UBC Theses and Dissertations

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UBC Theses and Dissertations

Dietary effects on the levels of serum cholesterol and serum total lipids in the growing chick Haqq, Samuel Ainul 1961

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DIETARY EFFECTS ON THE LEVELS OF SERUM CHOLESTEROL AND SERUM TOTAL LIPIDS IN THE GROWING CHICK by SAMUEL AINUL HAQQ B.S.A., University of British Columbia, 1957 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in Agriculture in the Department of Poultry Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1961 In presenting this thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t 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 representatives. It i s understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of / (x*~-^- f~^*-j ^€L/ C A ^ eg The University of British Columbia, Vancouver 8 , Canada. Date ( i i ) ABSTRACT An i n v e s t i g a t i o n was c a r r i e d o u t i n t o t h e e f f e c t s o f some d i e t a r y f a c t o r s on t h e serum c h o l e s t e r o l and serum t o t a l l i p i d l e v e l s i n b o t h n o r m a l and h y p e r t h y r o i d c h i c k s . D i e t a r y f a c t o r s i n v e s t i g a t e d were t h e e f f e c t s o f two l e v e l s o f p r o t e i n , two l e v e l s o f f a t , and two l e v e l s o f v i t a m i n s . B a s a l d i e t s were f e d a t e i t h e r a 20% o r 26% p r o t e i n l e v e l and when d i e t a r y f a t was i n v e s t i g a t e d t h e d e x t r o s e o f t h e b a s a l d i e t was s u b s t i t u t e d f o r a h y d r o g e n a t e d v e g e t a b l e o i l t o make up 12% o f t h e d i e t . The v i t a m i n s u p p l e m e n t i n t h e h i g h v i t a m i n d i e t s f e d c o n s i s t e d o f a d d i t i o n a l amounts o f t h e f o l l o w i n g B complex v i t a m i n s : c h o l i n e c h l o r i d e , c a l c i u m p a n t h o t h e n a t e , f o l a c i n , n i a c i n and r i b o f l a v i n . C h i c k s were r e n d e r e d h y p e r t h y r o i d b y f e e d i n g d i e t s c o n -t a i n i n g 0 . 0 2 $ i o d i n a t e d c a s e i n . H y p o t h y r o i d i s m was i n d u c e d b y t h e f e e d i n g o f 0.1% t h i o u r a c i l . N o r m a l c h i c k s showed l o w e r l e v e l s o f serum c h o l e s t e r o l and serum t o t a l l i p i d s when t h e y were f e d b a s a l d i e t s c o n -s i s t i n g o f 26% p r o t e i n l e v e l t h a n when f e d b a s a l d i e t s c o n -s i s t i n g o f a 20% p r o t e i n l e v e l . N o r m a l c h i c k s f e d h i g h f a t d i e t s showed h i g h e r l e v e l s o f serum c h o l e s t e r o l and serum t o t a l l i p i d s t h a n n o r m a l c h i c k s f e d low f a t d i e t s . N o r m a l c h i c k s f e d d i e t s low i n t h e B complex v i t a m i n s showed h i g h e r l e v e l s o f serum c h o l e s t e r o l t h a n n o r m a l c h i c k s f e d d i e t s h i g h i n t h e B complex v i t a m i n s . ( i i i ) The growth rate of chicks rendered hyperthyroid varied. In many instances hyperthyroid chicks grew at a significantly faster rate than normal chicks while in some instances no differences were noted. In some cases chicks rendered hyperthyroid showed depressed growth rates. No clear ex-planation could be given for such an effect on the growth rate of hyperthryoid chicks. It seems reasonable, however, to suspect that seasonal changes may affect thyroid activity and consequently the growth rate of the chicks in question. No differences were noted between the serum cholesterol and serum l i p i d levels from hyperthryoid chicks fed the basal diets containing 20%, and 26% protein. Hyperthyroid chicks, however, showed lower levels of serum cholesterol than normal chicks when the diet fed was low in the B complex vitamins, calcium panthothenate, choline chloride, folacin, niacin and riboflavin. The effect on the serum cholesterol and serum total l i p i d s when the chicks were rendered hyperthyroid varied. The results suggest some interaction between thyroid state and diet on the serum cholesterol and serum total l i p i d s . (iv) ACKNOWLEDGMENT The writer wishes to thank Professor Jacob Biely, Chairman and Head of the Department of Poultry Science for providing the f a c i l i t i e s used i n this study and for his encouragement and interest throughout the course of thi's work. To Mrs. Beryl E. March of the Department of Poultry Science, the writer would like to express his appreciation for the supervision, planning and execution of the experi-ments. He would also l i k e to express his thanks to many others for their help and useful suggestions. (v) TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF LITERATURE 3 MATERIALS AND METHODS 10 EXPERIMENT 1 17 Results - 18 Tables 22 EXPERIMENT 2 26 Results 27 Tables 30 EXPERIMENT 3 - 33 Results • 34-Tables 37 EXPERIMENT h • [fO Results H-2 Tables M-7 EXPERIMENT 5 • 50 Results 52 Tables 55 EXPERIMENT 6 58 Results 59 Tables 62 EXPERIMENT 7 65 Results 66 Tables 68 EXPERIMENT 8 73 Results 7h Tables 77 Diagrams "1 GENERAL DISCUSSION 87 Diagrams 100 GENERAL CONCLUSIONS 107 BIBLIOGRAPHY 109 INTRODUCTION T h e r e e x i s t s e x t e n s i v e l i t e r a t u r e on t h e e f f e c t s o f d i e t on t h e serum c h o l e s t e r o l l e v e l o f v a r i o u s a n i m a l s p e c i e s . I t i s an e s t a b l i s h e d f a c t t h a t a d m i n i s t r a t i o n o f f a t i n t h e d i e t a f f e c t s s erum c h o l e s t e r o l l e v e l . I n g e n e r a l , u n s a t u r a t i o n o f th e f a t has been a s s o c i a t e d w i t h l o w e r i n g o f t h e serum c h o l e s t e r o l v a l u e s . A l s o l a r g e q u a n t i t i e s o f s a t u r a t e d f a t s i n t h e a n i m a l s ' d i e t have been a s s o c i a t e d w i t h h i g h l e v e l s o f c h o l e s t e r o l i n i t s b l o o d serum. R e c e n t i n v e s t i g a t i o n s have d e m o n s t r a t e d an i n c r e a s e i n t h e s e r u m c h o l e s t e r o l l e v e l s o f c h i c k s when c o r n o i l ( a n u n s a t u r a t e d o i l ) has been i n c l u d e d i n t h e c h i c k s ' d i e t . I n v i e w o f t h e s e f i n d i n g s i t c o u l d no l o n g e r be g e n e r a l l y s a i d t h a t u n s a t u r a t e d f a t s a r e a s s o c i a t e d w i t h low l e v e l s o f se r u m c h o l e s t e r o l i n a l l a n i m a l s p e c i e s . I t a p p e a r s as has been s u g g e s t e d b y some i n v e s t i g a t o r s t h a t some f a c t o r , o t h e r t h a n t h e d e g r e e o f s a t u r a t i o n o f a f a t , i s r e s p o n s i b l e f o r i n f l u e n c i n g t h e serum c h o l e s t e r o l l e v e l o f some a n i m a l s p e c i e s . I t has a l s o been e s t a b l i s h e d t h a t an a d e q u a t e p r o t e i n l e v e l i n an a n i m a l ' s d i e t i s an i m p o r t a n t f a c t o r i n m a i n -t a i n i n g n o r m a l l e v e l s o f serum c h o l e s t e r o l i n t h e a n i m a l . B y i n c r e a s i n g t h e p r o t e i n l e v e l o f t h e d i e t , a d e c r e a s e i n serum c h o l e s t e r o l l e v e l c a n be p r o d u c e d . The f u n c t i o n o f t h e t h y r o i d g l a n d has been shown t o i n f l e u n c e l i p i d m e t a b o l i s m i n a n i m a l s p e c i e s . The g e n e r a l t r e n d a p p e a r s t o be t h a t l o w e r serum c h o l e s t e r o l l e v e l s a r e a s s o c i a t e d w i t h h y p e r t h y r o i d i s m itfhereas h y p o t h y r o i d i s m i s a s s o c i a t e d w i t h e l e v a t e d s e r u m c h o l e s t e r o l l e v e l s . I n g e n e r a l , t h e I n f l u e n c e o f v i t a m i n s on t h e serum c h o l e s t e r o l l e v e l o f c h i c k s i s u n c e r t a i n as t h e r e a p p e a r s t o be v e r y l i t t l e work done i n t h i s f i e l d . A number o f v i t a m i n d e f i c i e n c y s t a t e s a r e a s s o c i a t e d w i t h i n a n i t i o n and i t i s d i f f i c u l t t o d i f f e r e n t l a t e between a d i r e c t e f f e c t "Of v i t a m i n d e f i c i e n c y o r an i n d i r e c t e f f e c t due t o i n -a n i t i o n . I n v i e w of t h e above f i n d i n g s on t h e e f f e c t o f d i e t and t h y r o i d a c t i v i t y , on serum c h o l e s t e r o l l e v e l s i n c e r t a i n a n i m a l s p e c i e s , i t seemed o f i n t e r e s t t o s t u d y t h e e f f e c t s o f c e r t a i n d i e t a r y f a c t o r s on the serum c h o l e s t e r o l l e v e l s o f n o r m a l a n d h y p e r t h y r o i d c h i c k s . - 3 -LITERATURE REVIEW T h e r e i s a l a r g e volume o f e v i d e n c e s u g g e s t i n g t h a t d i e t has an e f f e c t on serum l i p i d l e v e l s . The c o n c e p t t h a t t h e r e may e x i s t a r e l a t i o n s h i p between l i p i d s and a t h e r o s c l e r o s i s has been e n t e r t a i n e d f o r o v e r f i f t y y e a r s , e v e r s i n c e r a b b i t a r t e r i a l l e s i o n s were p r o d u c e d by f e e d i n g c h o l e s t e r o l . A l -t h o u g h t h i s a s s o c i a t i o n o f l i p i d m e t a b o l i s m and a t h e r o s c l e r o s i s a p p e a r s t o be w e l l e s t a b l i s h e d , i t i s s t i l l n ot c e r t a i n w h e t h e r t h e a s s o c i a t i o n i s one o f cause and e f f e c t . I t s h o u l d be p o i n t e d out t h a t many f a c t o r s a f f e c t body l i p i d m e t a b o l i s m a s i d e f r o m d i e t . Age, s e x , h e r e d i t y , s t r e s s and o t h e r v a r i a b l e s have been shown t o a f f e c t l i p i d m e t a b o l i s m . D i e t s r i c h i n f a t b u t not n e c e s s a r i l y i n c h o l e s t e r o l have been- t h o u g h t t o be a s s o c i a t e d i n man w i t h h i g h l e v e l s o f p l a s m a c h o l e s t e r o l . Keys and h i s a s s o c i a t e s (1952) have been t h e c h i e f p r o p o n e n t o f t h i s c o n c e p t , a l t h o u g h r e c e n t l y Keys e t a l (1957) p r e s e n t e d d a t a t o show t h a t t h e t y p e as w e l l as t h e q u a n t i t y o f f a t may p l a y a r o l e i n d e t e r m i n i n g t h e l e v e l s o f c h o l e s t e r o l i n t h e serum o f b l o o d . P r i o r t o t h i s , a n i m a l and v e g e t a b l e f a t s were r e g a r d e d as i n t e r c h a n g e a b l e i n s o f a r as t h e i r e f f e c t on t h e serum c h o l e s t e r o l was c o n c e r n e d . W i t h f u r t h e r n u t r i t i o n a l s t u d i e s some s c i e n t i s t s have come t o a s s o c i a t e h i g h e r l e v e l s o f serum c h o l e s t e r o l w i t h t h e i n t a k e o f a n i m a l o r s a t u r a t e d f a t t y a c i d s , and t h e l a c k o f un-s a t u r a t e d f a t t y a c i d s . Among them a r e A h r e n s e t a l (1957)> Malmros and Wigand (1957). I n man, B e v e r i d g e e t a l (1955) have shown s t r o n g e v i d e n c e i n d i c a t i n g t h a t i n t a k e o f l a r g e q u a n t i t i e s o f - k -v e g e t a b l e f a t o r u n s a t u r a t e d o i l s i n t h e d i e t r e d u c e s t h e p l a s m a c h o l e s t e r o l s i g n i f i c a n t l y , a nd t h i s f a l l can be o v e r -come o r r e v e r s e d b y t h e s i m u l t a n e o u s f e e d i n g o f s i m i l a r amounts o f a n i m a l f a t s o r h y d r o g e n a t e d v e g e t a b l e o i l s . I n e x p e r i m e n t s u s i n g m ature c o c k e r e l s , K o k a t n u r e t a l (1958) have c o n c l u d e d t h a t serum c h o l e s t e r o l v a l u e s were i n -c r e a s e d more r a p i d l y i n c h i c k e n s w h i c h have been f e d f a t t h a n t h o s e f e d an e s s e n t i a l l y f a t - f r e e d i e t . As e a r l y as 1912 S t u c k e y c a r r i e d out c e r t a i n e x p e r i m e n t s f r o m w h i c h he was a b l e t o d i s c o v e r t h a t t h e p o r t i o n o f t h e a n i m a l s d i e t r e s p o n s i b l e f o r t h e a p p e a r a n c e o f l e s i o n s i n i t s b l o o d v e s s e l s was t h e l i p i d p o r t i o n and not the p r o t e i n p o r t i o n . Prom h i s e x p e r i m e n t s he n o t e d t h a t t h e egg y o l k f a t was r e s p o n s i b l e f o r the l e s i o n s p r o d u c e d i n t h e a n i m a l s ' b l o o d v e s s e l s . Newburg and C l a r k s o n (1922) however, were o f a d i f f e r e n t o p i n i o n , f o r t h e y were a b l e t o p r o d u c e a t h e r o s c l e r o s i s i n r a b b i t s b y f e e d i n g d i e t s r i c h i n p r o t e i n . T h e y a c h i e v e d t h i s c o n d i t i o n i n r a b b i t s b y f e e d i n g p r o t e i n l e v e l s o f 27% and 36%. Meeker a n d K e s t e n (19i|0) p r o d u c e d h y p e r c h o l e s t e r e m i a i n r a b b i t s b y f e e d i n g t h e s e a n i m a l s h i g h p r o t e i n d i e t s . I n d o g s , h y p e r c h o l e s t e r e m i a was p r o d u c e d b y L i , Hough, Monahan and Freeman (19^3) when t h e y f e d dogs a h i g h f a t c h o l e s t e r o l d i e t d e f i c i e n t i n p r o t e i n . An i n c r e a s e i n serum c h o l e s t e r o l o f 172$ t o 225$ was a c h i e v e d . When t h e y f e d a d i e t c o n -t a i n i n g f a t a n d c h o l e s t e r o l w i t h a d e q u a t e amounts o f p r o t e i n , - 5 -serum c h o l e s t e r o l c o n c e n t r a t i o n ^ a l t h o u g h i n c r e a s e d , was not i n c r e a s e d t o t h e e x t e n t i t w o u l d have been i f t h e p r o t e i n s u p p l i e d was i n a d e q u a t e . I n c r e a s e s i n s e r u m c h o l e s t e r o l l e v e l o f dogs when f e d (a) H i g h f a t d i e t s w i t h added c h o l e s t e r o l ; and (b) H i g h f a t d i e t s , d e f i c i e n t i n p r o t e i n , w i t h o r w i t h o u t c h o l e s t e r o l were a g a i n a c h i e v e d b y L i and Freeman (19i|6). Moyer, K r i t c h e v s k y , Logan a n d Cox (195°) r e p o r t e d a p r o g r e s s i v e d r o p i n serum c h o l e s t e r o l l e v e l s o f r a t s b y i n -c r e a s i n g t h e p r o t e i n c o n t e n t o f t h e d i e t . T h e y u s e d c a s e i n as t h e i r p r i n c i p a l p r o t e i n o f t h e d i e t and f e d i t a t f o u r l e v e l s i n a d d i t i o n t o 0.5$ c h o l i n e c h l o r i d e . I n a n o t h e r e x p e r i m e n t I s o l a t e d s o y a p r o t e i n was us e d a t l e v e l s o f 10$, i].0$, a n d 60$ and w i t h a l l t h e i r d i e t s c o n t a i n i n g 25$ l a r d , 5$ H.M.W. s a l t m ix, 1$ c h o l i c a c i d a n d 2$ c h o l e s t e r o l . F i l l i o s e t a l (1956) r e p o r t e d a s i m i l a r d r o p i n serum c h o l e s t e r o l c o n c e n t r a t i o n i n r a t s f e d a h i g h p r o t e i n l e v e l . T h e y f o u n d t h a t when t h e d i e t s o f 10$, 20$, and 60$ c a s e i n , and 20$ a l p h a p r o t e i n were f e d t o r a t s , t h e r e was a s i g n i f i c a n t d r o p i n serum c h o l e s t e r o l c o n c e n t r a t i o n o f t h e s e r a t s a f t e r b e i n g f e d a 20$ c a s e i n r a t i o n f o r a p e r i o d o f f i f t y - s i x d a y s . The g r o u p o f r a t s f e d 60$ c a s e i n d i e t s d i s p l a y e d c h o l e s t e r e m i c r e s p o n s e s w h i c h were s i g n i f i c a n t l y l o w e r t h a n any o f t h e t h r e e o t h e r g r o u p s . T h e s e w o r k e r s c o n c l u d e d f r o m t h e i r ex-p e r i m e n t s t h a t t h e h i g h e r t h e p r o t e i n l e v e l i n t h e d i e t , t h e l o w e r t h e b l o o d c h o l e s t e r o l l e v e l o f t h e a n i m a l s f e d t h e s e - 6 -d i e t s . J o n e s and Huffman (195°) d e m o n s t r a t e d i n r a t s t h a t r e -d u c t i o n o r e l e v a t i o n o f d i e t a r y c a s e i n b e y o n d a modest r a n g e (12-18$) w i l l l e a d t o u l t i m a t e e l e v a t i o n o f serum c h o l e s t e r o l a n d p h o s p h o l i p i d . T h e y o b t a i n e d u n u s u a l l y h i g h l e v e l s o f serum c h o l e s t e r o l i n o l d r a t s f e d 1*0$ c a s e i n . I n mature W h i t e L e g h o r n c o c k e r e l s K o k a t n u r , Rand, Kummerow and S c o t t (1958) r e p o r t e d a d r o p i n serum c h o l e s t e r o l l e v e l w i t h an i n c r e a s e i n p r o t e i n l e v e l . T h e s e b i r d s (12-18 weeks o l d ) were f e d d i e t s c o n t a i n i n g 7-5$* 15$, o r 30$ p r o t e i n and 0.1$ o r 15$ c o r n o i l t h r o u g h o u t an e x p e r i m e n t a l p e r i o d o f tw e n t y - o n e d a y s . The l a r g e s t i n c r e a s e i n se r u m c h o l e s t e r o l was n o t e d i n b i r d s w h i c h h a d consumed t h e l e a s t amount o f p r o t e i n . T h e r e seemed t o be no a p p a r e n t r e l a t i o n s h i p between t h e s e r u m c h o l e s t e r o l l e v e l s and d i f f e r e n c e s i n f e e d i n t a k e o r d i f f e r e n c e s i n t h e p e r c e n t a g e o f c a l o r i e s s u p p l i e d b y d i e t a r y f a t . The serum c h o l e s t e r o l l e v e l s o f h y p e r -c h o l e s t e r m i c c o c k e r e l s d r o p p e d r a p i d l y d u r i n g t h e t h r e e - w e e k e x p e r i m e n t a l p e r i o d . T h i s d e c r e a s e d i d not r e a c h t h e n o r m a l v a l u e i n t h i s p e r i o d o f t i m e u n l e s s t h e p r o t e i n l e v e l o f t h e d i e t was h i g h . I n New Hampshire f o u r - w e e k o l d c h i c k s , N i s h i d a , T a k e n a k a and Kummerow (1958) a l s o d e m o n s t r a t e d a d e c r e a s e i n serum c h o l e s t e r o l l e v e l when the p r o t e i n l e v e l o f th e d i e t f e d was h i g h . T h e s e w o r k e r s f e d p r o t e i n l e v e l s o f 15$, 20$, a n d 30$ t o c h i c k s , a n d c o n c l u d e d t h a t a h i g h p r o t e i n l e v e l i n c o m b i n a t i o n w i t h h e a t e d o i l was s u c c e s s f u l i n l o w e r i n g t h e ser u m c h o l e s t e r o l l e v e l o f t h e c h i c k s . F i l l i o s e t a l (1951|) s u g g e s t e d as a r e s u l t o f f u r t h e r e x p e r i m e n t a l work w i t h monkeys, t h a t m e t h i o n i n e i n t h e p r o t e i n o f t h e d i e t i s an i m p o r t a n t f a c t o r i n t h e r e g u l a t i o n o f c h o l e s t e r m i a i n t h e s e monkeys. They were n o t a b l e t o c o n -f i r m t h i s s t a t e i n r a t s , f o r a l t h o u g h h y p e r c h o l e s t e r e m i a was n o t e d when r a t s i^ere f e d a p r o t e i n d i e t d e f i c i e n t i n t h e s u l p h u r amino a c i d m e t h i o n i n e , t h e y were a b l e t o p r e v e n t an i n c r e a s e d h y p e r c h o l e s t e r m i a , o n l y p a r t i a l l y , by t h e s u p p l e -m e n t a t i o n o f d l - . m e t h i o n i n e . I n young c h i c k s , when h y p e r -c h o l e s t e r e m i a r e s u l t e d f r o m s u b o p t i m a l p r o t e i n i n t a k e s J o h n s o n e t a l (1958) d e m o n s t r a t e d t h a t b y s u p p l e m e n t i n g t h e d e f i c i e n t p r o t e i n w i t h amino a c i d s s u c h t h a t more p r o t e i n c o u l d be made a v a i l a b l e t o t h e c h i c k s , p r o t e i n d e f i c i e n c y was a l l e v i a t e d a n d n o r m a l l e v e l s o f p l a s m a c h o l e s t e r o l were o b s e r v e d . R e c e n t l y , M a r c h and B i e l y (1959) r e p o r t e d l o w e r l e v e l s o f serum c h o l e s t e r o l i n f i v e - w e e k o l d W h i t e L e g h o r n c o c k e r e l s f e d a 26$ p r o t e i n d i e t t h a n s i m i l a r c h i c k s f e d a 20$ p r o t e i n d i e t . I n a n i t i o n i s i n v o l v e d i n a number o f v i t a m i n d e f i c i e n c y s t a t e s and f o r t h i s r e a s o n i t i s o f t e n d i f f i c u l t t o d i f f e r -e n t i a t e between a d i r e c t e f f e c t o f v i t a m i n d e f i c i e n c y and an i n d i r e c t e f f e c t r e s u l t i n g f r o m i n a n i t i o n . The h y p e r c h o l e s t e r e m i a r e s u l t i n g f r o m i n a n i t i o n o r s t a r v a t i o n i n v a r i o u s l a b o r a t o r y a n i m a l s i s p r o b a b l y due t o a d e p r e s s i o n i n c h o l e s t e r o l s y n t h e s i s . Any r i s e In b l o o d c h o l e s t e r o l l e v e l may r e f l e c t i n c r e a s e d m o b i l i z a t i o n o f f a t r e s e r v e s . - 8 -R i n e h a r t a nd G r e e n b e r g ( I 9 J 4 9 ) n o t e d an e l e v a t e d p l a s m a c h o l e s t e r o l l e v e l i n p y r i d o x i n e d e f i c i e n t monkeys. I n a s e p a r a t e e x p e r i m e n t when t h e d i e t was c h a n g e d t o one o f h i g h f a t , b o t h t h e c o n t r o l a n d t h e p y r i d o x i n e d e f i c i e n t monkeys showed i n c r e a s e d l e v e l s o f serum c h o l e s t e r o l w i t h 1% a d d e d c h o l e s t e r o l i n t h e i r d i e t s . As a r e s u l t 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 h i g h f a t d i e t may have i n c r e a s e d a b s o r p t i o n o f t h e c h o l e s t e r o l . R i n e h a r t a nd G r e e n b e r g w o r k i n g w i t h monkeys and M u s c h e t t a n d Emerson (1956) w o r k i n g w i t h dogs d e m o n s t r a t e d a r t e r i o s c l e r o s i s c a u s e d b y p y r i d o x i n e d e f i c i e n c y b u t d i d not however measure t h e c h o l e s t e r o l c on--r c e n t r a t i o n i n t h e s e a n i m a l s . M i t t e n a n d H o l l m a n ( 1 9 5 2 ) s t u d y i n g t h e i n t e r - r e l a t i o n s h i p between p y r i d o x i n s a n d e s s e n t i a l f a t t y a c i d s i n c o r r e c t i n g t h e syndrome p r o d u c e d b y p y r i d o x i n e d e f i c i e n c y , f o u n d t h a t p y r i d o x i n e a nd l i n o l e a t e c o r r e c t e d a l l a s p e c t s o f t h e d e f i c i e n c y syndrome. In t h e c a s e o f v i t a m i n A a number o f w o r k e r s have d e m o n s t r a t e d h y p o l i p e m i a w i t h h i g h v i t a m i n A i n t a k e . W h i l e S m i t h ( 1 9 3 i |) f o u n d an i n c r e a s e i n b l o o d f a t t y a c i d s and c h o l e s t e r o l i n d e f i c i e n t r a t s . S u r e e t a l (1933) a n d G r e e n e t a l ( 1 9 5 5 ) f o u n d no s i g n i f i c a n t d i f f e r e n c e s i n b l o o d f a t t y a c i d s , c h o l e s t e r o l and p h o s p h o l i p i d s i n v i t a m i n A d e f i c i e n t a l b i n o r a t s . Van b r u g g e n e t a l (19l|.8) r e p o r t e d an i n c r e a s e d b l o o d c h o l e s t e r o l l e v e l w i t h a h i g h i n t a k e of v i t a m i n A i n humans. S i m i l a r r e s u l t s were r e p o r t e d b y R a l l i a n d M a t e r h o u s e ( 1 9 3 3 ) i n v i t a m i n d e f i c i e n t d o g s . M e i t z e l e t a l (1956) gave o r a l - 9 -d o s e s o f v i t a m i n A t o o l d a r t h e r o s c l e r o t i c h e n s , and r e p o r t e d o n l y s l i g h t changes i n the serum c h o l e s t e r o l o f t h e s e h e n s . Wood (I960) r e p o r t e d t h a t b o t h t h e a l c o h o l and a c e t a t e f o r m o f v i t a m i n A when added t o the c h i c k s d i e t , s i g n i f i c a n t l y l o w e r e d t h e serum c h o l e s t e r o l c o n c e n t r a t i o n o f c h o l e s t e r o l f e d W hite L e g h o r n c h i c k s . A l t s c h u l e t a l (1955) w o r k i n g w i t h humans, and P a r s o n s e t a l (1956, 1957) w o r k i n g w i t h r a b b i t s , r e p o r t e d d e c r e a s e d b l o o d c h o l e s t e r o l l e v e l s w i t h o r a l a d m i n i s t r a t i o n s o f n i c o t i n i c a c i d . W i t h r e g a r d t o t h e e f f e c t o f ' t h e B-complex g r o u p o f v i t a m i n s on t h e l e v e l o f serum c h o l e s t e r o l i n c h i c k e n s , S t a m l e r e t a l (1958) and P i c k e t a l (1957) r e p o r t e d t h e p o s s i b l e e x i s t e n c e of a n i n t e r - r e l a t i o n s h i p between t h e v i t a m i n s , f a t s , a n d c h o l e s t e r o l . The s e r u m c h o l e s t e r o l l e v e l s o f m a t u r e c o c k e r e l s f e d h i g h p r o t e i n , v i t a m i n B d e f i c i e n t d i e t s were not a f f e c t e d , but when c o c k e r e l s were f e d v i t a m i n B d e f i c i e n t d i e t s , low i n p r o t e i n , i n t e n s i f i e d h y p e r c h o l e s t e r e m i a was p r o d u c e d . Hsu and Chow (1957) r e p o r t e d h i g h e r l e v e l s o f s e r u m c h o l e s t e r o l i n e i g h t New Hampshire c h i c k s f e d h i g h f a t , 12 v i t a m i n B d e f i c i e n t d i e t s , t h a n c h i c k s f e d h i g h f a t d i e t s 12. s u p p l e m e n t e d w i t h v i t a m i n B - 10 -MATERIALS AND METHODS Throughout the experiments, the same experimental con-ditions were maintained. The chicks were started on ex-perimental diets when they were one day old. They were wing-banded and inoculated intraocularly against Newcastle Disease. A l l chicks were distributed at random into groups. They were housed in electri c a l l y heated Jamesway battery brooders. They were fed and watered ad libitum. Two basal diets, the composition of which i s shown in Table I were formulated to contain 20% and 26% protein respectively. It w i l l be noted that dextrose makes up 12% of each basal diet. When fat was added, i t was substituted for the dextrose. The source of thyroactive protein used in the experiments was iodinated casein. Blood samples, taken from the wing vein of the chick were allowed to stand overnight in a refrigerator at 10°C. The blood samples were then centrifugated, for approximately five minutes at 2500 revolutions per minute, in order to f a c i l i t a t e the separation of the serum. The serum was analysed for total cholesterol by the method of Zlatkis, Zak, and Boyle (1953) and for total l i p i d s by the method of Huerga et a l (1953). The justification for using the method of Zlatkis, Zak, and Boyle for serum cholesterol determination was based on the simplicity and ease with which this method could be carried out i n the laboratory, and because i t Is less time consuming. It must be realized, however, that this method has i t s limitations (Rhodes, 1959)* In this study, the knowledge of the relative change in the levels of serum cholesterol i s of. greater importance than the knowledge of the absolute amounts. - 11 -Consequently, i t i s worthwhile to s a c r i f i c e a l i t t l e accuracy i n the absolute serum cholest e r o l determination f o r want of a simple method. The method consists of adding a fixed volume of con-centrated sulphuric acid, g l a c i a l acetic acid, and f e r r i c chloride solution to 0.1 ml. of serum i n 3.0 mis. of g l a c i a l a c e t i c acid. F u l l development of the color reaction, which i s purple, requires approximately one minute. The resultant color obeys Beer's Law and remains stable over a period of several hours. METHOD Reagents I . Standard cholesterol solution (1 mg. per m i l l i t e r ) I I . F e r r i c chloride solution. Dissolve 10 mg. of f e r r i c chloride, reagent grade, i n 100 ml. of 100$ g l a c i a l a c e t ic a c i d . I I I . Color Reagent. Dilute 2.0 mis. of the f e r r i c chloride solution to 200 ml. with C P . concentrated sulphuric a c i d . PROCEDURE (a) Determination of Total Serum Cholesterol. Pipette 0.1 ml. of serum into a dry, clean 30 ml. test tube containing 3 ml* of g l a c i a l a c e t i c a c i d . Shake test tube to disperse p r e c i p i t a t e . Add 2.0 ml. of the color reagent, by c a r e f u l l y allowing i t to flow down the side of the tube, thus producing two l a y e r s . Mix thoroughly, by s t r i k i n g the bottom of the tube sharply while holding i t at the top between the thumb and fore-finger, to eff e c t mixing and even heat d i s t r i b u t i o n . Measure the abosrbancy of the solution a f t e r i t has cooled to room temperature, and determine the serum cholesterol concentration from the standard curve described below. The absorbancy of the sample i s measured against a blank prepared by mixing together 3.0 ml. of g l a c i a l a c e t i c acid, 0.1 ml. of d i s t i l l e d water, and 2 ml. of f e r r i c chloride color reagent. The absorbancy peak i s at 560 mu. (b) Standard Curve. Pipette 0.1, 0 .2 , 0 .3 , O.h, and 0.5 ml. aliquots of the standard cholesterol solution into clean, dry 30 ml. test tubes. Dilute each aliquot to 3.0 mis. with g l a c i a l a c e t i c acid. Add 0.1 ml. of d i s t i l l e d water to each tube. Add c a r e f u l l y , 2.0 ml. of f e r r i c chloride color reagent to each tube. Mix the solution thoroughly as explained above. When the solutions are cooled to room temperature, measure t h e i r absorbancies at 560 mu i n a spectrophotometer. The standard curve as shown i n ( F i g . l ) , Is obtained by p l o t t i n g absorbancy against cholesterol concentration. THE ESTIMATION OF SERUM TOTAL LIPIDS The method presented here, for the determination of serum t o t a l l i p i d s , has the advantage of being accurate f o r the purposes required. I t i s l e s s time consuming, and requires no special equipment. This method i s based on the t u r b i d i t y , of the extracted l i p i d s , i n h% sulphuric acid solution. - 13 -Reagents I. Bloor's mixture (1 v o l . ethyl ether: 3 vols of ethyl a l c o h o l ) . I I . P Dioxan. I I I . k-% sulphuric acid. METHOD Into a test tube graduated to 10 mis. transfer 9.5 mis. of Bloor's mixture. Add dropwise 0.5 nil. serum and place the tube i n a water bath at 50 - 60°C for at l e a s t half-an-hour. After cooling to room termperature, make up the tube to the 10 ml. mark with Bloor's mixture. Centrlfugate the tube to obtain a clear supernatant. Evaporate to dryness 1 cc aliquot of the supernatant. Cool the tube, and add 1.5 mis. p Dioxan to I t . Emulsify the l i p i d s dissolved i n the p Dioxan with 5 nils, of h% sulphuric a c i d . Allow the tube to stand for t h i r t y minutes at room temperature before reading i n a colorimeter at a wavelength of 650 mu or red f i l t e r , using water as a blank. The t u r b i d i t y formed i s stable for 20 - 60 minutes aft e r addition of the sulphuric a c i d . STANDARD CURVE The standard curve i s made by determining the con-centration of t o t a l l i p i d s i n various serums gravimetrically. Extract 5 mis. of serum with 95 mis. of Bloor's mixture. After incubation f o r half-an-hour i n a water bath at 50 - 60°C v c o o l to room temperature, the volume i s adjusted to 100 mis. with Bloor's mixture. This i s now f i l t e r e d and 80 mis. i s taken and evaporated to dryness i n a water bath. The impure l i q u i d residue i s taken up i n four portions of 7 ml. each of - 11+ -petroleum ether and f i l t e r e d . After evaporation of the solvent, the purified l i p i d s are weighed to a constant weight and the amount of l i p i d s i n milligrams per 1 0 0 mis. of serum i s calculated. Prom the same alcolol ether ex-tract, 1 ml. aliquots are also taken for determination by the turbidimetric method. The optical readings obtained by the turbidimetric method i s then plotted against the concentration of total l i p i d s , as determined gravimetrically. The standard curve as shown in (Fig 2 . ) could now be drawn. - i$ -16-14-1-2-i-oH >» S o-ei o o. o 0-4-0»2-1 1 1 1 1 50 100 150 200 250 Serum total cholesterol in mg % S t a n d a r d c u r v e f o r e s t i m a t i o n - o f t o t o l c h o l e s t e r o l • 300 F i g u r e 1. - 16 -Figure 2. E X P E R I M E N T 1 Experiment 1 was designed to test the ef f e c t s of ( i ) two l e v e l s of protein; ( i i ) two l e v e l s of f a t ; ( i i i ) two l e v e l s of B complex vitamins and the i n t e r a c t i o n of these fa c t o r s , on the serum cholesterol l e v e l s , serum t o t a l l i p i d l e v e l s , and growth of chicks. The e f f e c t s of the above factors was tested both on normal chicks and on chicks i n which hyperthyroidism was Induced. Hyperthyroidism was induced i n chicks by feeding them di e t s containing 0,02$ iodinated casein. The two l e v e l s of protein were 20% and 26% as shown i n Table 1. High f a t d i e t s contained 12% hydrogenated vegetable o i l which was substituted for the dextrose i n the basal d i e t s as shown i n Table 1. To make up d i e t s with a high vitamin l e v e l , the basal di e t as shown i n Table 1 was further supplemented with l+2.9 grams of choline chloride, 0.63 grams of calcium panthothenate, 0.375 grams of f o l a c i n , 0.2 grams of r i b o f l a v i n and 1.8 grams of n i a c i n per one hundred pounds of d i e t . Twelve groups of New Hampshire cockerels were used i n t h i s experiment. Each group, consisting of twenty-two chicks, was fed an experimental d i e t as described i n Table IA. Tne chicks were weighed i n d i v i d u a l l y at seven day i n t e r v a l s up to the age of twenty-eight days. Blood samples were taken from s i x chicks selected at random i n each group, when the chicks were twenty-eight, t h i r t y - f i v e , and forty-two days o l d . Serum cholesterol was determined by the method of Z l a t k i s , Zak and Boyle (1953)> and serum t o t a l l i p i d s by the method of Huerga et a l (1953). GROWTH As seen from the r e s u l t s i n Table IB chicks fed the higher protein l e v e l grew at a faster rate than chicks fed the lower protein l e v e l . Both the normal and hyperthyroid chicks grew at a faster rate, when they were fed a 26% protein d i e t than when they were fed a- 20%. protein d i e t . Chicks fed die t 3 (low protein, high fat) grew at a slower rate than chicks fed die t 1 (low protein, low f a t ) . High f a t d i e t s , low i n protein, did not produce any increase i n the growth rate of chicks fed these d i e t s over those fed di e t s low i n f a t and.low i n protein. Chicks fed die t 7 (high f a t , high protein) displayed faster growth rates than chicks fed d i e t 5 (low f a t , high pr o t e i n ) . This I l l u s t r a t e s that the amount of energy supplied In the basal d i e t at the low protein l e v e l was adequate, but at the higher protein l e v e l the chick required a greater amount of energy. In a l l cases, either normal or hyperthyroid chicks grew at a faster rate when fed high protein d i e t s supplemented with additional B complex vitamins than when they were fed di e t s with a low B complex vitamin supplement. The addition of f a t i n the basal d i e t apparently aggravated the vitamin deficiency induced by that d i e t on the basis of the growth response of the chicks. Chicks fed basal d i e t s d e f i c i e n t i n the B vomplex vitamins showed increased growth rates when - 19. -the B complex vitamin supplement was fed. Inclusion of 12$ fat i n the diet depressed growth in chicks fed a vitamin deficient diet. Hyperthyroid chicks fed diets 1 (low pro-tein, low fat, high vitamin), 3 (low protein, high fat, high vitamin), 7 (high protein, high fat, high vitamin), 9 (high protein, low fat, low vitamin) or II (high protein, high fat, low vitamin) grew at a slower rate than normal chicks fed similar diets. There were no differences between normal and hyper-thyroid chicks fed diet 5 (high protein, low fat, high vitamin). SERUM CHOLESTEROL No differences were obtained between the serum cholesterol levels of normal and hyperthyroid chicks fed the basal diet (diet 1) (low protein, low fat) and normal or hyperthyroid chicks fed diet 5 (high protein, low f a t ) . Hyperthyroid chicks fed diet 1 showed lower levels of serum cholesterol than hyperthyroid chicks fed diet 5« There were no differences between the serum cholesterol levels of normal chicks fed diet 3 (high fat, low protein) and normal chicks fed diet 7 (high fat, high protein). Hyperthyroid chicks fed diet 3 show higher levels of serum cholesterol than hyper-thyroid chicks fed diet 7. On the basis of this experiment i t appears that hyperthyroid chicks fed low protein, high fat diets tend to have higher levels of serum cholesterol than hyperthyroid chicks fed high protein, high fat diets. When high fat and low fat diets were fed to normal chicks, the chicks receiving high fat diets always had higher serum cholesterol levels than chicks receiving low fat diets. The serum cholesterol l e v e l s of chicks fed basal d i e t s high In B eomplex vitamins were lower than chicks fed basal d i e t s low i n the B complex vitamins. There are indicat i o n s i n the l i t e r a t u r e to suggest that chicks fed diets high i n B eomplex vitamins had lower serum cholesterol l e v e l s than chicks fed diets low i n the B complex vitamins (Hsu and Chow, 1957). This decrease i n serum cholesterol l e v e l i n chicks fed diets high i n B complex vitamins was not achieved except i n the case where chicks were fed basal d i e t s . In some cases hyperthyroid chicks fed high vitamin B complex d i e t s had lower serum cholesterol l e v e l s than normal chicks fed simi l a r d i e t s . This decrease i n serum cholesterol l e v e l was not as great as that reported In the l i t e r a t u r e . SERUM TOTAL LIPIDS Generally, chicks fed the 26% protein diet showed lower serum t o t a l l i p i d l e v e l s than chicks fed the 20% protein d i e t . Only i n one Instance both normal and hyperthyroid twenty-eight day old chicks showed higher l e v e l s of serum cholesterol when fed a 26% protein d i e t than chicks of the same age and fed a 20% protein d i e t . High protein diets fed to hyperthyroid chicks were inconsistent i n i t s e f f e c t on the serum t o t a l l i p i d s of the chicks. While twenty-eight day old hyper-thyroid chieks fed die t 5 produced higher serum t o t a l l i p i d s than hyperthyroid chicks fed die t I, the reverse e f f e c t was obtained i n chicks t h i r t y - f i v e days o l d . Normal chicks fed high f a t d i e t s showed higher l e v e l s of serum t o t a l l i p i d s than normal chicks fed low f a t d i e t s . Hyperthyroid twenty-eight day old chicks fed d i e t 3 gave higher l e v e l s of serum t o t a l l i p i d s than normal chicks of the same age and fed the same d i e t . In t h i r t y - f i v e day old hyperthyroid chicks the reverse e f f e c t was observed. Hyperthyroid chicks fed d i e t 7 showed lower l e v e l s of serum t o t a l l i p i d s than normal chicks fed the same d i e t at both sets of determinations. Generally, normal chicks fed high protein diets supplemented with a d d i t i o n a l B complex vitamins had lower l e v e l s of serum t o t a l l i p i d s than normal chicks fed high protein d i e t s of low B complex vitamin eontent. - 22 -Ingredients TABLE 1  Composition of Basal Diets Protein i n Diet Dextrose Ground Wheat Ground Yellow Corn Soya Bean O i l Meal (M+$ Protein) Protein) F i s h Meal Limestone Bone Meal Iodized S a l t ' Vitamin A Vitamin D Manganeese Sulphate Menadione Nicarbazine (25$) lb/100 l b s . 12.0 22.0 23.0 28.0 12.0 1.0 1.5 0.5 2000 i u / l b . 120 i c u / l b . 6.0 gms. 0.027 gms, 22.5 gms. 20$ Protein i n Diet  lb/100 lbs"! 12.0 30.0 28.75 20.0 6.0 1.0 1.75 0.50 2000 i u / l b . 120 i c u / l b . 6.0 gms. 0.027 gms. 22.5 gms. - 23 -TABLE IA DIET DESCRIPTION OF DIET I N "t" Basal I H Diet 1 4 0.02% iodinated casein. 3 N Diet 1 4 12% hydrogenated vegetable o i l . 3 H Diet 1 4- 12% hydrogenated vegetable o i l 4 0.02$ iodinated casein. 5 N ® Basal 5 H Diet 5 + 0.02$ Iodinated casein. 7 N Diet 5+ 12$ hydrogenated vegetable o i l . 7 H Diet 5+ 12$ hydrogenated vegetable o i l 4 0.02$ iodinated casein. 9 N o Basal 9 H Diet 9 0.02$ iodinated casein. I I N Diet 9 4- 12$ hydrogenated vegetable o i l . I I H Diet 9 4- 12$ hydrogenated vegetable o i l . 4 0.02$ iodinated casein. + 20$ Protein d i e t further supplemented with B complex vitamins. 26$ Protein d i e t further supplemented with B complex vitamins. o 26$ Protein d i e t without any further B complex vitamin supplement N» Normal chicks. H*Hyperthyroid chicks. TABLE IB Body W e i g h t s , Serum C h o l e s t e r o l L e v e l and T o t a l L i p i d L e v e l o f C h i c k s i n E x p e r i m e n t 1  ft * — « ^-Average W e i g h t (gms) 4 Serum C h o l e s t e r o l m g s / l o o c . c . ^ S e r u m T o t a l mgs/100 c . L i p i d s c . Age i n Days 28 28 35 28 35 DIET 1 N" 332 166 162 665 833 1 H 288 157 170 Hk 759 3 N 319 178 177 82J4 859 3 H 2^ 2 190 188 868 812 5 N 360 165 165 687 697 5 H 365 166 172 756 703 7 N 389 179 I8lf 739 717 7 H 356 166 171 700 611 9 N 31+2 Ilk 182 710 625 9 H 327 161 170 690 598 11 N 317 175 19l+ 755 756 11 H 299 li|.I(. 186 609 61*9 ^ A v e r a g e o f 20 C h i c k s . t A v e r a g e o f 6 D e t e r m i n a t i o n s . - 2 5 -ANALYSIS OP VARIANCE (GROWTH) EXPERIMENT 1 TABLE 1C S o u r c e o f V a r i a t i o n d f Sum o f S q u a r e s Mean S q u a r e s P -A 1 B 1 C 1 . 0 2 3 . 0 0 2 . 1 6 6 . 0 3 6 9 .0023 .1661]. 1 8 . 0 1 . 1 8 1 . 0 Vt * •?:- -x AB 1 BC 1 AC 1 . 0 1 2 . 0 1 5 . 0 0 6 .0121* .011*9 . 0 0 5 7 6 . 0 7 . 2 2 . 8 -» ABC 1 .000 . 0 0 0 1 . 0 T o t a l T r e a t m e n t 7 . 2 3 9 . . 0 3 l | l 1 6 . 6 E r r o r 1 2 0 . 2 1 * 7 . 0 0 2 1 T o t a l 1 2 7 . I | - 8 5 A - I o d i n a t e d C a s e i n B - P a t C - P r o t e i n S o u r c e o f V a r i a t i o n d f Sum o f S q u a r e s Mean S q u a r e s P A 1 B 1 C 1 .001+ . 0 0 0 . 0 5 0 . 0 0 3 9 . 0 0 0 0 . 0 1 * 9 7 1 . 7 . 0 2 1 . 2 Vt vt AB 1 BC 1 AC 1 .00l* . 0 2 9 . 0 0 3 . 0 0 3 9 " .0290 . 0 0 2 9 1 . 6 1 2 . 1 * 1 . 2 Vt # ABC 1 . 0 0 3 . 0 0 3 4 1 . 5 T o t a l T r e a t m e n t 7 . 0 9 3 . 0 1 3 2 5 . 7 E r r o r 1 2 0 . 2 8 1 . 0 0 2 3 T o t a l 1 2 7 . 3 7 4 A - I o d i n a t e d C a s e i n B = P a t C - V i t a m i n - 26 -EXPERIMENT 2 Experiment 2 was designed to study the effects of ( i ) two l e v e l s of f a t ; ( i i ) two l e v e l s of vitamins and the interactions of these factors on the serum t o t a l cholesterol l e v e l , serum t o t a l l i p i d l e v e l and growth of both the normal chick and the induced hyperthyroid chick. As described i n the previous experiment, hyperthyroidism was induced i n the chick by the administration of 0.02$ iodinated casein i n the d i e t . The high f a t di e t was formulated by substituting hydro-genated vegetable o i l for dextrose i n the 26$ protein basal d i e t shown i n Table 1. To obtain diets with a high vitamin l e v e l , the 26$ protein basal diet shown i n Table 1 was further supplemented with the following B vitamins; h2.9 grams of choline chloride, 0.63 grams of calcium panthothenate, 0.395 grams of f o l a c i n , 0.2 grams of r i b o f l a v i n , and 1.8 grams of n i a c i n per 100 pounds of d i e t . Eight groups of White Leghorn mixed male and female chicks were used i n t h i s experiment. Each group consisting of approximately twenty-two chicks was fed an experimental d i e t as described i n Table 2. Chicks were weighed i n d i v i d u a l l y at seven-day Intervals up to a period of t h i r t y - f i v e days. Blood samples were taken from six chicks selected at random i n each group when the chicks were twenty-eight, t h i r t y - f i v e and t h i r t y - e i g h t days old. Serum cholesterol was determined by the method of Z l a t k i s , Zak and Boyle (1953)» and serum t o t a l l i p i d s by the - 2-7 -method of Huerga et a l (1953). GROWTH The e f f e c t of fat on the growth rate of chicks was i n -consistent. Normal twenty-eight day old chicks fed d i e t 3 (high f a t , high vitamin) grew at a slower rate than normal chicks of the same age and fed diet 1 (low f a t , high vitamin);. The growth rate of t h i r t y - f i v e day old normal chicks fed diet 3 was similar to the growth rate of normal chicks fed d i e t 1. The growth rate of hyperthyroid chicks was also incon-s i s t e n t . Hyperthyroidism resulted i n an increased growth rate response i n twenty-eight day old chicks fed d i e t 1 over normal chicks of the same age and fed a similar d i e t , while no e f f e c t was obtained from hyperthyroidism i n t h i r t y - f i v e day old chicks fed the same d i e t . In both instances hyperthyroidism i n chicks fed diet 3 resulted i n increased growth rate response over normal chicks fed a similar d i e t . Generally, chicks when fed diets low i n the B complex vitamins grew at a slower rate than chicks fed d i e t s high i n B complex vitamins. High f a t d i e t s supplemented with a d d i t i o n a l B complex vitamins retarded the growth rate of twenty-eight day old chicks fed these d i e t s . Apparently the i n c l u s i o n of fat i n these diets increased the require-ment for B complex vitamins. Chicks fed diet 7 (high f a t , low vitamin) grew at a slower rate than chicks fed d i e t 5 (low f a t , low vitamins). Hyperthyroid chicks fed d i e t s low i n B complex vitamins did not d i f f e r i n growth rate from normal chicks fed similar d i e t s . - 28 -SERUM CHOLESTEROL The e f f e c t of high f a t d i e t on the serum cholesterol of the normal chick fed these diets was inconsistent. Twenty-eight day old normal chicks fed d i e t 1 (low f a t , high vitamin) showed higher serum cholesterol l e v e l s than normal chicks of the same age fed die t 3 (high f a t , high vitamin). There were no differences i n the serum cholesterol l e v e l s of t h i r t y -f i v e day old normal chicks fed diet 1 and normal chicks of the same age fed d i e t 3. S i m i l a r l y no differences i n the serum cholesterol l e v e l s of t h i r t y - e i g h t day old normal chicks fed di e t 1 and normal chicks of equal ages fed d i e t 3, were obtained. The e f f e c t of induced hyperthyroidism i n chieks fed diets 1 and 3> was inconsistent. T h i r t y - f i v e and t h i r t y - e i g h t day old hyperthyroid chicks had higher l e v e l s of serum cholesterol than normal chicks when they were fed die t 1. Twenty-eight day old hyperthyroid chicks fed diet 1 showed lower l e v e l s of serum cholesterol than normal chicks of the same age and fed simi l a r d i e t s . Twenty-eight and t h i r t y - f i v e day old hyper-thyroid chicks fed die t 3 showed higher l e v e l s of serum cholesterol than normal chicks of the same age fed similar d i e t s . Under similar conditions the reverse e f f e c t on the serum cholesterol l e v e l of hyperthyroid chicks was obtained. When the l e v e l of B complex vitamins was low, the inc l u s i o n of f a t i n the diet was not consistent In i t s e f f e c t on the serum cholesterol l e v e l of chicks fed these d i e t s . Twenty-eight day old normal chicks fed die t 7 (high f a t , low vitamin) showed lower serum cholesterol l e v e l s than normal chicks of the same age and fed diet 5. Hyperthyroid chicks fed die t 7 - -2.9 -showed lower levels of serum cholesterol than normal chicks fed similar diets. Normal chicks fed diets low in B complex vitamins generally showed higher levels of serum cholesterol than normal chicks fed diets high in the B complex vitamins. SERUM TOTAL L I P I D S Normal chicks fed high fat diets had higher serum total l i p i d levels than normal chicks fed low fat diets. Hyper-thyroid chicks fed diet 1 (low fat, high vitamin) had higher levels of serum total l i p i d s than normal chicks fed a similar diet. The serum total l i p i d level, however, of hyperthyroid twenty-eight and thirty-five day old chicks fed diet 3 (high fat, high vitamin) was higher than normal chicks of the same age and fed a similar diet. Thirty-eight day old hyper-thyroid chicks had lower levels of serum total l i p i d s than normal chicks equal in age and fed a diet high in the B complex vitamins (diet 1). The effect of feeding diets low in the B complex vitamins to.: hyperthyroid chicks on their serum total l i p i d s , was inconsistent. - 30 -TABLE 2 DIET DESCRIPTION OF DIET I N "t Basal I H Diet I + 0.02$ iodinated casein. 3 N Diet I + 12$ hydrogenated vegetable o i l . 3 H Diet I + 12$ hydrogenated vegetable o i l + 0.02$ Iodinated casein. 5 N o Basal 5 H Diet 5 4 0.02$ iodinated casein. 7 N Diet 5 + 12$ hydrogenated vegetable o i l . 7 H Diet 5 + 12$ hydrogenated vegetable o i l . -f 0.02$ iodinated casein. + 26$ Protein supplemented with add i t i o n a l B complex vitamins. O 26$ Protein diet low i n B complex vitamins. N » Normal chicks. H - Hyperthyroid chicks. TABLE 2A Body W e i g h t s , Serum C h o l e s t e r o l L e v e l and Serum T o t a l L i p i d L e v e l o f C h i c k s i n E x p e r i m e n t 2  Age i n Days A v e r a g e Weight i n Grams. Serum C h o l e s t e r o l mgs/100 c c . 3 erum T o t a l L i p i d s rags/100 c c . 28 35 28 35 38 28 35 38 DIET 1 N 32k kih 253 216 200 676 600 630 1 H 335 1*15 21+9 2l+8 222 713 620 63^ 3 N 309 1+18 197 222 198 696 623 771 3 H 319 1+35 230 235 171 781+ 61+7 629 5 N 221 308 262 233 181 672 591 658 5 H 227 309 233 203 190 61+7 600 66'1+ 7 N 202 285 229 233 198 61+7 673 705 7 H 20k 287 X9l+ 193 170 698 1+95 622 A v e r a g e o f 22 C h i c k s . A v e r a g e o f 6 D e t e r m i n a t i o n s . - 32 -ANALYSIS OP VARIANCE (GROWTH) EXPERIMENT 2 TABLE 2B S o u r c e o f V a r i a t i o n d f Sum o f S q u a r e s Mean S q u a r e s F A 1 .024 .0242 5.2 -is-B 1 .003 .0029 .6 C 1 .389 .3886 83.9 * AB 1 .000 .0000 .0 BC 1 .034 .0338 7.3 * AC 1 .014 .0141 3.0 ABC 1 .001 .0008 .2 T o t a l T r e a t m e n t 7 .461* .0664 14.3 * E r r o r 136 .630 .0046 T o t a l Hl3 1.094 B = P a t C s V i t a m i n - 33 -EXPERIMENT 3 Experiment 3 was designed to study the e f f e c t of ( i ) two l e v e l s of f a t ; ( i i ) two l e v e l s of B complex vitamins and the i n t e r a c t i o n of these factors on the serum cholesterol l e v e l , the serum t o t a l l i p i d l e v e l , and growth of the chick. The effects of the above fact o r s were studies on both the normal and the Induced hyperthyroid chick. Hyperthyroidism was Induced i n chicks by feeding them die t s containing 0.02$ iodinated casein. High f a t di e t s were formulated by substituting 12$ hydro-genated vegetable o i l for dextrose In the 26$ protein basal d i e t shown i n Table 1. To obtain diets with a high B complex vitamin l e v e l the 26$ protein basal d i e t shown i n Table 1 was further supplemented with the following B vitamins; 42.9 grams of choline chloide, 0.63 grams of calcium panthothenate, 0.395 grams of f o l a c i n , 0.2 grams of r i b o f l a v i n , and 1.8 grams of n i a c i n per 100 pounds of d i e t . Eight groups of White Leghorn cockerels were used i n t h i s experiment. Each group consisting of approximately twenty chicks was fed an experimental d i e t , the description of which i s given i n Table 3. A l l chicks were weighed i n d i v i d u a l l y at seven-day i n t e r v a l s throughout an experimental period of t h i r t y - f i v e days. Blood samples were taken from s i x chicks selected at random i n each group when the chieks were twenty-eight, t h i r t y - f i v e and forty-two days old. The blood samples were analysed for serum cholesterol by the method of Z l a t k i s , Zak and Boyle (1953) and for serum - 34-total l i p i d s by the method of Huerga et a l (1953). GROWTH Chicks fed high fat, high B complex vitamin diets showed higher growth rates than chicks fed low fat, high B complex vitamin diets. The inclusion of fat in diets low in the B complex vitamins resulted in a depression of growth rate in chicks fed these diets. The addition of the fat in the basal diet apparently aggravated the vitamin deficiency in-duced by that diet on the basis of the growth response of the chicks. Chicks fed dtets low in B complex vitamins grew at a slower rate than chicks fed high vitamin B complex diets. The effect of induced hyperthyroidism in chicks fed high fat, high vitamin B complex diets was inconsistent. Twenty-eight day old hyperthyroid chicks showed an increased growth response when fed diet 3 (high fat, high vitamin) than normal chicks of the same age and fed a similar diet. The thirty-five day old hyperthyroid chick, however, showed a decrease in growth response as compared to the normal chick when fed diet 3. Hyperthyroid chicks fed low vitamin B complex diets showed slower growth rates than normal chicks fed similar diets. SERUM CHOLESTEROL The inclusion of fat in the high B vitamin diets resulted in higher serum cholesterol levels of chicks fed these diets than of chicks fed low fat, high B complex vitamin diets. The high fat diets low in vitamin B complex had very l i t t l e effect on the serum cholesterol levels of chicks fed these - 35 -diets as compared to chicks fed low fat, low vitamin B complex diets. The effect of the diet on the serum cholesterol levels of induced hyperthyroid chicks was very inconsistent. Thirty-five day old hyperthyroid chicks fed diet 1 (low fat, high vitamin) showed higher levels of serum cholesterol than normal chicks of the same age and fed the same diet. No differences were obtained i n the serum cholesterol level between either twenty-eight day old or forty-two day old normal chicks and hyperthyroid chicks fed diet 1. No differences could be obtained in the serum cholesterol level between normal and hyperthyroid chicks fed diets low i n the B complex vitamins. SERUM TOTAL LIPIDS Chicks fed diets high i n fat generally showed higher serum total l i p i d levels than chicks fed diets low i n fat. Induced hyperthyroid chicks fed high fat high B complex vitamin diets had higher serum total l i p i d levels than the hyperthyroid chicks fed low fat high B complex vitamin diets. The effect of feeding vitamin high diets to hyperthyroid chicks was inconsistent. Twenty-eight day old hyperthyroid chicks fed either diets 1 (low fat, high vitamin), 5 (low fat, low vitamin), or 7 (blgh fat, low vitamin) showed lower serum total l i p i d levels than normal chicks of the same age and fed the same diets. Thirty-five day old hyperthyroid chicks fed diets 1 had higher levels of serum total l i p i d s than normal chicks similar in age and fed the same diet. Diet 3 (high fat, high vitamin) fed to thirty-five day old hyperthyroid chicks showed lower l e v e l s of serum t o t a l l i p i d s , while diet 3 fed to forty-two day old hyperthyroid chicks showed higher serum t o t a l l i p i d l e v e l s than normal chicks of s i m i l a r ages and fed similar d i e t s . Chicks fed basal diets low i n B complex vitamins generally showed higher l e v e l s of serum t o t a l l i p i d s than chicks fed basal d i e t s high i n B complex vitamins. - 37 -TABLE 3 DIET 1 N ^Basal 1 H Diet I +• 3 N Diet I + 3 H Diet I + 5 N oBasal 5 H Diet 5 + 7 N Diet 5 4 7 H Diet 5 4 DESCRIPTION OF DIET 0.02$ iodinated casein. 12$ hydrogenated vegetable o i l . 12$ hydrogenated vegetable o i l + 0.02$ iodinated casein. 0.02$ iodinated casein. 12$ hydrogenated vegetable o i l . 12$ hydrogenated vegetable o i l 4- 0.02$ iodinated casein. c s 26$Protein supplemented with additional B complex ^ vitamins. O • 26$ Protein diet low in B complex vitamins. U = Normal chicks. H = Hyperthyroid chicks. TABLE 3 A Body Weights, Serum Cholesterol Level and Serum Total Lipid Level of Chicks in Experiment 3  -^ -Average Weight in Grams -fSerum Cholesterol Level in mgs/100 c.c. erum Total Lipid Level in mgs/100 c.c. Age in Davs DIET 1 N 28 35 28 35 42 28 35 42 314 447 175 152 189 758 595 595 1 H 312 449 182 171 184 689 775 582 3 N 325 474 171 181 191 768 887 599 3 H 335 465 174 165 181 778 788 611 5 N 262 388 201 189 180 775 809 65o 5 H 232 333 181 186 173 573 708 600 7 N 258 355 191 201 174 724 895 692 7 H 219 302 204 199 182 677 854 638 * Average of 20 chicks. + Average of 6 determinations. - 39 -ANALYSIS OF VARIANCE (GROWTH)  EXPERIMENT 3 TABLE 3B S o u r c e o f V a r i a t i o n d f Sum o f S q u a r e s Mean S q u a r e s F A 1 .001 .0013 •4 B 1 .001 .0012 .4 C 1 .603 .6033 178.3 * AB 1 .000 .0002 .1 BC 1 .012 .0118 3.5 AC 1 .001 .0007 .2 ABC 1 .001 .0009 .3 T o t a l T r e a t m e n t 7 .619 .0885 26 .1 E r r o r 152 .0034 T o t a l 159 1.134 A «» I o d i n a t e d C a s e i n B = F a t C = V i t a m i n o _ LLO -EXPERIMENT 4 Experiment 4 was designed to study the effects of (i) two levels of fat; ( i i ) three levels of B complex vitamins on the serum cholesterol level, serum total l i p i d level and growth of the chick. The above factors were studied in the normal chick, induced hyperthyroid chick,, and the induced hypothyroid chick. Hyperthyroidism was induced in chicks by feeding them diets containing 0*02% iodinated casein. Hypothyroidism was induced by feeding chicks diets con-taining 0.1$ thiouracil. To formulate diets of a high fat level, 12$ hydrogenated vegetable o i l was substituted for the dextrose of the 26$ protein basal diet as shown in Table 1. Diets containing three levels of B complex vitamins were made up as follows (i) Level one - consisted of the 26$ protein basal diet as shown in Table 1. This diet was not supplemented with additional B complex vitamins. ( i i ) Level two - consisted of the 26$ protein basal diet as shown in Table 1, and to this diet 90 grams of choline chloride and 0.375 grams of folacin per 100 pounds of feed was added. ( i i i ) Level three- consisted of the 26$ protein basal diet as shown in Table 1 and to this diet a further B complex vitamin supplementation was made by the addition of the following - 90 grams of choline chloride, 0,375 grams of f o l a c i n , 0.02 grams of r i b o f l a v i n , O.63 grams of calcium p a n t o -thenate and 1.8 grams of n i a c i n per 100 pounds of feed. Eighteen groups of New Hampshire p u l l e t s were used i n t h i s experiment. Each group consisting of approximately t h i r t y chicks was fed an experimental d i e t as shown i n Table h. The chicks were weighed i n d i v i d u a l l y at seven day i n t e r v a l s throughout an experimental period of forty-nine days, while blood samples were taken when the chicks were thirty-two, t h i r t y - n i n e and forty-nine days old. Blood samples were taken from ten chicks selected at random i n each l o t . Serum cholesterol was determined by the method of Z l a t k i s , Zak and Boyle (1953) while serum t o t a l l i p i d s were determined by the method of Huerga et a l (1953). - 1*2 -GROWTH Chicks, thirty-five days old, fed high fat diets high in the B complex vitamins grew at a faster rate than chicks fed low fat diets low i n the B complex vitamins. Normal, hyper-thyroid and hypothyroid chicks fed diet 16 (high fat, high B complex vitamin) grew at a faster rate than normal chicks fed diet 13 (low fat, high vitamin). Hyperthyroid chicks grew at a faster rate than normal chicks fed diet 16, but no differences could be obtained between the growth rates of normal and hypothyroid chicks fed diet 16. Both hyper-thyroid and hypothyroid chicks, however, gave increased growth responses over normal chicks when they were a l l fed diet 13. When high fat diets supplemented with the B complex vitamins choline chloride and f o l i c acid were fed to chicks, an improvement of growth rate over chicks fed low fat diets, supplemented with choline chloride and f o l i c acid was seen only i n hyperthyroid chicks. Both normal and hypothyroid chicks were not able to u t i l i z e the high fat diets to any advantage in improving growth rate over chicks fed diets low in fat. Apparently as noted in previous experiments, the inclusion of fat in the diet aggravated the vitamin deficiency induced by that diet, on the basis of i t s growth response. Similar observations were noted when chicks were fed high fat diets very low in the B complex vitamins. Either normal, hyperthyroid or hypothyroid chicks when fed diet h (high fat, high B complex vitamin) showed a lower growth rate response than normal, hyperthyroid and hypothyroid chicks fed diet 1 - 43 -(low fat, vitamin level 1). The effect of increasing the vitamin level to hyper-thyroid chicks, on the basis of their growth response, was inconsistent. Hyperthyroid chicks fed diets 1, 10 (high fat, B complex vitamin, level 2) 13 (low fat, high B complex vitamin, level 3) and 16 (high fat, high B complex vitamin, level 3) showed higher growth rates than normal chicks fed similar diets. Hypothyroid chicks, however, showed increased growth rate responses over normal chicks, when fed diets supple-mented with the two additional B complex vitamins, choline chloride and folacin, or when the diets were further supple-mented with the B complex vitamins calcium panthothenate, folacin, choline chloride, niacin and riboflavin. When basal diets were not supplemented with additional B complex vitamins there was l i t t l e effect on the growth rates between normal and hypothyroid chicks fed these diets. SERUM CHOLESTEROL Normal chicks fed a basal diet high i n fat, low in the B complex vitamins showed higher levels of serum cholesterol than chicks fed a basal diet low in fat and low in the B com-plex vitamins. There was very l i t t l e change, however, in the levels of serum cholesterol of normal chicks fed high fat diets further supplemented with the B complex vitamins choline chloride and folacin alone, or high fat diets further supplemented with calcium panthothenate, choline chloride, folacin, riboflavin and niacin, and normal chicks fed low fat diets and with similar vitamin B complex content. - kk -Apparently the increase in serum cholesterol level, which ordinarily occurs when the level of fat in the diet is high, would tend to be reduced with the intake of the B complex vitamins. The effect of the diet modification on the serum cholesterol level of hyperthyroid chicks was inconsistent as shown in Table 4A. In some instances high levels of serum cholesterol were noted in hyperthyroid chicks than in normal chicks fed a similar diet. In other instances smai-l-er lower levels i n the serum cholesterol were noted i n hyper-thyroid chicks than in normal chicks fed similar diets. While thirty-two and thirty-nine day old hypothyroid chicks fed a basal diet low in the B complex vitamins showed higher levels of serum cholesterol than normal chicks of the same age and fed the same diet. Thirty-two and thirty-nine day old hypothyroid chicks fed diet 13 (low fat, high vitamin) showed lower levels of serum cholesterol than normal chicks of the same ages and fed similar diets. A similar observation was noted in thirty-two day old hypothyroid and normal chicks fed diet 16 (high fat, high vitamin). In other instances hypothyroid chicks showed lower leveis or similar levels of serum cholesterol than normal chicks when they were fed similar diets. SERUM TOTAL LIPIDS Normal chicks fed diets high in fat showed higher levels of serum total li p i d s than chicks fed diets low in fat. Thirty-nine day old hyperthyroid chicks fed high fat diets low in B complex vitamins showed a higher level of serum total li p i d s than hyperthyroid chicks fed low fat diets - 45 -low i n B complex vitamins. Hyperthyroid chicks fed high f a t die t s high i n B complex vitamins showed higher l e v e l s of serum l i p i d s than hyperthyroid chicks fed low f a t diets high i n B complex vitamins. Thirty-two day old hyperthyroid chicks fed di e t 7 (low f a t , vitamin l e v e l 2) showed higher l e v e l s of serum t o t a l l i p i d s than hyperthyroid chicks fed diet 10 (high f a t , vitamin l e v e l 2) whereas th i r t y - n i n e day old hyperthyroid chicks showed a lower l e v e l of serum t o t a l l i p i d s when fed diet 7 than hyperthyroid chicks fed di e t 10. The comparison of the serum t o t a l l i p i d s of the normal and the hyperthyroid chick shows a great deal of inconsistency i n the e f f e c t of the d i e t . Whereas a lower serum t o t a l l i p i d l e v e l was noted i n thirty-two day old hyperthyroid chicks fed di e t 7 than the normal chick fed a similar d i e t , the reverse e f f e c t was noted i n thirty-nine day old hyperthyroid and normal chicks fed d i e t 7. In a l l instances hypothyroid chicks fed low f a t die t s , regardless of the l e v e l of B complex vitamins, showed higher l e v e l s of serum t o t a l l i p i d s than normal chicks fed similar d i e t s . Hypothyroid chicks fed diets high i n f a t and high i n B complex vitamins also showed higher l e v e l s of serum total l i p i d s than normal chicks fed similar d i e t s . Thirty-two day old hypothyroid chicks fed d i e t 4 (high f a t , vitamin l e v e l 1) or 10 (high f a t , vitamin l e v e l 2) showed lower l e v e l s of serum t o t a l l i p i d s than normal chicks fed similar d i e t s , whereas thi r t y - n i n e day old hypothyroid chicks fed di e t k- or 10 showed higher l e v e l s of serum t o t a l l i p i d s than normal - 46 -chicks fed the same diet. A l l thirty-two day old chicks fed diets high in the B complex vitamins showed lower serum total l i p i d levels than thirty-two day old chicks fed diets low in the B vitamins. Apparently the increase in serum l i p i d level, which ordinarily occurs when the level of fat in the diet i s in-creased, would appear to be lessened with the intake of the B complex vitamins. - kT -TABLE h DIET DESCRIPTION OF DIET I N T Basal 1 HPR 1 + 0.02$ iodinated casein 1 HPO 1 +-0.1$ t h i o u r a s i l h N 1 +• 12$ hydrogenated vegetable o i l k HPR 1 4- 12$ hydrogenated vetetable o i l 4- 0.02$ iodinated casein h HPO 1 +»12$ hydrogenated vegetable o i l 4-0.1$ t h i o u r a c i l 7 N X Basal 7 HPR 7 4- 0.02$ iodinated casein 7 HPO 7 4 0.1$ t h i o u r a c i l 10 N 7 4* 12$ hydrogenated vegetable o i l 10 HPR 7 4- 12$ hydrogenated vegetable o i l + 0.02$ iodinated casein 10 HPO 7 4- 12$ hydrogenated vegetable o i l 4-0.1$ t h i o u r a c i l 13 N S Basal 13 HPR 134-0.02$ iodinated casein 13 HPO 13+*0.1$ thuouracil 16 N 134-12$ hydrogenated vegetable o i l 16 HPR 13-1- 12$ hydrogenated vegetable o i l + 0.02$ iodinated casein 16 HPO 134-12$ hydrogenated vegetable o i l 4-0.1$ t h i o u r a c i l 4- +- 26$ protein basal d i e t with no further supplementation with B complex vitamins X 26$ protein basal diet + choline chloride + f o l a c i n c 26$ protein basal d i e t + choline chloride + f o l a c i n ^ calcium panthothenate +• n i a c i n *- r i b o f l a v i n N •» Normal; HPR= Hyperthyroid; HP0= Hypothyroid TABLE 4A Body Weight, Serum Cholesterol Level and Total Lipid Levels of Chicks in Experiment 4  •Average Weight in Grams +.Serum Cholesterol mgm./lOO c.c. +Total Lipid Level rngm./lOO c.c. Age in Days 28 35 32 39 49 32 39 DIET i 1 tf 286 459 161 177 165 627 558 1 HPR 305 467 178 184 158 627 616 1 RTO 282 438 197 203 165 772 664 4 N 282 449 187 182 179 752 630 4 HPR 264 428 164 173 163 623 565 4 HPO 275 436 182 193 165 703 706 7 N 304 460 186 186 197 616 645 7 HPR 275 426 185 193 200 534 686 7 HPO 306 473 177 169 187 651 712 10 N 278 437 183 170 199 721 658 10 HPR 302 481 162 183 194 519 718 10 HPO 305 467 179 178 189 626 763 •a TABLE 4A (Cont'd) •Average Weight in Grams +Serum Cholesterol mgm./lOO c.c. +Total Lipid mgm./lOO c Level • c • Age in Days 28 35 32 39 49 32 39 DIET 13 N 311 419 177 171 193 434 570 13 HPR 322 481 172 182 190 498 605 13 HPO 310 469 194 185 185 588 675 16 N 319 478 174 187 188 582 670 16 HPR 323 502 167 178 190 530 712 16 HPO 318 485 201 183 188 591 806 I + Average of 10 determinations per group * Average of thirty chicks per group - Bo -EXPERIMENT 5 Experiment 5 was designed to study the effects of (i) two levels of fat; ( i i ) three levels of B complex vitamins. on the interaction of these factors on the serum cholesterol levels, and growth of the chick. The above factors were studied in both the normal and the induced hyperthyroid chick. Hyperthyroidism was induced in chicks by feeding them diets containing 0.02$ iodinated casein. The diet with a high fat level was formulated by sub-stituting 12$ hydrogenated vegetable o i l for the dextrose in the 26$ protein basal diet shown in Table 1. Diets of different B complex vitamin levels were formulated as follows: Vitamin level 1 - consisted of the 26$ protein basal diet as shown in Table 1. This diet was not supplemented with additional B complex vitamins. Vitamin level 2 - consisted of the 26$ protein basal diet as shown in Table 1. In addition this diet was further supple-mented with 90 grams of choline chloride, and 0.375 grams of folacin per 100 pounds of feed. Vitamin level 3 - consisted of the 26$ protein basal diet as shown in Table 1 and this diet was further supplemented with 90 grams of choline chloride, 0.375 grams of folacin, 0.63 grams of - £L -calcium panthothenate, 1,8 grams of niacin and 0,02 grams of ribo-flavin. Twelve groups of New Hampshire pullets were used in this experiment. Each group consisting of approximately twenty-five chicks was fed an experimental diet as shown i n Table 5. The chicks were weighed individually at seven day intervals up to a period of thirty-five days. Blood samples taken from ten chicks selected at random in each lot were analysed for serum cholesterol by the method of Zlatkis, Zak and Boyle (1953). - 52 -GROWTH Chicks fed high fat high vitamin B complex diets grew at a faster rate than chicks fed low fat diets high in vitamins (vitamin level 3) as seen in Table 5A , both normal and hyperthyroid chicks fed diet 1 (high fat, high vitamin) grew at a faster rate than normal or hyperthyroid chicks fed diet 9 (low fat, high vitamin). The growth rate of normal twenty-eight day old chicks fed basal diets high in fat and low in B complex vitamins (vitamin level 1 or vitamin level 2) was lower than normal chicks twenty-eight days old fed low fat, low in B complex vitamins. As seen in previous experiments, the inclusion of fat in the basal diet apparently aggravated the vitamin deficiency induced by that diet on the basis of the growth response of the chicks. The growth rate of normal chicks thirty-five days old was not depressed by the inclusion of a high fat level in their low B complex vitamin basal diets. Hyperthyroid chicks, fed low fat diets generally showed an increase in growth rate over normal chicks fed similar diets. The growth rate of hyperthyroid chicks fed high fat diets low in B complex vitamins was generally lower than the growth rate of normal chicks fed high fat diets low in B.complex vitamins. It seems that the inclusion of a high level of fat in the low B complex vitamin diets of hyperthyroid chicks, tend to aggravate the already existing vitamin B complex deficiency to a greater degree than in normal chicks, on the basis of their growth response. Generally chicks fed diets high in B complex - 53 -vitamins grew at a faster rate than chicks fed diets low in B complex vitamins. Chicks fed basal diets supplemented with the vitamins choline chloride and folacin alone (vitamin level 2) grew at a slower growth rate than chicks fed diets low in B complex vitamins (vitamin level 1). On the basis of this particular experiment i t might appear that an inter-relationship exists among the B vitamins. It appears as i f the presence of the two B complex vitamins, choline chloride and folacin alone in the diet, when fed to the chick may have aggravated the already existing deficiency of B complex vitamins. This probably accounts for the slower growth rates of chicks fed diets supplemented with choline chloride and folacin alone than chicks fed diets without any additional supplementation with the B complex vitamins (i.e., vitamin level 1). SERUM CHOLESTEROL Chicks fed basal diets high in B complex vitamins and high in fat showed higher levels of serum cholesterol than chicks fed basal diets high in B complex vitamins and low in fat. The serum cholesterol level of thirty day old chicks fed diet 7 (high fat, low vitamin-level 2) was lower than that of thirty day old chicks fed diet 5 (low fat, low vitamin-level 2 ) . in other instances there appeared to be no differences between the serum cholesterol levels of chicks fed high fat, low B complex vitamin level diets (vitamin levels 1 or 2) than the serum cholesterol level of chicks fed low fat, low B complex vitamin diets. - 5k -The serum cholesterol levels of hyperthyroid chicks were lower than those of normal chicks in some instances while in other instances the serum cholesterol level was higher in hyperthyroid chicks than that of normal chicks although fed the same diets. Twenty-five day old hyper-thyroid chicks fed diet 1 (low fat, low vitamin-level 1 ) was lower than normal chicks fed the same diet whereas the serum cholesterol level of hyperthyroid chicks fed diet 9 (low fat, high vitamin-level 3) was higher than normal chicks fed the same diet. TABLE 5 DIET DESCRIPTION OF DIET I N X Basal I H 1 +• 0.02$ iodinated casein 3 N 1 -f- 12$ hydrogenated vegetable o i l 3 H 1 +• 12$ hydrogenated vegetable o i l •4- 0.02$ iodinated casein 5 N ><. Basal 5 H 5 +" 0.02$ iodinated casein 7 N 5 +• 12$ hydrogenated vegetable o i l 7 H 5 +" 12$ hydrogenated vegetable o i l + 0.02$ iodinated casein 9 N S Basal 9 H 9 +" 0.02$ iodinated casein U N 9 12$ hydrogenated vegetable o i l II H 9 + 12$ hydrogenated vegetable o i l 4-0.02$ iodinated casein -+• 26$ protein basal diet - no additional B vitamins X 26$ protein basal diet +• choline chloride +• folacin S 26$ protein basal diet + choline chloride Ar folacin +• riboflavin 4 niacin +- calcium panthothenate. N = Normal H = Hyperthyroid TABLE 5A Body Weights, and Serum Cholesterol Levels of Chicks i n Experiment 5 +Average Weight i n Grams *Serum mgm. Cholesterol /100 c.c. Age i n Days 28 35 25 30 38 DIET 1 N 298 407 218 206 214 1 H 299 425 203 210 212 3 N 289 426 217 211 210 3 H 272 394 214 213 211 5 N 274 360 228 233 229 5 H 298 430 227 228 218 7 N 252 377 226 204 226 7 H 246 366 219 209 214 9 N 306 443 227 209 216 9 H 358 496 216 222 219 11 N 363 500 236 218 225 11 H 377 527 219 229 211 + Average of twenty-five chicks per group * Average of 10 determinations per group - 57 -ANALYSIS OF VARIANCE (GROWTH)  EXPERIMENT 5 TABLE 5B S o u r c e o f V a r i a t i o n d f Sum o f S q u a r e s Mean S q u a r e s F A 2 .,1+92 .21+58 51.1 * B 1 .002 .0017 . 3 C 1 .030 .0296 6.1 * AB 2 .054 .0270 5.6 # BC 1 .035 .031+5 7.2 * AC 2 .021+ .0118 2 . 5 ABC 2 . 0 0 3 .0013 . 3 T o t a l T r e a t m e n t 11 .638 .0580 12.1 * E r r o r 2l+0 1.151+ .001+8 T o t a l 251 1.792 A s V i t a m i n B = F a t C = I o d i n a t e d C a s e i n - 58 -EXPERIMENT 6 Experiment 6 was designed to study the effect of (i) two levels of fat; ( i i ) two levels of B complex vitamins and the interaction of these factors on the serum cholesterol level, serum total l i p i d level and growth of both the normal and the hyperthyroid chick. Hyperthyroidism was induced in the chick by feeding them diets containing 0.02$ iodinated casein. In previous experiments the effect of various diets on the serum cholesterol and serum total l i p i d level of hyper-thyroid chicks were inconsistent. It was suspected that hyperthyroidism was not always induced in the chick when diets containing 0.02$ iodinated casein was fed. As a result an additional diet was formulated to contain twice the level of iodinated casein as was previously used. In this experiment the above factors were studied in chicks rendered hyperthyroid by feeding them diets containing levels of 0.02$ and 0.04$ iodinated casein. Diets containing a high level of fat were formulated by substituting 12$ hydrogenated vegetable o i l for dextrose in the 26$ protein basal diets as shown i n Table 1. Diets con-taining a high vitamin B complex level were formulated by the addition of the following B complex vitamins to the 26$ protein basal diet shown in Table 1; 90 grams of choline chloride, 0.375 grams of folacin, 0.02 grams of riboflavin, 1.8 grams of niacin, and 0.63 grams of calcium panthothenate per 100 - 59 -pounds of diet. Twelve groups of New Hampshire cockerels were used in Experiment 6. Each group consisting of approximately twenty-five chicks was fed an experimental diet as described in Table 6. The chicks were weighed at seven day intervals up to an experimental period of thirty-one days. Blood samples were taken from ten chicks selected at random in each l o t . Serum cholesterol was determined by the method of Zlatkis, Zak and Boyle (1953) while serum total li p i d s was estimated by the method of Huerga et al (1953). RESULTS  GROWTH Chicks fed basal diets high in fat, high in B complex vitamins grew at a faster rate than chicks fed basal diets low in fat, and high in B complex vitamins. No differences were observed in the growth rate of chicks fed basal diets low in the B complex vitamins, and at either the high or low fat level. Hyperthyroid chicks fed high fat diets showed higher growth rates than hyperthyroid chicks fed low fat diets, provided the chicks were high in the B complex vitamins. Hyperthyroid chicks (level O.Qh%) fed diet 7 (low fat, low B complex vitamins) or diet 10 (high fat, low B complex vitamins) showed lower growth rates than normal chicks fed similar diets. The effect of the diet on the growth rate on chicks rendered hyperthyroid, when either level of thyroactive pro-tein was fed, was inconsistent. Whilst chicks rendered hyperthyroid by administration of the higher level of - 60 -thyroactive protein showed higher growth rates than chicks rendered hyperthyroid from a lower level of thyroactive protein when they were fed diets 1 (low fat, high B complex vitamins) or h (high fat, high B complex vitamins), similar effects could not he observed in hyperthyroid chicks fed lew vitamin diets. Hyperthyroid twenty-eight day old chicks (level0.02$) grew at a faster rate than hyperthyroid chicks (level 0.0*+$) of the same age and fed the same diet (diet 7). In twenty-eight day old hyperthyroid chicks the reverse effect was noted in the gro\\/th rate of these chicks fed diet 7-Chicks fed diets with a high B complex vitamin level grew at a faster rate than chicks fed diets of a low vitamin B complex level. Chicks fed high fat diets low in the B complex vitamins grew at a slower rate than chicks fed diets low in fat and low in the B complex vitamins. Apparently as previously noted the inclusion of fat in the low B complex vitamin diets aggravated the vitamin deficiency already existing on the basis of the growth response of the chicks fed these diets. SERUM CHOLESTEROL Chicks fed high fat diets generally showed higher levels of serum cholesterol than chicks fed low fat diets. Clicks rendered hyperthyroid from the administration of thyroactive protein showed higher levels of serum cholesterol than normal chicks when they were fed diet 1 (low fat, high vitamin). Twenty-eight day old hyperthyroid chicks (at both levels) fed diet h (high fat, high vitamin) showed higher levels of serum - 61 -cholesterol than normal chicks of the same age and fed the same diet. Wo differences were observed in thirty-one day old hyperthyroid chicks fed diet h. The effect of chicks rendered hyperthyroid when either level of thyroactive pro-tein was fed, was not consistent. Chicks fed diets low in B complex vitamins generally showed higher levels of serum cholesterol than chicks fed diets high in the B complex vitamins. SERUM TOTAL L I P I D S Chicks fed diets high in the B complex vitamins and high in fat showed higher levels of serum total l i p i d s than chicks fed diets low in fat and high in B complex vitamins. Twenty-five day old chicks fed basal diet 7 (low fat, low vitamin) showed higher levels of serum total l i p i d s than t twenty-five day old chicks fed diet 10 (high fat, low vitamins). The reverse effect on the serum total l i p i d s was noted in thirty-one day old normal chicks fed similar diets. Hyperthyroid chicks fed diets low in fat and high in B complex vitamins showed higher levels of serum total l i p i d s than normal chicks fed similar diets. The effect of feeding diets h (high fat, high vitamin) 7 (low fat, low vitamin) and 10 (high fat, low vitamin) to hyper-thyroid chicks was inconsistent. Chicks fed a basal diet low in B complex vitamins showed higher levels of serum total l i p i d s than chicks fed a basal diet high in B complex vitamins. - 62 -TABLE 6 DIET DESCRIPTION O P DIET I N + Basal 1 Hx 1 + 0.02$ iodinated casein 1 Hxx 1 •+- 0.04$ iodinated casein 4 N 1 «f 12$ hydrogenated vegetable o i l 4 Hx 1 +• 12$ hydrogenated vegetable o i l + 0.02$ iodinated casein 4 Hxx 1 •+• 12$ hydrogenated vegetable o i l +- 0.04$ iodinated casein 7 N O Basal 7 Hx 7* 0.02$ iodinated casein 7 Hxx 7 +• 0.04$ iodinated casein 10 N 7 *r 12$ hydrogenated vegetable o i l 10 Hx 7 4- 12$ hydrogenated vegetable o i l •+•0.02$ iodinated casein 10 Hxx 7 +" 12$ hydrogenated vegetable o i l + 0.04$ iodinated casein 1~ 26$ protein basal diet as shown in Table 1 supplemented with additional B complex vitamins. O 26$ protein basal diet as shown in Table 1. N = Normal chicks Hx = Chicks rendered hyperthyroid by feeding them diets con-taining 0.02$ iodinated casein Hxx=Chicks rendered hyperthyroid by feeding them diets con-taining 0.04$ iodinated casein RESULTS TABLE 6A Body Weights, Serum Cholesterol Levels and Tot a l L i p i d Levels of Chicks i n Experiment 6  ^•Average Weight i n Grams +Serum mgm Cholesterol ./100 c.c. +Total l i p i d s mgm./lOO c.c. Age i n Days 21 28 25 31 25 31 DIET 1 N 189 309 144 157 657 553 1 Hx 205 316 157 189 755 579 1 Hxx 219 344 152 205 710 730 4 N 216 347 156 202 814 819 4 Hx 223 332 186 194 848 715 4 Hxx 231 371 176 216 815 696 7 N 173 285 183 203 735 589 7 Hx 166 268 175 181 712 551 7 Hxx 152 287 186 202 784 639 10 N 167 284 213 212 720 682 10 Hx 179 312 208 224' 781 533 10 Hxx 160 267 187 209 754 632 + Average of 10 determinations per group * Average of twenty-five chicks per group - 6k -ANALYSIS OF VARIANCE (GROWTH)  EXPERIMENT 6 TABLE 6 B Source of Variation df Sum of Squares Mean Squares F A 2 . 0 0 8 .OOkl 1 . 7 B 1 . 0 1 6 . 0 1 6 3 6 . 9 # C 1 . 2 1 + 7 .21+67 1 0 3 . 6 » AB 2 . 0 0 0 . 0 0 0 1 . 1 BC 1 . 0 0 6 . 0 0 ^ 8 2.1+ AC 2 .021+ . 0 1 2 0 5 . 1 # ABC 2 . 0 0 2 . 0 0 1 2 . 5 Total Treatment 1 1 .301+ . 0 2 7 6 1 1 . 6 * Error 2 l + 0 . 5 7 1 .0021+ Total 2 5 1 . 8 7 5 A s Iodinated Casein B - Fat C - Vitamin - 65 -EXPERIMENT 7 Experiment 7 was designed to study the effect of (i) two levels of fat; ( i i ) two levels of riboflavin and the interaction of these factors on the serum cholesterol, serum total l i p i d s , and growth of the chick. The effect of the above factors were studied both in the normal chick and in the induced hyperthyroid chick. Hyperthyroidism was i n -duced in chicks by feeding them diets containing 0.02% iodinated casein.. Diets containing a high level of fat were formulated by substituting 12$ hydrogenated vegetable o i l for the dextrose in the 26$ protein basal diet shown in Table 1. A l l diets were supplemented with the following B complex vitamins: 90 grams of choline chloride 0.375 grams of folacin 0.8 grams of niacin 0.63 grams of calcium panthothenate 0.02 grams of riboflavin per 100 pounds of diet Diets containing a low level of riboflavin were formulated by the addition of the above B vitamins with the exception of riboflavin to the 26$ protein basal diet as shown in Table 1. It w i l l be noted that diets of both vitamin levels differ only in respect to the amount of additional riboflavin. Eight groups of New Hampshire cockerels were used in this experiment. Each group consisting.of approximately twenty-five chicks was fed an experimental diet as described - 66 -in Table 7C. The chicks were weighed individually at seven day intervals throughout an experimental period of twenty-eight days. Blood samples were taken from ten chicks selected at random from each group when the chicks were twenty-one and twenty-eight days old. Serum cholesterol was determined by the method of Zlatkis, Zak and Boyle (1953) and serum total l i p i d s by the method of Huerga et a l (1953). RESULTS GROWTH Chicks fed diets high in fat and high in riboflavin only grew at a faster rate than chicks fed low fat diets high in riboflavin. There were no differences in the growth rate of twenty-one day old chicks fed high fat basal diets, low in riboflavin and chicks fed low fat basal diets low in ribo-flavin. Twenty-eight day old normal chicks fed diet 3 (high fat, low riboflavin) grew at a slower rate than normal chicks fed diet 1 (low fat, low riboflavin). This observation agrees with those indicated in the literature, which state that the inclusion of fat in the diet of experimental animals results in an increased need for dietary riboflavin by those animals. (Harrill et a l , 1959). Hyperthyroid chicks fed the high fat diet supplemented with riboflavin grew at a faster rate than normal chicks fed a similar diet. Hyperthyroid chicks fed diet 3 grew at a faster rate than normal chicks fed the same diet and also grew faster than hyperthyroid chicks fed diet 1. SERUM CHOLESTEROL Normal chicks fed the basal diet high in fat and high - 67 -in riboflavin showed higher levels of serum cholesterol than chicks fed a basal diet low in fat and high i n riboflavin. Chicks fed a basal diet high in fat and low in riboflavin showed higher levels of serum cholesterol than chicks fed a basal diet low in fat and low in riboflavin, Hyperthyroid chicks fed diet 1 (low fat, low riboflavin), 3 (high fat, low riboflavin), 5 (low fat, high riboflavin) or 7 (high fat, high riboflavin) showed slightly lower levels of serum cholesterol than normal chicks fed similar diets. There were no differences- in the serum cholesterol levels of chicks fed diets low i n riboflavin and those fed diets high in riboflavin, SERUM TOTAL LIPIDS Chicks fed high fat diets either high or low in ribo-fl a v i n showed higher levels of serum total l i p i d s than chicks fed diets low in fat, Hyperthyroid chicks fed low fat diets showed lower levels of serum total lipids than normal chicks fed similar diets. In one instance, however, there were no differences between the serum total l i p i d levels of hyper-thyroid and normal chicks fed a low fat diet, viz. hyper-thyroid chicks fed diet 1 (low fat, low riboflavin). Chicks fed diets low in riboflavin showed lower levels of serum total lip i d s than chicks fed diets high in riboflavin. At this stage i t may be of interest to mention that although chicks were fed diets high in riboflavin, they s t i l l showed symptoms typical of chicks suffering from a riboflavin deficiency. Summary TABLE 7  of Results i n Experiment 7 Age i n Days DIET nr 1 H 3 N 3 H 5 N 5 H 7 N 7 H Average V/eight i n Grams Serum Cholesterol Level mgm./lOO c c . Total L i p i d Level mgm./loo c c . 21 28 21 28 21 28 198 322 206 185 377 270 195 314 195 176 301 270 197 310 211 208 386 329 217 343 203 201 405 366 207 340 192 181 419 321 214 348 188 174 396 303 227 366 210 202 490 382 255 406 191 191 486 389 o 03 TABLE 7A Serum Cholesterol Level from Individual Chicks in mgm./lOO c.c. in Experiment 7  DIET 21 day old chicks Average 1 N 208 200 245 245 196 206 208 194 196 158 206 1 H 188 241 184 228 180 210 184 194 184 160 195 3 N 225 200 194 228 307 180 200 200 203 173 211 3 H 164 178 203 160 164 203 265 231 249 215 203 5 N 186 191 180 184 203 241 203 173 184 170 192 5 H 200 164 184 208 196 192 170 182 172 210 188 7 N 220 200 192 215 208 231 203 208 196 228 210 7 H 172 213 173 192 173 194 184 212 208 184 191 28 day old chicks 1 N 163 197 184 180 166 173 213 191 188 196 188 1 H 166 180 168 186 153 166 203 172 194 172 176 3 N 180 194 203 206 245 192 200 220 228 212 208 3 H 192 170 241 184 184 220 180 215 227 194 201 5 N 175 145 180 182 172 194 168 184 197 215 181 5 H 149 173 191 173 170 237 192 180 138 138 174 7 N 186 203 203 186 194 208 228 191 212 208 202 7 H 203 170 196 197 191 178 158 194 227 191 191 TABLE 7 B Total L i p i d Level of Individual Chicks i n mgm./lOO c c . Serum i n Experiment 7 DIET 21 day old chicks Average 1 N 380 580 330 370 320 320 345 460 320 345 377 1 H 310 380 300 300 370 360 300 270 210 210 301 3 N 345 485 270 510 - 345 330 370 310 285 386 3 H 405 345 345 345 370 395 485 415 485 462 405 5 N 460 320 345 370 510 510 498 415. 380 380 419 5 H 395 345 370 415 498 370 330 395 370 476 396 7 N 560 510 462 580 440 580 440 415 450 462 490 7 H 450 535 395 535 485 550 462 485 510 450 486 28 day old chicks 1 N 235 310 225 285 225 270 320 320 310 200 270 1 H 270 225 235 320 270 265 200 246 345 320 270 3 N 360 246 360 310 405 345 320 302 360 285 329 3 H 330 300 370 370 370 320 415 395 300 485 366 5 N 320 345 345 300 260 360 380 300 285 310 321 5 H 320 320 330 330 260 310 380 260 270 246 303 7 N 440 285 330 450 476 285 395 450 360 345 382 7 H 330 345 380 300 450 380 370 345 485 450 389 - 71 -TABLE 7C DIET DESCRIPTION OP DIET I N "t Basal 1 H 1 + 0.02$ iodinated casein 3 N 1 12$ hydrogenated vegetable o i l 3 H 1 + 12$ hydrogenated vegetable o i l -4-0.02$ iodinated casein 5 N o Basal 5 H 5 0.02$ iodinated casein 7 N 5 +• 12$ hydrogenated vegetable o i l 7 H 5+ 12$ hydrogenated vegetable o i l 4- 0.02$ iodinated casein 26$ protein basal diet as shown in Table 1 and supple-mented with B complex vitamins with the exception of riboflavin. O 26$ protein basal diet as shown in Table 1 and supple-mented with B complex vitamins including riboflavin. N = Normal chick H = Hyperthyroid chick - 72 -ANALYSIS OF VARIANCE (GROWTH)  EXPERIMENT 7 TABLE 7D S o u r c e o f V a r i a t i o n d f Sum o f S q u a r e s Mean S q u a r e s F A 1 .012 .0117 5.7 * B 1 .023 .0229 11.2 * # C 1 .069 .0691 33.7 * * AB 1 .015 .0lk5 7.1 * BC 1 .008 .0081 3.9 # AC 1 .000 .OOOlj. .2 ABC 1 .000 .0000 .0 T o t a l T r e a t m e n t 7 .127 .0181 8.8 E r r o r 128 .262 .0021 T o t a l 135 .389 A - Iodine B = F a t C = R i b o f l a v i n - 73 -EXPERIMENT 8 Experiment 8 was designed to study the effects of (i) two levels of protein; ( i i ) two levels of fat and the interaction of these factors on the serum cholesterol levels, the serum total l i p i d levels and growth of the chick. The effects of the above factors were studied both in the normal and the hyperthyroid chick. Hyperthyroidism was i n -duced in the chick by feeding them diets containing 0.02$ iodinated casein. The two levels of protein used in this experiment were 20$ and 26$ as shown in Table 1. Diets with a high level of fat were formulated by substituting 12$ lard for dextrose in the 20$ and 26$ protein basal diets as shown in Table 1. Prom the previous experiments carried out i t was noted that there is a great variation in the serum cholesterol and serum total l i p i d levels of the chick at different intervals. It was therefore decided to study the effects of the above factors on the serum cholesterol and serum total l i p i d levels of the chick from an earlier age (five days old). Eight groups of White Leghorn cockerels were used in this experiment. Each group consisting of approximately twenty-five chicks was fed an experimental diet, the description of which is shown in Table 8. The chicks were weighed at approximately seven day intervals and blood samples were taken periodically from ten chicks selected at random in each group. Serum cholesterol was determined by the method of Zlatkis, Zak and Boyle (1953) while serum total lipi d s were - 71+ -determined by the method of Huerga et a l (1953). RESULTS  GROWTH As seen from the results in Table 8B, chicks fed a 26$ protein diet grew at a faster rate than chicks fed a 20$ protein diet. Only in one instance the reverse effect was noted viz. chicks fed diet 5 (high protein, low fat) grew at a slower rate than chicks fed diet 1 (low protein, low f a t ) . This effect was noted in chicks twenty-eight days old. Hyperthyroid chicks fed diets 1, 3 (low protein, high fat) or diet 7 (high protein, high fat) grew at a slower rate than normal chicks fed a similar diet. Apparently hyper-thyroid chicks gave an increased gorwth rate response only when the protein to energy ratio of the diet was high (see Table 8 B ) . Chicks fed 26$ protein diets high in fat grew at a faster rate than chicks fed 26$ protein diets low in fat. Normal chicks fed 20$ protein diets high in fat grew at a slower rate than normal chicks fed 20$ protein diets low in fat. Hyperthyroid chicks fed 20$ protein diets high in fat grew at a slower rate than hyperthyroid chicks fed 20$ protein diets low in fat. SERUM CHOLESTEROL Generally chicks fed 26$ protein diets showed lower levels of serum cholesterol than chicks fed 20$ protein diets. In one instance, thirty-three day old normal chicks fed diet 5 (high protein, low fat) showed higher levels of serum cholesterol than thirty-three day old normal chicks fed - 75 -diet 5 showed very l i t t l e difference in their serum cholesterol levels, from the twenty-six day old normal chicks fed diet 1 . Hyperthyroid chicks fed diet 5 showed slightly lower levels of serum cholesterol than hyperthyroid chicks fed diet 1. The serum cholesterol levels of hyperthyroid chicks fed diet 7 (high protein, high fat) were much lower than those of hyperthyroid chicks fed diet 3 (low protein, high f a t ) . The effect of induced hyperthyroidism on the serum cholesterol levels of the chicks fed diets 1 or 3 was inconsistent. As seen in Table 8A hyperthyroid chicks fed diets 1 or 3 showed higher levels of serum cholesterol in some instances than normal chicks fed similar diets, whilst in other instances the reverse effect was noted. Hyperthyroid chicks fed diets 5 or 7 generally showed lower levels of serum cholesterol than normal chicks fed similar diets. Forty-nine day old hyperthyroid chicks fed diet 7 showed higher levels of serum cholesterol than normal forty-nine day old chicks fed the same diet. Chicks fed high fat diets generally showed higher levels of serum cholesterol than chicks fed low fat diets. TOTAL LIPIDS Except for thirty-three day old normal chicks, the total l i p i d levels of normal chicks recorded at a l l other ages, were lower in those chicks fed a 26$ protein diet than those fed a 20$ protein diet. Thirty-three day old normal chicks fed 26$ protein diets showed higher levels of serum total lip i d s than normal chicks of the same age and fed the 20$ protein diet. Normal chicks fed diet 3 (low protein, high fat) generally showed higher levels of serum total li p i d s than - 76 -normal chicks fed diet 7 (high protein, high f a t ) . Twenty-six and thirty-three day old normal chicks fed diet 3 showed lower levels of serum total lipi d s than normal chicks of similar ages and fed diet 7« Hyperthyroid chicks fed diets 5 (high protein, low fat) or 7 generally showed lower levels of serum total l i p i d s than normal chicks fed diets 5 or 7. The effect of hyperthyroidism on the serum total l i p i d s of chicks fed diets 1 or 3 was in-consistent. Hyperthyroid chicks fed diet 1 generally showed lower levels of serum total lipi d s than normal chicks fed similar diets, while forty day old hyperthyroid chicks showed slightly higher serum total l i p i d levels than normal chicks of a similar age. There were no differences between the serum total l i p i d levels of twenty-six day old hyperthyroid chicks and normal chicks fed diet 1. Hyperthyroid chicks fed diet 7 generally showed lower levels of serum total lipids than normal chicks fed the same diet. Forty-six day old hyperthyroid chicks, however, showed higher levels of serum total lip i d s than forty-six day old normal chicks when they were fed diet 7. Formal chicks fed high fat, high protein diets generally showed higher serum total l i p i d levels than normal chicks fed low fat, high protein diets. Five day old and twenty-six day old normal chicks fed diet 3 showed lower levels of serum total lipids than normal chicks of similar ages and fed diet 1. - -7-7 -TABLE 8 DIET DESCRIPTION OF DIET I N + Basal 1 H 14- 0.02$ iodinated casein 3 N 1 4 12$ lard 3 H 1 4 12$ lard + 0.02$ iodinated casein 5 N o Basal 5 H 5 + 0.02$ iodinated casein 7 N 5+12$ lard 7 H 5 + 12$ lard + 0.502$ iodinated casein T 20$ protein basal diet as shown in Table 8. O 26$ protein basal diet as shown in Table 8. N - Normal chicks. H = Hyperthyroid chicks. TABLE 8A Body Weights, Serum Cholesterol Level and To t a l L i p i d Level of Chicks i n Experiment 8 *Total Cholesterol - 20% Protein Age i n Days 5 12 19 26 33 40 46 DIET -1 N 227 210 200 175 175 179 182 1 H 255 202 182 172 184 193 184 3 N 278 206 238 199 216 202 197 3 H 252 220 233 205 26$ Protein 196 197 195 5 N 169 163 189 182 197 183 157 5 H 170 135 180 181 188 172 178 7 N 205 169 216 204 201 190 179 7 H 198 158 210 186 189 180 166 •Total Lipids - 20$ Protein 1 N 473 304 326 340 324 291 294 1 H 429 291 297 341 270 301 285 3 N 421 315 415 264 339 285 378 3 H 402 301 380 330 26$ Protein 306 291 408 5 N 279 239 333 318 350 246 291 5 H 275 197 306 276 336 264 302 7 N 370 247 380 348 401 236 314 7 H 346 227 337 307 352 221 348 TABLE 8B •Average Weights i n Grams - 20% Protein Age i n Days DIET 5 12 19 26 33 40 46 1 N 72 126 180 272 369 488 614 1 H 68 118 168 260 375 461 612 3 N 69 122 167 245 338 432 550 3 H 66 109 150 220 26% Protein 308 409 518 5 N 74 128 185 258 374 493 613 5 H 77 133 186 282 395 524 647 7 N 77 142 211 310 439 580 708 7 H 77 131 200 296 430 559 692 * Average of 10 determinations per group + Average of twenty-five chicks per group - 8 0 -ANALYSIS OF VARIANCE (GROWTH) EXPERIMENT 8 TABLE 8C S o u r c e o f V a r i a t i o n d f Sum o f S q u a r e s Mean S q u a r e s F A 1 .007 . 0 0 7 3 .9 B 1 . 0 0 7 . 0 0 7 5 - .9 C 1 . 2 3 3 . 2 3 3 0 2 8 . 8 # * AB 1 . 0 3 7 . 0 3 7 1 U.6 # BC 1 . 1 3 2 . 1 3 2 5 16.1+ % % AC 1 .000 . 0 0 0 2 .0 ABC 1 . 0 1 5 . 0 1 5 2 1.9 T o t a l T r e a t m e n t 7 .1+33 . 0 6 1 8 7.6 # * E r r o r 15*2 1.229 . 0 0 8 1 T o t a l 1 5 9 1.662 A B C I o d i n e L e v e l F a t L e v e l P r o t e i n L e v e l - 81 -Diagram VIII(a)  Diagram to Show the Growth Rate of Chicks in Experiment 8 7-, GROWTH ( 2 0 % Protein diet.) - 82 -D i a g r a m V I I I ( b )  D i a g r a m t o Show t h e Growth R a t e o f C h i c k s i n E x p e r i m e n t 8 GROWTH ( 2 6 % Protein). Diagram VIII(c) Diagram to Show the Serum Cholesterol Level of Chicks in Experiment 8  30CH co 250 8 I <x> co | 2 0 0 - j £ 3 k_ CD (rt 150-2 0 % Protein \ \ \ \\ -2 Basal. 3^ o — o Basal + 0 0 2 % iod. casein. E A <& Basal + fat-4 -A Basal + fat + 0-02% iod- casein. 100 i . 0 10 20 30 Age in days. — r — 40 50 Diagram VIII(d) Diagram to Show the Serum Cholesterol Level of Chicks i n Experiment 8  -d-co 25*0 o-« | 200-1 o U E : 'so-o> E 100 0 2 6 % Protein. e-o-_# Basal. -O Basal + 0 0 2 % iod. casein. - A Basal + fat. -A. Basal +f&t * 0 0 2 % iod- casein. 0 A - 2 0 Age in days — i — 30 40 50 Diagram VHI(e) Diagram to Show the Serum To t a l L i p i d Level of Chicks in Experiment Q  5 0 0 -400-300-o o 2 0 % Protein. E | 200-1 a> S o-^ Basal. o B a s a l . <f 0 . 0 2 % iod. cos • i n * Basal • fat. t> A. Basal + fat + 0 0 2 % iod casein. 100 3b~ 4 '0 ^5 Age in days D i a g r a m V I I I ( f ) D i a g r a m t o Show t h e Serum T o t a l L i p i d L e v e l o f C h i c k s i n E x p e r i m e n t 8  CO •o CL « * Basal. o o Basal 4 0 0 2 % iod. casein. 4 , ^ Basal + fat. & 6 Basal + fat + 0 0 2 % iod.casein 26,% Protein. ~ 400-o E 3 v. v> 300-200-^  a 1001 0 20 Age in 30 days. 40 50 - 8? -GENERAL DISCUSSION  GROWTH The results of experiments 1 and 8 indicate that (i) Normal chicks fed a 26$ protein diet grew at a si g n i f i c -antly faster rate than normal chicks fed a 20$ protein diet. ( i i ) When 12$ fat was included in the diets of normal chicks, either in the form of hydrogenated vegetable o i l or lard, normal chicks fed the fat diet containing a 26$ protein level grew at a significantly faster rate than normal chicks fed a 20$ protein diet. ( i i i ) Induced hyperthyroidism increased the growth rate of chicks fed a 26$ protein diet, low in fat content, as compared to normal chicks fed a similar diet. (iv) Administration of the thyroactive protein to chicks fed a 20$ protein diet at either fat level depressed the growth of these chicks. (v) The administration of thyroactive protein retarded the growth of chicks fed a 26$ protein diet, high in fat content, as compared to normal chicks fed a similar diet. The above findings agree with those reported in the literature. Biely and March (1954) reported that the effect of fat supplements in chick diets depends in part upon the protein content of the diets. The addition of fat to low protein diets depresses growth and feed efficiency whereas the addition of fat to high protein diets has been shown to stimulate growth - 88 -and t o improve e f f i c i e n c y o f f e e d u t i l i z a t i o n . B a r n e s , P r i m r o s e and B u r r (I9I4I+) showed t h a t a l o w e r p r o t e i n c o n t e n t i n t he d i e t f e d t o r a t s was a s s o c i a t e d w i t h a l o w e r f a t d i g e s t i b i l i t y . The e f f e c t on t h e g r o w t h r a t e o f c h i c k s r e n d e r e d h y p e r -t h y r o i d was i n c o n s i s t e n t . Whereas a s i g n i f i c a n t i n c r e a s e i n th e g r o w t h r a t e o f c h i c k s f e d t h y r o a c t i v e p r o t e i n was o b t a i n e d i n e x p e r i m e n t s 2, 5, and 19 and a h i g h l y s i g n i f i c a n t i n c r e a s e i n g r o w t h r a t e o f h y p e r t h y r o i d c h i c k s f e d a 26$ p r o t e i n d i e t i n e x p e r i m e n t 1, no e f f e c t was o b t a i n e d i n t h e g r o w t h r a t e o f h y p e r t h y r o i d c h i c k s f r o m e x p e r i m e n t s 3 , 6, and 8 . I n e x p e r i -ment 1 g r o w t h r a t e was r e t a r d e d i n h y p e r t h y r o i d c h i c k s f e d a 20$ p r o t e i n d i e t . D a t a i s a v a i l a b l e t o i n d i c a t e t h a t r e -q u i r e m e n t s f o r a number o f n u t r i e n t s a r e m a r k e d l y i n c r e a s e d i n t h e h y p e r t h y r o i d a n i m a l . D r i l l e t a l ( I9I+3) have shown t h a t t h e r e q u i r e m e n t s f o r t h e t h r e e B complex v i t a m i n s , t h i a m i n , p y r i d o x i n e , and panthottoenic a c i d a r e i n c r e a s e d d u r i n g e x p e r i m e n t a l h y p e r t h y r o i d i s m o f t h e r a t . M a r t i n ( 1952) has d e m o n s t r a t e d an i n c r e a s e r e q u i r e m e n t f o r f o l i c a c i d , w h i l e N i c h o l e t a l ( 191+9) have d e m o n s t r a t e d an i n c r e a s e d r e q u i r e m e n t 12 f o r v i t a m i n B 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 l a r g e d o s e s o f t h y r o a c t i v e s u b s t a n c e s i n t h e r a t . I f the a d m i n i s t r a t i o n o f t h y r o a c t i v e s u b s t a n c e s i n t h e c h i c k has s i m i l a r p h y s i o l o g i c a l e f f e c t s as have been shown t o o c c u r i n t h e r a t , t h e n i t i s l i k e l y t h a t the e f f e c t o f f e e d i n g t h y r o a c t i v e p r o t e i n has i n -c r e a s e d t h e r e q u i r e m e n t s o f the c h i c k f o r c e r t a i n v i t a m i n s o r - 89 -e s s e n t i a l g r o w t h f a c t o r s . I t i s p o s s i b l e t h a t a d e c r e a s e i n t h e g r o w t h r a t e o f c h i c k s f e d a 20$ p r o t e i n b a s a l d i e t t o g e t h e r w i t h t h e a d -m i n i s t r a t i o n o f t h y r o a c t i v e p r o t e i n , o v e r n o r m a l c h i c k s f e d s i m i l a r d i e t s , can be e x p l a i n e d b y a p o s s i b l e v i t a m i n d e f i c i e n c y w h i c h may have r e s u l t e d f r o m i n d u c i n g h y p e r t h y r o i d i s m i n t h e young c h i c k . E x p e r i m e n t s 1, 2, 3> 5, 6, and 7 show t h a t when c h i c k s were f e d d i e t s c o n t a i n i n g a d d i t i o n a l amounts o f t h e B com-p l e x v i t a m i n s , c a l c i u m p a n t h o t h e n a t e , f o l a c i n , n i a c i n , r i b o f l a v i n and c h o l i n e c h l o r i d e , s i g n i f i c a n t i n c r e a s e s i n g r o w t h r a t e were o b t a i n e d f r o m c h i c k s f e d t h e s e d i e t s as compared t o c h i c k s f e d d i e t s low i n t h e above B complex v i t a m i n s . W i t h r e g a r d t o t h e e f f e c t o f t h e q u a n t i t y o f f a t i n t h e d i e t , on t h e g r o w t h r a t e s o f c h i c k s , e x p e r i m e n t s 1 and 8 show a s i g n i f i c a n t i n c r e a s e i n t h e g r o w t h r a t e s o f c h i c k s f e d h i g h f a t d i e t s , p r o v i d e d t h e p r o t e i n c o n t e n t o f t h e d i e t was a d e q u a t e . In e x p e r i m e n t s 3, h* 5> 6, and 7, t h e p r o t e i n c o n -t e n t o f t h e d i e t s u s e d was 26$. A l t h o u g h i n c r e a s e s i n g r o w t h r a t e o f a l l c h i c k s were o b t a i n e d when 12$ f a t was I n c l u d e d i n t h e i r d i e t s , t h e i n c r e a s e d g r o w t h r a t e s shown by c h i c k s f r o m e x p e r i m e n t s 1, 6, 7, and 8 were s i g n i f i c a n t . I t i s a p p a r e n t t h a t t h e e n e r g y c o n t e n t o f t h e d i e t s , c o n t a i n i n g 12$ h y d r o g e n a t e d v e g e t a b l e o i l as i n e x p e r i m e n t 1, o r 12$ l a r d as i n e x p e r i m e n t 8, was a d e q u a t e when t h e p r o t e i n - 90 -l e v e l o f t h e d i e t was 20$. The e n e r g y c o n t e n t o f t h e h i g h f a t d i e t s was not a d e q u a t e when t h e p r o t e i n l e v e l was 26$. T h i s may e x p l a i n t h e i n c r e a s e i n g r o w t h o b t a i n e d i n young c h i c k s f e d a h i g h f a t , h i g h p r o t e i n d i e t o v e r t h o s e f e d a h i g h f a t , low p r o t e i n d i e t . T h i s f i n d i n g i s s u p p o r t e d b y X t h o s e o f Dam e t a l (1959) a n d B a l d i n i and R o s e n b e r g (1957) i n c h i c k s . Dam e t a l o b t a i n e d h i g h l y s i g n i f i c a n t g r o w t h i n c r e a s e s w i t h c h i c k s , when t h e h y d r o g e n a t e d v e g e t a b l e o i l o r l a r d c o n t e n t o f t h e d i e t was i n c r e a s e d f r o m 2-5$ t o 10$. The p r o t e i n c o n t e n t o f t h e d i e t was a d j u s t e d t o t a k e c a r e o f t h e i n c r e a s e d e n e r g y c o n t e n t . - 91 -SERUM TOTAL CHOLESTEROL By i n c r e a s i n g t h e p r o t e i n l e v e l o f a d i e t , a d e c r e a s e i n s e r u m . c h o l e s t e r o l c o n c e n t r a t i o n was o b t a i n e d b y Moyer e t a l (1956) i n r a t s , K o k a t n u r e t a l (1958) i n mature c o c k e r e l s , N i s h i d a e t a l (1958), J o h n s o n e t a l (1958), and M a r c h e t a l (1959) i n t h e young c h i c k . The r e s u l t s p r e s e n t e d i n e x p e r i m e n t 8 c o n f i r m t h e f i n d i n g s o f t h e above w o r k e r s . When b a s a l d i e t s o f 20$ a n d 26$ p r o t e i n l e v e l r e s p e c t i v e l y were f e d t o young c h i c k s , l o w e r serum c h o l e s t e r o l and l i p i d l e v e l s were o b t a i n e d f r o m c h i c k s f e d t h e 26$ p r o t e i n d i e t t h a n f r o m t h o s e f e d t h e 20$ p r o t e i n d i e t . Diagrams I X ( a ) and I X ( b ) compare t h e serum c h o l e s t e r o l l e v e l o b t a i n e d i n c h i c k s f e d b o t h a 20$ and a 26$ p r o t e i n b a s a l d i e t . I n e x p e r i m e n t 1, no d i f f e r e n c e s i n t h e serum c h o l e s t e r o l l e v e l o f b o t h n o r m a l c h i c k s and c h i c k s r e n d e r e d h y p e r t h y r o i d c o u l d be o b t a i n e d f r o m f e e d i n g e i t h e r a 20$ o r 26$ p r o t e i n b a s a l d i e t . I n e x p e r i m e n t 1 and 8, however, c h i c k s r e n d e r e d h y p e r t h y r o i d and f e d a h i g h f a t d i e t c o n t a i n i n g a p r o t e i n l e v e l of 20$, showed h i g h e r serum c h o l e s t e r o l l e v e l s t h a n t h o s e c h i c k s r e n d e r e d h y p e r t h y r o i d and f e d a h i g h f a t d i e t c o n s i s t i n g o f a 26$ p r o t e i n l e v e l . Much work has been done on t h e e f f e c t o f e x p e r i m e n t a l l y p r o d u c e d changes i n t h e t h y r o i d s t a t u s . An i n c r e a s e i n t h y r o i d a c t i v i t y has been f o u n d t o d e c r e a s e t h e serum c h o l e s t e r o l and serum t o t a l l i p i d l e v e l i n some e x p e r i m e n t a l a n i m a l s . The d a t a o b t a i n e d i n t h e v a r i o u s e x p e r i m e n t s a n d - 92 -i l l u s t r a t e d i n d i a g r a m X ( a ) a nd X ( b ) show t h a t t h e r e i s a g r e a t amount o f v a r i a b i l i t y i n t h e serum c h o l e s t e r o l l e v e l , o f c h i c k s f e d 0.02$ i o d i n a t e d c a s e i n . As s e e n i n d i a g r a m X ( a ) t h e l e v e l s o f ser u m t o t a l c h o l e s t e r o l and serum t o t a l l i p i d s o f i n d u c e d h y p e r t h y r o i d c h i c k s a r e compared. I n e x p e r i m e n t s 2, 3, and 6 h y p e r -t h y r o i d c h i c k s showed somewhat l a r g e r l e v e l s o f serum c h o l e s t e r o l t h a n n o r m a l c h i c k s w h i l e , i n e x p e r i m e n t s l\. a n d 5 no d i f f e r e n c e s c o u l d be s e e n . However, i n e x p e r i m e n t s 7 and 8 serum c h o l e s t e r o l l e v e l s were l o w e r i n h y p e r t h y r o i d c h i c k s t h a n i n n o r m a l c h i c k s . An i n c r e a s e i n c h o l e s t e r o l l e v e l due to. a h y p e r t h y r o i d s t a t e i s i n c o n t r a d i c t i o n w i t h t h e work r e p o r t e d i n t h e l i t e r a t u r e . B e r t h e i l e t a l (191+7) have d e m o n s t r a t e d a d e c r e a s e i n serum c h o l e s t e r o l l e v e l i n t h e h y p e r t h y r o i d r a t w h i l e S m i t h (1935) was t h e o n l y w o r k e r t o f i n d t h e o p p o s i t e e f f e c t i n r a b b i t s . I t seemed r e a s o n -a b l e t o s u s p e c t t h a t t h e l e v e l o f t h y r o a c t i v e p r o t e i n f e d may n ot have b e e n e f f e c t i v e i n p r o d u c i n g a h y p e r t h y r o i d s t a t e i n t h e c h i c k s a t a l l t i m e s . T h i s p o s s i b i l i t y was i n -v e s t i g a t e d f u r t h e r a nd i n e x p e r i m e n t 5 t h e e f f e c t o f f e e d i n g t w i c e t h e l e v e l o f t h y r o a c t i v e p r o t e i n (0.0lj.$ i o d i n a t e d c a s e i n ) was d e t e r m i n e d . The e f f e c t o f t h e i n c r e a s e d l e v e l o f t h y r o a c t i v e p r o t e i n on t h e serum c h o l e s t e r o l c o n c e n t r a t i o n was s t i l l i n c o n s i s t e n t . W h i l e i n some i n s t a n c e s a h i g h c h o l e s t e r o l c o n c e n t r a t i o n was o b t a i n e d , on o t h e r o c c a s i o n s a d e c r e a s e d l e v e l was p r o d u c e d . T h e s e o b s e r v a t i o n s w o u l d t e n d t o r u l e o ut t h e p o s s i b i l i t y t h a t t h e l e v e l o f 0.02$ - 93 -i o d i n a t e d c a s e i n was i n e f f e c t i v e i n p r o d u c i n g a h y p e r t h y r o i d s t a t e i n young c h i c k s . 0 .02$ i o d i n a t e d c a s e i n may have h a d a s e a s o n a l e f f e c t i n p r o d u c i n g i n c r e a s e d t h y r o i d a c t i v i t y i n th e young c h i c k . I n o r d e r t o c o n f i r m t h i s p o s t u l a t e f u r t h e r e x p e r i m e n t a l work s h o u l d be done w h i c h w o u l d i n v o l v e l o n g -t e r m e x p e r i m e n t s . U s i n g t h e r a t as the e x p e r i m e n t a l a n i m a l Rosenman e t a l (1952) have shown t h a t t h e r a t e o f s y n t h e s i s o f c h o l e s t e r o l has i n c r e a s e d i n h y p e r t h y r o i d i s m , a n d c o n v e r s e l y t h e r e i s a l o w e r s y n t h e s i s r a t e i n h y p o t h y r o i d i s m . B y e r s (1958) has s t a t e d t h a t t h e s e changes i n t h e r a t e o f s y n t h e s i s a r e o p p o s i t e i n d i r e c t i o n t o t h e b l o o d c h o l e s t e r o l c o n c e n t r a t i o n c h a n g e s . The p l a s m a c h o l e s t e r o l l e v e l has been shown t o f a l l i n t h e h y p e r t h y r o i d s t a t e o f t h e r a t and r i s e i n t h e h y p o t h y r o i d s t a t e . B y e r s (1958) s u g g e s t e d t h a t t h e r a t e o f e x c r e t i o n o f p l a s m a c h o l e s t e r o l d u r i n g h y p e r t h y r o i d i s m i s i n -c r e a s e d w h i l e d u r i n g h y p o t h y r o i d i s m t h e r a t e o f c h o l e s t e r o l e x c r e t i o n i s d e c r e a s e d . The l o w e r l e v e l s o f seru m c h o l e s t e r o l o b t a i n e d i n h y p e r t h y r o i d c h i c k s t h a n i n n o r m a l c h i c k s as s e e n I n e x p e r i m e n t s 7 and 8 c o n f i r m s t h e s u g g e s t i o n s o f B y e r s (1958) . I n e x p e r i m e n t s 7 and 8, i t i s p o s s i b l e t h a t t h e r a t e o f ex-c r e t i o n o f c h o l e s t e r o l i s i n c r e a s e d i n t h o s e c h i c k s r e n d e r e d h y p e r t h y r o i d . The above r e a s o n i n g , however, does not e x p l a i n why h i g h e r l e v e l s o f serum c h o l e s t e r o l were o b t a i n e d i n h y p e r -t h y r o i d c h i c k s f r o m e x p e r i m e n t s 2 , 3 , a n d 6 . The e f f e c t o f h y p e r t h y r o i d i s m on serum t o t a l l i p i d s f o l l o w e d a l m o s t t h e same p a t t e r n as t h a t f o r serum t o t a l - 91+ -c h o l e s t e r o l . D i a g r a m X I ( c ) shows t h i s p a t t e r n and t h e same e x p l a n a t i o n w h i c h h e l d f o r serum c h o l e s t e r o l c an h o l d f o r t h e e f f e c t o f t h y r o a c t i v e p r o t e i n on t h e t o t a l l i p i d l e v e l s . I n e x p e r i m e n t I4. w h i l e no d i f f e r e n c e s were o b t a i n e d between t h e l e v e l s o f c h o l e s t e r o l i n t h e h y p e r t h y r o i d c h i c k and t h e n o r m a l c h i c k , t h e l i p i d l e v e l s o f t h e h y p e r t h y r o i d c h i c k was much h i g h e r t h a n t h o s e o f t h e n o r m a l c h i c k . E f f e c t s o f F a t A n i m a l e x p e r i m e n t s have c o n s i s t e n t l y d e m o n s t r a t e d d u r i n g t h e p a s t h a l f c e n t u r y t h a t d i e t a r y c h o l e s t e r o l i s one o f t h e most i m p o r t a n t f a c t o r s w h i c h i n f l u e n c e t h e serum c h o l e s t e r o l c o n c e n t r a t i o n . We a r e unaware o f any mammalian o r a v i a n s p e c i e s s y s t e m a t i c a l l y s t u d i e d w h i c h under c e r t a i n c i r c u m -s t a n c e s does not r e s p o n d t o d i e t a r y c h o l e s t e r o l w i t h i n -c r e a s e d serum c h o l e s t e r o l l e v e l s . T h i s i s an e s t a b l i s h e d f a c t . I n man Keys and h i s a s s o c i a t e s have p r e s e n t e d d a t a t o show t h a t t h e q u a l i t y as w e l l as t h e q u a n t i t y o f f a t may p l a y a r o l e i n d e t e r m i n i n g t h e l e v e l s o f c h o l e s t e r o l i n the serum o f t h e b l o o d . W i t h f u r t h e r n u t r i t i o n a l s t u d i e s A h r e n s e t a l (1957), Malmros and Wigand (1957) have come t o a s s o c i a t e h i g h e r l e v e l s o f serum c h o l e s t e r o l w i t h t h e i n t a k e o f a n i m a l o r s a t u r a t e d f a t t y a c i d s . U s i n g mature c o c k e r e l s , K o k a t n u r e t a l (1958) have c o n c l u d e d t h a t serum c h o l e s t e r o l v a l u e s were i n c r e a s e d i n c h i c k e n s w h i c h have been f e d f a t o v e r t h o s e f e d an e s s e n t i a l l y f a t f r e e d i e t . I n young c h i c k s M a r c h and B i e l y (1959) have shovm t h a t d i e t s s u p p l e m e n t e d w i t h s a t u r a t e d - 95 -f a t increase serum cholesterol l e v e l to a greater degree than those supplemented with unsaturated f a t . The saturated f a t used in experiments 1, 2, 3, k> 5> a n ^ 6 was hydrogenated vegetable o i l . In experiment 8 lard was the saturated fat used (see diagram XIII(a) and XIII(b). The effects on the l e v e l of serum cholesterol of chicks fed saturated f a t , as seen from experiments car r i e d out, con-firm those obtained by other workers. The res u l t s as shown in diagram XlII(a) i l l u s t r a t e s t h i s . Experiments 1, 3, k> 5, 6, 7, and 8 show increases in serum cholesterol l e v e l of chicks fed high f a t basal diets over those fed basal diets without supplemental f a t . In experiment 2 a lower l e v e l of serum cholesterol was obtained from normal chicks fed high fat basal diets than from normal chicks fed low fat basal d i e t s . When hyperthyroidism was induced, chicks from ex-periment 6 were the only ones to show a reasonably high l e v e l of serum cholesterol, when they were fed high fat diets (see diagram X l l ( c ) ) . E f f ects of Vitamins Diagram XII(d) summarizes the effect on the serum cholesterol levels when low B complex vitamin diets were fed to chicks. In experiments 1+ and 5 small decreases in serum cholesterol concentration were obtained when B complex vitamin low diets were fed. Apparently there is no evidence in the l i t e r a t u r e to show the effects obtained on the serum cholesterol and l i p i d l e v e l of chicks when t h e i r diets were defi c i e n t or low in the B complex vitamins, choline chloride, - 96 -f o l i c a c i d , n i c o t i n i c a c i d , r i b o f l a v i n , a n d c a l c i u m p a n t h o t h e n a t e , as a g r o u p . Herman (1952) showed t h a t b y o r a l a d m i n i s t r a t i o n o f n i c o t i n i c a c i d a l o n e , t h e s e r u m c h o l e s t e r o l c o n c e n t r a t i o n o f r a b b i t s .was r e d u c e d . The e f f e c t o f d e f i c i e n c y o f any one p a r t i c u l a r v i t a m i n i s l i k e l y t o i n f l u e n c e t h e serum c h o l e s t e r o l c o n c e n t r a t i o n and t o t a l l i p i d c o n c e n t r a t i o n i n one d i r e c t i o n , b u t t h i s i n -f l u e n c e may not be m a i n t a i n e d when o t h e r v i t a m i n s a r e d e f i c i e n t as w e l l . A l t h o u g h a v i t a m i n i n t e r - r e l a t i o n s h i p has b e en known t o e x i s t f o r s e v e r a l y e a r s now, t h e r e i s s t i l l v e r y l i t t l e i n -f o r m a t i o n as t o t h i s r e l a t i o n s h i p , and t h i s i s t r u e e s p e c i a l l y among t h e B complex v i t a m i n s . I t i s d i f f i c u l t t o s a y w hether t h e d i e t a r y l e v e l o f one B complex v i t a m i n e f f e c t s t h e g r o w t h o f a n i m a l s r e c e i v i n g i n a d e q u a t e amounts o f a s e c o n d B v i t a m i n . G y o r g y e t a l (1931+), C h i c k e t a l ( 1 9 3 5 ) and H a r r i s ( 1 9 3 5 ) b y e x p e r i m e n t a l p r o d u c t i o n o f d e f i c i e n c y d i s e a s e s i n r a t s , f o u n d t h a t a d e f i c i e n c y o f r i b o f l a v i n and p y r i d o x i n e d i d not e x h i b i t f l o r i d d e r m a t i t u s , c h a r a c t e r i s t i c o f p y r i d o x i d e d e f i c i e n c y , u n l e s s a d e q u a t e r i b o f l a v i n was a d d e d t o the d i e t . L e p k o v s k y e t a l ( 1 9 3 6 ) a l s o showed t h a t symptoms o f a c u t e p y r i d o x i n e d e f i c i e n c y d i d not a p p e a r i n t h e r a t u n l e s s a d e q u a t e p a n t h o t e n i c a c i d was p r e s e n t i n t h e d i e t . As a r e s u l t i t i s d i f f i c u l t t o p i n - p o i n t t h e e f f e c t o f a s p e c i f i c v i t a m i n . When i n a n i t i o n o c c u r s i n a n i m a l s an i n c r e a s e i n serum c h o l e s t e r o l a n d serum l i p i d , c o n c e n t r a t i o n r e s u l t s (Cook, 1 9 5 8 ) . - 97 -Whether t h i s i n c r e a s e i n c h o l e s t e r o l and l i p i d s c o n c e n t r a t i o n i s a d i r e c t e f f e c t o f i n a n i t i o n or w h e t h e r i t i s i n d i r e c t l y r e l a t e d t o a v i t a m i n i n a d e q u a c y , i s d i f f i c u l t t o d e t e r m i n e . C h i c k s u s e d i n t h e s e e x p e r i m e n t s were not d e p l e t e d f o r a n y p a r t i c u l a r v i t a m i n p r i o r t o t h e e x p e r i m e n t a l p e r i o d and t h e r e i s a p o s s i b i l i t y t h a t the c a r r y o v e r o f v i t a m i n s f r o m t h e egg t o the n e w l y h a t c h e d c h i c k , Is a d e q u a t e t o m a i n t a i n n o r m a l g r o w t h and d e v e l o p m e n t f o r a s h o r t p e r i o d o f t i m e . T h e r e may a l s o e x i s t t h e c a s e where t h e c a r r y o v e r of some v i t a m i n s i s so low t h a t t h e amount s u p p l e m e n t e d may not be a d e q u a t e t o p r e v e n t an e f f e c t of v i t a m i n d e f i c i e n c y . I n e x p e r i m e n t 7 t h i s i s p r o b a b l y what h a p p e n e d , f o r symptoms o f r i b o f l a v i n d e f i c i e n c y ( c u r l e d t o e p a r a l y s i s ) were n o t e d not o n l y i n t h e c h i c k s of r i b o f l a v i n low d i e t s but a l s o i n c h i c k s f e d t h e c o n t r o l d i e t s . Of t h e s e v e n e x p e r i m e n t s c a r r i e d out where low B complex v i t a m i n d i e t s were f e d , f i v e e x p e r i m e n t s showed i n c r e a s e d serum c h o l e s t e r o l c o n c e n t r a t i o n w h i c h may be due t o the e f f e c t s o f l o w l e v e l s o f t h e B complex v i t a m i n s , c h o l i n e c h l o r i d e , f o l i c a c i d , c a l c i u m p a n t h o t h e n a t e , n i c o t i n i c a c i d , and r i b o f l a v i n . When the d i e t s were s u p p l e m e n t e d w i t h c h o l i n e c h l o r i d e and f o l i c a c i d o n l y , serum c h o l e s t e r o l l e v e l was s t i l l i n c r e a s e d . I n e x p e r i m e n t s ij. and 5 a s m a l l i n c r e a s e i n serum c h o l e s t e r o l was o b t a i n e d . E f f e c t o f T h i o u r a c i l The o p p o s i n g e f f e c t o f t h i o u r a c i l a nd t h y r o x i n e were f i r s t u s e d b y Dempsey and A s t w o o d (191+3) t o e s t i m a t e t h e - 98 -rate of thyroid secretion in rats and by Mixner et a l (19M+) for the assay of thyroid activity in chicks. These workers found that thiouracil decreased the metabolic rate of animals treated. Diagrams XV(a), XV(b) and X(c) compare the results obtained by induing hypothyroidism in young chicks. Both the serum cholesterol and serum total l i p i d s are higher In induced hypothyroid chicks than normal chicks. These results confirm those already found by Fleischmann et al (191+5) in the thiouracil treated chicks, and those of Roseman et a l (1952) in the thiouracil treated rat. In experiment h in most instances i t was found that by pro-ducing a hypothyroid state in the young chick the serum cholesterol and serum total l i p i d levels were elevated. When hypothyroidism was induced in chicks fed high fat diets, no differences were noted in the cholesterol levels of these chicks and those of normal chicks fed diets low in the B complex vitamins, calcium panthothenate, niacin, f o l i c acid, riboflavin and choline chloride or in the B complex vitamins choline chloride and riboflavin only. Hyperthyroid chicks fed basal diets low in B complex vitamins showed lower serum cholesterol levels than normal chicks fed basal diets low in B complex vitamins. This result was achieved in experiments 1, 2, 3, 5> 6, and J7. Experiment *f showed higher levels of serum cholesterol in hyperthyroid chicks than in normal chicks, fed basal diets low in vitamin B complex (see diagram XIV(a)). - 99 -Prom the r e s u l t s o b t a i n e d i t seems r e a s o n a b l e t o s a y t h a t i n most c a s e s when h y p e r t h y r o i d i s m i s i n d u c e d i n t h e y o u n g c h i c k t h e r e i s a t e n d e n c y f o r t h e s e c h i c k s t o have l o w e r serum c h o l e s t e r o l l e v e l s t h a n n o r m a l c h i c k s , p r o v i d e d t h e b a s a l d i e t s f e d were low i n t h e B complex v i t a m i n s , c a l c i u m p a n t h o t h e n a t e , n i c o t i n i c a c i d , r i b o f l a v i n , f o l a c i n a n d c h o l i n e c h l o r i d e . E x p e r i m e n t s 1 , i\.t 6, a n d 7 showed h i g h e r l e v e l s o f serum c h o l e s t e r o l when c h i c k s were f e d b a s a l d i e t s h i g h i n f a t and low i n t h e B complex v i t a m i n s t h a n when t h e y were f e d b a s a l d i e t s low i n f a t a n d low i n B complex v i t a m i n s , ( d i a g r a m X I V ( b ) ) . No e f f e c t was o b t a i n e d i n e x p e r i m e n t s 3 and 5 . W h i l e i n e x p e r i m e n t 2 a l o w e r l e v e l o f serum c h o l e s t e r o l was o b t a i n e d b y f e e d i n g c h i c k s low v i t a m i n B complex b a s a l d i e t s , h i g h i n f a t , t h a n f e e d i n g c h i c k s b a s a l d i e t s l o w i n B complex v i t a m i n s and low i n f a t c o n t e n t . I n a l m o s t a l l c a s e s , however, h y p e r t h y r o i d c h i c k s f e d h i g h f a t low v i t a m i n B complex d i e t s showed l o w e r l e v e l s o f ser u m c h o l e s t e r o l t h a n n o r m a l c h i c k s f e d s i m i l a r d i e t s , ( d i a g r a m X l V ( c ) ) . Diagram IX 2 6 % p r o t e i n . 2 0 % E 8 N E 8 H EL N E l H E l NF] p r o t e i n . " E 8 HF E I m HF NF N - normal c h i c k s H - hyperthyroid c h i c k s N F - n o r m a l c h i c k s f e d higtv f a f d i e t s . HF - h y p e r t h y r o i d c h i c k s f e d h i g h f a t d i e t s . E l - e x p e r i m e n t I • E 8 - e x p e r i m e n t 8 * Diagram to show the effect of the protein level-on the s-erum cholesterol concentration of chicks in experiments I 8* 8 . Diagram X Serum tofol cholesterol Serum total lipids. 6 Serum total cholesterol 8 total lipid C s control. in induced hyperthyroid chicks. , . 8 s experiments. Diagram X I D i a g r a m t o Show E f f e c t o f 0.02$ I o d i n a t e d C a S e i n on Growth, Serum C h o l e s t e r o l and Serum T o t a l L i p i d L e v e l s i n C h i c k s f r o m E x p e r i m e n t s 1-8 6 5 A-Effect of 0 0 2 % B-Effect of 0 0 2 % C-Effect of 0 0 2 % iod- coseinon . iod. casein on iod. casein on serum cholesterol. growth. total lipids. D i a g r a m X I I D i a g r a m t o Show E f f e c t s o f H i g h P a t D i e t s , 0.02$ I o d i n a t e d C a s e i n and V i t a m i n D e f i c i e n c y on C h i c k s i n E x p e r i m e n t s 1-8 C = control. 6 I -8 • experiments. 8 6 o H 0 0 2 % iod. casern Ugh energy on chol. 0 0 2 % iod. casein Vlt. low diet on chol. on chol- level in level in chicks fed in chicks fed high level. chicks i9d vit. low vit. supplemented diet, energy diet. *= inter- vit. level. diet. D i a g r a m XIII High fat diet on the cholesterol level and lipid level of normal chicks. Diagram XIV Serum cholesterol level of chicks fed vit. low diet. 4 6 4 40 i I . I I 3 6 j i o H I A. effect of 0 0 2 % B.effeci of high fat diet. C- effect of 0'02% iod- casein. iod-casein high fat diet. C = control. 1-7= experiments. Diagram XV D i a g r a m t o Show t h e E f f e c t o f T h i o u r a c i l on Serum C h o l e s t e r o l and Serum T o t a l L i p i d L e v e l s and Growth o f C h i c k s i n E x p e r i m e n t ij.  o r-i C- control. I - thiouracil-2 - thiourocil+fot. 3- thiouracil + vit. deficient diet. 4- thiouracil • vit-def. diet*fat. 5- thiouracil + inter, vit. def. 6- thiouracil • inter, vit. def. • fat Effect of thiouracil on A - cholesterol. B - total lipids' C - growth - 107 -GENERAL CONCLUSIONS A s e r i e s o f e x p e r i m e n t s was c o n d u c t e d t o o b s e r v e t h e e f f e c t o f c e r t a i n d i e t a r y f a c t o r s on t h e serum c h o l e s t e r o l and serum t o t a l l i p i d l e v e l s i n t h e g r o w i n g c h i c k . Among t h e f a c t o r s t e s t e d were d i e t a r y p r o t e i n , d i e t a r y f a t and a g r o u p o f B complex v i t a m i n s c o n s i s t i n g o f c h o l i n e c h l o r i d e , c a l c i u m p a n t h o t h e n a t e , n i a c i n , f o l a c i n and r i b o f l a v i n . The above d i e t a r y f a c t o r s were t e s t e d i n s e v e r a l ex-p e r i m e n t s on b o t h t h e h y p e r t h y r o i d and t h e n o r m a l c h i c k . I n one e x p e r i m e n t t h e h y p o t h y r o i d c h i c k was u s e d . H y p e r t h y r o i d i s m was i n d u c e d i n t h e c h i c k s by f e e d i n g them d i e t s c o n t a i n i n g 0.02$ i o d i n a t e d c a s e i n , whereas h y p o t h y r o i d i s m was i n d u c e d b y f e e d i n g c h i c k s d i e t s c o n -t a i n i n g 0 . 1 $ t h i o u r a c i l . The growth r a t e o f c h i c k s r e n d e r e d h y p e r t h y r o i d v a r i e d . I n many i n s t a n c e s h y p e r t h y r o i d c h i c k s grew a t a s i g n i f i c a n t l y f a s t e r r a t e t h a n n o r m a l c h i c k s . I n o t h e r i n s t a n c e s however no d i f f e r e n c e was n o t e d between t h e growth r a t e o f h y p e r -t h y r o i d and n o r m a l c h i c k s , whereas on some o c c a s i o n s when h y p e r t h y r o i d i s m was i n d u c e d i n c h i c k s , t h e growth o f t h e s e c h i c k s was d e p r e s s e d . When a 26$ p r o t e i n b a s a l d i e t was f e d t o n o r m a l c h i c k s , t h e serum c h o l e s t e r o l o b s e r v e d i n t h e s e c h i c k s was l o w e r t h a n t h o s e f e d a 20$ p r o t e i n b a s a l d i e t . H y p e r t h y r o i d c h i c k s f e d a h i g h f a t d i e t c o n t a i n i n g 26$ - 108 -p r o t e i n showed l o w e r serum c h o l e s t e r o l and serum l i p i d l e v e l s t h a n h y p e r t h y r o i d c h i c k s f e d a h i g h f a t d i e t c o n -t a i n i n g a p r o t e i n l e v e l o f 20$. N o r m a l c h i c k s f e d a h i g h l e v e l o f f a t showed h i g h e r l e v e l s o f serum c h o l e s t e r o l and t o t a l l i p i d s t h a n n o r m a l c h i c k s f e d a low l e v e l o f f a t . N o r m a l c h i c k s f e d d i e t s low i n t h e B complex v i t a m i n s ( c h o l i n e c h l o r i d e , f o l a c i n , c a l c i u m p a n t h o t h e n a t e , n i a c i n , and r i b o f l a v i n ) showed h i g h e r l e v e l s o f serum c h o l e s t e r o l t h a n c h i c k s f e d d i e t s h i g h i n t h e above B c o m p l e x v i t a m i n s . C h i c k s r e n d e r e d h y p o t h y r o i d showed h i g h e r l e v e l s o f serum c h o l e s t e r o l and t o t a l l i p i d s t h a n n o r m a l c h i c k s . - 109 -BIBLIOGRAPHY A h r e n s , E.H. J r . , B l a n k e n h o r n , D.H. and T s a l t a , T.T. -" E f f e c t on human serum l i p i d s o f s u b s t i t u t i n g p l a n t f o r a n i m a l f a t i n t h e d i e t " . P r o c . S o c . E x p . B i o l . Med., 86 :872-878, 1954. A h r e n s , E.H. J r . , H i r s c h , J . , I n s u l l , W. J r . , T s a l t a s , T.T., B l o w s t r a n d , R. a n d P e t e r s o n , M.L. - "The I n f l u e n c e o f d i e t a r y f a t s on serum l i p i d l e v e l s i n man". L a n c e t , 1:943-953, 1957. A l t s c h u l , R., H o f f e r , A. a n d S t e p h e n , J.D. - " i n f l u e n c e o f n i c o t i n i c a c i d on serum c h o l e s t e r o l i n man". A r c h i v . o f B i o c h e m . and B i o p h y s i c s , 54 :558 , 1955. B a l d i n i , f J . , Rosenberg,H.R. - "The e f f e c t o f c a l o r i e scu r c e i n a c h i c k d i e t on g r o w t h , f e e d u t i l i z a t i o n and body c o m p o s i t i o n " . P o u l t r y S c i e n c e , 36, i+32-434, 1957• B a r n e s , R.H., P r i m r o s e , M.P., and B u r r , G.O. -"The i n -f l u e n c e o f the p r o t e i n c o n t e n t o f t h e d i e t upon f a t d i g e s t i b i l i t y " . J . N u t r i t i o n , 27 :179-184, 1944. B e t h e i l , J . J . , W i e b e l h a u s , V.D., and L a r d y , H.A. - "A n u t r i t i o n a l f a c t o r w h i c h a l l e v i a t e s t h e t o x i c i t y o f i n g e s t e d t h y r o i d s u b s t a n c e " . J . N u t r i t i o n , 34^431-441, 1947. B e v e r i d g e , J.M.R., C o n n e l l , W.F., Mayer, G.A., F i r s t b r o o k , J.B., and D e w o l f e , M.S. - "The e f f e c t s o f c e r t a i n v e g e t a b l e and a n i m a l f a t s on t h e p l a s m a l i p i d s o f humans". J . N u t r i t i o n , 56 :311-320, 1955. B i e l y , J . , a n d M a r c h , B . - " P a t s t u d i e s i n p o u l t r y " . " P o u l t r y  S c i e n c e , 33:1220-1227, 1954-B i e l y , J . , and March, B. - " P a t s t u d i e s i n p o u l t r y " . P o u l t r y S c i e n c e , 36:1236-1240, 1957. B r o n t e e - S t e w a r t , B., A n t o n i s , A., E a l e s , L . and B r o c k , J . F . " E f f e c t s of f e e d i n g d i f f e r e n t f a t s on serum c h o l e s t e r o l l e v e l " . L a n c e t , 1:521-526, 1956, B y e r s , S.O. - "The mechanism f o r changes i n b l o o d c h o l e s t e r o l i n d e r a n g e d t h y r o i d s t a t e s " . Am&r. J o u r . o f C l i n . N u t . , 6 : 6 4 2 , 1958. C h i c k , H., C o p p i n g , A.M., and E d g a r , G.E. - "The Water S o l u b l e B V i t a m i n . ( i v ) The components o f v i t a m i n B12". B i o c h e m . J . , 29 :722-734, 1935. Combs, G.F., and Romoser, G.L. - "A new a p p r o a c h t o p o u l t r y f e e d f o r m u l a t i o n " . M a r y l a n d A g r . E x p . S t a t . M i s c . Pub., 226: 1955. - 1 1 0 -Cook, R.P. - C h o l e s t e r o l * U n i v e r s i t y o f S t . Andrews, New Y o r k A c a d e m i c P r e s s , 1 9 5 8 . p.288. Dam, R i c h a r d , L e a c h , R.M. J r . , N e l s o n , T.S., N o r r l s , L.C., and H i l l , F.W. - " S t u d i e s on t h e e f f e c t o f q u a n t i t y and t y p e o f f a t on c h i c k g r o w t h " . J . o f Nut., 68:615-631, 1959. Dempsey, E.M., and A s t w o o d , E.B. - " D e t e r m i n a t i o n o f t h e r a t e o f t h y r o i d hormone s e c r e t i o n a t v a r i o u s en-v i r o n m e n t a l t e m p e r a t u r e s " . E n d o c r i n o l , 3 2 : 5 0 9 > 1 9 ) 4 3 . D o n a l d s o n , W.E., Combs, G.F., Romoser, G.L., and S u p p l e e , W.G. - "Body c o m p o s i t i o n , e n e r g y i n t a k e , f e e d e f f i c i e n c y , g r o w t h r a t e a n d f e a t h e r c o n d i t i o n o f g r o w i n g c h i c k e n s as i n f l u e n c e d b y c a l o r i e p r o t e i n r a t i o o f t h e r a t i o n " . P o u l t r y S c i e n c e , 3l+:1190, 1 9 5 5 . D r i l l , V.A., arid Overman, R. - " i n c r e a s e d r e q u i r e m e n t s o f p a n t h o t h e n i c a c i d a n d v i t a m i n B6 d u r i n g e x p e r i -m e n t a l h y p e r t h y r o i d i s m " . Am. J . P h y s i o l . , 1 3 5 : k l k - k l l s 191+1. F i l l i o s , L.C., and Mann, G.V. - " i n f l u e n c e o f s u l p h u r -amino a c i d d e f i c i e n c y on c h o l e s t e r o l m a t a b o l i s m " . M e t a b o l i s m , 3 : 1 6 - 2 6 , 1951+. F i l l i o s , L.C., Mann, G.V., and S t a r e , F . J . - " D i e t s low i n p r o t e i n f a v o u r h y p e r c h o l e s t e r e m i a i n r a t s " . J . E x p . Med., 1 0 l + : 5 3 9 - 5 5 1 , 1 9 5 6 . F l e i s c h m a n n , W., a n d F r i e d , I.A. - " S t u d i e s on the mechanism o f t h e h y p e r o l e s t e r o l e m i a and h y p e r -c o l c e r a i a i n d u c e d b y e s t r o g e n i n immature c h i c k s " . , 3 5 : 1 + 0 6 , 191+5. G r e e n , B., Lowe, J . S . , and M o r t o n , R.A. - "The e f f e c t o f v i t a m i n A d e f i c i e n c y on t h e c h o l e s t e r o l l e v e l o f the p l a s m a and l i v e r o f t h e r a t " . B i o c h e m . J . , 61:1+1+7-1+53, 1 9 5 5 . G r e e n b e r g , L.D., and R i n e h a r t , J.D. - " P l a s m a c h o l e s t e r o l l e v e l s o f c h o l e s t e r o l f e d c o n t r o l and p y r i d o x i n e d e f i c i e n t monkeys". P r o c . S o c . E x p . B i o l . Med., 7 6 : 5 8 0 - 5 8 3 , 1 9 5 1 . G y o r g y , P. - " V i t a m i n B 1 2 and t h e p e l l e g r a - l i k e d e r m a t i t i e s i n r a t s " . N a t u r e , 133 :1+98, 1931+. H a r d i n g e , M.G., and S t a r e , F . J . - " D i e t a r y and Serum l e v e l s o f c h o l e s t e r o l " . Am. J . C l i n . N u t . , 2:83, 1951+. - I l l -H a r r i l l , I n e z , K y l e n , A.M., W e i s , A., and D y a r , E . " R e l a t i o n o f d i e t a r y f a t and s u p p l e m e n t a r y r i b o -f l a v i n t o t i s s j e l e v e l s o f c h o l e s t e r o l , r i b o f l a v i n and t o t a l l i p i d s i n t h e r a t " . J . N u t r i t i o n , 69:356-361+, 1959. H a r r i s , L . J . - " F l a v i n and t h e p e l l e g r a - p r e v e n t i n g f a c t o r as s e p a r a t e c o n s t i t u e n t s o f complex B2". Bloc h e m . J . , 29:776-781, 1935. Herman, George - " M e t h i o n i n e d e c h o l e s t e r o l i z a t i o n i n o l d h e n s " . U n i v e r s i t y o f Texas B u l l e t i n , 1952. H i l d r e a t h , E.A., M e l l i n k o f f , S.M., B l a i r , G.M., and H i l d r e a t h , D.M. - "The e f f e c t o f v e g e t a b l e f a t i n g e s t i o n on human ser u m c h o l e s t e r o l c o n c e n t r a t i o n " . C i r c , 3:61+1-61+6, 1951. Hsu, J.M., a n d Chow, B.F. - " V i t a m i n B12 d e f i c i e n c y and h y p e r c h o l e s t e r o l e m i a " . F e d . P r o c , 16:63, 1957. H u e r g a , J . de l a , C h a r l o t t e , Y., and P o p p e r , H. " E s t i m a t i o n o f t o t a l serum l i p i d s b y a t u r b i -d i m e t r i c method". Am. J . C l i n . P a t h . , 23:1163-1167, 1953. I r w i n , M.R., R e i n e k e , E.P., and T u r n e r , C.W. - " E f f e c t o f f e e d i n g t h y r o a c t i v e i o d o c a s e i n on growth, f e a t h e r i n g , and w e i g h t o f g l a n d s o f young c h i c k s " . P o u l t r y S c i e n c e , 22:37l+-380, 191+3. J o h n s o n , D. J r . , L e v e i l l e , G.A., and F i s h e r , H. - " i n -f l u e n c e o f amino a c i d d e f i c i e n c i e s and p r o t e i n l e v e l on the p l a s m a c h o l e s t e r o l o f t h e c h i c k " . J . N u t r i t i o n , 66:367-376, 1958. J o n e s , R . J . , a n d Huffman, S. - " C h r o n i c e f f e c t o f d i e t a r y p r o t e i n on h y p e r c h o l e s t e r m i a i n the r a t " . S o c . f o r  E x p . B i o l . Med., 93:519, 1956. K e y s , A., A n d e r s o n , J . T . , F i d a n z a , F . , K e y s , M.H., a n d S w a l e n , B. - " E f f e c t s o f d i e t on b l o o d l i p i d s i n man, p a r t i c u l a r l y c h o l e s t e r o l and l i p o p r o t e i n " . C l i n . Chem., l:3i+-52, 1955. K e y s , A., A n d e r s o n , J . T . , a n d G r a n d e , F. - "Serum C h o l e s t e r o l R e s p o n s e t o N a t u r a l a n d H y d r o g e n a t e d F a t s " . C i r c , 16:1+80 ( A b s t r . ) . , 1957. K i n s e l l , L.W., P a t r i d g e , J . , B o l i n g , L., M a r g e n , S., a n d M i c h a e l s , G.D. - " D i e t a r y m o d i f i c a t i o n o f s e r u m c h o l e s t e r o l a n d p h o s p h o l i p i d l e v e l s " . J . C i j n . E n d o c r i n a l a n d M e t a b o l i s m , 12:909-913, 1953. - 112 -K o k a t n u r , M., Rand, R.T., - Kummerow, P.A., and S c o t t , H.M. " E f f e c t o f d i e t a r y p r o t e i n a n d f a t on changes o f serum c h o l e s t e r o l i n mature b i r d s " . J. N u t r i t i o n , 6I+: 177-183, 1958. L e p k o v s k y , S., Dukes, T.H., a n d K r a u s e , M.E. - "The m u l t i p l e n a t u r e o f the t h i r d f a c t o r o f t h e v i t a m i n B complex". J.B . C , 115:557-565, 1936. L e o n g . K.C., Sunde, M.L., B i r d , H.R., a n d E l v e h j e m , C.A. " E f f e c t o f e n e r g y : p r o t e i n r a t i o and g r o w t h r a t e , e f f i c i e n c y , f e a t h e r i n g and f a t d e p o s i t i o n i n c h i c k e n s " . P o u l t r y S c i e n c e , 3l+:1206, 1955. L i , T., a n d Freeman, S. - " E f f e c t o f p r o t e i n d e f i c i e n c y and c h o l e s t e r o l f e e d i n g on t h e l i v e r o f d o g s " . Am. J. P h y s i o l . , l[+5:61+6-659, 191+6. L i , T., and Freeman, S. - " E x p e r i m e n t a l l i p e m i a and h y p e r -c h o l e s t e r m i a b y p r o t e i n d e p l e t i o n a n d b y c h o l e s t e r o l f e e d i n g i n d o g s " . Am. J. P h y s i o l . , li+5:660-665, 191+6. Malmros, H., a n d Wigand, G. - " T r e a t m e n t o f H y p e r -c h o l e s t e r e m i a " . M i n n . Med. In symposium on a r t e r o s c l e r o s i s . U. M i n n . , 38:861)., 1955. Malmros, H., a n d ^ i g a n d , G. - "The e f f e c t on serum c h o l e s t e r o l of d i e t s c o n t a i n i n g d i f f e r e n t f a t s " . L a n c e t , 2:1-7, 1957. Mann, G.V., A n d r u s , S.B., M c N a l l y , A., and S t a r e , F . J . " E x p e r i m e n t a l a t h e r o s c l e r o s i s i n Cebus monkeys". J. E x p . Med., 98:195-217, 1953. M a r c h , B.E., and B i e l y , J. - "The e f f e c t o f d i e t a r y f a t l e v e l on t h y r o i d a c t i v i t y - i n the g r o w i n g c h i c k " . P o u l t r y S c i e n c e , 36:1270-1277, 1957. March, B.E., a n d B i e l y , J. - " D i e t a r y m o d i f i c a t i o n o f serum c h o l e s t e r o l I n t h e . c h i c k " . J. N u t r i t i o n , 62:105-110, 1959. M a r t i n , G.T. - " i n c r e a s e d f o l i c a c i d r e q u i r e m e n t s r e -s u l t i n g f r o m t h y r o x i n i n f e c t i o n s " . Am. D i g e s t P i s . , ll+:3l+l, 1952. M a t t e r s o n , L.D., P o t t e r , L.M., S t i n s o n , L.D., a n d S i n g s e n , E.P. - " S t u d i e s on t h e e f f e c t o f v a r y i n g p r o t e i n a n d e n e r g y l e v e l s i n p o u l t r y r a t i o n s on g r o w t h and f e e d e f f i c i e n c y " . P o u l t r y S c i e n c e , 3i+:1210, 1955. - 113 -Meeker, B.R., a n d K e s t e n , H.D., - "The e f f e c t o f h i g h p r o t e i n d i e t on e x p e r i m e n t a l a t h e r o s c l e r o s i s o f r a b b i t s " . A ^ c h i v . o f P a t h . , 31:11*7-162, 191+0. M i x n e r , J.B., R e i n e k e , E.P., a n d T u r n e r , C.W., -" E f f e c t o f T h i o u r a c i l a n d T h i o u r e a on t h e t h y r o i d g l a n d of t h e c h i c k " . E n d o c r i n o l , 3^:168, 19l|.'l+. Moyer, A.W., K r i t c h e v s k y , D., Lo g a n , J.B., and Cox, H.R. " D i e t a r y p r o t e i n a nd serum c h o l e s t e r o l i n r a t s " , P r o c . S o c . E x p . B i o l . Med., 92:736-737, 1956. 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