n d e m o n s t r a t e d i n b o t h t h e Se p o s i t i v e a n d Se d e f i c i e n t p r e g n a n t e w e s . F u r t h e r m o r e i t was f o u n d t h a t t h e f e t a l l a m b s h a v e o n l y a l i m i t e d Se s t o r i n g c a p a b i l i t y . 172 GENERAL SUMMARY AND CONCLUSIONS Although the n u t r i t i o n a l e s s e n t i a l i t y of Se had been w e l l e s t a b l i s h e d f o r sheep and other domestic animals, very l i t t l e a t t e n t i o n was p a i d to the t r a n s a c t i o n s of Se metabolism i n the whole animal with regard to k i n e t i c s , a b s o r p t i o n and t i s s u e u t i l i z a t i o n . Therefore the present study was undertaken to address these p o i n t s i n nonpregnant and pregnant ewes fed e i t h e r Se p o s i t i v e or d e f i c i e n t r a t i o n s . The b i d i r e c t i o n a l t r a n s f e r and q u a n t i t a t i o n of Se exchanges across the p l a c e n t a were a l s o i n v e s t i g a t e d . The major accomplishments of the cu r r e n t study were as f o l l o w s : 1. The i s o t o p e d i l u t i o n technique, i n v o l v i n g a s i n g l e 75 i n j e c t i o n of S e - s e l e n i t e , was employed to determine the k i n e t i c parameters of Se metabolism. The i r r e v e r s i b l e d i s p o s a l r a t e , plasma c l e a r a n c e , plasma Se pool s i z e and volume of d i s t r i b u t i o n were determined i n nonpregnant as w e l l as i n pregnant ewes (Expt I and I I A ) . Regardless of the f a c t whether ewes were pregnant or not the plasma Se turnover was higher i n those fed Se p o s i t i v e r a t i o n s than i n the d e f i c i e n t ones. 75 2. A c h a r a c t e r i s t i c plasma Se clearance which i n v o l v e d a very r a p i d disappearance i n i t i a l l y f o l l o w e d by an i n c r e a s e i n the plasma r a d i o a c t i v i t y f o r 3-4 hr a f t e r 173 t h e t r a c e r i n j e c t i o n was d e m o n s t r a t e d f o r t h e f i r s t t i m e i n b o t h n o n p r e g n a n t ( E x p t . I ) and p r e g n a n t ( E x p t . I I A) ewes. 75 T h i s s u g g e s t e d an a v i d u p t a k e o f Se by t h e l i v e r a n d e r y t h r o c y t e s and t h a t t h e p r o t e i n s s y n t h e s i z e d b y t h o s e t i s s u e s may be i n v o l v e d i n t h e t r a n s p o r t and m e t a b o l i s m o f Se i n t h e v e r y e a r l y p e r i o d f o l l o w i n g t r a c e r i n j e c t i o n . Of i n t e r e s t was t h e f i n d i n g t h a t s u c h a m e c h a n i s m o f p l a s m a 75 Se c l e a r a n c e was n o t as p r o n o u n c e d i n t h e f e t u s e s ( E x p t . I I B ) . T h i s may be e x p l a i n e d on t h e b a s i s t h a t t h e f e t a l t i s s u e s may n o t h a v e y e t d e v e l o p e d t h e m e c h a n i s m s r e s p o n s i b l e f o r t h e s y n t h e s i s o f t h o s e s p e c i f i c p r o t e i n s i n v o l v e d i n t h e p l a s m a Se t r a n s p o r t and m e t a b o l i s m . 3. The Se b a l a n c e t e c h n i q u e c o u p l e d w i t h t h e r a d i o t r a c e r m e t h o d o l o g y e n a b l e d t h e c a l c u l a t i o n o f n e t Se a b s o r p t i o n . An i n v e r s e r e l a t i o n s h i p b e t w e e n t h e Se i n t a k e and t h e n e t a b s o r p t i o n e x p r e s s e d a s a p e r c e n t o f Se i n t a k e was o b s e r v e d b o t h i n t h e n o n p r e g n a n t and p r e g n a n t ewes. As t h e Se i n t a k e i n c r e a s e d , t h e e f f i c i e n c y o f n e t a b s o r p t i o n (% Se i n t a k e ) d e c r e a s e d (r=0.90 and r=0.89 i n t h e n o n p r e g n a n t and p r e g n a n t ewes r e s p e c t i v e l y ) . However t h e n e t amount o f Se a b s o r b e d ( u g / d ) i n c r e a s e d w i t h t h e Se i n t a k e e x h i b i t i n g a p o s i t i v e l i n e a r r e l a t i o n s h i p . Thus i t was c o n c l u d e d t h a t t h e d i e t a r y Se i n t a k e ( u g / d ) was t h e m a j o r f a c t o r w h i c h r e g u l a t e d t h e a v a i l a b i l i t y o f t h i s t r a c e m i n e r a l t o b o t h t h e p r e g n a n t and n o n p r e g n a n t ewes. 174 4. The Se b a l a n c e t e c h n i q u e a l s o p r o v e d t o be a v a l u a b l e t o o l i n a s s e s s i n g t h e c o n t r i b u t i o n o f t h e m a j o r e x c r e t o r y p a t h w a y s n a m e l y t h e g a s t r o i n t e s t i n a l t r a c t a n d t h e k i d n e y t o t h e o v e r a l l Se m e t a b o l i s m . The f e c a l Se l o s s e s were s i g n i f i c a n t l y r e d u c e d when t h e d i e t a r y Se s u p p l y was l o w s h o w i n g t h a t t h e f e c a l r o u t e e x e r t e d a m a j o r h o m e o s t a t i c c o n t r o l on Se m e t a b o l i s m u n d e r d e f i c i e n c y c o n d i t i o n s i n b o t h t h e p r e g n a n t a n d n o n p r e g n a n t ewes . Si The c a l c u l a t i o n o f t h e t i s s u e f r a c t i o n a l r a t e c o n s t a n t o f Se u t i l i z a t i o n a n d T i / 2 v a l u e s f r o m t h e w h o l e o r g a n / t i s s u e r a d i o a c t i v i t y d a t a p r o v i d e d a m e t h o d o f e x a m i n i n g t h e m e t a b o l i s m o f Se i n i n d i v i d u a l t i s s u e s i n a q u a n t i t a t i v e m a n n e r . The t u r n o v e r o f Se was h i g h e r i n t h e t i s s u e s o f b o t h t h e p r e g n a n t a n d n o n p r e g n a n t ewes u n d e r c o n d i t i o n s o f Se d e f i c i e n c y . T h u s t h e s t o r e d Se i n t h e t i s s u e s w o u l d be e x h a u s t e d a t a f a s t e r r a t e l e a d i n g t o d e f i c i e n c y s y n d r o m e s when t h e d i e t a r y i n t a k e s a r e d r a s t i c a l l y r e d u c e d . 6. By e m p l o y i n g t h e i s o t o p e d i l u t i o n , i n v o l v i n g a s i n g l e 75 i n j e c t i o n o f S e - s e l e n i t e , a n d t h e c h r o n i c v a s c u l a r c a t h e t e r i z a t i o n t e c h n i q u e s i t was p o s s i b l e t o d e t e r m i n e t h e k i n e t i c s o f Se m e t a b o l i s m q u a n t i t a t i v e l y i n t h e f e t a l l a m b s i n u t e r o . The k i n e t i c p a r a m e t e r s were c a l c u l a t e d a n d t h e v a l u e s o f t h e s e p a r a m e t e r s i n t h e f e t u s e s were g e n e r a l l y h i g h e r t h a n i n t h e a d u l t e w e s . T h e r e f o r e , i t was 175 s u g g e s t e d t h a t t h e f e t a l Se t u r n o v e r was h i g h e r t h a n i n t h e a d u l t . 7 . The c o m p a r i s o n o f t h e t i s s u e Se c o n c e n t r a t i o n s o f t h e f e t u s a n d ewe p r o v i d e d a means o f a s s e s s i n g t h e Se s t o r a g e c a p a b i l i t y o f t h e i n d i v i d u a l t i s s u e s . The s i g n i f i c a n t l y l o w e r Se c o n c e n t r a t i o n v a l u e s i n t h e f e t u s i n d i c a t e d t h a t t h e f e t a l t i s s u e s h a v e a l i m i t e d Se s t o r a g e c a p a b i l i t y a s c o m p a r e d t o t h e a d u l t . 8 . The p l a c e m e n t o f t h e c h r o n i c i n d w e l l i n g c a t h e t e r s i n t h e f e t a l a n d m a t e r n a l b l o o d v e s s e l s c o u p l e d w i t h t h e r a d i o i s o t o p e e x p e r i m e n t s f a c i l i t a t e d t h e i n v e s t i g a t i o n s 7 5 on p l a c e n t a l Se t r a n s f e r . When S e - s e l e n i t e was i n j e c t e d e i t h e r i n t o ewe o r f e t u s , t h e t r a c e r c r o s s e d t h e p l a c e n t a a n d a p p e a r e d i n t h e c o r r e s p o n d i n g f e t a l o r m a t e r n a l c o m p a r t m e n t s r e s p e c t i v e l y . T h u s t h e b i d i r e c t i o n a l t r a n s f e r o f Se a c r o s s t h e p l a c e n t a i n u t e r o was d e m o n s t r a t e d f o r t h e f i r s t t i m e . 75 K i n e t i c a n a l y s i s o f t h e p l a s m a Se s p e c i f i c a c t i v i t y d a t a p e r m i t t e d t h e c a l c u l a t i o n o f t h e p l a c e n t a l Se t r a n s f e r r a t e s f r o m ewe t o f e t u s and v i c e v e r s a . The r a t e s o f Se t r a n s f e r f r o m ewe t o f e t u s a n d f e t u s t o ewe were 53 a n d 24 u g / d r e s p e c t i v e l y i n t h e Se p o s i t i v e ewes , w h e r e a s , t h e c o r r e s p o n d i n g v a l u e s were 29 a n d 12 u g / d i n t h e d e f i c i e n t o n e s . The n e t p l a c e n t a l Se t r a n s f e r f r o m ewe 176 t o f e t u s d e c l i n e d f r o m 29 ug/d i n p o s i t i v e ewes t o 17 u g / d i n t h e d e f i c i e n t o n e s . Thus t h e m a t e r n a l Se s t a t u s was f o u n d t o i n f l u e n c e t h e n e t Se t r a n s f e r f r o m ewe t o f e t u s . Though t h e r e s u l t s o f t h e p r e s e n t s t u d y h a v e c l e a r l y d e m o n s t r a t e d t h a t a) t h e f e t a l t i s s u e c o n c e n t r a t i o n s w e r e s i g n i f i c a n t l y r e d u c e d u n d e r Se d e f i c i e n c y c o n d i t i o n s , b) t h a t t h e p l a c e n t a l t r a n s f e r r a t e o f Se f r o m ewe t o f e t u s was r e d u c e d when m a t e r n a l d i e t a r y Se i n t a k e was l o w and c ) t h a t t h e f e t a l t i s s u e Se s t o r a g e c a p a b i l i t y was l o w e r t h a n i n t h e a d u l t , t h e b a s i c q u e s t i o n - "How t o p r e v e n t Se d e f i c i e n c y i n t h e o f f s p r i n g ?" n e e d s t o be a n s w e r e d . Se s u p p l e m e n t a t i o n o f t h e p r e g n a n t ewe i n l a t e g e s t a t i o n was r e p o r t e d t o i n c r e a s e t h e Se s t a t u s o f t h e f e t u s ( Y o u n g e t a l . 1 9 6 1 ; Hamdy e t a l . 1 9 6 3 ; H i d i r o g l o u e t a l . 1 9 6 9 ) . F u r t h e r m o r e t h e s e w o r k e r s s u g g e s t e d t h a t t h e m a t e r n a l Se t r e a t m e n t p r e v e n t e d t h e o c c u r r e n c e o f j u v e n i l e NMD i n t h e lamb. I n v i e w o f t h e l i m i t e d Se s t o r a g e c a p a b i l i t y o f t h e f e t u s o b s e r v e d i n E x p t . I I B , t h e Se s u p p l e m e n t a t i o n o f t h e p r e g n a n t ewe may n o t e n s u r e a d e q u a t e s t o r a g e o f m i n e r a l i n t h e f e t a l t i s s u e s t o p r e v e n t t h e o c c u r r e n c e o f w h i t e m u s c l e d i s e a s e i n t h e n e o n a t e . A s i m i l a r o b s e r v a t i o n s u g g e s t i n g l i m i t e d r e t e n t i o n o f Se i n t h e f e t a l t i s s u e s was made by H i d i r o g l o u e t a l . (1969) i n t h e i r s t u d i e s w i t h p r e g n a n t ewes f e d d y s t r o p h o g e n i c h a y and i n j e c t e d w i t h s e l e n i u m a t t h e t i m e o f b r e e d i n g . H o wever, t h e s u p p l e m e n t a t i o n o f t h e p r e g n a n t ewe w o u l d 177 e n h a n c e t h e s t o r a g e o f Se i n t h e m a t e r n a l t i s s u e s a s o b s e r v e d i n t h e p r e s e n t s t u d y ( T a b l e 1 6 ) . The mammary g l a n d h a s b e e n shown t o t r a n s f e r s e l e n i u m f r o m b l o o d t o m i l k i n d o g ( M c C o n n e l l and R o t h , 1 9 6 4 ) , s h e e p ( H i d i r o g l o u e t a l . 1 9 6 9 ) , g o a t ( A l l e n and M i l l e r , 1 9 8 1 ) , b e e f c a t t l e ( R o l l e r e t a l . 1984b) and d a i r y cows (de T o l e d o and P e r r y , 1 9 8 5 ) . I n t h e p r e s e n t s t u d y t h e r a d i o a c t i v i t y i n t h e m i l k o f one o f t h e p r e g n a n t ewes (# 818) w h i c h r e c e i v e d 75 S e - s e l e n i t e i n j e c t i o n and d e l i v e r e d n o r m a l l y was m o n i t o r e d d a i l y . Though t h i s r e s u l t was n o t r e p o r t e d u n d e r 75 t h e e x p e r i m e n t a l s e c t i o n i t was f o u n d t h a t t h e Se r a d i o a c t i v i t y i n t h e m i l k c o n t i n u e d t o be p r e s e n t a s l o n g a s 41 d a f t e r t r a c e r i n j e c t i o n . T h e r e f o r e i t a p p e a r s t h a t m i l k c o u l d be a s o u r c e o f Se f o r t h e n e o n a t e . F u r t h e r s t u d i e s a r e w a r r a n t e d t o a s s e s s t h e i m p o r t a n c e o f mammary g l a n d i n t h e Se n u t r i t i o n o f n e o n a t e i n q u a n t i t a t i v e t e r m s . B a s e d on t h e p r e s e n t f i n d i n g s i t may be s u g g e s t e d t h a t t h e b e s t p r a c t i c a l method t o p r e v e n t t h e d e f i c i e n c y o f Se i n t h e n e o n a t e i s t o s u p p l e m e n t t h e dam o r a l t e r n a t i v e l y t r e a t t h e n e o n a t e i t s e l f e i t h e r p a r e n t e r a l l y o r o r a l l y w i t h Se compounds. 178 BIBLIOGRAPHY A l l e n , J.C. and W.J. M i l l e r . 1981. T r a n s f e r of selenium from blood to milk i n goats and no n i n t e r f e r e n c e of copper wit h selenium metabolism. J . Da i r y Sc. 64:814-821. A l l e n , W.M., P.R. Moore and B.F. Sansom. 1981. C o n t r o l l e d Release Glass e s (CRG) f o r selenium supplementation. In "Trace element metabolism i n man and animals", TEMA 4, J.M. Gawthorne, J.M. Howell and C L . White (eds.) Pp. 195, S p r i n g e r - V e r l a g , B e r l i n . A l l e n , W.M. and C.B. M a l l i n s o n . 1984. 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Reprod. 20:793-798. X i a , Y., J.E. O l d f i e l d , M.A. B i e l s t e i n 7 5 a n d P.D. Whanger. 1985. Metabolism of o r a l l y administered S e - s e l e n i t e by the p i g . Nutr. Res. 5:545-553. Young, S., W.W. Hawkins, J r . and K.F. Swingle. 1961. N u t r i t i o n a l muscular dystrophy i n lambs - The e f f e c t of a d m i n i s t e r i n g selenium to pregnant ewes. Am. J . Vet. Res. 22:416-418. Young, M. and E.M. Widdowson. 1975. The i n f l u e n c e of d i e t s d e f i c i e n t i n energy, or i n p r o t e i n , on conceptus weight, and the p l a c e n t a l t r a n s f e r of a non-metabolizable amino a c i d i n the guinea p i g . B i o l . Neonate, 27:184-191. Y u d i l e v i c h , D.L., B.M. Eaton, A.H.A. Short and H.P. L e i c h t w e i s s . 1979. Glucose c a r r i e r s at the maternal and f e t a l s i d e of the t r o p h o b l a s t i n the guinea p i g p l a c e n t a . Am. J . P h y s i o l . 237:C205-C212. Zhang, W.R., P.K. Ku, E.R. M i l l e r and D.E. U l l r e y . 1986. S t a b i l i t y of g l u t a t h i o n e peroxidase i n swine plasma samples under v a r i o u s storage c o n d i t i o n s . Can. J . Vet. Res. 50:390-392. 202 APPENDIX 203 Appendix 1 . Plasma Se s p e c i f i c a c t i v i t y ( f r a c t i o n of dose/ug Se) changes i n Se p o s i t i v e and d e f i c i e n t nonpregnant ewes. Time Se p o s i t i v e ewes Se d e f i c i e n t ewes a f t e r t r a c e r i n j e c t i o n 6 2 2 6 5 9 9 1 0 0 9 9 9 9 9 8 1 2 3 3 2 5 2 min . 0 0 0 9 5 2 . 0 0 2 8 1 8 . 0 0 0 7 0 9 . 0 0 1 3 9 8 . 0 0 7 0 1 3 . 0 0 4 7 0 9 . 0 0 6 1 7 8 . 0 0 1 4 0 8 5 i t . 0 0 0 7 5 3 . 0 0 1 3 9 6 . 0 0 0 7 0 9 . 0 0 0 9 1 8 . 0 0 5 5 7 9 . 0 0 3 8 4 8 . 0 0 5 0 2 9 . 0 0 1 2 2 4 1 0 n . 0 0 0 5 7 5 . 0 0 1 0 6 0 . 0 0 0 4 5 1 . 0 0 0 7 5 1 . 0 0 4 3 5 1 . 0 0 2 9 3 7 . 0 0 3 3 7 7 . 0 0 0 9 0 0 1 5 n . 0 0 0 4 7 6 . 0 0 0 8 5 3 . 0 0 0 3 8 7 . 0 0 0 6 2 6 . 0 0 3 7 3 7 . 0 0 2 4 3 1 . 0 0 2 7 3 0 . 0 0 0 7 3 9 3 0 M . 0 0 0 3 9 6 . 0 0 0 5 6 9 . 0 0 0 2 9 0 . 0 0 0 4 3 8 . 0 0 2 7 6 4 . 0 0 1 9 2 4 . 0 0 1 7 2 4 . 0 0 0 6 2 3 4 5 II . 0 0 0 3 7 7 . 0 0 0 4 6 5 . 0 0 0 3 2 2 . 0 0 0 4 3 8 . 0 0 2 5 0 6 . 0 0 2 1 7 7 . 0 0 1 7 9 6 . 0 0 0 6 0 0 6 0 It . 0 0 0 3 9 6 . 0 0 0 5 4 3 . 0 0 0 3 3 9 . 0 0 0 4 1 7 . 0 0 2 6 1 1 . 0 0 2 1 7 7 . 0 0 1 8 6 8 . 0 0 0 6 2 3 2 hr . 0 0 0 4 1 6 . 0 0 0 8 2 7 . 0 0 0 3 8 7 . 0 0 0 5 0 1 . 0 0 2 8 1 5 . 0 0 2 8 8 6 . 0 0 2 3 7 1 . 0 0 0 6 9 3 3 n . 0 0 0 4 3 6 . 0 0 0 8 7 9 . 0 0 0 3 8 7 . 0 0 0 4 3 8 . 0 0 2 9 1 8 . 0 0 2 6 8 6 . 0 0 2 4 4 3 . 0 0 0 7 1 6 4 n . 0 0 0 4 3 6 . 0 0 0 8 7 9 . 0 0 0 4 3 5 . 0 0 0 5 0 1 . 0 0 2 7 6 4 . 0 0 2 8 3 6 . 0 0 2 5 1 4 . 0 0 0 6 7 0 5 n . 0 0 0 4 1 6 . 0 0 0 8 5 3 . 0 0 0 4 1 9 . 0 0 0 5 0 1 . 0 0 2 6 6 2 . 0 0 2 6 8 4 . 0 0 2 4 4 3 . 0 0 0 6 4 6 6 n . 0 0 0 4 3 6 . 0 0 0 8 2 7 . 0 0 0 3 8 7 . 0 0 0 5 2 2 . 0 0 2 7 1 3 . 0 0 2 5 8 2 . 0 0 2 2 9 9 . 0 0 0 6 7 0 7 n . 0 0 0 4 1 6 — — — . 0 0 2 5 5 9 — — — 8 n . 0 0 0 4 1 6 — — — . 0 0 2 3 0 3 — — — 9 tt . 0 0 0 3 9 6 — — — . 0 0 2 3 5 5 — — — 1 0 n . 0 0 0 3 7 7 — — — . 0 0 2 2 0 1 — — — 1 1 t i . 0 0 0 3 7 7 — — — . 0 0 2 1 5 0 — — — 1 2 n . 0 0 0 3 7 7 — — — . 0 0 2 1 5 0 — — — 1 d . 0 0 0 2 9 7 . 0 0 0 4 4 0 . 0 0 0 2 4 2 . 0 0 0 3 1 3 . 0 0 1 6 8 9 . 0 0 1 6 7 1 . 0 0 1 9 4 0 . 0 0 0 4 8 5 2 n — . 0 0 0 3 8 8 . 0 0 0 1 9 3 . 0 0 0 2 7 1 — . 0 0 1 2 6 6 . 0 0 1 7 9 6 . 0 0 0 3 2 3 3 n — . 0 0 0 3 1 0 . 0 0 0 1 6 1 . 0 0 0 2 2 9 — . 0 0 1 0 6 3 . 0 0 1 5 8 1 . 0 0 0 3 0 0 4 i t — . 0 0 0 2 8 4 . 0 0 0 1 6 1 . 0 0 0 2 2 9 — . 0 0 1 0 1 3 . 0 0 1 2 9 3 . 0 0 0 2 5 4 5 t i . 0 0 0 2 5 9 . 0 0 0 1 4 5 . 0 0 0 1 8 8 — . 0 0 0 8 6 1 . 0 0 1 1 4 9 . 0 0 0 2 3 1 6 tt — — . 0 0 0 1 2 9 . 0 0 0 1 6 7 — — . 0 0 1 1 4 9 . 0 0 0 2 5 4 7 tt — — . 0 0 0 1 2 9 . 0 0 0 1 6 7 — — . 0 0 1 0 0 6 . 0 0 0 2 3 1 8 tt — — . 0 0 0 1 1 3 . 0 0 0 1 4 6 — — . 0 0 1 0 0 6 . 0 0 0 2 3 1 9 tt — — . 0 0 0 1 1 3 . 0 0 0 1 4 6 — — . 0 0 1 0 0 6 . 0 0 0 2 0 8 1 0 n — — — . 0 0 0 1 4 6 — — — . 0 0 0 2 0 8 1 1 tt — — — . 0 0 0 1 4 6 — — — . 0 0 0 2 0 8 1 2 t i — — — . 0 0 0 1 4 6 — — — . 0 0 0 1 8 5 1 3 t i — • — — . 0 0 0 1 2 5 — — — . 0 0 0 1 8 5 1 4 n — — — . 0 0 0 1 0 4 — — — . 0 0 0 1 8 5 204 Appendix 2 . Plasma Se s p e c i f i c a c t i v i t y ( f r a c t i o n of dose/ug Se) changes i n Se p o s i t i v e and d e f i c i e n t pregnant ewes. Time Se p o s i t i v e ewes Se d e f i c i e n t ewes a f t e r t r a c e r i n j e c t i o n 9 7 7 9 8 2 8 1 8 8 4 0 58 7 0 7 7 2 5 2 min . 0 0 1 1 2 7 . 0 0 0 8 9 7 . 0 0 1 4 0 0 . 0 0 1 8 4 6 . 0 0 4 1 4 4 . 0 0 2 9 9 0 . 0 0 2 5 9 3 5 n . 0 0 0 9 3 5 . 0 0 0 6 8 2 . 0 0 1 1 9 2 ** . 0 0 3 4 7 4 . 0 0 2 5 3 3 . 0 0 2 2 8 5 1 0 n . 0 0 0 7 9 4 . 0 0 0 5 4 8 . 0 0 0 9 6 1 . 0 0 1 2 9 1 . 0 0 2 8 6 5 . 0 0 1 9 0 0 . 0 0 1 9 5 9 1 5 n . 0 0 0 7 5 6 . 0 0 0 4 6 0 . 0 0 0 8 5 6 . 0 0 1 0 5 1 . 0 0 2 4 9 9 . 0 0 1 5 1 3 . 0 0 1 6 3 2 3 0 n . 0 0 0 6 0 2 . 0 0 0 3 5 7 . 0 0 0 6 1 4 . 0 0 0 8 5 6 . 0 0 2 1 3 3 . 0 0 1 1 9 6 . 0 0 1 3 2 5 4 5 n . 0 0 0 5 5 1 . 0 0 0 3 8 9 . 0 0 0 5 9 0 . 0 0 0 9 0 1 . 0 0 2 1 3 3 . 0 0 1 4 7 8 . 0 0 1 2 4 8 6 0 n . 0 0 0 4 9 9 . 0 0 0 4 2 1 . 0 0 0 6 4 8 . 0 0 0 9 7 6 . 0 0 2 1 3 3 . 0 0 1 4 4 2 . 0 0 1 1 5 2 2 hr . 0 0 0 6 2 8 . 0 0 0 5 4 8 , . 0 0 0 6 8 3 . 0 0 1 0 5 1 . 0 0 2 3 7 7 . 0 0 1 6 8 9 . 0 0 1 1 3 3 3 fl . 0 0 0 6 5 3 . 0 0 0 5 7 1 . 0 0 0 7 2 9 . 0 0 1 1 8 6 . 0 0 2 5 6 0 . 0 0 1 7 2 4 . 0 0 1 2 1 0 4 n . 0 0 0 7 1 7 . 0 0 0 5 6 3 . 0 0 0 7 4 1 . 0 0 1 2 6 1 . 0 0 2 4 3 8 . 0 0 1 7 2 4 . 0 0 1 2 1 0 5 n . 0 0 0 6 9 2 . 0 0 0 5 5 5 . 0 0 0 7 2 9 . 0 0 1 1 5 6 . 0 0 2 4 3 8 . 0 0 1 7 2 4 . 0 0 1 1 9 1 6 n . 0 0 0 6 5 3 . 0 0 0 5 1 6 . 0 0 0 8 1 0 . 0 0 1 1 2 6 . 0 0 2 3 7 7 . 0 0 1 6 8 9 . 0 0 1 1 3 3 1 d . 0 0 0 4 1 0 . 0 0 0 3 0 9 . 0 0 0 4 5 1 . 0 0 0 7 2 1 . 0 0 1 5 8 5 . 0 0 1 0 2 0 . 0 0 0 8 8 3 2 n . 0 0 0 2 9 5 . 0 0 0 2 8 6 ** . 0 0 0 5 4 0 . 0 0 1 0 9 7 . 0 0 0 7 7 4 . 0 0 0 7 1 1 3 n . 0 0 0 2 4 3 . 0 0 0 1 7 5 . 0 0 0 2 6 6 . 0 0 0 4 0 5 . 0 0 0 9 7 5 . 0 0 0 6 6 8 . 0 0 0 6 5 3 ** Blood samples hemolysed and therefore plasma r a d i o a c t i v i t y could not be determined. 205 Appendix 3. Plasma Se s p e c i f i c a c t i v i t y ( f r a c t i o n of dose/ug Se) changes i n Se p o s i t i v e and d e f i c i e n t f e t u s e s . Time Se p o s i t i v e f e t u s e s Se d e f i c i e n t f e t u s e s a f t e r t r a c e r i n j e c t i o n 5 7 2 5 8 7 6 5 4 6 6 5 7 0 7 7 2 4 9 0 9 1 0 0 0 2 min . 0 2 8 9 0 1 . 0 2 0 7 0 6 . 0 3 1 2 2 0 . 0 2 3 4 1 4 . 0 2 6 5 2 0 . 0 2 9 4 1 3 . 0 6 1 7 8 2 . 0 4 5 8 1 3 5 n . 0 1 9 6 3 1 . 0 1 2 2 2 6 ** . 0 1 8 6 2 1 . 0 2 5 3 4 1 . 0 2 3 7 3 4 . 0 5 5 5 3 5 . 0 3 9 0 8 2 1 0 n . 0 1 8 1 7 7 . 0 0 9 2 6 8 . 0 3 0 5 2 1 . 0 1 6 5 9 3 . 0 2 2 5 9 1 . 0 1 9 6 5 7 . 0 3 9 1 0 6 . 0 3 2 1 3 4 1 5 n . 0 1 7 8 1 3 . 0 0 8 0 8 5 . 0 2 5 1 6 2 . 0 1 4 7 4 9 . 0 1 8 0 7 3 . 0 1 7 0 3 6 . 0 3 2 1 6 4 . 0 2 6 7 0 6 3 0 n . 0 1 4 5 4 2 . 0 0 6 5 0 8 . 0 2 1 4 3 4 . 0 1 1 0 6 2 . 0 1 3 7 5 1 . 0 1 2 5 2 2 . 0 2 5 6 8 5 . 0 2 0 6 2 7 4 5 n . 0 1 2 9 0 6 . 0 0 5 5 2 2 . 0 2 0 7 3 5 . 0 0 9 9 5 6 . 0 1 3 1 6 2 . 0 1 0 6 2 9 . 0 2 3 3 7 1 . 0 1 8 2 3 8 6 0 n . 0 1 1 4 5 2 . 0 0 5 3 2 4 . 0 1 9 5 7 1 . 0 0 9 7 7 1 ** . 0 1 0 7 7 5 . 0 2 1 2 8 8 . 0 1 8 6 7 3 2 hr . 0 0 9 6 3 4 . 0 0 4 3 3 8 . 0 1 9 3 3 8 . 0 0 7 5 5 9 ** . 0 0 9 6 1 0 . 0 2 0 5 9 4 . 0 1 8 4 5 5 3 tl . 0 0 8 9 0 7 . 0 0 4 3 3 8 . 0 1 6 0 7 6 . 0 0 7 0 0 6 . 0 1 1 0 0 1 . 0 0 9 6 1 0 . 0 2 0 8 2 5 . 0 1 8 4 5 5 4 n . 0 0 7 8 1 6 . 0 0 3 9 4 4 . 0 1 1 4 1 6 . 0 0 6 0 8 4 . 0 0 9 6 2 6 . 0 1 0 3 3 8 . 0 2 0 5 9 4 . 0 1 8 8 9 0 5 n . 0 0 7 0 8 9 . 0 0 3 7 4 7 ** . 0 0 5 7 1 5 . 0 0 8 4 4 7 . 0 0 9 4 6 5 . 0 2 0 5 9 4 . 0 1 7 1 5 3 6 n . 0 0 6 5 4 4 . 0 0 3 1 5 5 ** . 0 0 5 3 4 7 . 0 0 7 8 5 8 . 0 0 9 1 7 3 . 0 1 8 7 4 3 . 0 1 5 6 3 3 1 d . 0 0 5 4 5 3 . 0 0 2 5 6 4 . 0 0 8 8 5 3 . 0 0 2 5 8 1 . 0 0 5 5 0 0 . 0 0 5 5 3 3 . 0 1 1 3 3 8 . 0 0 8 9 0 2 2 n . 0 0 4 3 6 2 . 0 0 2 3 6 6 . 0 0 7 6 8 8 *** . 0 0 3 5 3 6 . 0 0 4 0 7 7 . 0 0 9 0 2 4 . 0 0 6 2 9 7 3 M . 0 0 3 8 1 7 . 0 0 2 1 6 9 . 0 0 6 7 5 6 1 *** . 0 0 3 1 4 3 . 0 0 2 9 1 2 . 0 0 8 3 3 0 . 0 0 5 4 2 8 ** Blood samples hemolysed and t h e r e f o r e plasma r a d i o a c t i v i t y c ould not, be determined. *** Catheters q u i t and hence no blood samples were c o l l e c t e d . 206 Appendix 4 . Plasma Se s p e c i f i c a c t i v i t y ( f r a c t i o n of dose/ug Se) changes i n Se p o s i t i v e and d e f i c i e n t fetuses f o l l o w i n g maternal t r a c e r i n j e c t i o n . Time a f t e r Se p o s i t i v e fetuses Se d e f i c i e n t f e t u s e s maternal t r a c e r i n j e c t i o n 9 7 7 9 8 2 8 1 8 8 4 0 58 7 0 7 7 2 5 2 min . 0 0 0 0 2 7 . 0 0 0 0 0 9 . 0 0 0 0 1 4 . 0 0 0 0 0 5 . 0 0 0 0 2 0 . 0 0 0 0 0 5 . 0 0 0 0 1 0 5 n . 0 0 0 0 2 1 . 0 0 0 0 0 9 . 0 0 0 0 1 6 . 0 0 0 0 0 6 . 0 0 0 0 2 2 . 0 0 0 0 0 7 . 0 0 0 0 1 2 1 0 it . 0 0 0 0 2 0 . 0 0 0 0 1 1 . 0 0 0 0 1 3 . 0 0 0 0 0 8 . 0 0 0 0 2 5 . 0 0 0 0 0 5 . 0 0 0 0 1 2 1 5 n . 0 0 0 0 2 3 . 0 0 0 0 1 1 . 0 0 0 0 1 5 . 0 0 0 0 0 7 . 0 0 0 0 4 5 . 0 0 0 0 0 6 . 0 0 0 0 1 2 3 0 H . 0 0 0 0 2 0 . 0 0 0 0 1 4 . 0 0 0 0 2 3 . 0 0 0 0 0 9 . 0 0 0 0 6 3 . 0 0 0 0 0 5 . 0 0 0 0 1 3 4 5 n . 0 0 0 0 2 7 . 0 0 0 0 1 5 . 0 0 0 0 2 4 . 0 0 0 0 1 2 . 0 0 0 0 3 6 . 0 0 0 0 0 6 . 0 0 0 0 1 5 6 0 n . 0 0 0 0 3 3 . 0 0 0 0 1 4 . 0 0 0 0 3 1 . 0 0 0 0 1 1 . 0 0 0 0 3 5 . 0 0 0 0 0 8 . 0 0 0 0 1 6 2 hr . 0 0 0 0 3 2 . 0 0 0 0 1 8 . 0 0 0 0 3 7 . 0 0 0 0 1 3 . 0 0 0 0 3 9 . 0 0 0 0 0 9 . 0 0 0 0 2 0 3 ft . 0 0 0 0 3 8 . 0 0 0 0 1 9 . 0 0 0 0 2 8 . 0 0 0 0 1 3 . 0 0 0 0 3 8 . 0 0 0 0 0 7 . 0 0 0 0 2 2 4 n . 0 0 0 0 4 7 . 0 0 0 0 2 3 . 0 0 0 0 3 1 . 0 0 0 0 1 4 . 0 0 0 0 4 2 . 0 0 0 0 0 9 . 0 0 0 0 1 7 5 n . 0 0 0 0 5 1 . 0 0 0 0 2 5 . 0 0 0 0 3 2 . 0 0 0 0 1 3 ** . 0 0 0 0 1 0 . 0 0 0 0 1 9 6 n . 0 0 0 0 4 4 . 0 0 0 0 2 7 . 0 0 0 0 3 8 . 0 0 0 0 1 5 . 0 0 0 0 5 1 . 0 0 0 0 0 9 . 0 0 0 0 1 5 1 d . 0 0 0 0 9 6 . 0 0 0 0 3 8 . 0 0 0 0 6 6 . 0 0 0 0 2 8 . 0 0 0 1 0 2 . 0 0 0 0 1 9 . 0 0 0 0 2 0 2 n . 0 0 0 0 9 9 . 0 0 0 0 5 2 . 0 0 0 0 4 9 . 0 0 0 0 3 4 . 0 0 0 1 2 2 . 0 0 0 0 3 0 . 0 0 0 0 4 6 3 tt . 0 0 0 0 9 4 . 0 0 0 0 4 6 . 0 0 0 0 4 4 . 0 0 0 0 3 0 . 0 0 0 1 1 5 . 0 0 0 0 2 3 . 0 0 0 0 3 8 1 ** Plasma ~*^Se r a d i o a c t i v i t y could not be determined as a r e s u l t of hemolysis. 207 Appendix 5. Plasma Se s p e c i f i c a c t i v i t y ( f r a c t i o n of dose/ug Se) changes i n Se p o s i t i v e and d e f i c i e n t ewes fo l l o w i n g f e t a l t r a c e r i n j e c t i o n . Time a f t e r f e t a l Se p o s i t i v e ewes Se d e f i c i e n t ewes t r a c e r i n j e c t i o n 572 587 654 665 707 724 909 1000 2 min .000007 .000003 .000003 ** .000005 .000208 .000052 .000070 5 " .000006 ** .000004 .000019 .000007 .000201 .000057 .000084 10 " .000007 .000003 .000004 .000015 ** .000196 .000054 .000074 15 " .000009 .000004 .000004 .000011 .000008 .000272 .000049 .000081 30 " .000009 .000003 .000006 .000012 .000009 .000213 .000056 .000074 45 H .000009 .000002 .000004 .000010 .000016 .000265 .000078 .000079 60 " .000012 .000005 .000014 .000017 .000017 .000251 .000064 .000092 2 hr .000015 .000007 .000020 .000023 .000050 .000324 .000084 .000125 3 " .000014 .000009 .000031 .000028 .000046 .000353 .0001B7 .000179 4 " .000024 .000013 .000032 .000032 ** .000466 .000151 .000165 5 " .000032 .000013 .000051 .000034 ** .000473 .000122 .000171 6 " .000040 .000014 .000061 .000034 .000087 .000467 .000112 .000163 1 d ** .000027 .000080 .000053 .000117 .000602 .000428 .000283 2 " .000059 .000026 ** .000052 .000110 .000658 .000293 .000433 3 " .000054 .000019 .000056 .000046 .000101 .000612 .000151 .000297 Blood samples hemolysed and therefore plasma r a d i o a c t i v i t y could not be determined. Catheters q u i t and hence no blood samples were c o l l e c t e d . Appendix 6. Tissue Se s p e c i f i c a c t i v i t y (fraction of dose/ug Se/whole tissue) of the Se positive and def i c i e n t nonpregnant ewes. Se positive ewes Se d e f i c i e n t ewes Tissue 622(1) 65(5) 99(9) 100(14) 999(1) 998(5) 123(9) 325(14) Liver .063357 .073988 .040981 .037820 .145598 .155491 .138462 .028950 Kidney .065668 .039201 .020142 .017459 .342729 .142651 .240378 .069782 Skeletal muscle .083720 .190352 .140608 .155131 .412112 .516230 .574994 .216103 Heart .010119 .007381 .006369 .006235 .022100 .015792 .030706 .008443 Lung .025288 .029269 .017851 .012962 .081880 .083359 .117573 .026096 Spleen .007541 .008367 .006853 .028620 .021847 .033451 .037472 .004938 Pancreas .004889 .002902 .002234 .002235 .009994 .010538 .012921 .003168 Mammary gland .005920 .005085 .002561 .003332 .005559 .002580 .015768 .006900 Day of s a c r i f i c e after tracer injection i s indicated in brackets against each animal. * Weight of skeletal muscle was assumed to be 40% of body weight (Arnal, 1977) . 7 5 Appendix 7 . Tissue Se sp e c i f i c a c t i v i t y (fraction of dose/ug Se/whole tissue) of the Se positive and def i c i e n t pregnant ewes. Se positive ewes Se d e f i c i e n t ewes Tissue 9 8 2 ( 6 ) 840 (14 ) 977 (18 ) 8 1 8 ( 4 1 ) 5 8 ( 4 ) 7 0 7 ( 6 ) 7 2 5 ( 1 2 ) Liver . 080844 .063958 .040817 .017735 . 2 2 3 5 5 7 . 0 8 4 2 2 7 . 0 6 7 0 9 5 Kidney . 061905 .023028 .024614 .006134 . 3 1 8 8 6 3 . 1 2 7 4 8 5 . 0 8 1 2 6 4 Skeletal muscle . 169488 .097120 .088673 .093744 . 3 3 1 8 7 3 . 2 4 4 7 3 7 . 2 3 9 9 0 4 Heart . 008092 .006488 .006633 .003143 . 0 1 4 8 4 3 . 0 1 4 5 6 5 . 0 0 8 3 9 6 Lung . 037652 .026950 .015724 .005833 . 0 9 5 3 7 7 . 0 5 9 2 6 4 . 0 3 5 6 2 2 Spleen . 011239 .005555 .010674 .005934 . 0 2 6 6 0 6 . 0 2 6 1 8 1 . 0 0 9 9 8 3 Day of s a c r i f i c e after tracer injection i s indicated i n brackets against each animal. * Weight of skeletal muscle was assumed to be 40% of body weight (Arnal, 1 9 7 7 ) . Ewe #818 gave b i r t h to l i v e , healthy twin lambs and was l a c t a t i n g at the time of s a c r i f i c e . Appendix 8 . Tissue Se spe c i f i c a c t i v i t y (fraction of dose/ug Se/whole tissue) of the Se positive and def i c i e n t fetuses. Tissue Se positive fetuses 6 5 4 ( 7 ) 587(9) 572(23) Se d e f i c i e n t fetuses 9 0 9 ( 5 ) 1 0 0 0 ( 7 ) 7 2 4 ( 2 1 ) Liver Kidney . 167627 .757988 Skeletal . 182707 muscle Heart . 024250 .003899 .165513 .031110 .004042 .015078 .187594 .028298 .003546 .128048 . 0 9 7 4 2 1 . 0 2 5 1 1 2 . 0 2 6 6 6 9 . 0 1 3 6 3 1 . 0 0 9 8 3 7 .223293 . 2 7 7 5 0 6 . 0 5 7 1 7 4 . 009917 . 0 0 9 0 1 5 . 0 0 3 7 6 8 Day of s a c r i f i c e after tracer injection i s indicated in brackets against each animal. Tissues from fetuses 6 6 5 , 707 were not col l e c t e d . * Weight of skeletal muscle was assumed to be 40% of body weight (Arnal, 1 9 7 7 ) . 

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