V I T A M I N I* II. S T U D I E S IN THE L AMB R i b o f l a v i n D e f i c i e n c y and Requirement V ita m in E D e f i c i e n c y and E s t im a te d Requirement by Rudolf C u lik M A THESIS S u b m itted t o the School o f G raduate S t u d i e s of Michigan S t a t e C o l le g e o f A g r i c u l t u r e and A p p l i e d S c ie n c e i n P a r t i a l F u l f i l l m e n t of the r e q u i r e m e n t s f o r th e Degree of DOCTOR OF P H I L O S O P H Y D epartm ent o f Animal Husbandry 1951 ProQuest Number: 10008286 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008286 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 A ffe c tio n a te ly dedicated to my dec ea sed Mother and F a t h e r ACKNOWLEDGMENTS The a u t h o r w ishes t o e x p r e s s h i s r e s p e c t f u l a p p r e c i a t i o n and s i n c e r e t h a n k s t o P r o f e s s o r G. A. Brown, p a s t Head, and to P r o f e s s o r R. H. N e ls o n , p r e s e n t Head of th e Animal Husbandry D e p a rtm e n t, f o r t h e i r c o n t i n u o u s g u i d a n c e , encouragem ent, i n t e r e s t and h e l p th r o u g h o u t th e whole p e r i o d o f s t u d i e s a t M ichigan S t a t e C o l l e g e . The a u t h o r i s g r e a t l y i n d e b t e d t o Dr. Frank Thorp, J r . f o r h i s c o n s t a n t h e l p , g u i d a n c e , s u p e r v i s o n , c r i t i c i s m and i n t e r e s t t h r o u g h o u t t h e c o u r s e of t h e s e i n v e s t i g a t i o n s . He, a s a t e a c h e r , s c i e n t i s t and f r i e n d o f a s t u d e n t , w i l l always be remembered w ith a d m i r a t i o n and g r a t i t u d e . S i n c e r e th a n k s and acknowledgment a r e due t o Dr. R. W. Luecke f o r h i s genuine work on a d a p t a t i o n of m ilk fo rm u la used i n t h i s i n v e s t i g a t i o n and f o r h i s v a l u a b l e h e l p and s u g g e s t i o n s th r o u g h o u t th e work; t o P r o f e s s o r L. H. B l a k e s l e e f o r h i s un­ f a i l i n g i n t e r e s t and many s u g g e s t i o n s ; t o Miss S y l v i a L . L a i n e f o r t e c h n i c o l o g i c a l t r a i n i n g and h e r k in d h e l p i n p r e p a r a t i o n o f t h e numerous s l i d e s ; t o Dr. R. L. Johnson f o r h i s k in d g u id a n c e and v a l u a b l e h e l p i n c h e c k in g t h e p a t h o l o g i c a l t i s s u e s t o Mr. M. L. Gray f o r h i s i n v a l u a b l e h e l p i n t a k i n g t h e p h o to ­ m ic ro g r a p h s and p i c t u r e s and f o r r e a d i n g the m a n u s c r i p t ; t o my c o l l e a g u e , coworker and f r i e n d , A. F. B a c ig a lu p o , g r a d u a t e s t u d e n t from P e r u , who, working on b io c h e m i c a l and p h y s i o l o g i c a l a s p e c t s of v i t a m i n d e f i c i e n c i e s , s h a r e d w ith the a u t h o r the burd en o f t a k i n g c a re o f t h e animals*. G r a t e f u l t h a n k s a r e ex tended to p a s t Dean o f School of G ra d u ate S t u d i e s , P r o f e s s o r R. C. Huston; t o Mr. and Mrs. Kavan; t o t h e S t a f f o f Animal Husbandry D epartm ent; to th e S t a f f o f P h y s io lo g y and Pharmacology D epartm ent; and t o many f r i e n d s on t h e Campus and th r o u g h o u t the S t a t e f o r t h e i r v e r y k in d words and h e l p i n making t h i s c o u n t r y my home. The a u t h o r d e e p ly a p p r e c i a t e d t h e f i n a n c i a l s u p p o r t g iv e n to him by M ichigan S t a t e C ollege i n form o f s c h o l a r s h i p and a s s i s t a n t s h i p t h r o u g h o u t th e e n t i r e p e r i o d of s t u d i e s a t t h i s In stitu tio n * The a u t h o r i s i n d e b t e d to S w i f t and Company, Chicago, 111. f o r the g r a n t i n a i d which g r e a t l y f a c i l i t a t e d t h i s i n v e s t i g a t i o n t h e a u t h o r i s a l s o i n d e b t e d to Merck & C o ., I n c . and L e d e r l e L a b o r a t o r i e s D i v i s i o n , American Cyanamid Co. f o r the su p p ly of B v i t a m i n s u se d i n t h i s work* Rudolf C u lik c a n d i d a t e f o r th e d e g r e e of Doctor o f P h ilo s o p h y F i n a l e x a m i n a t i o n , August 1 6, 1951, 9:30 A.M., 306 Animal P a t h o l o g y L i b r a r y Room, Anatomy B u i l d i n g D issertation : V ita m in S t u d i e s i n t h e Lamb I . R i b o f l a v i n D e f i c i e n c y and Requirem ent I I . V ita m in E D e f i c i e n c y and E s t i m a t e d R equirem ent O u t l i n e of S t u d i e s Major s u b j e c t : Animal Husbandry ( N u t r i t i o n ) Minor s u b j e c t s : D airy N u t r i t i o n , P h y s i o l o g y , Animal P a th o lo g y B i o g r a p h i c a l I te m s Born, J u l y 14 , 1917, L e s t i n a , C z e c h o slo v a k ia U n d e r g r a d u a te S t u d i e s , C o lle g e o f A g r i c u l t u r e , Brno, Czecho­ s l o v a k i a , 1 9 3 7 -3 9 , E cole N a t i o n a l e d ' A g r i c u l t u r e de Grignon, G rignon, F r a n c e , 1939, C ollege of A g r i c u l t u r e , P ra h a , C z e s h o s l o v a k i a , 1945 G raduate S t u d i e s , Michigan S t a t e C o l l e g e , 1946-51 E xperience: G raduate A s s i s t a n t , Animal Husbandry D e p a rtm en t, C o l le g e of A g r i c u l t u r e , P r a h a , C z e c h o s l o v a k i a , 1945-4 6, R e s e a rc h Graduate A s s i s t a n t , Animal Husbandry D ep a rtm en t, Michigan S t a t e C o l l e g e , 1 9 48-51, O f f i c e r Free C z e c h o s lo v a k i a n Army i n F ra n c e and G re a t B r i t i a n 1939-1945 Member o f I n s t i t u t e E r n e s t Denise ( F r e n c h ) , I n s t i t u t e of I n t e r n a t i o n a l E d u c a t i o n , American S o c i e t y o f Animal P r o d u c t i o n , S o c i e t y o f th e Sigma Xi V I T A M I N I« II* S T U D I E S IN THE LAMB R i b o f l a v i n D e f i c i e n c y and Requirement V ita m in E D e f i c i e n c y and E s t i m a t e d Requirement Rudolf C u l ik AN ABSTRACT S ubm itted t o t h e School of G raduate S t u d i e s o f M ic higan S t a t e C o lle g e o f A g r i c u l t u r e and A p p lie d S cience i n P a r t i a l F u l f i l l m e n t o f th e r e q u i r e m e n t s f o r the Degree of DOCTOR OF P H I L O S O P H Y Department o f Animal Husbandry 1951 Approved, R u d o lf C u lik VITAMIN STUDIES IN THE LAMB P a r t I* R i b o f l a v i n D e f i c i e n c y and Requirem ent A p u r i f i e d l i q u i d d i e t ( s y n t h e t i c m ilk) was s u c c e s s f u l l y use d t o r a i s e lambs two to e l e v e n days o l d . This r a t i o n was made up t o c o n t a i n 16 p e r c e n t s o l i d s and was composed of (on d r y m a t t e r b a s i s ) c a s e i n 30$; gluc ose 28$; l a c t o s e 10$; l a r d 10 %\ s a l t m i x t u r e 6$. I n a d d i t i o n th e r a t i o n c o n t a i n e d a l l of t h e f a t and w a te r s o l u b l e v i t a m i n s . E i g h t lambs fed t h i s r a t i o n grew n o r m a ll y w i t h an av e ra g e d a i l y g a i n 0 . 4 4 l b . A group o f se ven lambs were fed t h e r i b o f l a v i n d e f i c i e n t sy n th etic d ie t. At a b o u t the f o u r t h to s i x t h day on t h e d e f i c i e n t d i e t th e lambs d e v e lo p e d d i a r r h e a and ceased to g a in i n w e i g h t . For t h e n e x t s i x to seven days no g a in or l o s s was n o t e d . S hortly a f t e r , th e lambs l o s t t h e i r a p p e t i t e s c o m p l e t e l y , showed symptoms o f pneumonia and d i e d . U s u a l l y th e lambs d i d n o t l i v e beyond t h e t w e n t i e t h day o f the e x p e r i m e n t a l p e r io d .I n 'tw o lambs showing symp­ toms o f r i b o f l a v i n d e f i c i e n c y , i n c l u d i n g pneumonia, were r a p i d l y a l l e v i a t e d and cured w ith a d m i n i s t r a t i o n of c r y s t a l l i n e r i b o f l a v i n P a t h o l o g i c a l s t u d i e s of the r i b o f l a v i n d e f i c i e n t lambs showed l u n g involve m e nt w i t h b i l a t e r a l , p r o g r e s s i v e pneumonia, g e n e r a l f a t t y d e g e n e r a t i o n and n e c r o s i s o f th e l i v e r , f a t t y degen e r a t i o n o f t h e k id n e y and c o n g e s t i o n and f a t t y d e g e n e r a t i o n of the a d r e n a ls . The growth r e s p o n s e of t e n lambs on f i v e d i f f e r e n t l e v e l s o f r i b o f l a v i n (5, 50, 150, 250 and 350 micrograms p e r pound of body w eig ht p e r day) was d i r e c t l y p r o p o r t i o n a l to t h e l e v e l of R u d o lf C u lik r ib o f la v in adm inistered. The two h i g h e s t l e v e l s o f r i b o f l a v i n a d m i n i s t e r e d proved t o be a d e q u a te t o s u p p o r t good h e a l t h and t o promote normal growth o f t h e young lamb i n th e p r e r u m in a n t state. I t was o b se rv e d t h a t t h e r e q u i r e m e n t f o r r i b o f l a v i n i n t h e young lamb was somewhere between 250-350 micrograms p e r pound o f body w e ig h t p e r day. P art II.. V ita m in E D e f i c i e n c y and E s t i m a t e d Requirem ent A d i e t s i m i l a r to t h a t i n P a r t I was used t o s tu d y v i t a m i n E d e f i c i e n c y i n 51 young s u c k l i n g lambs. The so u rc e o f f a t i n t h e d i e t o f t h e f i r s t group was commercial l a r d . symptoms a p p e a r e d i n 30-75 d a y s . The d e f i c i e n c y I n th e second and t h i r d groups m o le c u l a r d i s t i l l e d l a r d , v ery low i n Vitamin £ , was used and t h e symptoms were o b s e rv e d i n 20-55 d a y s . The symptoms o f v i t a m i n E d e f i c i e n c y were c h a r a c t e r i z e d by d i s t u r b a n c e s i n lo c o m o t i o n , s t i f f n e s s , p a r a l y s i s , pneumonia and sudden d e a t h . showed low t o c o p h e r o l l e v e l s . Blood a n a l y s i s P o s t mortem e x a m in a tio n s r e v e a l e d d y s t r o p h y o f th e s k e l e t a l and c a r d i a c m u sc le. The l e s i o n s i n th e h e a r t were i n v a r i a b l y most pronounced i n t h e r i g h t v e n t r i c l e . M ic ro s c o p ic e x a m i n a t i o n s o f t h e t i s s u e s showed d e g e n e r a t i o n and n e c r o s i s of t h e s k e l e t a l and c a r d i a c m u sc le, t o g e t h e r w i t h f a t t y d e g e n e r a t i o n o f t h e l i v e r and k id n e y . A d d i t i o n o f 100 mg o f a l p h a - t o c o p h e r o l t o t h e d i e t ev e ry o t h e r day p r o t e c t e d 12 p o s i t i v e c o n t r o l lambs from m usc ular d y s tr o p h y . An i n i t i a l dose o f 500 mg o f a l p h a - t o c o p h e r o l and 100 mg a d m i n i s t e r e d e v e ry o t h e r day t o e i g h t d e p l e t e d lambs a l l e v i a t e d the symptoms i n 3-5 d a y s . R u d o lf C u lik M ic r o s c o p i c e x a m i n a tio n o f the t i s s u e s from lambs t r e a t e d f o r 30-40 d ay s r e v e a l e d a l m o s t com plete r e g e n e r a t i o n o f th e m u s c le s . While t h i s e x p e r i m e n t a l work was i n p r o g r e s s , th e symptoms and d e g e n e r a t i v e changes of t h e m u s c u la tu re of lambs a f f e c t e d w i t h s o - c a l l e d 11S tif f - L a m b " d i s e a s e under f i e l d c o n d i t i o n s were a l s o studied. The c o n d i t i o n known as "S tiff-L arab " d i s e a s e as o b s e rv e d i n th e f i e l d a p p e a r e d to be i d e n t i c a l i n e v e r y r e s p e c t w i t h n u t r i t i o n a l m uscular d y s tr o p h y produced on v i t a m i n E d e f i c i e n t diet. TABLE OF CONTENTS PART I . RIBOFLAVIN DEFICIENCY AND REQUIREMENT INTRODUCTION Page 1 OBJECT 3 REVIEW OF LITERATURE 4 Rat P i g , C a l f and Lamb O ther L a b o r a t o r y Animals EXPERIMENTAL PROCEDURE Anim als Used F e e d in g and Care C om position and P r e p a r a t i o n of S y n t h e t i c Milk Blood S t u d i e s U rine S t u d i e s Gross P a th o lo g y S t u d i e s M ic ro s c o p ic S t u d i e s R i b o f l a v i n Requirement RESULTS Growth Symptoms o f D e f i c i e n c y Blood S t u d i e s U rine S t u d i e s Gross P a th o lo g y M i c r o s c o p ic P a th o lo g y R i b o f l a v i n Requirement 4 8 IS 14 14 14 16 20 21 21 22 22 25 25 32 33 35 36 39 42 DISCUSSION 47 SUMMARY AND CONCLUSION 56 BIBLIOGRAPHY 59 PLATES 64 PART I I . VITAMIN E DEFICIENCY AND ESTIMATED REQUIREMENT INTRODUCTION Page 71 OBJECT 72 REVIEW OF LITERATURE 73 N u t r i t i o n a l Muscular Dystrophy i n L a b o r a t o r y Anim als "S tif f - L a m b " D ise a se EXPERIMENTAL PROCEDURE Anim als Used F e e d in g and Care Blood S t u d i e s Gross P a th o lo g y M ic ro s c o p ic P a th o lo g y E s t i m a t e d A lpha-T ocophe rol R equirem ent, T re atm ent and Recovery RESULTS Growth and H e a l t h Symptoms of D e f i c i e n c y Blood S t u d i e s Gross P a th o l o g y M ic ro s c o p ic P a th o lo g y E s t i m a t e d A lpha-T ocophe rol R e quirem e nt, T re a tm e n t and Recovery 11S tif f - L a m b " D isease S t u d i e s 73 78 81 81 81 84 84 85 85 87 87 88 90 95 98 102 104 DISCUSSION 106 SUMMARY AND CONCLUSIONS 115 BIBLIOGRAPHY 117 PLATES 123 Part I . RIBOFLAVIN DEFICIENCY AND REQUIREMENT INTRODUCTION Since many of th e i n f e c t i o u s and epidem ic d i s e a s e s of d o m e s tic a n i m a l s here i n the U n ite d S t a t e s have been e r a d i ­ c a t e d or f a i r l y w e l l c o n t r o l l e d , more and more em phasis i s b e i n g p l a c e d on s t u d i e s of n u t r i t i o n a l d i s e a s e s or d i s e a s e s s u s p e c t e d t o be o f n u t r i t i o n a l o r i g i n . B e fo re the problem o f n u t r i t i o n a l d i s e a s e s could be a t t a c k e d i t was e s s e n t i a l t h a t b a s i c s t u d i e s i n animal n u t r i t i o n such as th e i n d i s p e n s a b i l i t y and q u a n t i t a t i v e r e q u i r e m e n t s o f c e r t a i n n u t r i e n t s be d e t e r m in e d . By means o f s e m i - p u r i f i e d and p u r i f i e d d i e t s deve loped d u r in g t h e l a s t tw enty y e a r s , e s s e n t i a l n u t r i e n t s of most a n im a ls and q u a n t i ­ t a t i v e r e q u i r e m e n t s o f some a n im a ls have been s t u d i e d and estab lish ed . P r a c t i c a l l y no i n f o r m a t i o n i s a v a i l a b l e c o n c e r n in g t h e r e q u i r e m e n t s of the s u c k l i n g lamb f o r s p e c i f i c n u t r i e n t s . V enkatachalam et_ al^. (1949) found t h a t th e h ig h m o r t a l i t y of lambs d u r i n g th e s u c k l i n g p e r i o d was dependent on b o th e n v i r o n ­ m e n tal and h e r e d i t a r y f a c t o r s . B e l l (1947) r e p o r t e d "poor m o th e rin g and l a c k of milk" o f the ewe as a s i g n i f i c a n t i f n o t t h e major l i m i t i n g f a c t o r i n sheep p r o d u c t i o n . Therefore i t seemed d e s i r a b l e t o s tu d y the e s s e n t i a l n u t r i e n t s and n u t r i ­ t i o n a l r e q u i r e m e n t s o f the young s u c k l i n g lamb and p o s s i b l y c o r r e l a t e t h i s high m o r t a l i t y w ith n u t r i t i o n . - 2 - I n r e l a t i o n t o i t s b i r t h w eig h t and compared w ith the c a l f t h e lamb grows more r a p i d l y ; t h e r e f o r e r e q u i r e s e x t r a n u t r i e n t s t o meet i t s needs* Lambs no rm ally double t h e i r b i r t h w eight i n a b o u t 14 days while th e c a l f r e q u i r e s 47 days ( P e a r s o n e t a l *, 1946)* S in c e m il k from th e dam i s t h e o n ly c o n s t i t u e n t o f t h e d i e t o f b o th young lambs and c a l v e s f o r a t l e a s t t h e f i r s t few weeks o f l i f e P e a r s o n s t u d i e d t h e c o m p o s i t i o n o f c o l o s tr u m and m ilk o f b o th cows and ewes* The c o l o s tr u m and m ilk o f the ewe was found r i c h e r i n r i b o ­ f l a v i n , t h i a m i n e , n i c o t i n i c a c i d and p a n t o t h e n i c a c i d th a n t h e c o l o s tr u m and m ilk o f th e cow* R iboflavin le v e ls for t h e ewe were found two t o t h r e e tim es h i g h e r t h a n i n th e c o l o s t r u m and m ilk o f t h e cow. R i b o f l a v i n was t h e n th o u g h t t o be o f a s p e c i a l s i g n i f i c a n c e i n the d i e t of a young s u c k l i n g lamb and th e p r e s e n t i n v e s t i g a t i o n was u n d e r t a k e n t o d e t e r m i n e t h e i n d i s p e n s a b i l i t y and q u a n t i t a t i v e r e q u i r e ­ ments o f t h i s v i t a m i n f o r young s u c k l i n g lambs fed a p u r i f i e d liq u id d ie t (sy n th etic m ilk). OBJECT The o b j e c t o f t h i s i n v e s t i g a t i o n was t h r e e f o l d . F i r s t , t o d e t e r m in e whether a l i q u i d p u r i f i e d d i e t c ould be p r e p a r e d i n t h e l a b o r a t o r y which would s u p p o r t normal growth and h e a l t h o f the young lamb. Second, t o s tu d y t h e e f f e c t s o f r i b o f l a v i n d e f i c i e n c y by th e use o f the same d i e t when r i b o f l a v i n was o m i t t e d . T hird, to d e t e r ­ mine t h e q u a n t i t a t i v e r e q u i r e m e n t o f r i b o f l a v i n f o r t h e young s u c k l i n g lamb. REVIEW OF LITERATURE R ats As e a r l y as 1928, b e f o r e t h e e x i s t e n c e o f r i b o f l a v i n a s a v i t a m i n was d e f i n i t e l y e s t a b l i s h e d , Chick and Roscoe (1928) used r a t s f o r t h e b i o l o g i c a l a s s a y o f v i t a m i n Bg. R a t s on a v i t a m i n Bg f r e e d i e t l o s t a p p e t i t e , grew slo w ly and d e v e lo p e d d e r m a t i t i s c h a r a c t e r i z e d by l o s s o f h a i r e s ­ p e c i a l l y from t h e e y e l i d s . Infla m ed and l a t e r bloody n o s t r i l s , e a r s and paws were a l s o c h a r a c t e r i s t i c o f t h i s d eficiency. Small amounts of v i t a m i n Bg p r e v e n t e d or c u r e d th e symdrome and r e s t o r e d normal growth. Sherman and Derbigny (1932) s t u d i e d v i t a m i n G (Bg) i n r e l a t i o n t o th e c o m p o s itio n o f t h e d i e t p a r t i c u l a r l y to p rotein le v e ls. R a ts on a low p r o t e i n d i e t (6 p e r c e n t ) d e v e lo p e d t h e d e f i c i e n c y symptoms much e a r l i e r and more s e v e r e l y th a n r a t s on a h i g h p r o t e i n d i e t (18 p e r c e n t ) . Day and L a n g s t o n (1934) produced c a t a r a c t i n a d d i t i o n t o th e a l r e a d y e s t a b l i s h e d symptoms o f v i t a m i n G (Bg) d e ­ f i c i e n c y i n t h e r a t i n n e a r l y 100 p e r c e n t o f a l b i n o r a t s f e d v i t a m i n G (Bg) f r e e d i e t . I n a f u r t h e r s tu d y o f c a t a r a c t i n t h e r a t a s a c h a r a c ­ t e r i s t i c symptom of f l a v i n d e f i c i e n c y Day et_ al_. (1937) fed r a t s a d i e t d e f i c i e n t i n a l l th e th e n known B v i t a m i n s b u t su p p le m en ted w ith r i c e p o l i s h e x t r a c t and a d d i t i o n a l amounts - 5 - of la c to f la v in . T h is d i e t p r o t e c t e d th e a n i m a l s from a l o ­ p e c i a and c a t a r a c t w h ile t h e l i t t e r mates on th e same d i e t w i t h o u t l a c t o f l a v i n developed th e c h a r a c t e r i s t i c symptoms of d e fic ie n c y . By i n t r a m u s c u l a r i n j e c t i o n of 120 gamma of c r y s t a l l i n e r i b o f l a v i n tw ic e weekly Day e t a l . (1938) were a b l e t o a r r e s t and c l e a r up t h e c a t a r a c t i n most of th e a ffe c te d anim als. I n th e same y e a r Day and Darby (1938) s t u d i e d t h e r i b o f l a v i n r e q u ir e m e n t o f th e r a t by growth response. These a u t h o r s e s t a b l i s h e d th e minimum l e v e l of r i b o f l a v i n f o r normal growth as 120 gamma o f r i b o f l a v i n p e r week. Bessey and Wolbach (1939) o b se rv e d t h a t r a t s on a r i b o f l a v i n d e f i c i e n t d i e t e x h i b i t e d i n a d d i t i o n to l o s s of a p p e t i t e , poor growth and a l o p e c i a a new syndrome; v a s c u l a r ­ i z a t i o n o f the c o r n e a c h a r a c t e r i z e d by r a d i a l in g r o w th of c a p i l l a r i e s from t h e lim bus v e s s e l s and t u r b i d i t y due to le u co cy tic i n f i l t r a t i o n . S u p p le m e n ta tio n o f the d i e t w ith sm a ll amounts o f r i b o f l a v i n c l e a r e d th e t u r b i d i t y i n few days and t h e in g ro w th o f v e s s e l s d i s a p p e a r e d i n a few weeks. A x e lr o d et_ al_. (1940) s t u d i e d t h e mode and s i t e of a c tio n of r ib o f la v in . A n a l y s i s o f the l i v e r and kid n e y of r i b o f l a v i n d e f i c i e n t r a t s showed a d e c r e a s e i n r i b o f l a v i n and d-amino a c i d o x id a se c o n t e n t . A d m in istratio n of r ib o ­ f l a v i n p r o m p tly r e s t o r e d t h e r i b o f l a v i n l e v e l s i n b o t h o r g a n s a s w e ll as th e d-amino a c i d o x id a s e l e v e l of t h e k id n e y s and r a i s e d t h e l e v e l o f d-amino a c i d o x i d a s e of t h e l i v e r . - 6 - The f o l l o w i n g y e a r i n f u r t h e r s t u d i e s on r i b o f l a v i n and i t s r o l e i n r e s p i r a t o r y enzymes A xelrod and Elveh^em (1941) showed a d e f i n i t e d e c r e a s e o f x a n t h i n e o x id a s e l e v e l i n th e t i s s u e s o f r i b o f l a v i n d e f i c i e n t a n i m a l s . Shaw and P h i l l i p s (1941) k e p t r a t s on a r i b o f l a v i n d e f i c i e n t d i e t very high i n f a t c o n te n t. R a ts on t h i s d i e t d e v e lo p e d n e u r o p a t h o l o g i c a l changes o f t h e main p e r i p h e r a l nerv e t r u n k s s i m i l a r t o th o s e o b se rv e d i n c h i c k s . S u l l i v a n and N i c h o l l s (1941) d e s c r i b e d t y p i c a l , un­ c o m p l i c a t e d symptoms o f r i b o f l a v i n d e f i c i e n c y i n t h e r a t . Among th e f i r s t symptoms were l o s s of a p p e t i t e , r e t a r d e d growth and l a t e r l o s s o f weight and e m a c i a t i o n . d e v e lo p e d i n a b o u t 5-6 weeks. Skin changes The h a i r was uneven, u n t h r i f t y and c r u s t e d w i t h a d a r k r e d d i s h brown s u b s t a n c e . H air was l o s t from t h e e y e l i d s and l a t e r p r o g r e s s i v e a l o p e c i a a f f e c t e d most o f t h e body. N o s t r i l s , e a r s and paws were blo ody and sw ollen. M i c r o s c o p i c a l l y , t h e e p i d e r m i s was a t r o p h i e d and showed h y p e r k e r a t o s i s w i t h o u t in f l a m m a t i o n . were s w o lle n and l a t e r a t r o p h i e d . t o u s and s w o l l e n . The sebaceous g la n d s The l i p s became erythe m a­ I f the animal was n o t t r e a t e d a t t h i s s t a g e o f d e f i c i e n c y d e a t h would f o l l o w . Mannering and Elvehjem (1944) s t u d i e d th e i n f l u e n c e of t h e c o m p o s iti o n o f t h e d i e t on r i b o f l a v i n r e q u i r e m e n t o f th e rat. D e x t r i n and s t a r c h showed s p a r i n g a c t i o n on r i b o f l a v i n w h ile s u c r o s e d i d n o t . L actose e s p e c i a l l y spared r i b o f l a v i n - 7 - a s measured by growth r e s p o n s e and e x c r e t i o n o f r i b o f l a v i n i n t h e f e c e s* L a c t o s e , d e x t r i n and s t a r c h e v i d e n t l y s e rv e d a s v e r y good s u b s t r a t e s f o r b a c t e r i a and promoted i n t e n s i v e i n t e s t i n a l s y n t h e s i s of t h i s vitam in* High f a t d i e t s a c c e l ­ e r a t e d and produced more se v e r e d e f i c i e n c y symptoms th a n d i e t s low i n f a t . L i m i t e d i n f o r m a t i o n i s a v a i l a b l e on r i b o f l a v i n e s s e n ­ t i a l i t y and r e q u i r e m e n t f o r a d u l t r a t s . Ti/arkany and S c h r a f - f e n b e r g e r (1944) k e p t b r e e d i n g s t o c k r a t s on low r i b o f l a v i n d iet. D e s p i te t h e f a c t t h a t t h e mothers d id not show any t y p i c a l symptoms of c h r o n i c r i b o f l a v i n d e f i c i e n c y a number o f young were b o rn w ith v a r i o u s c o n g e n i t a l m a lfo r m a tio n s and d e f e c t s o f the s k e l e t o n . I n f l u e n c e o f t h e d i e t , p a r t i c u l a r l y the e f f e c t s of p r o t e i n and f a t on r i b o f l a v i n r e q u i r e m e n t were s t u d i e d a g a i n by Czaczkes and Guggenheim (1 9 4 6 ). These a u t h o r s confirm ed t h e e a r l i e r f i n d i n g s o f Shaw and P h i l l i p s (1941) t h a t a low f a t d i e t would s p a r e r i b o f l a v i n b u t d i d n o t a g r e e w ith Sherman and Derbigny (1932) t h a t a h ig h p r o t e i n d i e t h a s s p a r i n g a c t i o n on r i b o f l a v i n . On the c o n t r a r y b o th h ig h p r o t e i n and h i g h f a t d i e t s d e c r e a s e d th e e x c r e t i o n of r i b o ­ f l a v i n i n u r i n e and f e c e s and d im i n is h e d t h e r i b o f l a v i n l e v e l i n v a r i o u s t i s s u e s and o r g a n s . Low f a t d i e t showed a d e f i n i t e s p a r i n g a c t i o n on r i b o f l a v i n w h ile low p r o t e i n d i e t d id not a f f e c t e x c r e t i o n or s t o r a g e o f r i b o f l a v i n i n the r a t . - 8 - P i g , C a l f and Lamb There i s p r a c t i c a l l y no i n f o r m a t i o n c o n c e r n in g v i t a ­ min B complex r e q u i r e m e n t s f o r th e a d u l t p i g , cow and sh e ep , £L I t h a s been d e m o n s tr a te d t h a t few members of t h e v i t a m i n B complex i f n o t a l l o f them a r e s y n t h e s i z e d i n t h e rumen of t h e a d u l t r u m i n a t i n g animal or i n the i n t e s t i n a l t r a c t of t h e p i g (McElroy and Goss, 1940a, Wegner ejb a l^ ., 1940 and Hunt e t a l . , 1 9 4 3 ) . Under farm c o n d i t i o n s t h e n a t u r a l f e e d s t u f f s o r d i n a r i l y c o n t a i n a d e q u ate amounts o f th e B v i t a m i n s t o supplem ent t h o s e s y n t h e s i z e d i n t h e rumen. However i n th e young lamb, c a l f and p i g when th e rumen i s n o n - f u n c t i o n a l o r i n t e s t i n a l s y n t h e s i s has not ta k e n p l a c e , th e amount of B v i t a m i n s i n the d i e t i s o f g r e a t im p o r ta n c e . P u r i f i e d d i e t s used i n l a r g e animal i n v e s t i g a t i o n s were d e v e lo p e d r e c e n t l y . Hughes (1939) use d a p u r i f i e d d i e t t o d e m o n s t r a t e th e i n d i s p e n s a b i l i t y of r i b o f l a v i n and o t h e r f a c t o r s o f t h e v i t a m i n B complex f o r th e young growing pig* P i g s on a d e f i c i e n t d i e t grew slo w ly and were u n t h r i f t y w h ile p i g s on t h e same d i e t supplemented w ith r i c e bran f i l t r a t e made normal g a i n s and sta y e d i n good c o n d i t i o n . The a u t h o r concluded t h a t b o th th ia m in e and r i b o f l a v i n a r e e s s e n t i a l f o r normal growth and h e a l t h . The f o l l o w i n g y e a r th e same a u t h o r (Hughes, 1940a) used the same p u r i f i e d d i e t and e s t a b l i s h e d t h a t t h e minimum r e q u ir e m e n t o f r i b o f l a v i n f o r young growing p i g s was 3 m i l l i g r a m s o f c r y s t a l l i n e r i b o ­ f l a v i n p e r 100 pounds o f body w e ig h t . - 9 - P a t e k ej^ a l . (1941) produced and d e s c r i b e d th e symp­ toms of r i b o f l a v i n d e f i c i e n c y i n the p i g . L oss o f a p p e t i t e and r e t a r d e d growth were f o llo w e d by c o r n e a l o p a c i t y , derma­ titis, changes i n h a i r and h o o f s and e v e n t u a l l y d e a t h . A d d i t i o n o f r i b o f l a v i n to the d i e t a l l e v i a t e d t h e symptoms rapidly, Wintrobe and a s s o c i a t e s (1944) i n a d d i t i o n to the symptoms d e s c r i b e d by P a te k o b se rv e d m y e lin d e g e n e r a t i o n of th e s c i a t i c and b r a c h i a l n e r v e s , c a t a r a c t , moderate normoc y t i c anemia and f a t t y l i v e r and k id n e y . Van Poucke and K r i d e r (1946) and K rid e r £ t al_. (1949) used a h i g h l y p u r i f i e d d i e t to e s t a b l i s h the q u a n t i t a t i v e r e q u i r e m e n t of r i b o f l a v i n f o r growing p i g s . Feed e f f i c i e n c y and d a i l y g a i n s c l o s e l y follow ed t h e l e v e l s of r i b o f l a v i n i n th e d i e t . The minimum r i b o f l a v i n i n t a k e p e r day was 1 , 4 mg p e r l b of r a t i o n . P ig s on d i e t s w ith l e s s t h a n 1 . 3 mg d i d n o t make normal g a i n s . Feed e f f i c i e n c y was improved 18 p e r c e n t when th e r i b o f l a v i n l e v e l was i n c r e a s e d from 0 . 3 5 t o 1 . 5 0 mg p e r l b of r a t i o n p e r day. M i t c h e l l ejt a l . (1950) n o t i c e d t h a t t h e u r i n a r y e x c r e ­ t i o n o f r i b o f l a v i n o f growing p i g s i n c r e a s e d i n a h o t e n v i r ­ onment, w h ile a c o l d environment te nded t o d e c r e a s e t h e o u t p u t . Wiese and coworkers (1947) f i r s t approached the problem o f t h e r e q u i r e m e n t f o r s p e c i f i c n u t r i e n t s of th e young s u c k l i n g calf. These w orkers dev eloped a p u r i f i e d , l i q u i d d i e t (syn­ t h e t i c m ilk ) composed o f v i t a m i n f r e e c a s e i n , l a r d , c e r e l o s e and s a l t s supplem ented w i t h wheat germ o i l and a l l known f a t 10 - and w a te r s o l u b l e v i t a m i n s . Calves on t h i s d i e t made normal d a i l y g a i n s and m a in ta in e d good h e a l t h c o n d i t i o n . A fter s u c c e s s f u l l y r a i s i n g th e c a l v e s on t h e p u r i f i e d l i q u i d d i e t th e same group s t u d i e d the r e q u ir e m e n t of a number of B vitam ins. Among t h o s e f i r s t s t u d i e d was r i b o f l a v i n (Wiese Qt a l . . 1 947b ). Calves fed a r i b o f l a v i n d e f i c i e n t l i q u i d p u r i f i e d d i e t deve loped t h e f i r s t symptoms a f t e r about two weeks. These c o n s i s t e d of hyperemia o f t h e b u c c a l mucosa and t o n g u e , l e s i o n s i n th e c o r n e r s of t h e mouth, on t h e l i p s and around t h e n a v e l . At a b o u t the same time th e c a l v e s l o s t t h e i r a p p e t i t e , developed s c o u r s and became e m a c i a te d . D e f i c i e n t c a l v e s e x h i b i t e d e x c e s s i v e s a l i v a t i o n and l a c r i m a tio n and p r o g r e s s i v e l o s s o f h a i r e s p e c i a l l y i n th e r e g i o n o f t h e abdomen. ritated . The navel r e g i o n became inflamfed, and i r ­ A s i n g l e i n j e c t i o n of 5 mg o f c r y s t a l l i n e r i b o ­ f l a v i n improved t h e c o n d i t i o n i n one day. The a p p e t i t e was improved and s a l i v a t i o n and l a c r i m a t i o n d e c r e a s e d . t h e symptoms r e a p p e a r e d a f t e r a few d a y s . However A d i e t supplemented w ith 5 mg o f c r y s t a l l i n e r i b o f l a v i n p e r day r e s t o r e d th e a p p e t i t e and t h e c a l v e s g ained i n w eight and c o n d i t i o n . t h e symptoms g r a d u a l l y d i s a p p e a r e d . h e a l and new h a i r s t a r t e d to grow. A ll The l e s i o n s s t a r t e d to The a u t h o r s d id not o b s e rv e v a s c u l a r i z a t i o n o f th e cornea or o p a c i t y of t h e l e n s . P o s t mortem e x a m i n a tio n of t h e cu re d a n im a ls d i d n o t show lesio n s. The a u t h o r s concluded t h a t t h e v e r y young non­ r u m i n a t i n g c a l f r e q u i r e d a d i e t a r y s o u r c e of r i b o f l a v i n . - 11 - Warner and S u t t o n (1948) checked th e work o f Wiese Si.« (1947b) by f e e d i n g c a l v e s w ith n a t u r a l c o lo stru m and m ilk i n which most o f the r i b o f l a v i n had been d e s t r o y e d by e x p o s u re to s u n l i g h t or to a mercury vapor lamp. About 96 p e r c e n t o f the r i b o f l a v i n i n th e m ilk was destro y e d * R i b o f l a v i n d e f i c i e n c y symptoms were s i m i l a r t o t h o s e o b s e r v e d by Wiese and h i s group. P o s t mortem e x a m in a t i o n s showed a m ild c a t a r r h a l e n t e r i t i s , m ild edema o f th e cerebrum and some p a t h o l o g i c a l changes i n th e k id n e y . A dm inistration o f 2 mg o f r i b o f l a v i n d a i l y t o one d e f i c i e n t c a l f p ro m p tly r e s t o r e d t h e a p p e t i t e , a r r e s t e d t h e d i a r r h e a and i n tim e com pletely r e s t o r e d a t h r i f t y c o n d itio n . One c a l f on t h e same d i e t supplem ented w ith 2 ,9 9 mg o f r i b o f l a v i n d a i l y th r o u g h o u t th e e x p e rim e n t grew n o rm a lly w ith o u t showing any d i s o r d e r o r symptom o f d e f i c i e n c y . Very l i t t l e work has been done on th e r e q u ir e m e n t of th e s u c k l i n g lamb f o r s p e c i f i c n u t r i e n t s . P e a r s o n and S c h w e ig e r t (1947) a t t e m p t e d t o i n c r e a s e t h e l e v e l of r i b o ­ f l a v i n i n m il k o f ewes by su p p le m en tin g the n a t u r a l r a t i o n w ith e x t r a c r y s t a l l i n e r i b o f l a v i n . The r e s u l t s showed t h a t t h e r i b o f l a v i n l e v e l was not g r e a t l y a f f e c t e d . The s i t e o f d e s t r u c t i o n of o r a l l y a d m i n i s t e r e d r i b o ­ f l a v i n and p a n t o t h e n i c a c i d i n t h e sheep was i n v e s t i g a t e d by O lcese and P e a r s o n (1948). They found t h a t b o th v i t a m i n s were v e r y rapidly and e f f e c t i v e l y d e s t r o y e d i n t h e g a s t r o ­ in te stin al tra c t. - 12 - Other L a b o r a t o r y Animals The r e q u i r e m e n t o f r i b o f l a v i n as an e s s e n t i a l d i e t a r y f a c t o r i n th e r a t i o n has been d e m o n s tr a te d f o r a number of l a b o r a t o r y a n i m a l s and man. Lepkovsky and Jukes (1 9 3 6 ), S t o k s t a d and Manning (1938) and P h i l l i p s and Engel (1938) produced r i b o f l a v i n d e f i c i e n c y symptoms i n c h i c k s and t u r k e y s . I n c h i c k s the d e f i c i e n c y might be a c u t e , c h a r a c t e r i z e d by g e n e r a l p a r a l y s i s and a n e u r o m a l a c i a - l i k e d i s o r d e r or c h r o n i c t y p e c h a r a c t e r i z e d by "curled toe" p a r a l y s i s . Both c o n d i t i o n s were accompanied by d e g e n e r a t i v e changes i n t h e m y e lin s h e a t h s of t h e main p e r i ­ p h e r a l nerve t r u n k s , s w e l l i n g and f r a g m e n t a t i o n of a x i s c y l i n d e r and p o s s i b l e d e g e n e r a t i o n o f motor end p l a t e s and muscle f i b e r s . I n t u r k e y s th e symptoms c o n s i s t e d o f slow grow th, d i a r r h e a , e m a c i a t i o n and se vere d e r m a t i t i s . There was no agreement c o n c e r n in g c h a r a c t e r i s t i c symp­ toms o f r i b o f l a v i n d e f i c i e n c y i n the dog. Zimmerman and Burack (1934) , S t r e e t and Cowgill (1939) and S t r e e t et_ al_. (1941) d e s c r i b e d th e symptoms a s d i a r r h e a , v o m i t i n g , , marked m u sc u la r weakness and sudden c o l l a p s e accompanied by a f a l l i n body t e m p e r a t u r e , f a l l i n r e s p i r a t o r y r a t e and v a r i a b l e heart ra te . In chronic r i b o f l a v i n d eficien cy the au th o rs o b s e r v e d n e u r o l o g i c a b n o r m a l i t i e s c h a r a c t e r i z e d by d e g e n e r a ­ t i o n of p e r i p h e r a l n e r v e s and p o s t e r i o r columns o f th e s p i n a l cord. The a u t h o r s a l s o obse rv e d o p a c i t y of th e cornea i n a few cases* 13 - P o t t e r e_t a l . (1942) produced symptoms o f r i b o f l a v i n d e f i c i e n c y i n t h e dog s i m i l a r t o those o b s e rv e d i n the r a t ; th at is , derm atitis. The a u t h o r s a l s o o b se rv e d a mild m i c r o c y t i c hypochromic anemia and f a t t y d e g e n e r a t i o n of t h e liv er. R i b o f l a v i n was a l s o shown to be e s s e n t i a l f o r t h e mouse (Jones e t a l . . 1 9 4 5 ) , c o t t o n r a t ( M c l n t i r e e t a l . . 1 944), h a m s te r (Routh and Houchin, 1 9 4 2 ) , monkey (Waisman, 1944 and Cooperman e t a l . . 1945) and man ( S e b r e l l and B u t l e r , 1938). EXPERIMENTAL PROCEDURE Animals Used A l t o g e t h e r 25 lambs were u s e d . The v a r i o u s t r e a t m e n t s and d i s t r i b u t i o n of b r e e d s , s e x e s , ages and w e ig h ts when p u t on th e e x p e r i m e n t a l d i e t s a r e summarized i n t a b l e 1. Lambs o f b o th m utton and f i n e wool t y p e r e p r e s e n t i n g s i x d i f f e r e n t p u r e b r e d b r e e d s and one c r o s s b r e d lamb ( S u f f o l k and Hampshire) were o b t a i n e d from th e Experim ent S t a t i o n f l o c k a t Michigan S t a t e C o l l e g e . The system used i n a s s i g n ­ i n g lambs f o r d i f f e r e n t t r e a t m e n t s c o n s i s t e d of random a l l o t ­ ment as t h e lambs were born d u r i n g J a n u a r y and F e b r u a r y i n pens i n th e c l i n i c and d u r in g s p r i n g months i n the c o l l e g e experim ental b arn s. Lambs o f b o th s e x e s , s i n g l e or tw in s were used w ith o u t d i s c r i m i n a t i o n and t h e only p r e r e q u i s i t e was h e a l t h and normal c o n d i t i o n . The age of the lambs when p u t on the e x p e r i m e n t a l d i e t v a r i e d from two to e l e v e n d a y s . A l l lambs r e c e i v e d c o l o s tr u m b e f o r e b e i n g ta k e n from th e ewes • F e ed in g and Care Throughout th e e xpe rim ent a l l t h e a n im a ls were k e p t in c o n c re te -flo o re d pens. f a i r l y constant. The t e m p e r a tu r e and h u m id ity was U s u a l l y two or t h r e e lambs were i n one 12 x 4 f o o t pen and were changed from one pen to a n o t h e r e v e r y o t h e r d ay. The pens were t h e n scrubbed and c l e a n e d w i t h h o t w a t e r and d i s i n f e c t e d w ith a l y e s o l u t i o n to e l i m i n - - 15 - TABLE 1 DISTRIBUTION OF LAMBS IN THE EXPERIMENTAL GROUPS T rial Lamb M 2 4 5 6 7 1 10 11 1 3 8 9 12 2 T re atm en t Breed R i b o f l a v i n d efi- -Cotswold cient diet ii Oxford ii Cotswold ii Shropshire ii ii D e p le te d Suffolk Treated H Shropshire P o s i t i v e c o n t r o l Ramb o u illet it Suffolk x Hampshire it Ramb o u illet n ti n ii 13 5 micrograms r i b o ­ Ramfla v in per lb b o u illet p e r day ii " 14 5 ii 15 50 " D orset 16 50 " Ram17 150 " b o u illet D o rs e t 18 150 " Ram" 19 250 b o u illet D o rs et 21 350 " ii 23 250 " n 25 350 M 20 P o s i t i v e c o n t r o l Ramb ou illet n n 22 it ii 24 Sex Age when Weight when Put on ex­ p u t on e x ­ p e r im e n t p e r im e n t (lbs) (Days) F 2 F F M M M 3 2 2 2 1 0 .0 3 1 0 .2 F M 3 9 1 1 .0 F 3 9 .4 M 11 1 5 .0 M M 3 8 11.5 1 3 .0 F 3 9 .5 M F M M 10 1 1 .8 3 4 3 7 .0 7.5 8 .5 M F 4 5 9.2 F M M M 6 1 0 .0 4 3 11 7 .0 7.6 13 .1 5 4 1 0 .6 1 2 .8 F F 11.5 9 .0 7 .2 7.0 9.5 8 .0 - 16 - a t e as f a r a s p o s s i b l e c o m p l i c a t i o n s w ith o u t s i d e d i s e a s e s . B u r la p bags f i l l e d w ith a s m a ll q u a n t i t y of wood s h a v in g s served fo r bedding. These were a l s o changed ev e ry o t h e r day. The lambs were f e d from n u r s i n g b o t t l e s f i v e tim e s a day a t 7 and 11 A.M. and 3, 7 and 11 P.M. The milk was k e p t under r e f r i g e r a t i o n and an e s t i m a t e d q u a n t i t y was t h o r o u g h l y shaken and warmed t o about 80° F. p r i o r t o f e e d i n g . The amount o f m ilk each lamb consumed p er f e e d i n g was r e c o r d e d t o g e t h e r w i t h o b s e r v a t i o n s and remarks r e g a r d i n g h e a l t h , a p p e t i t e and general behavior. A l l th e a n im als were weighed ev e ry o t h e r day b e f o r e t h e morning f e e d i n g . The w e ig h ts were e s t i m a t e d t o the t e n t h of a pound and r e c o r d e d f o r f u r t h e r a n a l y s i s . Com position and P r e p a r a t i o n of S y n t h e t i c Milk The c o m p o s iti o n and method of p r e p a r a t i o n of th e p u r i ­ f i e d d i e t used i n t h i s s tu d y were s i m i l a r to th o se d e s c r i b e d by Wiese _et al_. , (1947b). The s a l t m ix tu re was s i m i l a r t o t h a t d e s c r i b e d by Johnson et^ al^. (1948). a r e shown i n t a b l e s 2 and 3 . The c o m p o sitio n s The s y n t h e t i c milk c o n t a in e d 16 p e r c e n t s o l i d s which was s l i g h t l y h i g h e r t h a n the a v e ra g e p e r c e n t of s o l i d s i n m ilk o f normal l a c t a t i n g ewes. The B v i t a m i n s were d i s s o l v e d i n a 20 p e r c e n t a l c o h o l s o l u t i o n i n such a p r o p o r t i o n t h a t 10 ml o f th e s t o c k s o l u t i o n s u p p l i e d th e n e c e s s a r y v i t a m i n s f o r one l i t e r of m ilk . S to ck s o l u t i o n of f a t s o l u b l e v i t a m i n s was p r e p a r e d by f o r t i f i c a t i o n of cod l i v e r o i l w ith pure v i t a m i n A and D and by add in g v i t a m i n E - 17 - TABLE 2 COMPOSITION OF THE PURIFIED DIET Component Per c e n t V ita m in s ■n— Mg. p er kg. of L i q u i d D ie t i m .wssg aassssssg s C a s e i n (Labco) 30 Thiamine 1*4 Glucose 28 R iboflavin 3.0 Lactose 10 N icotinic acid 4 .0 Lard 26 Calcium p a n t o ­ thenate 5.0 P y rid o x in e 2.0 P t e r o y l g lu ta m ic acid 0.2 p-Amino b en zoic acid 4 .0 S a l t m ix tu re 6 Inositol B iotin Choline 40.0 0.04 400.0 A lp ha-T ocopherol acetate 2 .0 2-Methyl nap h th o ­ quinone 0 .4 V itamin A 4000 I . U . V itam in D 500 I . U . - 18 - TABLE 3 COMPOSITION OF SALT MIXTURE (5 Kgs.) Component NaCl Grams 594.00 K2 HPO4 1288.00 CaHP04 1690.00 Ca l a c t a t e 1144.00 MgS04 177.00 FeS0 4 96.50 KI 3 .00 MnS04 5.00 ZnClg 1 .0 0 CuS04 . 5Hg0 1 .0 0 COClg 0 .5 0 5000.00 - 19 - and K i n such manner t h a t 1 ml o f t h e s t o c k s o l u t i o n was u s e d f o r one l i t e r of m il k . Both s t o c k s o l u t i o n s were p r e ­ p a r e d i n l a r g e q u a n t i t i e s and k e p t under r e f r i g e r a t i o n . A f t e r a few t r i a l s th e method f i n a l l y a d a p te d f o r p r e p a r i n g th e m ilk was a s f o l l o w s . D i s t i l l e d w ater (29,500 ml) was measured i n t o an o r d i n a r y 10 g a l l o n m ilk can and h e a t e d w i t h steam t o 80-85° C. Sodium b i c a r b o n a t e (82 gms) was d i s s o l v e d i n t h i s w a te r and v i t a m i n f r e e c a s e i n (Labco)# was b r o u g h t i n s u s p e n s i o n by means o f an e l e c t r i c s t i r r e r modi­ f ie d fo r th is purpose. The c a s e i n was added slo w ly w ith c o n s t a n t r a p i d s t i r r i n g which was c o n t in u e d 30 to 45 m in u te s t o o b t a i n a complete d i s s o l u t i o n o f c a s e i n . Glucose (1613 gms) and l a c t o s e (576 gms) were t h e n d i s s o l v e d i n t h i s homogenaousssuspension. Meanwhile, commercial l a r d (1498 gms) was h e a t e d to 80-85° C on the steam b a th and mixed w ith 36 ml o f stock s o lu tio n of f a t soluble vitam ins. This l a r d - f a t soluble v i t a m i n s o l u t i o n was slo w ly added to the c a s e i n s o l u t i o n and the s t i r r i n g a c c e le ra te d . I n t h i s s t a t e the s o l u t i o n of c a s e i n , s u g a r s and l a r d was homogenized i n a Manton-Gaulin homogenizer## a t 3 , 2 0 0 - 3 ,5 0 0 pounds p r e s s u r e p e r sq u a re i n c h . The s a l t m ix tu re (345 gms) was d i s s o l v e d i n a p a r t of th e a l r e a d y homogenized m ilk u s i n g Waring b l e n d o r s . Not more #The Borden Company, Labco and V ita m in P r o d u c ts D e p a r t ­ ment, 350 Madison Avenue, New York 17, N.Y. #«Manton G a u li n M a n u f a c tu r in g C o ., I n c . , E v e r e t t 49, Mass. 20 - t h a n a b o u t 50 gin o f s a l t were added to an a p p r o p r i a t e amount o f m il k i n th e b l e n d o r and a g i t a t e d f o r 7 to 10 m inutes a t f u l l speed t o i n s u r e a complete d i s s o l u t i o n of a l l m i n e r a l s . S a l t i n t h i s form and th e a p p r o p r i a t e amount of B v i t a m i n s s t o c k s o l u t i o n were added to t h e homogenized m i l k , s t i r r e d a g a i n f o r 2-3 m in u te s b e f o r e t h e m ilk was b o t t l e d i n t o t h e c l e a n and s t e r i l i z e d g a l l o n b o t t l e s . The m ilk was k e p t under r e f r i g e r a t i o n a t 42° F e x c e p t when used f o r f e e d i n g . S y n th e ­ t i c m ilk p r e p a r e d and s t o r e d under t h e above c o n d i t i o n s k e p t w e l l f o r o v e r two weeks. p a r e d a t one tim e . U s u a l l y two cans o f milk were p r e ­ T h is q u a n t i t y was s u f f i c i e n t f o r 7 to 10 days. Blood S t u d i e s To d e t e r m in e th e i n i t i a l and presumably normal blood p i c t u r e a l l th e lambs were b l e d as soon as p o s s i b l e and never l a t e r t h a n f i v e days a f t e r b eing p l a c e d on th e e x p e r i ­ m ental d i e t s * The blood was c o l l e c t e d e v e ry week t h e r e a f t e r and t h e hemoglobin c o n c e n t r a t i o n , t o t a l and d i f f e r e n t i a l l e u k o c y t e c o u n ts were d e t e r m i n e d . About one ml o f blood was drawn from th e j u g u l a r v e i n i n t o a v i a l c o n t a i n i n g a s m a ll amount of dry p o ta s s iu m o x a l a t e as a n t i - c o a g u l a n t . A bloo d smear f o r d i f f e r e n t i a l l e u k o c y t e count was p r e p a r e d and s t a i n e d w ith W rig h ts s t a i n . The hemoglobin c o n c e n t r a t i o n was d e t e r m in e d by MC e n c o - S h e a r e d - S a n f o r d - P h o t e l o m e t e r "* and tific #The C e n c o - S h e a r e d - S a n fo r d P h o t e l o m e t e r , C e n t r a l ScienCompany, C hicago, I l l i n o i s . - 21 - e x p r e s s e d i n grams p e r 100 ml o f b lo o d . The t o t a l l e u k o c y t e c o u n t was d e te r m in e d by u s i n g a one p e r c e n t s o l u t i o n 0 .1 N HC1 t o which was added one or two drops o f g e n t i a n v i o l e t . A hemoeytometer w ith Neubau&r.ruling was used f o r the a c t u a l e n u m e r a tio n . The o i l immersion o b j e c t i v e was used to c l a s s i f y th e l e u k o c y t e s c o v e r i n g as many as p o s s i b l e d i f f e r e n t a r e a s of th e f i l m i n o r d e r t o o b t a i n a r e l i a b l e c o u n t . U rin e S t u d i e s A f t e r two weeks on th e e x p e r i m e n t a l d i e t b o th d e f i ­ c i e n t and c o n t r o l lambs were p l a c e d i n a 2 | ' x 4 ' m etabolism cage w i t h a f i n e w ire mesh f l o o r and a 24-hour u r i n e sample collected for rib o fla v in assay. The u r i n e was c o l l e c t e d i n a brown g l a s s b o t t l e c o n t a i n i n g ab o u t 5 ml o f t o l u e n e and a c e t i c a c i d a s p r e s e r v a t i v e s . The 24-hour e x c r e t i o n o f u r i n e was measured and im m ediately a s s a y e d i n t h e A g r i ­ c u l t u r a l C hem istry l a b o r a t o r y u s i n g L a c t o b a c i l l u s c a s e i . Gross P a th o lo g y S t u d i e s Lambs were o b se rv e d c l o s e l y every day a t each f e e d i n g and th e o b s e r v a t i o n s r e c o r d e d . Whenever i t was e v i d e n t t h a t th e lamb would die i n t h e p e r i o d between f e e d i n g s or d u r i n g th e n i g h t t h e animal was s a c r i f i c e d by e x s a n g u i n a t i o n under e t h e r a n e s t h e s i a and n e c r o p s i e d . s a c r i f i c e d i n a s i m i l a r manner. The c o n t r o l lambs were I n a few i n s t a n c e s t h e d e f i ­ c i e n t lambs d i e d s u d d e n ly and nec ro p sy was perform ed a s soon as p o s s i b l e . As soon as th e c a r c a s s was opened, t h e l u n g s , - 22 - l i v e r and h e a r t - b l o o d were c u l t u r e d f o r b a c t e r i a on b lo o d agar p l a t e s . These were th e n i n c u b a t e d a t 37° C f o r two or t h r e e days and th e r e s u l t s r e c o r d e d . A f t e r t h e organs have been c u l t u r e d t h e whole c a r c a s s and e v e r y organ were c a r e ­ f u l l y examined f o r g ro ss l e s i o n s * M ic ro s co p ic S t u d i e s S e c t i o n s f o r h i s t o p a t h o l o g i c a l s t u d i e s were ta k e n from t h e l i v e r , l u n g , k i d n e y , h e a r t , a d r e n a l s , stomach ( p y l o r u s ) duodenum, je ju n u m , ile u m , i l eo c e c a l v alv e and c o l o n . These t i s s u e s were f i x e d i n Z e n k e r ’s f l u i d , embedded i n p a r a f i n , c u t a t 7 raicra and s t a i n e d w ith h e m a t o x y l i n - e o s i n . Sections from t h e l i v e r , k id n e y , h e a r t and a d r e n a l s were a l s o f i x e d i n 10 p e r c e n t f o r m o l - s a l i n e s o l u t i o n , c u t a s f r o z e n s e c t i o n s and s t a i n e d w ith Sudan IV, T is s u e from t h e c o r n e r o f t h e mouth, b r a i n , s c i a t i c n e r v e , s p i n a l c o r d , and eye were p r e ­ p a r e d f o r h i s t o p a t h o l o g i c a l s t u d i e s from t h r e e d e f i c i e n t and two c o n t r o l la m b s. R i b o f l a v i n Requirement The f i r s t t r i a l c o n s i s t e d of f i v e c o n t r o l and seven lambs on th e r i b o f l a v i n d e f i c i e n t d i e t . Two lambs when de­ p l e t e d and showiflgadvanced symptoms of d e f i c i e n c y were t r e a t e d and c u r e d w ith r i b o f l a v i n . I n t h e second t r i a l were t h r e e c o n t r o l and t e n e x p e r i ­ m e n ta l lambs. C a l c u l a t e d from t h e l e v e l of r i b o f l a v i n i n t h e m ilk consumed by t h e c o n t r o l lambs i n th e f i r s t t r i a l th e - 23 - a p p r o x im a te r e q u i r e m e n t of r i b o f l a v i n p e r pound o f body w e ig h t p e r day was 250 t o 300 micrograms ( t a b l e 4 ) . The 1 0 e x p e r i m e n t a l lambs were d i v i d e d a t random i n t o f i v e sub­ groups. The l e v e l s of r i b o f l a v i n were s e t a r b i t r a r i l y a t 5, 50, 1 5 0 , 250 and 350 micrograms p e r pound of body w eig h t per day. T h is was added t o t h e milk consumed a t one f e e d i n g per day. S o l u t i o n s o f c r y s t a l l i n e r i b o f l a v i n i n 20 p e r c e n t a l c o h o l were made and t h e e x a c t amount of r i b o f l a v i n , c a l c u ­ l a t e d from body w eight ta k e n e v e r y o t h e r d ay , was measured w i t h a p i p e t t e and mixed w ith the milk f o r t h e i n d i v i d u a l lamb j u s t b e f o r e f e e d i n g . Care was e x e r c i s e d t h a t a l l m ilk so p r e p a r e d was consumed, i n s u r i n g the f u l l i n t a k e of r i b o ­ flav in . I f n e c e s s a r y t h e lamb was f o r c e d t o d r i n k t h e amount o f m ilk p r e p a r e d . - 24 - TABLE 4 AVERAGE DAILY RIBOFLAVIN INTAKE OF FIVE CONTROL LAMBS CALCULATED FROM AN AVERAGE DAILY FEED CONSUMPTION AT DIFFERENT BODY WEIGHTS Body Weight in lbs Milk Consumed oz ml Micrograms R ib o fla v ; p e r i o hoay weignv p e r Day 10 30 887 266 11 32 946 258 13 36 1064 277 13 38 1124 259 14 41 1212 259 15 44 1301 260 16 50 1478 276 17 57 1685 297 18 57 1685 279 19 58 1715 270 30 59 1745 261 21 57 1685 240 22 56 1656 226 23 56 1656 215 24 57 1685 211 RESULTS Growth The g a i n i n body w eight and th e e f f i c i e n c y of f e e d u t i l i z a t i o n of f i v e c o n t r o l lambs, and f i v e lambs on t h e r i b o f l a v i n d e f i c i e n t d i e t a r e i n d i c a t e d i n t a b l e s 5 and 6 * T a b le 7 shows th e g a i n i n body weight and f e e d u t i l i z a t i o n o f two lambs d u r i n g the d e p l e t i o n p e r i o d and d u r i n g t h e period of r ib o f l a v in therapy. The i n d i v i d u a l growth c u r v e s o f a l l seven lambs on the e x p e rim e n ta l d i e t and one curve r e p r e s e n t i n g t h e av e ra g e growth of the f i v e c o n t r o l lambs a r e i l l u s t r a t e d i n graph 1 . A stu d y of t a b l e 5 d i s c l o s e d t h a t t h e a v e r a g e d a i l y g a in of the c o n t r o l lambs over t h e t w e n t y - e i g h t - d a y p e r i o d was 0 . 4 4 l b . Over th e same p e r i o d th e c o n t r o l lambs r e q u i r e d 1 . 0 7 l b . of dry m a t t e r to p r o ­ duce a one l b g a i n i n body w eight and consumed an average o f 0 . 5 4 l b o f dry m a t t e r d a i l y . The average d a i l y gain o f the f i v e lambs f e d th e r i b o f l a v i n d e f i c i e n t d i e t over a p e r i o d o f 19 days was 0 .0 5 4 l b and t h e d ry m a t t e r r e q u i r e d t o produce a one l b g a i n i n body w eight was 5.47 l b s (Table 6 ) . S in c e the av e ra g e s u r v i v a l time o f t h e lambs f e d the r i b o ­ f l a v i n d e f i c i e n t d i e t was 18 d a y s , a more a c c u r a t e com parison o f th e c o n t r o l and d e f i c i e n t groups would n e c e s s i t a t e t a k i n g i n t o a c c o u n t th e growth p erform ance of the c o n t r o l lambs f o r o n ly th e f i r s t e i g h t e e n d ay s . T h is i s i l l u s t r a t e d in - 26 - < J> bO O P. © fa­ 's: 0- m * rH rH 00 O ♦ to rH co CQ las < CM rH P P O CO • o> CO tP • o • SP rH c^ o • rH eo> cn • o ffi P E-* © S -pi o n o *o (x, o 3 CJ1 s ■H o> lO * rH i— 1 to o O to • -d* rH in C M to • o rH co C M o • to (— I to 00 • to rH in o • i— i o M E-* TABLE in CtO d •X P C! rH CO ■ — 1 rH o • in rH to X* • C T > to • C M rH • o H *H Q > W (0 W rH f t Vt o P X> S -H <2 u ft CO s to o rH • rH rH 0> a. to cc E-» C O 8 § O 55 X> 8 © ► 4 60 e t>> O ccj 73 P 3 « ftp a G © © s X1 H St u © © ft bD X c © • P 01 3 .a f t rH £2 *> ©P xs G & © s P fH X! G bO © -H f t © X *5 © p a © e •HI g © ft X © c 03 • CO rH o o • • in rH o rH o • rH 00 o • rH • 01 x> 1— 1 • © XI 1 — 1 *» G •rH © bO o © >> © Q o in • o cr> rH • rH rH © P O Eh « g a •rH © bQ © p • P 01 © X) El rH rH •H © •a >> " P. 'O ■a © s rH 3 © 01 • > < P o E-* o o g I* © P P 3 © tH s © hO >> G • 'd x> rH • © G x> © - 27 - to 00 S3 <4 53 04 M O M 0 t*0 jm 9 03 P © !> • CO 03 in o • o Q > <4 fd O OQ M 01 rH oo o> • o CO 03 to 03 • tO 03 o £*rH • o o in CO o• o <=* o> • CO O CO • CO in • in CO o • CO in in CO • o or; TABLE to &H o S3 o n lO o• o 03 rH CO rH in • rH CO CO o• o • fH 3 w PI M E-< O « o• X* o> CO 00 • rH 03 o o o• o to m • • in C*in • rH c♦ CO 04 B Q !3 <4 03 to • rH rH 03 CO rH in • rH 04 CO 5 O Ph CQ P4 cs £«H 3 fg Ci> 01 £3 !>> o a n P o o S3 43 a P • P as 3 43 P*rH d ® P xs d & ® s p *rl X i Li fcD o> •rH U , © X « d <0 £ •rl P © Pi X © d o 03 I—1 m d •H cd oo rH r d •rl od W t» rH *rl ai rH © P O £H TJ • > <4 Li © P . P (0 ctS X> 8 rH >> * Li TJ «o as E rH 3 Cd 03 p d o o IH O p © p d id iH S cd bO >3 p * T3 X» rH • ® P 43 to r-. fi = -I H 9- K r § 8 3 I # I z0 2 < Q r S < _i 5 • ■ 6 i £ 0 z i i a r 5 E f g < -1 J I A U *£ !S S £ «! s SONOOd u! JX0I3A S S - 30 - tab le 8 * The d a t a from t h r e e c o n t r o l lambs from th e second t r i a l are also included in t h i s t a b l e . The av e ra g e d a i l y g a i n of 0 ,0 4 3 l b f o r th e r i b o f l a v i n d e f i c i e n t lambs as com­ p a r e d t o t h e a v e ra g e d a i l y g a i n o f 0 . 3 9 l b f o r the c o n t r o l group i n d i c a t e d t h a t t h e young lamb h a s a d e f i n i t e r e q u i r e m e n t for rib o fla v in . I t can be se en from graph 1 t h a t the lambs fed th e r i b o f l a v i n d e f i c i e n t d i e t grew a t a normal r a t e f o r a b o u t 4 to 6 days. At t h i s time the a p p e t i t e of the an im als d e c l i n e d f o r ab o u t 3 to 4 days w hile t h e i r w eig h ts remained the same or s l i g h t l y decreased. The a p p e t i t e improved to some e x t e n t between t h e e i g h t h and f o u r t e e n t h day and the lambs made some g a i n i n body w e i g h t . A f t e r 12 to 14 days on the experim ent th e lambs l o s t t h e i r a p p e t i t e s c o m p l e t e l y , l o s t w eight r a p i d l y , became e m a c ia te d and, on th e s e v e n t e e n t h t o t w e n t y - t h i r d day, a l l d e f i c i e n t lambs were i n a moribund c o n d i t i o n . Two lambs d i e d , t h r e e were s a c r i f i c e d and two lambs were t r e a t e d w ith r i b o f l a v i n on the s i x t e e n t h and n i n e t e e n t h day on t h e e x p e r i ­ mental p e r i o d (Table 7 ) . The g e n e r a l c o n d i t i o n of the t r e a t e d lambs improved i n h o u r s a f t e r t h e i n j e c t i o n of r i b o f l a v i n and the normal a p p e t i t e was r e s t o r e d i n two d a y s . The a n im a ls were g a i n i n g w e ig h t by th e second day and i n one week a l l th e symptoms, i n c l u d i n g e m aciatio n , d i s a p p e a r e d . W hile, d u r i n g the p e r i o d o f s e v e n t e e n days on t h e d e f i c i e n t d i e t , t h e a v e ra g e d a i l y g a i n was 0 .0 3 5 l b and 9 . 8 2 l b s o f dry m a t t e r was needed t o produce one l b of g a i n ; d u r i n g th e s e v e n t e e n day p e r i o d - 31 - TABLE 8 GROWTH RESPONSE OF THE CONTROL AND DEFICIENT LAMBS FOR THE FIRST EIGHTEEN DAYS OF THE TRIAL Number a n i m a l s R iboflavin D eficient 8 7 to i 3-11 C V 3 I n i t i a l age days C o n t ro l 9 .3 Av. i n i t i a l w e i g h t , l b s 12 Av. e i g h t e e n day w eight, lb s * 19.1 1 0 .0 Days on ex p e rim e n t 18 18 Av. d a i l y g a i n , l b s 0.39 0.0 4 3 Av. d a i l y d ry m a t t e r consumed, l b s 0 .4 1 0.14 Lbs dry m a t t e r l b g a i n 1.18 7.63 #The c o n t r o l lambs were fed t h e same d i e t f o r 23 t o 36 days. 32 - o f r i b o f l a v i n t r e a t m e n t , t h e a v e ra g e d a i l y g a i n i n body w eight was 0*48 l b , and o n ly 0 .9 7 l b . o f dry m a t t e r was r e q u i r e d t o p r o d u c e one l b . o f w e i g h t . The growth r e s p o n s e to r i b o f l a v i n of the two lambs (No. 10, 11) i s a l s o i l l u s t r a t e d i n graph 1* Symptoms o f D e f i c i e n c y The growth o f t h e c o n t r o l lambs was u n e v e n t f u l e x c e p t f o r o c c a s i o n a l m ild d i a r r h e a , p r o b a b l y due t o o v e r f e e d i n g . The d i a r r h e a soon d i s a p p e a r e d w i t h o u t t r e a t m e n t . A f t e r ab o u t s i x days th e lambs f e d t h e d e f i c i e n t d i e t d ev e lo p ed a s e v e r e d i a r r h e a accompanied by l o s s of a p p e t i t e . t h r i f t y , d e p r e s s e d and moved s l o w l y . The lambs became un­ The d i a r r h e a r e c e d e d a f t e r a b o u t f o u r to s i x days and the a p p e t i t e improved tempo­ rarily . A f t e r the t w e l f t h day the d e f i c i e n t lambs d e v e lo p e d d i f f i c u l t i e s i n b r e a t h i n g which p r o g r e s s i v e l y became t h e t y p i c a l symptoms o f pneumonia c h a r a c t e r i z e d by r a p i d and l a b o r e d b r e a t h ­ i n g and d u l l n e s s . The a n i m a l s r e f u s e d t o e a t , became l e t h a r g i c , would not walk o r s t a n d and th e c o n d i t i o n t e r m i n a t e d by a c o m p lete c o l l a p s e and d e a t h . The lambs were o b s e rv e d ev e ry day b u t t h e o r a l l e s i o n s , e x c e s s i v e s a l i v a t i o n and l a c h r y m a t i o n , l o s s of h a i r and i n ­ f la m m a tio n o f th e n a v e l a r e a , t h e t y p i c a l symptoms o f r i b o f l a v i n d e f i c i e n c y i n c a l v e s d e s c r i b e d by Wiese e t a l . (1947) and Warner and S u t t o n (1948) were n o t note d i n r i b o f l a v i n d e f i c i e n t la m b s. The p r i n c i p l e and o n ly symptoms o f r i b o f l a v i n d e f i c i e n c y i n th e lamb were l o s s o f a p p e t i t e , d i a r r h e a , r e t a r d e d growth, 33 - f o llo w e d by e m a c i a t i o n , d u l l n e s s , pneumonia and d e a t h . Blood S t u d i e s The r e s u l t s of the i n i t i a l and weekly blood a n a l y s e s o f th e e i g h t c o n t r o l and seven d e f i c i e n t lambs were summarized i n t a b l e 10 (page 52) and graph 2. The average i n i t i a l hemo­ g l o b i n l e v e l s o f th e c o n t r o l and e x p e r i m e n t a l groups were 1 2 . 6 and 1 2 . 8 gm p e r HlQ&c of b l o o d , r e s p e c t i v e l y , w i t h v a l u e s r a n g ­ i n g from 1 0 .8 t o 1 4 .2 gm p e r c e n t . The l e v e l s of hemoglobin rem ained f a i r l y c o n s t a n t th r o u g h o u t th e e x p e ri m e n t. The i n i t i a l l e u k o c y t e count p e r mm3 and th e r a t i o of polymorpho­ n u c l e a r l e u k o c y t e s t o lymphocytes of both c o n t r o l and d e f i c i e n t lambs showed o n ly s l i g h t v a r i a t i o n . The v a l u e s of t h e l e u k o ­ c y t i c c o u n t s were 1 0,125 and 10,950 p e r mm3 , and t h e r a t i o s of n e u t r o p h i l s to lym phocytes were 42:55 and 46 :5 2 . The most s i g n i f i c a n t d i f f e r e n c e between the c o n t r o l and r i b o f l a v i n d e p l e t e d lambs was found i n th e le u k o c y t e c o u n t . D uring th e e x p e r i m e n t a l p e r i o d t h e l e u k o c y t e count of t h e c o n t r o l lambs d e c r e a s e d from t h e i n i t i a l 10,125 t o 7 ,7 0 0 p er mm3 w h ile t h e number of l e u k o c y t e s of t h e d e f i c i e n t lambs g r a d u a l l y i n c r e a s e d and r e a c h e d 14,650 p er mm3 by t h e t h i r d week. T h is i n c r e a s e , no d o u b t, was due t o th e pneumonia which a l s o was r e f l e c t e d i n a r e v e r s a l of t h e d i f f e r e n t i a l count. During th e ex p e rim e n t th e n e u t r o p h i l s of t h e c o n t r o l lambs g r a d u a l l y d e c r e a s e d i n f a v o r o f t h e lymphocytes and a t t h e end o f t h e e x p e rim e n t t h e r a t i o was 3 5 : 6 3 , which i s w i t h i n Graph 2 . H e m o g lo b in , l e u k o c y t e and d i f f e r e n t i a l c o u n t s o f c o n t r o l and r i b o f l a v i n d e f i c i e n t l a m b s . - 34 - «uio i- o- oeficient o XL id 60 I— z uUI oc ui Q . a 0O co '’E £ 1 _ i * - CONTROL o — DEFICIENT a . 3 0 1 I- c (A U J t>- 2 3 LlI -J *— o - CONTROL DEFICIENT Z 03 O _J 0) o E ui X a 5 W EEKS on EX P E R IM E N T -t - 35 - normal range f o r young h e a l t h y lam bs. However, i n the d e f i ­ c i e n t la m b s, th e r a t i o r e v e r s e d and, e s p e c i a l l y d u r i n g t h e f i n a l s t a g e s o f d e f i c i e n c y , t h e number of n e u t r o p h i l s g r e a t l y increased. By t h e t h i r d week, j u s t b e f o r e t h e lambs d i e d , were s a c r i f i c e d or t r e a t e d , t h e r a t i o was 62 p olym orphonuclear l e u k o c y t e s t o 35 ly m p h o c y te s. Monocytes, e o s i n o p h i l s and b a s o p h i l s showed no changes i n numbers. U rine S t u d i e s I t was d i f f i c u l t to make an a c c u r a t e comparison of th e u r i n a r y e x c r e t i o n of r i b o f l a v i n from th e c o n t r o l and d e p l e t e d anim als. Only two metabolism cages were a v a i l a b l e f o r c o l ­ l e c t i o n of t h e 2 4 - hour u r i n e sam ples. As a r e s u l t , the a s s a y s were s t a g g e r e d over t h e l a s t two weeks o f th e experim ent which r e p r e s e n t e d two t h i r d s o f the t o t a l s u r v i v a l p e r i o d of t h e d e f i c i e n t group. These c i r c u m s t a n c e s e l i m i n a t e d the p o s s i b i l i t y o f s i m u l t a n e o u s l y comparing t h e two g ro u p s. The a v e ra g e u r i n ­ a r y e x c r e t i o n p e r 24 h ours was th e n c a l c u l a t e d by p o o l i n g the v a l u e s o f r i b o f l a v i n e x c r e t e d from a l l lambs i n a p a r t i c u l a r group o b t a i n e d d u r i n g the two week p e r i o d . U s u a l l y one c o n t r o l and one d e f i c i e n t lamb were s e l e c t e d a t random f o r u r i n e c o l ­ l e c t i o n i n one day. The v a r i a t i o n i n u r i n a r y e x c r e t i o n of r i b o f l a v i n w i t h i n the group was r a t h e r marked b u t t h e r e was always a d e f i n i t e and s t r i k i n g d i f f e r e n c e between the v a l u e s of t h e c o n t r o l and d e p l e t e d a n i m a l s . The av e ra g e u r i n a r y e x c r e t i o n o f r i b o f l a v i n o f the e i g h t c o n t r o l lambs was 0 .5 0 mg. - 56 - p e r 24 h o u r s w h ile t h a t o f the seven d e f i c i e n t lambs was 0 .0 1 mg p e r 24 h o u r s . Gross P a th o lo g y C ontrols. One of th e f i v e c o n t r o l lambs was r e l e a s e d from th e e x p e rim e n t when ab o u t 25 l b s i n w eight and d id not show any s i g n s of i l l h e a l t h . a f t e r two weeks. One c o n t r o l lamb d ie d suddenly P o s t mortem e x a m in a tio n r e v e a l e d t h a t the ca u se of d e a t h was o v e r e a t i n g and not d i r e c t l y r e l a t e d t o th e d i e t nor t o i t s c o m p o s iti o n . The c a r c a s s was w ell n o u r i s h e d . The i n t e s t i n a l t r a c t showed se v e r e e n t e r i t i s which extended from the duodenum to th e rectum . were bloody. The i n t e s t i n a l c o n t e n t s Soon a f t e r b e in g p u t on the e x p e r i m e n t a l d i e t th e lamb showed some c l i n i c a l symptoms o f pneumonia. However, a t t h e time o f d e a t h , no evid en ce of pneumonia was o b s e r v e d . C u l t u r e s p r e p a r e d from l u n g s , l i v e r , g a l l b l a d d e r , s p l e e n and h e a r t blood were n e g a t i v e . Three r i b s had been broken i n e a r l y l i f e , p r o b a b l y a t lam b ing, which would e x p l a i n the labored b re a th in g . The o t h e r t h r e e c o n t r o l lambs were sa c ­ r i f i c e d f o r g r o s s and h i s t o p a t h o l o g i c a l s t u d i e s a f t e r t h r e e , f o u r and f i v e weeks on th e e x p e r i m e n ta l d i e t . The c a r c a s s e s were i n e x c e l l e n t c o n d i t i o n and d i d n o t show any g r o s s p a t h o l o g i c a l changes e x c e p t two broken r i b s i n one lamb. The l u n g s , l i v e r s , g a l l b l a d d e r s and s p l e e n s were c u l t u r e d on blood a g a r p l a t e s and were found n e g a t i v e a f t e r a s u i t a b l e period of incubation. 37 - R i b o f l a v i n d e f i c i e n t la m b s . Of t h e seven r i b o f l a v i n d e f i c i e n t la m b s , two d i e d sudden ly and n e c r o p s i e s were p er f o rm e d . Three lambs were s a c r i f i c e d when i n a moribund c o n d i t i o n as a r e s u l t o f complete d e p l e t i o n of r i b o f l a v i n . Two lambs were t r e a t e d w i t h r i b o f l a v i n f o r 17 days a f t e r a 17 day p e r i o d o f d e p l e t i o n and t h e n s a c r i f i c e d f o r s t u d y . The g r o s s p a t h o lo g y o f th e f i v e r i b o f l a v i n d e f i c i e n t lambs was s i m i l a r w i t h only minor v ariatio n . The p r i n c i p a l f e a t u r e s were (1) l u n g involvem ent w ith bronchopneumonia, (2) f a t t y d e g e n e r a t i o n of th e l i v e r and (3) f a t t y d e g e n e r a t i o n o f th e k id n e y . The l u n g s o f a l l f i v e d e p l e t e d a n i m a l s showed b i l a t e r a l bronchopneumonia which seemed t o be p r e g r e s s i v e , r a p i d l y s p r e a d i n g from a p i c a l to c a r d i c and d i a p h r a g m a t i c l o b e s o f b o th l u n g s . There was u s u a l l y a marked zone o f c o n g e s t i o n between th e pneumonic a r e a s and the normal p a r t o f the l u n g . I n one lamb, g r a y i s h f i b r i n o u s ex u d a te was p r e s e n t between t h e lu n g s and the t h o r a c i c w all and th e l u n g s adh e re d to t h e r i b s . When c u t and s q u e e z e d , t h e c o n s o l i d a t e d a r e a s showed f r o t h y mucous e x u d a te i n t h e t r a c h e a , b ro n c h i and bronchioles. C u l t u r e s p r e p a r e d from lu n g s and b ro n c h i y i e l d e d p le o m o rp h ic gram n e g a t i v e rods r e s e m b lin g P a s t e u r e l l a e o r g a n is m s . P o s t mortem e x a m in a tio n of th e two r i b o f l a v i n t r e a t e d lambs, p r e v i o u s l y d e p l e t e d on r i b o f l a v i n d e f i c i e n t d i e t , showed " c o ld pneumonia" i n th e r i g h t a p i c a l lobe i n b o th lambs. The l i n e of d e m a r k a tio n between t h e normal and t h e pneumonic p a r t was n o t d i s t i n c t and r e s o l u t i o n was i n p r o g r e s s . from t h e s e a r e a s were n e g a tiv e * Cultures prepared - 38 - M a croscopic e x a m i n a t i o n of the l i v e r from d e f i c i e n t a n i m a l s showed p a t h o l o g i c a l changes r a n g in g from mild de­ g e n e r a t i o n to n e c r o s i s . I n t h r e e lam bs, s e v e r a l n e c r o t i c a r e a s were o b se rv e d i n d i f f e r e n t p a r t s o f the l i v e r . O th e r ­ w ise t h e changes c o n s i s t e d of g e n e r a l i z e d f a t t y i n f i l t r a t i o n and d e g e n e r a t i o n c h a r a c t e r i z e d by enlargem e nt of t h e o r g a n , y e l l o w i s h o r brown g r a y i s h c o l o r , smooth s u r f a c e , s o f t con­ s i s t e n c y and i n c r e a s e d f r i a b i l i t y (F ig . 1 ) . The l i v e r of t h e two t r e a t e d a n i m a l s d id not show m a croscopic l e s i o n s * The changes i n th e k id n e y s o f a l l th e r i b o f l a v i n d e f i c i e n t lambs f o llo w e d the same p a t t e r n . There was a marked f a t t y d e g e n e r a t i o n and t h e c o r t e x was always much more a f f e c t e d t h a n t h e m e d u lla . The l i n e of d e m a r c a t io n between t h e c o r t e x and th e m ed ulla was v ery pronounced. The m e d u lla ry zone was u s u a l l y c o n g e s te d and s l i g h t edema was p r e s e n t i n th e pelv is. The o u t e r s u r f a c e o f the k id n e y was of a d u l l , y e l lo w c o l o r and i n some i n s t a n c e s p a t c h y . When c u t , the s u r f a c e bulged and had a p rom inent p a r b o i l e d a p p e a r a n c e . I n more s e v e r e c a s e s t h e c u t s u r f a c e e a s i l y f l u c t u a t e d on p r e s s u r e and t h e s t r e a k s r u n n i n g from t h e o u t e r s u r f a c e t o the p e l v i s were pro m inent (Fig. 1 ) . The k id n e y s o f th e two r i b o f l a v i n t r e a t e d lambs showed a v e r y m ild f a t t y i n f i l t r a t i o n c h a r a c t e r i z e d by a s l i g h t l y yellow c o rte x . The m edulla and p e l v i s were normal. The h e a r t o f th e r i b o f l a v i n d e f i c i e n t lambs appe ared n o r m a l. The d u c t u s a r t e r i o s u s and foramen ovale o f a l l th e lambs ware c l o s e d . Due to t h e p r e s e n c e o f pneumonia i n p r a c ­ 39 - t i c a l l y e v e r y c a s e , t h e r e was an i n c r e a s e o f f l u i d i n th e p e r ic a r d ia l sac. G r o s s l y , t h e a d r e n a l s were e n l a r g e d . The c u t s u r f a c e showed a marked l i n e o f d e m a r c a t io n between t h e c o r t e x and m edulla. I n most i n s t a n c e s hemorrhage was c l e a r l y v i s i b l e . On s e c t i o n th e o u t e r s u r f a c e and t h e c o r t e x were p a l e and flab b y (Fig. 1 ) . The o t h e r o r g a n s , i n c l u d i n g t h e whole d i g e s t i v e t r a c t , t h y r o i d , eye and s p l e e n , were normal. The same was t r u e i n r e g a r d t o the s k e l e t a l , nervous and m uscular s y stem . M ic ro s c o p ic P ath o lo g y C ontrols. M ic ro s c o p ic s t u d i e s of th e t i s s u e s , t a k e n from the c o n t r o l la m b s, f a i l e d t o r e v e a l p a t h o l o g i c a l c ha nges. In two lambs f r o z e n s e c t i o n s from th e l i v e r , s t a i n e d w ith Sudan IV, showed a m ild f a t t y i n f i l t r a t i o n which was c o n s i d e r e d to be a p h y s i o l o g i c a l i n c r e a s e of h e p a t i c f a t . R i b o f l a v i n d e f i c i e n t la m b s . The m ic ro s c o p ic changes i n th e I d e f i c i e n t lambs were c o n f in e d t o the l i v e r , k id n e y and a d r e n a l s . The changes i n t h e l i v e r ranged from cloudy s w e l l i n g to f o c a l necro sis. N e i t h e r o f t h e s e extreme c o n d i t i o n s were t y p i c a l o f th e d e f i c i e n c y . The most prom inent f e a t u r e , common i n a l l d e f i c i e n t la m b s , was d i f f u s e f a t t y d e g e n e r a t i o n , a f f e c t i n g most o f the h e p a t i c c e l l s i n t h e l o b u l e . U s u a l l y the most pronounced f a t t y d e g e n e r a t i o n was i n th e c e n t e r of t h e l o b u l e ( F i g . 2) w h ile on th e p e r i p h e r y th e h e p a t i c c e l l s were s t i l l - 40 - i n t a c t o r u n d e r g o in g v a r i o u s d e g r e e s o f cloudy s w e l l i n g . One or more f a t g l o b u l e s o f v a r y i n g s i z e f i l l e d t h e h e p a t i c c e l l s and pushed t h e n u c l e i a g a i n s t one s id e of t h e c e l l w a l l , de­ p r i v i n g t h e c e l l o f n u t r i t i o n which r e s u l t e d i n k a r y o l y s i s , k a r y o r r h e x i s and p y k n o s i s . In most i n s t a n c e s t h e l i v e r showed a c u t e venous c o n g e s t i o n which was g e n e r a l i z e d and v ery marked. The s i n u s o i d s were g r e a t l y d i s t e n d e d and o c c a s i o n a l l y f i l l e d w ith an e x c e s s i v e number of blood c e l l s . N e c r o s i s obse rve d i n few i n s t a n c e s was f o c a l i n c h a r a c t e r , l o c a t e d u s u a l l y on th e s u r f a c e o f th e l i v e r and on th e p e r i p h e r y of t h e l o b u l e . The s i n u s o i d s were damaged. The n e c r o t i c a r e a s were p a r t l y o r c o m p l e t e l y su rro u n d e d by h e p a t i c c e l l s u n d erg o in g f a t t y d e g e n e r a t i o n and cloudy s w e l l i n g . The most u n ifo rm m i c r o s c o p ic l e s i o n s were found i n the kidney. The c o r t e x showed e x t e n s i v e and d i f f u s e f a t t y de­ g e n e r a t i o n w h ile t h e medulla underwent v a r i o u s d e g re e s of cl o u d y s w e l l i n g o r mild f a t t y c h a n g es. The k id n e y was markedly c o n g e s te d and hem orrhagic and i n v o l v e d both parenchyma and in te stin a l tissu es. The r e n a l c o r p u s c l e s , i n c l u d i n g Bowman c a p s u l e s and g l o m e r u l i , seemed t o be normal. I n two lambs t h e r e was a te n d e n c y toward h y a l i n e changes w i t h i n the glom­ e r u l i and c o n v o l u te d t u b u l e s . The basement membrane o f the c a p i l l a r y l o o p s seemed to be s l i g h t l y t h i c k e n e d and the c a p i l ­ la rie s slig h tly dilated . The e p i t h e l i u m of t h e co n v o lu te d t u b u l e s showed marked cloudy s w e l l i n g and s e v e r e f a t t y changes (Fig. 3 ). The c e l l s were s w o lle n , the cytoplasm a p p e a re d - 41 - g r a n u l a r and p r o j e c t e d i n t o the lumen o f the t u b u l e s . In some t u b u l e s t h e c e l l s p r o b a b l y r u p t u r e d and th e m a t e r i a l formed h y a l i n e c a s t s i n t h e c o n v o lu te d t u b u l e s . These t u b u l e s were d i l a t e d , th e e p i t h e l i a l c e l l s were a t r o p h i e d and t h e n u c l e i d e g e n e r a t e d . F roz en s e c t i o n s s t a i n e d w ith Sudan IV the showed t h a t most o f f a t had accum ulated i n the c o n v o lu te d tu b u le s (Fig, 4 ), Some c e l l u l a r i n f i l t r a t i o n and p r o l i f e r a ­ t i o n of i n t e r s t i t i a l t i s s u e i n th e medulla was observed i n one lamb. Lymphocytes were predom inant b u t a few macrophages a l s o were p r e s e n t . I t was d i f f i c u l t to d e l i n e a t e between t h e p h y s i o l o g i c a l and p a t h o l o g i c a l f a t t y i n f i l t r a t i o n of the a d r e n a l s . Micro­ s c o p i c s t u d i e s o f th e f r o z e n s e c t i o n s from the a d r e n a l s o f t h e c o n t r o l lambs r e v e a l e d the p r e s e n c e of f a t d r o p l e t s be­ tween t h e g lo m e ru lo s a and f a s c i c u l a t a o f th e c o r t e x . S ections from th e d e f i c i e n t lambs showed a s l i g h t l y i n c r e a s e d amount o f f a t i n th e same r e g i o n . I n two d e f i c i e n t lambs both t h e c o r t e x and m e d ulla were i n f i l t r a t e d w ith f a t ( F ig . 5, 6 ) . Otherw ise t h e a d r e n a l s from th e d e f i c i e n t lambs were c o n g e s te d and t h e s i n u s o i d s , e s p e c i a l l y around th e c e n t r a l v e i n , were d i l a t e d t o some e x t e n t . Both t h e c e n t r a l v e i n and a d j a c e n t s i n u s o i d s were f i l l e d w ith b lo o d . However, the margins of th e s i n u s o i d s were i n t a c t . M ic r o s c o p ic e x a m in a tio n of the h e a r t m uscle, i n t e s t i n a l t r a c t , s k i n from th e c o r n e r of t h e mouth, e y e , b r a i n , s p i n a l c o r d , b r a c e n i a l and s c i a t i c n e r v e s , s t a i n e d w ith h em ato x y lin - 42 - e o s i n and K u l t s c h i t z k y * s h e m a to x y l in s o l u t i o n , f a i l e d t o r e v e a l p a t h o l o g i c a l changes. T r e a t e d Lambs, M ic ro s c o p ic changes o f the l i v e r and k id n e y from th e two r i b o f l a v i n t r e a t e d a n i m a l s , p r e v i o u s l y d e p l e t e d o f t h e v i t a m i n , were s i m i l a r i n n a t u r e to th o s e o f th e d e f i ­ c i e n t anim als. The l i v e r s t i l l showed g r a n u l a r i t y o f t h e h e p a t i c c e l l s and some f a t d r o p l e t s w i t h i n th e c e l l s . The s i n u s o i d s were l e s s d i s t e n d e d and t h e r e was l e s s c o n g e s tio n th r o u g h o u t t h e l i v e r . The e p i t h e l i a l c e l l s of the co n v o lu te d t u b u l e s o f the k id n e y were sw ollen and i n some p l a c e s t h e c e l l s were desquam ated. Albuminous g r a n u l a r m a t e r i a l i n the lumen of t h e t u b u l e s was o c c a s i o n a l l y o b se rv e d . The g lo m e r u l i a p p e a r e d normal. R i b o f l a v i n Requirement The r e s u l t s o f t h e second t r i a l i n r e g a r d t o growth and f e e d u t i l i z a t i o n a r e shown i n t a b l e 9 and graph 3, The t r i a l c o n s i s t e d o f t h r e e p o s i t i v e c o n t r o l and t e n lambs on f i v e d i f f e r e n t l e v e l s of r i b o f l a v i n . The growth cu rv es a r e a v e r a g e s o f the g a i n s i n body w eight o f two lambs i n each subgroup w h ile t h e growth curve of th e c o n t r o l s r e p r e s e n t s t h r e e lambs. The t h r e e c o n t r o l lambs on th e complete p u r i f i e d d i e t grew n o r m a ll y and th e g a i n s i n body weight and f e e d u t i l i z a t i o n compared w i t h the f i v e c o n t r o l lambs from the f i r s t t r i a l . Two c o n t r o l lambs were r e l e a s e d from th e experim ent a f t e r 27 and 35 d a y s . The t h i r d lamb was s a c r i f i c e d f o r g ross and h isto p ath o lo g ical stu d ies. -4 3 - iH O CO © 03 iH 03 •P d o o rH O S* -P d o o CO • orH CO 03 03 O 03 i—1 * CO i—I rH rH i—i o Ch -p u o in co co rH c03 -cP CO • O • 03 rH vP • in c*• CO i—1 o 03 • CO rH oCO • o 00 CO • CO rH in • in co • rH C03 • rH O • i—1 • CO t—1 o o CO 03 CO rH 03 GROWTH RESPONSE AND FEED UTILIZATION OF CONTROL LAMBS AND LAMBS ON VARIOUS LEVELS OF RIBOFLAVIN CO • c*- o in CO o• orH CO o in CO o• co 03 o t** o> i—1 O in 03 o• CO H <*P o oo in rH cH to • 00 CO c03 o in CO • t- • in m 03 03 • o> LO in • co rH o rH co co O • CO in • in sp 03 in • CO 03 CO • CO o> • 03 rH ■sP 03 • rH a 03 • co CO 03 • i—I o • co o CO 03 • oo in • 03 ■—1 03 • rH co rH • coo • 00 sP tP • iH 00 o> • o rH -*P sP • 03 in c- • co rH • 03 rco • CO CO CTJ • CT> cr> 'CP • 03 o o ot—1 • o in rH • o rH rH o in LO • ts (O rH in rH rH o in O • t*- CO o in o• <>3 sp o• 00 • .H rH orH rl in 03 • rH in CO CO o• o in o> o• o rH co • 'CP o o• o cr> • t- CO CO • 03 CO t"• in 03 o> • in o- CO rH CO a o O to t» cd T3 •P 3 ** & .-P d d d o • -P 09 d x> f tr H d • © +9 xi d gs © a •P •C & aO*©•H d r © X ^ © in d •f"4 > >> cd cd rH TJ iH H \ U © S cd O U to rH O © fcr t> o rH 03 rH o© o -P d © a d •H cd •rl s_ © dr X © d o © t>> © *H cd ►4 S Q d •rl © to rH aS •P o Eh to >4 iH -M © © -p •p © a © •p •p d © »rH © taO a SU T3 TJ © a rH 3 © © • > •P d O O c Eh O >4 u • TJ X> rH • © u x» © J CH Graph 3* Growth c u r v e s o f c o n t r o l la m b s and la m b s on v ario u s le v e ls of r ib o f la v in . 44 - E X P E R IM E N T to t0 DAYS on cm' < OI CM to CO CD cD — 3 2? S b £ ! s £ ! S f i t SQ N DO d «! 1 H 9 I 3 / & s S a » « K - 45 - The p o s t mortem e x a m in a tio n d i d not show l e s i o n s . The same was t r u e f o r th e m ic r o s c o p ic s t u d i e s of th e t i s s u e s from v a r i o u s o r g a n s . The f o u r lambs on the two lo w e s t l e v e l s of r i b o f l a v i n (5 and 50 micrograms p e r l b of body weight p e r day) r e s e m b le d t h e lambs on th e r i b o f l a v i n d e f i c i e n t d i e t i n grow th, f e e d u t i l i z a t i o n , symptomat, o l o g y , g r o s s and m ic ro ­ scopic p ath o lo g y . The growth was p o o r , due to th e i n t e r m i t t e n t l o s s o f a p p e t i t e and d i a r r h e a . One lamb died a f t e r 21 days on t h e e x p e r i m e n t a l d i e t and th e o t h e r t h r e e anim als were necropsied fo r f u r th e r s tu d ie s . C onge stion o f th e lu n g s and pneumonia were found i n two lambs. A l l f o u r lam bs, however, showed m ild t o s e v e r e f a t t y d e g e n e r a t i o n of th e l i v e r and kid n e y and e n la r g e m e n t o f t h e a d r e n a l s . A f t e r 37 days on t h e e x p e r i m e n t a l d i e t , th e l i v e r and kidney of t h e lamb on th e 5 micrograms r i b o f l a v i n l e v e l showed a s e v ere f a t t y de­ g e n e r a t i o n , c h a r a c t e r i z e d by an i n t e n s e y e l lo w appe aranc e and m o t t l i n g o f the l i v e r and by prom inent s t r e a k s and y e llo w c o l o r of t h e c o r t e x o f t h e k id n e y . An i n t e n s e zone of c o n g e s t i o n a t t h e j u n c t i o n of t h e c o r t e x and medulla was a l s o o b s e rv e d . The ileum from t h e same lamb was t h i c k e n e d and w r i n k le d i n some a r e a s and showed some c o n g e s t i o n . The growth re sp o n se of t h e lambs on 15 0 , 250 and 350 micrograms of r i b o f l a v i n was d i r e c t l y p r o p o r t i o n a l to th e amount of r i b o f l a v i n a d m i n i s t e r e d t o the r e s p e c t i v e lamb. 0 .3 0 5 .lb . The a v e ra g e d a i l y g a in was 0 . 1 2 5 , 0.205 and The amount of 2 . 2 5 , 1 .3 2 and 1 .2 5 l b of dry m a t t e r were needed r e s p e c t i v e l y t o produce one l b o f body w e ig h t. The growth - 46 - of th e lambs on 150 and 250 micrograms was r a t h e r slow b u t the a n im a ls showed l e s s d i a r r h e a and r e t a i n e d a f a i r a p p e t i t e t h r o u g h o u t t h e e x p e r i m e n t. A l l s i x lambs remained i n good h e a l t h and f a i r l y good c o n d i t i o n . The lambs on 350 micrograms and one lamb on 250 micrograms were r e l e a s e d from the e x p e r i ­ ment and t h e o t h e r t h r e e an im als were s a c r i f i c e d . The lambs on 150 micrograms of r i b o f l a v i n showed f a t t y d e g e n e r a t i o n of the k id n e y and l i v e r and e n l a r g e d a d r e n a l s . No pneumonia or c o n g e s t i o n of th e lu n g was o b s e rv e d . The r e s u l t s of the weekly blood a n a l y s e s d id not f o llo w any con­ s i s t e n t p a t t e r n e x c e p t ^ h e two lambs on the lo w e s t r i b o f l a v i n le v el. The l e u k o c y t e count o f t h o s e lambs i n c r e a s e d toward t h e end o f th e e x p e r i m e n t a l p e r i o d . however, was unchanged. The d i f f e r e n t i a l c o u n t , I f t h e growth r e s p o n s e , e x p r e s s e d i n g a i n of body w e ig h t, was used as a c r i t e r i o n f o r e s t a b l i s h i n g th e r i b o f l a v i n r e q u ir e m e n t of the young lamb, i t seemed t h a t a l e v e l of 350 micrograms p er l b of body weight p e r day was h ig h enough to i n s u r e a normal growth o r growth comparable w ith t h e c o n t r o l a n i m a l s . There was no d i f f e r e n c e between th e fo u r lambs on th e l o w e s t l e v e l s o f r i b o f l a v i n (5 and 50 m i c r o g r a m s / l b / d a y ) and th e d e f i c i e n t lambs as f a r as t h e m ic ro s c o p ic p a t h o lo g y was concerned. One lamb on 50 micrograms of r i b o f l a v i n a c t u a l l y showed much more pronounced changes i n the k id n e y th a n any o t h e r d e f i c i e n t lamb. The g l o m e r u l i were markedly c o n g e ste d and p r a c t i c a l l y a l l o f them were i n a s t a t e of e a r l y h y a l i n e - 47 - d e g e n e r a t i o n (Fig* 7 ) . H yaline d r o p l e t s and p r o t e i n m a t e r i a l were e s c a p i n g from th e c a p i l l a r y lo o p s and were c o n c e n t r a t e d on th e s id e o p p o s i t e t o th e v a s c u l a r p o le of the r e n a l c o r ­ p u s c l e s between t h e p a r i e t a l and v i s c e r a l l a y e r s of Bowman's capsule. There was d e g e n e r a t i o n of t h e c a p i l l a r y lo o p s and i n a few i n s t a n c e s t h e g lo m e r u l i were c o m p le te ly a t r o p h i e d . The l i v e r showed v a r i o u s d e g r e e s o f cloudy s w e l l i n g and f a t t y degeneration. Of th e f o u r lambs on the two h i g h e s t r i b o f l a v i n l e v e l s (250, 350 m i c r o g r a m s / l b / d a y ) , o n ly one (350 micrograms) was sa c rific e d for fu rth e r studies. Only s l i g h t changes i n th e l i v e r and kid n e y were o b s e rv e d . However, t h e s e were n ot c o n s i d e r e d t o be s i g n i f i c a n t . F a i r growth and c o n d i t i o n and good h e a l t h s u g g e s te d t h a t th e l e v e l was ade q u ate to su p p o r t t h e v i t a l p r o c e s s e s o f t h e kid n e y and l i v e r . DISCUSSION The growth d a t a and growth curves (Tables 5 and 9, Graphs 1 and 3) r e p r e s e n t i n g e i g h t c o n t r o l lambs on a com­ p l e t e p u r i f i e d l i q u i d d i e t (Tables 2 and 3) f o r a p e r i o d o f 18 t o 35 days i n d i c a t e d t h a t the p u r i f i e d d i e t ( s y n t h e t i c m ilk ) p r e p a r e d i n t h e l a b o r a t o r y was a d e q u a te i n a l l th e n u t r i e n t s t o s u p p o r t normal growth and h e a l t h o f t h e young lam b. The av e ra g e d a i l y g a i n of th e c o n t r o l lambs was 0 .4 0 l b . and r e q u i r e d 1.17 l b s . of dry m a t t e r t o produce 1 l b . g a i n i n body w e i g h t . Kean and Henning (1949) i n a stu d y of t h e d a i l y g a i n s o f 787 lambs from b i r t h to 32-36 pounds i n body w eig h t r e p o r t e d an ave ra ge g a in o f 0 . 5 3 l b . p er day. I n s p i t e o f th e f a c t t h a t the lambs were fed only f i v e t im e s a day and r e c e i v e d a r a t h e r l i m i t e d amount o f the r a t i o n t o a v o i d o v e r f e e d i n g and t h e c o m p l i c a t i o n s r e s u l t i n g from i t , t h e growth o f t h e c o n t r o l lambs compared f a v o r a b l y w ith the growth o f lambs r u n n i n g w ith ewes and n u r s i n g a t w i l l . Five o f th e c o n t r o l lambs were s a c r i f i c e d a t th e end o f t h e e x p e r i ­ ment. Gross and m ic r o s c o p i c e x a m in a tio n of t h e c a r c a s s e s and o r g a n s d i d n o t show s i g n i f i c a n t changes. W illia m s et_ al_. (1950) d e f i n e d t h e B v i t a m i n s a s " t h o s e o r g a n i c s u b s t a n c e s which a c t c a t a l y t i c a l l y i n a l l l i v i n g c e l l s and which f u n c t i o n n u t r i t i o n a l l y f o r a t l e a s t some o f t h e h i g h e r a n i m a l s " . S in ce t h e f i r s t s t u d i e s of 49 - r i b o f l a v i n d e f i c i e n c y i n th e r a t by Chick and Roscoe i n 1928 and t h e e s t a b l i s h m e n t o f r i b o f l a v i n c o n s t i t u t i o n and i t s s y n t h e s i s i n 1 9 3 5 t t h e f u n c t i o n o f r i b o f l a v i n a s an e s s e n t i a l n u t r i e n t f o r a number o f h i g h e r an im als was s t u d i e d and established* R i b o f l a v i n has been shown to be an e s s e n t i a l n u tr ie n t for the r a t (Chick and Roscoe, 1928; Day and L a n g sto n , 1934; Bessey and Wolbach, 1939); mouse (Jones et. al_., 1945); cotton r a t ( M c l n t i r e e t a l . , 194 4); ham ster (Routh and Houchin, 1 9 4 2 ); monkey (Waisman, 1 9 4 4 ); c h i c k s and t u r k e y s (Lepkovsky and J u k e s , 1936; P h i l l i p s and E n g e l, 1938); dog (Zimmerman and B urac k, 1934; P o t t e r et, a l *, 1942); p i g (Hughes, 1939; P a t e k e t a l . . 1941; Wintrobe et. a l . , 1944; K r i d e r e t a l . * 1949) and c a l f (Wiese e t a l . . 1947b; Warner and S u tto n , 1948). S t u d i e s o f t h e symptomathology of r i b o f l a v i n d e f i c i e n c y i n t h e above mentioned s p e c i e s o f anim als r e v e a l e d some f e a t u r e s common and c h a r a c t e r i s t i c to a l l . This o b s e r ­ v a t i o n agree d w ith t h e g e n e r a l l y a c c e p te d i d e a t h a t t h e B v i t a m i n s were r e q u i r e d by ev e ry l i v i n g c e l l of animal t i s s u e s and, i f n o t a v a i l a b l e , the d e f i c i e n c i e s would m a n i f e s t them­ s e l v e s by s i m i l a r symptoms i n d i f f e r e n t s p e c i e s o f a n i m a l . The s t u d i e s o f r i b o f l a v i n d e f i c i e n c y i n t h e lamb, however, r e v e a l e d t h a t , e x c e p t f o r l o s s of a p p e t i t e and r e t a r d e d grow th, none o f th e t y p i c a l symptoms of a r i b o f l a v i n o s i s , such a s a l o p e c i a erythema and c o r n e a l v a s c u l a r i z a t i o n i n t h e r a t and dog, d e r m a t i t i s i n t h e ham ster and swine and c a t a r a c t i n t h e r a t and sw ine, were o b se rv e d i n t h e r i b o f l a v i n d e f i - - 50 c i e n t lamb. F u r th e r m o r e , none o f t h e c h a r a c t e r i s t i c symptoms o f r i b o f l a v i n d e f i c i e n c y n£n t h e c a l f (Wiese e t a l , , 1947b) were o b s e rv e d i n th e r i b o f l a v i n d e p l e t e d lamb. Some i n d i c a ­ t i o n f o r the e x p l a n a t i o n o f the s t r i k i n g d i f f e r e n c e i n the symptomatology of t h e r i b o f l a v i n d e f i c i e n t lambs was s u g g e s te d by markedly i n c r e a s e d r i b o f l a v i n l e v e l i n the co lo stru m and m il k o f th e ewe a s compared w ith co lo str u m and m ilk of the cow. Moreover, t h e r a p i d growth o f t h e lamb i n i t s e a r l y p o s t n a t a l l i f e would n e c e s s i t a t e a d i f f e r e n t arrangem ent i n r e g a r d to r i b o f l a v i n s u p p ly , d i s t r i b u t i o n and a v a i l a b i l i t y t o d i f f e r e n t o r g a n s and t i s s u e s . The p r e s e n c e of pneumonia i n a l l lambs on th e r i b o f l a v i n d e f i c i e n t or r e s t r i c t e d d i e t s s u g g e s t e d t h a t t h e pneumonia was i n some way r e l a t e d to rib o fla v in deficiency. I t may be t h a t lu n g t i s s u e has a h i g h e r r i b o f l a v i n r e q u i r e m e n t t h a n any o t h e r t i s s u e i n the body and t h a t t h e p a t h o l o g i c a l changes of the l u n g developed b e f o r e t h o s e of t h e s k i n and eye. I t was d i f f i c u l t t o d e t e r ­ mine i f th e p a t h o l o g i c a l changes of th e l i v e r , kidney and a d r e n a l s r e s u l t e d from a l i m i t e d i n t a k e of t h e r a t i o n and l a t e r from g e n e r a l i n a n i t i o n or from the l o s s of f u n c t i o n due t o th e u n av a ila b ility of rib o flav in i t s e l f . The normal ap pearance of t h e l i v e r and k id n e y of th e two d e f i c i e n t lambs, t r e a t e d w ith r i b o f l a v i n f o r 17 d a y s , s u g g e s te d t h a t th e changes were due t o r i b o f l a v i n 0 r a ^ r th a n to g e n e r a l i n a n i t i o n . In regard t o th e l u n g s o f t h e s e two lam bs, t h e e v id en ce of p a s t pneu­ monia and t h e p r o c e s s of h e a l i n g w i t h o u t any t r e a t m e n t e x c e p t 51 r i b o f l a v i n i n d i c a t e d t h a t t h i s type of pneumonia was somehow r e l a t e d to r i b o f l a v i n deficiency* Whether t h e pneumonia p r o ­ duced by a r i b o f l a v i n d e f i c i e n c y d i e t i n the l a b o r a t o r y has any r e l a t i o n s h i p to the pneumonia o c c u r i n g under f i e l d con­ d i t i o n s i s n o t known* Venkatachalam e t al^. (1949) i n a s t u d y of t h e v a r i o u s c a u s e s of d e a t h i n young la m b s , found t h a t , o u t o f 483 lambs, pneumonia was r e s p o n s i b l e f o r a lm o st o n e - t h i r d o f th e d e a t h s . The d a t a f o r hemoglobin, t o t a l and d i f f e r e n t i a l l e u k o ­ c y t e count of th e c o n t r o l and r i b o f l a v i n d e f i c i e n t lambs were shown i n t a b l e 10* C o n t r a r y to a m i c r o c y t i c hypochromic anemia i n d o g s, ob served by S p e c to r e t a l * (1943), and a moderate norm o cy tic anemia i n sw ine, r e p o r t e d by Wintrobe e t a l * ( 1 9 4 4 ), t h e blood p i c t u r e of t h e d e f i c i e n t lamb, i n g e n e r a l , was unchanged. The hemoglobin l e v e l remained normal and c o n s t a n t th r o u g h o u t th e e x p e r i m e n t , p r o v i d e d t h a t t h e v a l u e s o f t h e f i r s t blood d e t e r m i n a t i o n were c o n s i d e r e d norm al. T hat t h e i n i t i a l blood v a l u e s r e p r e s e n t e d th e normal v a l u e s of th e new b orn lamb was s u p p o r te d by a h i g h e r l e u k o c y t e co u n t accompanied by a h i g h e r p e r cent o f n e u t r o p h i l s a t th e b e g i n n i n g o f the e x p e rim e n t and by a g r a d u a l drop o f t h e l e u k o c y t e s and n e u t r o p h i l s and an i n c r e a s e o f lymphocytes a s t h e lambs became o l d e r . lam bs. This was observed i n t h e c o n t r o l D ec re ase o f l e u k o c y t e count and n e u t r o p h i l s i n f a v o r of lym phocytes toward m a t u r i t y i s a normal phenomenedof any - 52 - 01 (O CQ rH rH O o o o rH 03 rl 00 m 03 m in co o in ■d* 03 o in 03 O in a «S P CJ 0) O •rl SUMMARY OF BLOOD ANALYSES OF THE CONTROL AND RIBOFLAVIN DEFICIENT LAMBS •r-t © P a. !> o d CQ o in o> • • CO CO © • .—l o O •H cd rH 03 ■— 1 rH 00 CO 01 03 03 rH rH • 03 i— 1 CQ o O rH o p rH rH 03 03 C M 03 rH O in in » O 00 o a o jy o C D © m p • « o X X r CJ *rl X M C D P >> o • • o 3 d o X CL, u o a i>> *—i o a, • * ra © p o o X cm a >> p • • a © p o o d o S © * — i •rl 01 X cm o c •H © o P X! Q, O . 01 O S CQ • • • • •o • <0 rH rH cm p q X p cs d C D X! rH If) rH O 5 I t (0 P rH LO >i © cd *H p p W I ^ O CO I rH CM Xi ta o CQ P CQ - 53 - young a n i m a l . The i n c r e a s e d l e u k o c y t e count and t h e r e v e r s a l o f th e d i f f e r e n t i a l count ( i n c r e a s e d p e r c e n t of n e u t r o p h i l s ) i n t h e r i b o f l a v i n d e f i c i e n t lamb was e x p l a i n e d by t h e p r e s e n c e o f pneumonia. I t seemed more p r o b a b le t o r e l a t e both t h e i n ­ c r e a s e o f th e l e u k o c y t e s and n e u t r o p h i l s t o the i n f e c t i o n ( a c u t e bronchopneumonia) t h a n to r i b o f l a v i n d e f i c i e n c y . An i n c r e a s e o f l e u k o c y t e s and e s p e c i a l l y of n e u t r o p h i l s i s a c h a r a c t e r i s t i c f e a t u r e of a c u t e in flam m atio n or i n f e c t i o n * The pneumonia o b s e rv e d among th e r i b o f l a v i n d e f i c i e n t lambs was a c u t e , r a p i d l y s p r e a d i n g and t e r m i n a t i n g i n death* I t was r a t h e r u n f o r t u n a t e t h a t th e r e l a t i v e l y s h o r t p e r i o d of r i b o f l a v i n d e p l e t i o n i n th e lamb and th e l i m i t e d f a c i l i t i e s a v a i l a b l e to c o l l e c t the u r i n e samples p r e v e n t e d more th o r o u g h s t u d y o f u r i n a r y e x c r e t i o n of r i b o f l a v i n . These l i m i t a t i o n s were f u r t h e r a g g r a v a t e d by a g r e a t v a r i a t i o n i n t h e u r i n a r y r i b o f l a v i n l e v e l s w i t h i n the groups. I t might be e x p l a i n e d on th e b a s i s of v a r y i n g s t o r a g e c a p a c i t y of t h e v i t a m i n i n th e lamb b e f o r e b e i n g p l a c e d on th e d e f i c i e n t d i e t an d , p e r h a p s , by i n d i v i d u a l a b i l i t i e s o f th e lamb to economize t h e l i m i t e d amount o f r i b o f l a v i n . The v a r i a t i o n s i n th e u r i n a r y e x c r e t i o n of r i b o f l a v i n , no d o u b t, were a l s o g r e a t l y i n f l u e n c e d by th e age o f t h e lamb when ta k e n away from th e ewe. The age of t h e lambs when p u t on th e e xpe rim ent ranged from two t o e l e v e n d a y s . However, the av erage u r i n a r y r i b o ­ f l a v i n e x c r e t i o n f o r th e c o n t r o l lambs was 0*50 mg p er 24 h o u r s , compared w ith 0 .0 1 mg p e r 24 h o u r s f o r t h e r i b o f l a v i n 54 - d e f i c i e n t g roup . The d is a p p e a r a n c e o f r i b o f l a v i n from th e u r i n e o f th e lambs on t h e r i b o f l a v i n d e f i c i e n t d i e t i n d i c a t e d t h a t t h e v i t a m i n was not s y n t h e s i z e d e i t h e r i n the rumen or i n t e s t i n a l t r a c t or i n t h e t i s s u e s . I t was i n t e r e s t i n g to note t h a t t h e rumen o f th e c o n t r o l lamb was not developed and e v i d e n t l y was n o t y e t f u n c t i o n i n g even a f t e r 34 days on th e l i q u i d p u r i f i e d d i e t . I t h a s been p o i n t e d out by o t h e r s t h a t th e symptoms, g r o s s and m ic r o s c o p ic changes i n any v i t a m i n d e f i c i e n c y , seemed t o be the r e s u l t of a d im i n is h e d f u n c t i o n o f t h e body c e l l s and n o t the r e s u l t o f s p e c i f i c b io c h em ical p r o c e s s e s . Very l i t t l e i s known ab out th e b io c h e m ic a l changes o c c u r i n g i n t h e v a r i o u s t i s s u e s of d i f f e r e n t o rgans o f t h e animal when on a r i b o f l a v i n d e f i c i e n t d i e t . A xelrod e t a l . (1940, 1941) r e p o r t e d a d e c r e a s e d d-amino a c i d oxida se a c t i v i t y i n th e l i v e r and kid n e y of r i b o f l a v i n d e f i c i e n t r a t s and markedly low ered a c t i v i t y of x a n t h i n e o x id a se of d e f i c i e n t r a t s measur­ ed by oxygen consumption r a t e s . C o n s i s t e n t and r a t h e r u n i ­ form g r o s s and m ic r o s c o p i c l e s i o n s of t h e l u n g , l i v e r , kidney and a d r e n a l s s u g g e s t e d t h a t the t i s s u e s of t h e s e o r g a n s were th e f i r s t d e p l e t e d of r i b o f l a v i n . The c e l l u l a r d y s f u n c t i o n m a n i f e s t e d by t h e symptomat o l o g y , g r o s s and m ic ro s c o p ic p a t h o l o g y d e s c r i b e d , was th e n a d e f i c i e n c y of one and only one n u t r i t i o n a l f a c t o r , t h a t i s r i b o f l a v i n , and not compound d e f i c i e n c i e s due t o poor i n t a k e o f a s u p p o se d ly a d e q u ate d i e t . S i m i l a r growth r e s p o n s e , symptoms, s u r v i v a l t i m e , p a t h o l o g y , - 55 - a. r a t h e r s h o r t p e r i o d o f i n a n i t i o n , d r a m a tic re sp o n se to r i b o f l a v i n t h e r a p y i n r e s p e c t to growth and h e a l t h and a r a t h e r l i b e r a l amount of a l l o t h e r B v i t a m i n s i n th e d i e t s u g g e s t e d t h a t t h e d e f i c i e n c y , a s o b s e rv e d , could have been of r ib o fla v in . The a v e ra g e d a i l y i n t a k e o f r i b o f l a v i n p e r pound o f body w e ig h t o f the c o n t r o l lambs was around 250 mg». T h is was c a l c u l a t e d from t h e 3 mg r i b o f l a v i n l e v e l i n one l i t e r of p u r i f i e d m ilk a s shown i n t a b l e 2 , The av erage i n t a k e i n c r e a s e d up t o 297 micrograras d u r i n g th e f i r s t t h r e e weeks on th e d i e t . and A f t e r t h r e e weeks the lambs were growing f a s t e r r e q u i r e d l e s s d r y m a t t e r t o produce one pound C o n s e q u e n t ly , the of gain. r i b o f l a v i n i n t a k e , d i r e c t l y dependent on t h e amount o f m ilk consumed, was d e c r e a s e d . The a v e ra g e d a i l y g a in of t h e c o n t r o l lambs receiving a round 250 mgnof r i b o f l a v i n p er pound of body w eight per d ay, mixed i n t h e m ilk and fed a t each f e e d i n g , was 0 . 4 0 l b . w h ile t h e av e ra g e d a i l y g a i n of the two lambs on 250 and 350 micrograms o f r i b o f l a v i n , a d m i n i s t e r e d once a day, was 0 .2 0 5 and 0 .3 0 5 l b s . r e s p e c t i v e l y . The only e x p l a n a t i o n f o r t h i s d i f f e r e n c e i n growth r e sp o n se was t h e mode of v i t a m i n adm inistration. ratio n . The c o n t r o l lambs were always fed a complete The amount of r i b o f l a v i n (3 mg per l i t e r of m ilk) was added t o t h e milk i n th e l a b o r a t o r y and t h e c o n t r o l lambs r e c e i v e d a p a r t o f the d a i l y i n t a k e a t each f e e d i n g . In th e lambs on t h e v a r i o u s l e v e l s o f r i b o f l a v i n , the amount - 56 - was a d m i n i s t e r e d once a day. I t was p o s s i b l e t h e n t h a t r i b o f l a v i n a d m i n i s t e r e d w ith each f e e d i n g i n c r e a s e d the u t i l i z a t i o n of th e f e e d which was r e f l e c t e d i n f a s t e r g a i n s of t h e c o n t r o l lam bs. I t was a l s o f e l t t h a t th e l e v e l of 3 mg o f r i b b f l a v i n i n one l i t e r of m ilk, s e t a r b i t r a r i l y f o r t h e c o n t r o l la m b s, was adequate to su p p o r t th e h e a l t h and g e n e r a l c o n d i t i o n of th e young lamb, and to produce a normal growth and g a in i n body w e ig h t. SUMMARY A p u r i f i e d l i q u i d d i e t ( s y n t h e t i c milk) c o n t a i n i n g 16 p e r c e n t s o l i d s , composed of v i t a m i n f r e e c a s e i n (3 0 # ), g l u ­ cose ( 2 8 # ) , l a c t o s e ( 1 0 # ), l a r d (26 # ), s a l t m ix tu re (6#) and a l l f a t and w a te r s o l u b l e v i t a m i n s , was adequate to s u p p o r t good h e a l t h and normal growth of two to e l e v e n day o ld lambs. I t was shown t h a t th e e x c r e t i o n of t h e u r i n a r y r i b o f l a v i n i n r i b o f l a v i n d e f i c i e n t lambs was g r e a t l y lo w ered . g l o b i n c o n c e n t r a t i o n was unchanged. The hemo­ The le u k o c y t e count was i n c r e a s e d and was r e f l e c t e d i n an i n c r e a s e d p er c e n t of n e u t r o p h i l s i n th e d i f f e r e n t i a l c o u n t. R i b o f l a v i n d e f i c i e n c y i n the young lamb was c h a r a c t e r i z e d by lo w ered a p p e t i t e , i n t e r m i t t e n t d i a r r h e a , poor growth, em acia­ t i o n , pneumonia and d e a t h . The s u r v i v a l p e r i o d was 16-21 d a y s . P a t h o l o g i c a l s t u d i e s of t h e r i b o f l a v i n d e f i c i e n t lambs r e v e a l e d l u n g involvem ent w ith b i l a t e r a l pneumonia, g e n e r a l f a t t y d e g e n e r a t i o n and n e c r o s i s o f th e h e p a t i c c e l l s , f a t t y d e g e n e r a t i o n of th e c o r t e x and medulla o f th e k i d n e y , conges­ t i o n and f a t t y d e g e n e r a t i o n o f t h e a d r e n a l s . Symptoms of r i b o f l a v i n d e f i c i e n c y were r a p i d l y a l l e v ­ i a t e d and c u r e d w ith a d m i n i s t r a t i o n of c r y s t a l l i n e r i b o f l a v i n . The growth r e s p o n s e of t e n lambs on f i v e d i f f e r e n t l e v e l s of r i b o f l a v i n (5, 50, 150, 250 and 350 micrograms p e r pound o f body w eight p e r day) was d i r e c t l y p r o p o r t i o n a l t o t h e l e v e l - 58 - of r ib o f l a v i n ad m in istered . The two h i g h e s t l e v e l s o f r i b o ­ f l a v i n a d m i n i s t e r e d once a day even though comparable to th e r i b o f l a v i n i n t a k e o f th e c o n t r o l lambs, proved t o be adequate t o s u p p o r t good h e a l t h and c o n d i t i o n of the young lamb but i n a d e q u a t e t o promote normal or o ptim al growth. I t was f e l t t h a t t h e optimum r e q u ir e m e n t o f the young lamb was somewhere between 250-350 micrograms o f r i b o f l a v i n p e r pound o f body w e ig h t p e r day and t h a t t h e lamb i n th e p re ru m in a n t s t a t e had a d e f i n i t e r e q u ir e m e n t f o r r i b o f l a v i n . BIBLIOGRAPHY A x e l r o d , A. 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R e in h o ld Pub­ l i s h i n g C o r p o r a t i o n , New York, pp. 3 - 1 7 , 395-437. 1950. W in tro b e , M. M ., W. Buschke, R. H. F o l l i s , J r . and S. Humph­ reys. R i b o f l a v i n D e f i c i e n c y i n Swine. B u l l . Johns Hopkins H o s p . , 75: 1 0 2-114, 1944. Zimmerman, H. M. and E. B urack. S t u d i e s on the Nervous System i n D e f i c i e n c y D i s e a s e s . I I . L e s i o n s Produced i n t h e Dog by D i e t s L ac king the Waters o l u b l e , H e a t - s t a b l e V ita m in Bg (G). J o u r . Exp. Med., 59: 2 1 - 3 4 , 1934. Fig* 1 . Lamb No* 4* L i v e r , k i d n e y s and a d r e n a l from r i b o f l a v i n d e f i ­ c i e n t lamb* neys. Note p r o m i n e n t s t r e a k s i n t h e k i d ­ The a d r e n a l i s e n l a r g e d and t h e l i n e o f d e m a r k a t i o n b e t w e e n t h e c o r t e x an d m e d u l l a i s lo st. - 64 - F ig . 2. Lamb N o. 7 . E x ten siv e f a t t y i n f i l t r a t i o n o f the l i v e r . The h e p a tie c e l l s are f i l l e d with f a t g lo b u le s . The n u c le i are pyknotic and the s in u s o id s , e s p e c i a l l y c lo s e to the c e n tr a l v e in , are d i s ­ tended. Hematoxylin and E o sin s t a i n x 600 - 65 - F ig . 3. Lamb N o . 2 . Cortex o f the kidney showing severe f a t t y i n f i l ­ t r a t i o n of convoluted tu b u les and tendency toward h y a lin e degeneration in g lo m eru li. Note the d isap p earin g c a p illa r y loops* Hematoxylin and E osin s t a i n x 600 - 66 - Kinll F ig . 4. Lamb N o. 2 D is t a l convoluted tu b u les with e x c e s s iv e amount of f a t g lo b u le s . Frozen s e c t io n , Sudan IV s t a i n x 850 - 67 - \ •12 ‘O F ig . 5. Lamb N o . 5 . Pronounced f a t t y i n f i l t r a t i o n of the eo r tex and medulla of the ad ren al. The prominent and rather d i f f e r e n t f a t t y i n f i l t r a t i o n between glomerulosa and f a s c i e u l a t a was considered p h y s i o l o g i c a l . Frozen s e c t i o n , Sudan IV s t a i n x 40 - 68 - mi F ig . 6. Lamb N o . 5 . Higher m a g n ifica tio n o f adrenal showing f a t t y d egen era tio n . The s in u s o id s are distended but the borders are i n t a c t . Frozen s e c t i o n , Sudan IV s t a i n x 125 - 69 - HR f&9W lift T * * ? * * 1 ± V V V •..-» F ig . 7. Lamb N o . 1 0 . Cortex o f the kidney showing glomerulus in early sta g e o f h y a lin e d egen eration. The h ya lin e d r o p le ts are being accumulated on one s i d e , w hile the c a p illa r y loop i s d isap p earin g . Con­ v o lu ted tu b u les show pronounced cloudy s w e llin g . Hematoxylin and Eosin s t a i n x 850 Part I I . VITAMIN E DEFICIENCY AND ESTIMATED REQUIREMENT INTRODUCTION S in c e 1934 v i t a m i n E d e f i c i e n c y h as been s u s p e c t e d to be t h e cause of so c a l l e d " S tiff-L a m b " d i s e a s e . T h is d i s e a s e of lambs i s c h a r a c t e r i z e d by s t i f f n e s s , p a r a l y s i s and muscle d e g e n e r a t i o n s i m i l a r to th e n u t r i t i o n a l muscular d y s tr o p h y ( Z e n k e r ' s d e g e n e r a t i o n ) produced i n l a b o r a t o r y a n im a ls on d i e t s low or d e f i c i e n t i n v i t a m i n E. However, t h e r e was no deficiency d i r e c t e v id e n c e t o s u b s t a n t i a t e t h e i d e a t h a t v i t a m i n E was t h e p r im a r y and t h e o n ly f a c t o r r e s p o n s i b l e f o r the d i s e a s e . I n p r e v i o u s work a p u r i f i e d d i e t i n l i q u i d form ( s y n t h e t i c m ilk) proved t o be a d e q u a te to s u p p o r t t h e growth and h e a l t h o f t h e young lamb and was u se d t o s tu d y th e r i b o f l a v i n r e ­ q u ir e m e n t o f la m b s. I t seemed l o g i c a l th e n t h a t u s i n g a p u r i f i e d d i e t , low i n v i t a m i n E ( t o c o p h e r o l s ) , th e r o l e of t h i s v i t a m i n i n animal n u t r i t i o n and p a r t i c u l a r l y i t s r e l a ­ t i o n s h i p t o m uscular d y s t r o p h y of lambs c ould be s t u d i e d and possibly elu c id a te d . OBJECT The o b j e c t o f t h e s e s t u d i e s was a) t o produce v i t a m i n E d e f i c i e n c y i n t h e lamb by th e use o f a l i q u i d p u r i f i e d d i e t , low o r d e f i c i e n t i n t o c o p h e r o l s , b) t o stu d y and compare the symptoms and l e s i o n s o f v i t a m i n E d e f i c i e n c y , produced i n the l a b o r a t o r y w ith t h e symptoms and l e s i o n s of t h e so c a l l e d “S t i f f - L a m b " d i s e a s e o c c u r i n g u nder f i e l d c o n d i t i o n s , and c) t o d e te r m in e t h e q u a n t i t a t i v e r e q u i r e m e n t o f a l p h a - t o c o p h e r o l f o r th e young s u c k l i n g lamb. REVIEW OF LITERATURE N u t r i t i o n a l Muscular Dystrophy i n L a b o r a t o r y Animals U n t i l 1928 v i t a m i n E was th o u g h t t o be e n t i r e l y con­ c e r n e d w ith r e p r o d u c t i o n . Evans and Burr (1928) observed and d e s c r i b e d p a r a l y s i s o f young r a t s s u c k l i n g mothers k e p t on a v i t a m i n E d e f i c i e n t d i e t . At f i r s t i t was th o u g h t t h a t t h e p a r a l y s i s was n e u r o g e n ic i n o r i g i n . G o e t t s c h and Pappenheimer (19 3 1 ), u s i n g a s e m i - p u r i f i e d d i e t c o n s i s t i n g o f o a t s , wheat b r a n , c a s e i n , l a r d , skimmed m i l k and s a l t m i x t u r e , produced a d y s tr o p h y o f t h e s k e t e t a l m u s c u la t u r e o f t h e g u in e a p i g and r a b b i t . The d i e t was t h o u g h t t o be com plete and th e s k e l e t a l muscle changes were n o t due t o s t a r v a t i o n , i n f e c t i o n or s c u r v y . H istopatholo- g i c a l l y , t h e l e s i o n s of the s k e l e t a l muscles were d e s c r i b e d a s Z e n k e r ' s d e g e n e r a t i o n or n u t r i t i o n a l muscular d y s tr o p h y . No h e a r t l e s i o n s or changes i n th e c e n t r a l nervous system were o b s e r v e d . Madsen et, al^. (1935) produced a d y s tr o p h y o f s k e l e t a l and h e a r t muscle i n guine a p i g s , r a b b i t s , g o a t s and sheep k e p t on n a t u r a l or s y n t h e t i c d i e t s supplemented w i t h l a r g e do se s o f cod l i v e r o i l . The a u t h o r s su g g e s te d t h a t t h e s a p o n i f i a b l e p a r t o f t h e cod l i v e r o i l was t o x i c to t h e a n i m a l s and ca u sed th e l e s i o n s . The f o l l o w i n g y e a r Madsen - 74 - ( 1 9 3 6 ) , comparing v a r i o u s s o u r c e s o f f a t on a s e m i - p u r i f i e d d i e t and t h e e f f e c t o f cod l i v e r o i l , o b served t h a t guinea p i g s d ev e lo p ed d y s tr o p h y of t h e s k e l e t a l muscle sooner on a d i e t c o n t a i n i n g l a r d and cod l i v e r o i l t h a n on a d i e t con­ t a i n i n g c o t t o n seed o i l and v i t a m i n A and D c o n c e n t r a t e . Cummings and M a t t i l l (1931) and M a t t i l l (1938b) d id n o t a g r e e w ith th e t o x i c t h e o r y of cod l i v e r o i l . They p o s t u l a t e d t h a t t h e muscle d y s tr o p h y was due to v i t a m i n E d e f i c i e n c y which r e s u l t e d from d e s t r u c t i o n of v i t a m i n E by o x i d a t i v e r a n c i d i t y o f cod l i v e r o i l and animal f a t . S h im o to r i e t a l » (1940) p r o d u c e d , cured and p r e v e n t e d n u t r i t i o n a l m uscular d y s tr o p h y i n th e guinea p i g by s u p p l e ­ m e n ting t h e Madsen et, a l . . (1935) d i e t w ith wheat germ, wheat germ o i l or a l p h a - t o c o p h e r o l . com plete i n a l l B v i t a m i n s . T h is d i e t was a d e q u ate and The a u t h o r s p o s t u l a t e d t h a t v i t a m i n E was t h e s p e c i f i c f a c t o r which cured and p r e v e n t e d t h e muscle d e g e n e r a t i o n . Muscular d e g e n e r a t i o n , s i m i l a r t o t h a t o f l a b o r a t o r y a n i m a l s on a v i t a m i n E low d i e t , and t e s t i c u l a r d e g e n e r a t i o n c h a r a c t e r i s t i c o f v i t a m i n E d e f i c i e n c y were o bserved i n dogs and r e p o r t e d by Brinkhouse and Warner (1941). This c o n d i t i o n was produced by means o f a c h r o n i c b i l i a r y f i s t u l a i n a p e r i o d o f s e v en t o n in e months. E v i d e n t l y t h e b i l e was e s s e n t i a l f o r v i t a m i n E a b s o r p t i o n and u t i l i z a t i o n . T e l f o r d (1941) s t u d i e d t h e h i s t o p a t h o l o g y o f the degen­ e r a t e d s k e l e t a l m uscles from r a t s on a v i t a m i n E d e f i c i e n t - 75 - diet* I n a d d i t i o n t o Zenker*s d e g e n e r a t i o n , t h e nerve e n d i n g s were d e s tro y e d * On t r e a t m e n t w ith v i t a m i n E, r e ­ g e n e r a t i o n o f the s k e l e t a l muscle was accompanied by r e ­ g e n e r a t i o n o f n e r v e endings* The muscular d y s tr o p h y o f r a b b i t s produced by l a r g e d o s e s o f cod l i v e r o i l was t r e a t e d w ith t h e disodium s a l t o f a l p h a - t o c o p h e r o l pho sphate a d m i n i s t e r e d p a r e n t e r a l l y and w ith a l p h a - t o c o p h e r o l a c e t a t e i n j e c t e d i n t r a m u s c u l a r l y . E p p s t e i n and M o rg u lis (1942) found th e p a r e n t e r a l a d m in is ­ t r a t i o n more e f f e c t i v e t h a n th e i n ^ s t i o n . C reatinuria was r e p o r t e d t o be an in d e x o f v i t a m i n E d e f i c i e n c y . Mason (1942) a n a l y s e d t i s s u e s from r a t s on a low t o c o ­ p h e r o l d i e t and found t h a t th e h e a r t muscle had p r o p o r t i o n ­ a l l y tw ic e a s much v i t a m i n E as d id th e s k e l e t a l m u sc le . Pappenheimer (1943) i n a rev ie w on v i t a m i n E emphasized t h a t v i t a m i n E was e s s e n t i a l i n m etabolism of s k e l e t a l muscles o f a l l mammals, b u t d i d n o t seem t o be e s s e n t i a l i n metabolism, o f th e nervous sy ste m . H e a r t l e s i o n s i n t h e r a b b i t s i m i l a r t o Zenker*s de­ g e n e r a t i o n o f t h e s k e l e t a l m uscle, inditced by a v i t a m i n E d e f i c i e n t d i e t , and r e s p o n s i b l e f o r sudden d e a th due t o m y o c a r d ia l f a i l u r e were f i r s t r e p o r t e d by Houchin and Smith (1 9 4 4 ) . These a u t h o r s a l s o o b served an i n c r e a s e d s e n s i t i v i t y o f t h e d e p l e t e d a n i m a l s t o p i t r e s s i n , g r e a t e r r e s i s t a n c e to t o x i c e f f e c t s o f c a r d i a c g l u c o s i d e s , c a r d i a c d i l a t a t i o n and an i n c r e a s e d oxygen consumption. - 76 - G u l l i c k s o n and C a l v e r l e y (1946) s t u d i e d t h e cause o f sudden d e a t h o f c a t t l e i n th e f i e l d and c a l v e s on a v i t a m i n E d e f ic ie n t diet* The e l e c t r o c a r d i o g r a p h s p r e p a r e d d u r in g t h e p e r i o d o f d e f i c i e n c y showed d e f i n i t e changes i n th e v a r i o u s i n t e r v a l s and p o t e n t i a l s w ith i n c o n s i s t e n t d e f l e c ­ tions* The h i s t o p a t h o l o g i c a l e x a m in a tio n of the myocardium showed a t r o p h y and s c a r r i n g of th e c a r d i a c muscle f i b e r s and an i n c r e a s e d c e l l u l a r i n f i l t r a t i o n * E lectrocardiographs of r a b b i t s on v i t a m i n E d e f i c i e n t s y n t h e t i c d i e t s t u d i e d by Bragdon and L evine (1949) showed some changes i n waves* M ic r o s c o p ic e x a m in a tio n of th e myocardium r e v e a l e d f o c i of n e c r o s i s and an inflam m atory r e a c t i o n . The sequence of e v e n t s was a c o a g u l a t i v e n e c r o s i s of sa rc o p la sm w ith l o s s o f s t r i a t i o n , p y k n o s is and k a r y o r r h e x i s of muscle n u c l e i , edema, hem orrhages and i n f i l t r a t i o n of t h e a r e a s w ith mono­ c y t e s and p o l y m o r p h o n u c l e a r l e u k o c y t e s . Some c a l c i f i c a t i o n was a l s o o b s e r v e d . Menschik et. a l . (1949) s t u d i e d t h e d i s t r i b u t i o n o f f a t and f a t changes i n mice on a d i e t w ith and w i t h o u t v i t a m i n E* Anim als on v i t a m i n E supplem ented d i e t i n c r e a s e d th e amount o f n e u t r a l f a t i n t h e body, i n c r e a s e d t h e amount o f a d i p o s e t i s s u e and showed p h y s i o l o g i c a l f a t t y i n f i l t r a t i o n t h r o u g h ­ o u t th e body. Animals on a v i t a m i n E d e f i c i e n t d i e t soon became l e a n , n e u t r a l f a t d i s a p p e a r e d from th e t i s s u e s and was r e p l a c e d by a c i d f a s t l i p o - p r o t e i c m a t e r i a l composed o f un- - 77 - s a t u r a t e d f a t t y a c i d s , p h o s p h o l i p i d s , c h o l e s t e r o l and p r o t e i n . The a u t h o r s s u g g e s t e d t h a t v i t a m i n E was in v o l v e d i n f a t m e ta b o l is m . Tobin (1950) used 2, 10 and 20 p e r c e n t o f cod l i v e r o i l i n a d i e t t o produce v a r i o u s d e g r e e s of muscle d e g e n e r a ­ t i o n i n m ice. Animals on th e 2 p e r c e n t cod l i v e r o i l d i e t d i d n o t show locom otor d i s t u r b a n c e s or any o t h e r v i s i b l e symptoms of d e f i c i e n c y . Only 15 p e r ce n t o f th e a n im a ls r e v e a l e d s l i g h t h y a l i n e d e g e n e r a t i o n o f i n d i v i d u a l muscle f i b e r s c h a r a c t e r i z e d by c e n t r a l l y l o c a t e d n u c l e i or groups of n u c l e i a r r a n g e d i n rows or columns. Animals on th e 10 p e r c e n t cod l i v e r o i l d e v e lo p e d a c u te p a r a l y s i s i n 16 to 30 d ay s . M i c r o s c o p i c a l l y th e muscles were n e c r o t i c , showed l o s s of s t r i a t i o n , s e g m e n t a t i o n and clumping of t h e s a r c o p la s m , c o l l a p s e o f sarcolemraal s h e a t s , p r e s e n c e of g i a n t c e l l s and some c a l c i f i e d a r e a s . R e g e n e r a t i v e changes were c h a r a c t e r i z e d by p r o l i f e r a t i o n of sarcolemmal n u c l e i , r e g e n e r a t i o n o f s a r c o ­ plasm and renewed s t r i a t i o n . V ita m in E d e f i c i e n c y i n c h i c k s c h a r a c t e r i z e d by c e r e ­ b e l l a r d i s o r d e r ( e n c e p h a lo m a la c ia ) was produced and d e s c r i b e d by Pappenheimer and G o e tt s c h (1931) and J u n g h e rr (19 4 9 ). D u c k lin g s on t h e same h ig h f a t v i t a m i n E d e f i c i e n t d i e t d e v e l ­ oped d y s t r o p h y of t h e s k e l e t a l muscle (Pappenheimer and G o e t t s c h 1934) w h ile t u r k e y s showed myopathy of t h e g i z z a r d ( J u n g h e r r and Pappenheimer 1 9 3 7 ) . - 78 - S t i f f Lamb D is e a s e The s o - c a l l e d " S tiff-L a m b " d i s e a s e was f i r s t r e p o r t e d and d e s c r i b e d by Metzger and Hagan (1927). The symptoms were a d i s t u r b a n c e i n l o c o m o tio n , s t i f f n e s s of t h e h in d and f o r e l e g s , p a r a l y s i s and d ea th by s t a r v a t i o n due to i n a b i l i t y of t h e lamb t o f o llo w t h e mother. The g r o s s and m ic ro s c o p ic l e s i o n s of b o t h the s k e l e t a l and c a r d i a c muscles were d e s ­ c r i b e d a s n o n i n f e c t i o u s and noninflam matory i n n a t u r e . J u n g h e r r and Welch (1927) d e s c r i b e d th e d e g e n e r a t i v e changes i n th e muscle as se c o n d a ry to c o c c i d i o s i s . n e x t r e p o r t on " S tif f- L a m b " d i s e a s e , Welch e t a l . In his (1929) s t a t e d t h a t lambs p r e v i o u s l y c o n fin e d to small pens when s u d d e n ly p u t on p a s t u r e were e s p e c i a l l y s u s c e p t i b l e t o the disease. Severe e x e r c i s e i n p a s t u r e was su g g e sted as the cause of th e d i s e a s e . U m b ili c a l i n f e c t i o n as t h e p rim a ry cause o f " S t i f f Lamb" o r "White Muscle" d i s e a s e was s u g g e s t e d by Marsch (19 29). Willman ejb al_. (1931) produced " s t i f f lambs" experim en­ t a l l y by f e e d i n g p r e g n a n t ewes a r a t i o n c o n s i s t i n g o f a g r a i n m ix tu re h ig h i n p r o t e i n and a l f a l f a or c l o v e r hay. A lack o f e x e r c i s e was a l s o c o n s i d e r e d as a p o s s i b l e e t i o l o g i c a l factor. Marsch (1932) e x p l a i n e d th e w h i t i s h l e s i o n s o f t h e m usc les a s d e p o s i t s o f t r i c a l c i u m p h o sp h a te caused by a d i s ­ t u r b a n c e o f t h e calcium and phosphorus r a t i o i n the a f f e c t e d lamb. - 79 - I n 1934 th e d i s e a s e among lam bs, c a l v e s and h e i f e r s was r e p o r t e d from Norway by S la n g s v o l d and Lund-Larsen (19 3 4 ). The d e g e n e r a t i v e changes i n s k e l e t a l and h e a r t muscles were s i m i l a r t o h y a l i n e d e g e n e r a t i o n w ith i n c r e a s e d d e p o s i t i o n of calcium . A b a l a n c e d r a t i o n w ith adeq uate amounts of m i n e r a l s and v i t a m i n s was s u g g e s t e d a s a p r e v e n t i v e measure. Willman et^ a l . (1934) summarized the f o u r y e a r s tu d y on " S t i f f - L a m b " d i s e a s e . The a u t h o r s c o n s i s t e n t l y were a b l e t o produce " s t i f f lambs" from ewes fed a r a t i o n con­ s i s t i n g of o a t s , b a r l e y , c u l l beans and second c u t t i n g a l f a l f a h ay . P r e v i o u s l y s u s p e c t e d c a u ses o f t h e c o n d i t i o n such a s e x e r c i s e o f t h e ewes d u r i n g preg n an c y , heavy f e e d i n g of c o n c e n t r a t e s and c r e e p f e e d i n g of t h e lambs were d i s ­ carded. Willman et, al.. (1935) t r e a t e d the " s t i f f lambs" w ith v i t a m i n C w i t h o u t s u c c e s s , and concluded t h a t v i t a m i n C was not r e l a t e d to the d is e a se . Lee and S c r i v n e r (1935) s t u d i e d the h i s t o p a t h o l o g y of t h e d i s e a s e and found w h i t i s h l e s i o n s i n t h e kid n e y s i m i l a r t o t h o s e i n t h e s k e l e t a l and c a r d i a c m uscle. The a u t h o r s r e p r o d u c e d th e c o n d i t i o n e x p e r i m e n t a l l y i n lambs and r a b b i t s by i n o c u l a t i n g th o s e a n im als w i t h b a c t e r i a i s o l a t e d from t h e h e a r t of a s t i f f lamb. Chemical a n a l y s i s o f th e d e g e n e r a t e d t i s s u e s showed i n c r e a s e d calcium and p h o sp h o r u s. To d e t e r m in e i f t h e c o n c e n t r a t e s or th e hay was th e - 80 - " S t i f f - L a m b " p r o d u c i n g f a c t o r i n t h e r a t i o n , Willman a t a l . (1936) r e p l a c e d t h e a l f a l f a hay by mixed c l o v e r and tim o th y hay* Lambs became s t i f f a s b e f o r e , p r o v i n g t h a t t h e g r a i n m ix t u r e and n o t t h e hay was th e " S tiff-L a m b " f a c t o r . S c h o f i e l d and Bain (1939) s u c c e s s f u l l y t r e a t e d a num­ b e r o f " s t i f f lambs" w ith p h o s p h o r ic a c i d added to milk* Phosphorus d e f i c i e n c y as the cause of t h e c o n d i t i o n was sug­ g e s t e d by Wawter and Records (1939). a l o n e produced " s t i f f lam bs". Ewes fed a l f a l f a hay When r o l l e d o a t s were f e d w i t h a l f a l f a hay t h e d i s e a s e was p r e v e n t e d . S h o l l (1939) r e p o r t e d "S tif f- L a m b " d i s e a s e i n M ic higan. The m o r t a l i t y was up t o 30 p e r c e n t . Blood a n a l y s e s showed v e r y low c r e a t i n i n e and h ig h n o n - p r o t e i n n i t r o g e n v a l u e s . Willman a t a l . (1939) d i s p r o v e d t h e phosphorus d e f i ­ c i e n c y a s t h e cause o f t h e c o n d i t i o n . Added phosphorus t o t h e " S tif f - L a m b " r a t i o n f a i l e d to p r e v e n t th e development of symptoms. Willman a t al.. (1940) d e f i n i t e l y proved and r e c o g n i z e d the d is e a s e as n u t r i t i o n a l in o r ig in . The cause was some­ t h i n g o t h e r t h a n phosphorus and a s c o r b i c a c i d d e f i c i e n c y . The a u t h o r s ob se rv e d t h a t t h e i n c i d e n c e of " s t i f f lambs" was much lo w e r when wheat germ meal was added t o the " S t i f f Lamb" r a t i o n . The f o l l o w i n g y e a r Willman a t al_. (1941) sup­ p le m e n te d t h e " S tif f - L a m b " r a t i o n w ith u n e x t r a c t e d wheat germ m e a l. None o f t h e lambs born from ewes on t h i s r a t i o n be­ came s t i f f w h ile e l e v e n o u t o f tw e n t y - s e v e n lambs from t h e - 81 ewes w i t h o u t wheat germ meal developed th e d i s e a s e * Thorp (1942) d e s c r i b e d " S tiff-L a m b " d i s e a s e and sug­ g e s t e d p r e v e n t i v e methods. Willman et_ a l . (1944, 1945, 1946) s u s p e c te d v i t a m i n £ a s a f a c t o r r e s p o n s i b l e f o r " S tiff-L a m b " d i s e a s e . The a u t h o r s p r e v e n t e d th e c o n d i t i o n e i t h e r by a d m i n i s t r a t i o n o f an o i l s o l u t i o n o f mixed t o c o p h e r o l s to t h e ewes b e f o r e and to t h e i r lambs a f t e r p a r t u r i t i o n or by f e e d i n g an o i l s o l u t i o n o f d-1 a l p h a - t o c o p h e r o l a c e t a t e to th e lambs im­ m ediately a f t e r b i r t h . S t i f f lambs a l s o responded and were s u c c e s s f u l l y t r e a t e d by sub cutaneo us i n j e c t i o n o f a d i ­ sodium s a l t of d-1 a l p h a - t o c o p h e r o l p h o s p h o r ic a c i d e s t e r i n an aqueous* s o l u t i o n . W h i t t i n g ejt al_. (1949), u s i n g th e "S tiff-L am b" p r o d u c in g r a t i o n o f Willman e t a l . (1 9 3 4 ), compared t h e to c o p h e r o l l e v e l s of blood plasma from ewes and lambs and a l s o th e c o l o s tr u m and m ilk t o c o p h e r o l l e v e l s from the ewes w ith a group of normal c o n t r o l s . The r e s u l t s i n d i c a t e d a r e d u c t i o n from 15 t o 48 p e r c e n t i n th e t o c o p h e r o l l e v e l s o f the bloo d p la s m a , m ilk and c o lo s tr u m i n t h e ewes and lambs on the " S tif f - L a m b " p r o d u c in g r a t i o n . EXPERIMENTAL PROCEDURE Animals Used B e g in n in g w ith t h e f a l l of 1950, 51 lambs 3-4 days o ld were u se d f o r t h i s s t u d y . Lambs of b o th sexes of the mutton and f i n e wool ty p e were o b t a i n e d from th e Experim ent S t a t i o n f l o c k a t Michigan S t a t e C o lleg e or purchased from farms i n th e a r e a . P u re b re d o r c r o s s b r e d , s i n g l e or tw in lambs were u se d and th e o n ly p r e r e q u i s i t e was good h e a l t h and normal condition. A l l lambs r e c e i v e d c o lo s tr u m b e fo re b eing ta k e n from t h e ewes. E le v e n o u t o f t h e 51 lambs d ie d e a r l y i n t h e e x p e r i m e n t due t o c a u s e s n o t r e l a t e d to v i t a m i n E d e f i c i e n c y and were d i a g n o s e d as o v e r e a t i n g (2 l a m b s ) , i n t e s t i n a l ob­ s t r u c t i o n (2 l a m b s ) , c o c c i d i o s i s (1 lamb) and pneumonia (3 lam bs). The cause of d e a t h i n t h r e e lambs was not i d e n t i f i e d . F e ed in g and Care The c o m p o s iti o n and t h e method o f p r e p a r a t i o n o f t h e s y n t h e t i c m ilk and t h e care of the animals were d e s c r i b e d in d e t a i l in P a r t I of t h i s t h e s i s . were made however: A few m o d i f i c a t i o n s th e lambs were changed from one pen t o a n o t h e r e v e r y day i n s t e a d of e v e ry o t h e r day t o p r e v e n t any outside d isease. Ten p e r c e n t o f l a c t o s e was r e p l a c e d by 10 p e r c e n t g l u c o s e t o e l i m i n a t e a p o s s i b l e s y n t h e s i s of any v i t a m i n i n t h e i n t e s t i n a l t r a c t o f t h e lamb. f e d f o u r tim e s a day i n s t e a d o f f i v e . The lambs were The amount of m ilk - 83 - e a c h lamb consumed p e r f e e d i n g was r e c o r d e d as w ell as a l l t h e o b s e r v a t i o n s of symptoms, h e a l t h , a p p e t i t e and g e n e r a l behavior. A l l the lambs were weighed ev e ry f o u r days. The work r e p o r t e d h e r e i n c o n s i s t e d of t h r e e t r i a l s . I n t h e f i r s t t r i a l were f i v e p o s i t i v e c o n t r o l and t e n e x p e r i ­ m ental lam bs. The source of f a t i n t h e p u r i f i e d l i q u i d d i e t was commercial l a r d p u rc h a s e d l o c a l l y . A f t e r 40 days on t h i s d i e t two c o n t r o l and t h r e e anim als on supposedly v i t a ­ min E low d i e t were t r e a t e d d a i l y f o r 25 days w ith 3 ml. of f r e s h cod l i v e r o i l . A l l lambs were s a c r i f i c e d when d e p l e t e d and showing symptoms. The second t r i a l c o n s i s t e d o f f o u r c o n t r o l and s i x lambs on t h e d e f i c i e n t d i e t . The source of f a t i n t h i s group was m o l e c u l a r d i s t i l l e d l a r d (low t o c o p h e r o l animal f a t ) # , c o n t a i n i n g l e s s th a n f i v e micrograms of t o c o p h e r o l s p e r gram, and e s p e c i a l l y s u i t a b l e f o r e x p e r i m e n ta l work w ith v i t a m i n E. Two lambs a f t e r d e p l e t i o n showed advanced symptoms o f d e f i ­ c i e n c y and were t r e a t e d w ith a l p h a - t o c o p h e r o l . The c o n t r o l and t h e o t h e r f o u r d e f i c i e n t lambs were n e c r o p s i e d f o r h i s t o p a th o lo g ic a l study. The t h i r d t r i a l comprised t h r e e c o n t r o l and tw elve lambs on t h e s y n t h e t i c m ilk w ith d i s t i l l e d l a r d as th e so u rc e of fat. Four a n i m a l s were d e p l e t e d and s a c r i f i c e d w hile t h e o t h e r e i g h t lambs were t r e a t e d and cu red a f t e r showing symp- # D i s t i l l a t i o n P r o d u c t s , I n c . , R o c h e s t e r , 13, New York 84 - toms o f d e f i c i e n c y . Blood S t u d i e s The b lo o d s t u d i e s and t h e te c h n i q u e of blood c o l l e c ­ t i o n were s i m i l a r t o th o se d e s c r i b e d i n P a r t I . The lambs were b l e d im m edia tely when p u t on th e p u r i f i e d d i e t t o d e t e r ­ mine th e i n i t i a l blood p i c t u r e . On a l t e r n a t e weeks an a d d i ­ t i o n a l 20 ml. of blood was drawn f o r a n a l y s i s o f t o c o p h e r o l s . The t o c o p h e r o l c o n t e n t of th e blood plasma was d ete r m in e d by t h e h y d r o g e n a t i o n method of Quife and B i e h l e r (1945). The blood was c e n t r i f u g e d im m edia tely a f t e r i t was brought t o t h e l a b o r a t o r y and t h e to c o p h e r o l d e t e r m i n a t i o n was made as soon a s p o s s i b l e . At l e a s t two d e t e r m i n a t i o n s were c a r r i e d o u t f o r each sample. G ross P ath o lo g y Care was e x e r c i s e d t o s a c r i f i c e th e d e p l e t e d anim als whenever p o s s i b l e b e f o r e d y in g , i n o r d e r t o a v o i d p o s s i b l e p o s t mortem c h a n g es. However, i t was d i f f i c u l t t o p r e d i c t th e f i n a l s t a g e s o f d e f i c i e n c y , e s p e c i a l l y i f i t was n o t c o m p l i c a t e d w ith pneumonia. I n a number o f i n s t a n c e s d e a t h was sudden and t h e a n i m a l s d i s p l a y e d no s e r i o u s s i g n s o f i l l h e a l t h o t h e r th a n symptoms o f v i t a m i n E d e f i c i e n c y . Necropsy was perfo rm e d as soon a s p o s s i b l e . A l l c o n t r o l lambs were s a c r i f i c e d by e x s a n g u i n a t i o n under eth er a n e s th e s ia . in P a rt I . The a u t o p s y t e c h n i q u e was d e s c r i b e d In order to p reserve the h e a rt i n t a c t fo r gross 85 - and m ic r o s c o p i c s t u d i e s , the h e a r t blood was n o t c u l t u r e d . M ic ro s c o p ic S t u d i e s T i s s u e s f o r h i s t o p a t h o l o g i c a l stu d y were ta k e n from v a r i o u s muscles of th e r e a r and f r o n t l e g s , from th e h e a r t , from t h e l o n g i s s i m u s d o r s a l i s and o c c a s i o n a l l y from the abdominal m u s c le s , diaphragm, tongue and m a s s e t e r . specim ens were f i x e d i n Z e n k e r ' s f l u i d . The P a ra ffin sections were c u t se ven micra t h i c k and s t a i n e d w ith h e m a t o x y l i n - e o s i n . The t i s s u e s from th e l i v e r , kidney and a d r e n a l s were f i x e d b o t h i n Z e n k e r ' s f l u i d and 10$ formol s a l i n e and s t a in e d » r e s p e c t i v e l y , w ith h e m a t o x y l i n - e o s i n and Sudan IV. H e a rt specimens were a l s o f i x e d i n f o r m a l i n , c u t f r o z e n and s t a i n e d w i t h Sudan IV f o r f a t . E s t i m a t e d A lp h a - t o c o p h e r o l R equirem ent, T reatm ent and Recovery The p u r i f i e d v i t a m i n E low d i e t of th e IB c o n t r o l lambs was supple mented w i t h 100 mg. of a l p h a - t o c o p h e r o l a d m i n i s t e r e d o r a l l y on a l t e r n a t e d a y s . A r a t h e r h ig h l e v e l of a l p h a - t o c o ­ p h e r o l was chosen t o e l i m i n a t e any p o s s i b l e doubt t h a t l e s i o n s , i f found i n the c o n t r o l lam bs, were due t o a b o r d e r l i n e le v e l of the v itam in. A l t o g e t h e r e i g h t d e p l e t e d lambs were t r e a t e d w ith alpha-tocopherol. The anim a ls were d e p l e t e d u n t i l the y showed p a r a l y s i s and were unable to s t a n d . I n t h i s c o n d i t i o n the e i g h t a n im a ls were g iv e n an i n i t i a l dose o f 500 mg. of a l p h a — - 86 - t o c o p h e r o l f o llo w e d by 100 mg. e v e ry o t h e r day. The v i t a m i n was d i l u t e d i n f r e s h co rn o i l and a d m i n i s t e r e d o r a l l y by means o f a s y r i n g e f i t t e d w ith a r u b b e r h o se . The lambs were s a c r i f i c e d a t v a r i o u s i n t e r v a l s of t h e r a p y t o s tu d y t h e p r o c e s s of muscle r e g e n e r a t i o n . RESULTS Growth and H e a l t h I t was u n d e r s t o o d from th e work o f Willman e^t a l . (1945, 1946) and from t h e r e p o r t s on " s t i f f lambs" from t h e f i e l d t h a t t h e a p p e t i t e of t h e a f f e c t e d lambs was n o t g r e a t l y d e p r e s s e d , a t l e a s t i n t h e f i r s t s t a g e s of d e f i c i e n c y . It was t h u s f e l t t h a t t h e growth r a t e would n o t be a major c r i t e r i o n of d e f i c i e n c y and t h a t f e e d i n g t h e lambs f o u r tim e s a day i n s t e a d o f f i v e tim e s was j u s t i f i e d . However, th e m ilk consumption p e r f e e d i n g was r e c o r d e d and th e w eights o f t h e lambs were t a k e n e v e ry f o u r d a y s . D e s p i te t h i s l i m i ­ t a t i o n i n f e e d i n g , t h e c o n t r o l lambs on both commercial and d i s t i l l e d l a r d d i e t grew n o r m a ll y , made moderate d a i l y g a in s and s t a y e d i n good h e a l t h and c o n d i t i o n * The a v e ra g e d a i l y g a i n o f a l l 12 c o n t r o l lambs was 0 . 2 8 l b . over p e r i o d s on the d i e t . The c o n t r o l lambs r e q u i r e d an a v e ra g e of 1 .4 6 l b s . o f dry r a t i o n t o produce a one pound g a i n i n body w eig h t. T h i s r a t h e r low d a i l y g a i n o f the c o n t r o l lambs i n d i c a t e d t h a t f e e d i n g th e newborn lamb f o u r tim es a day was i n a d e q u a t e t o o b t a i n an o p tim a l growth r a t e (Kean _et al_. 1949) or a t l e a s t g a in s comparable to t h o s e r e p o r t e d i n P a r t I , The lambs on th e v i t a m i n E low d i e t w i t h commercial and d i s t i l l e d l a r d grew n o r m a ll y , rem ained h e a l t h y and made g a i n s i n body w eight s i m i l a r to the c o n t r o l s f o r a p e r i o d of - 88 - a b o u t 28 and 14 days r e s p e c t i v e l y . A f t e r t h i s time a m a j o r i t y o f t h e d e f i c i e n t lambs began to show symptoms o f pneumonia which was a l s o r e f l e c t e d i n an i n c r e a s e d l e u k o c y t e count# The a f f e c t e d lambs were t r e a t e d w ith 100,000 u n i t s of p r o c a i n e p e n i c i l l i n i n j e c t e d i n t r a m u s c u l a r l y i n th e neck r e g i o n every o t h e r day. None of th e lambs f u l l y r e c o v e r e d and t h i s t h e r a p y had to be c o n t i n u e d u n t i l the end o f the experim ent to keep th e a n im a ls a l i v e . The o t h e r d e f i c i e n t lambs w ith no com pli­ c a t i o n s of pneumonia, d e s p i t e th e e x i s t i n g symptoms o f d e f i ­ c i e n c y , r e t a i n e d a v e r y good a p p e t i t e and remained i n good c o n d i t i o n u n t i l th e y d i e d s u d d e n ly due to h e a r t f a i l u r e or were s a c r i f i c e d f o r f u r t h e r s tu d y . Symptoms o f D e f i c i e n c y The f i r s t n o t i c e a b l e symptoms o f v i t a m i n E d e f i c i e n c y i n t h e lambs on th e d i e t c o n t a i n i n g commercial l a r d as th e so u rc e o f f a t ap p e ared i n f o u r t o s i x weeks, w h ile th e a n im a ls on the m o l e c u l a r d i s t i l l e d l a r d d i e t showed th e f i r s t symptoms t hs i n t h r e e t o f o u r weeks a f t e r b e i n g p l a c e d on r e s p e c t i v e r a t i o n s . The lambs became d u l l and t i r e d and p r e f e r r e d to remain i n a lay in g p o sitio n . They showed some d i f f i c u l t y i n r i s i n g , were r e l u c t a n t t o walk and l a t e r were r e l u c t a n t to c r o s s small ob stacles. T h is was accom plished w ith d i f f i c u l t y or by f a l l i n g . S t i l l l a t e r a d e f i n i t e s t i f f n e s s , e s p e c i a l l y o f the h in d l e g s , was n o t i c e a b l e . w alk. The anim als were h a r d l y a b l e t o g e t up and When a s s i s t e d t o a s t a n d i n g p o s i t i o n , t h e y assumed a - 89 - c h a r a c t e r i s t i c a r c h e d back p o s i t i o n ( F ig . 8 ) . A fter standing f o r a s h o r t time e q u i l i b r i u m was l o s t and t h e a n im a ls f e l l . The h in d p a r t of th e body was u s u a l l y t h e f i r s t t o go down (Fig. 9). I n t h e l a t e r s t a g e s th e lambs were p a r a l y z e d i n b o t h h in d and f r o n t l e g s and were l a i d p r o s t r a t e . D ifficu lty i n sw allow ing th e l i q u i d d i e t was o b se rv e d i n two lambs. Twenty f o u r o u t o f 28 d e f i c i e n t lambs developed a r e s p i r a t o r y d i s t r e s s which l a t e r d eve lop ed i n t o pneumonia. were im m e d ia te ly t r e a t e d w ith p e n i c i l l i n . These lambs T his was the t y p i c a l sequence of e v e n t s d u r i n g th e e x p e r i m e n t. S e v e r a l a n im a ls showed o n ly a mild s t i f f n e s s and d id n o t d eve lo p the sequence of symptoms c h a r a c t e r i s t i c o f c h r o n ic deficiency. These lambs d ie d sud d e n ly w i t h o u t showing any s e v e r e s t i f f n e s s or p a r a l y s i s . P o s t mortem e x a m in a tio n c l e a r l y i n d i c a t e d c a r d i a c f a i l u r e due t o e x t e n s i v e l e s i o n s i n th e h e a r t . The s o u r c e of f a t i n t h e d i e t of lambs i n t h e f i r s t t r i a l , a s mentioned above, was commercial l a r d . D uring t h e e x p e rim e n t i t was e v i d e n t t h a t th e amount of v i t a m i n £ i n commercial l a r d was s u f f i c i e n t to g r e a t l y d e l a y t h e d e f i c i e n c y symptoms• Madsen e t a l . (1 9 3 5 ), Pamukcu (1948) and o t h e r s p r o ­ duced th e m u s c u la r d y s tr o p h y c h a r a c t e r i s t i c o f v i t a m i n E d e f i c i e n c y i n l a b o r a t o r y an im als by su p p le m e n tin g t h e d i e t w ith cod l i v e r o i l . To check the p o s s i b i l i t y of in d u c in g - 90 - t h e same m usc ular d y s t r o p h y o r s p e e d in g up th e app e a ra n c e o f v i t a m i n E d e f i c i e n c y symptoms w ith cod l i v e r o i l i n lam bs, two c o n t r o l and t h r e e d e f i c i e n t a n im a ls p r e v i o u s l y on commer­ c i a l l a r d d i e t f o r 40 days were t r e a t e d d a i l y f o r 25 days w i t h 3 ml o f f r e s h cod l i v e r o i l . There was no s i g n i f i c a n t d i f f e r e n c e between t h e t r e a t e d and n o n - t r e a t e d lambs i n r e ­ gard t o th e time r e q u i r e d to produce th e symptoms or th e s e v e r i t y o f the m uscular d y s t r o p h y . The amount of cod l i v e r o i l a d m i n i s t e r e d a p p a r e n t l y was too s m a l l . The time r e q u i r e d t o p r o d u c e symptoms o f d e f i c i e n c y on commercial and m o le c u la r d i s t i l l e d l a r d d i e t s and th e e f f e c t of cod l i v e r o i l i s i n d i c a t e d i n t a b l e 11. Blood S t u d i e s The a v e r a g e hemoglobin v a l u e of a l l 51 lambs d e t e r ­ mined w i t h i n f i v e days a f t e r b e i n g p u t on the e x p e r i m e n t a l d i e t s was 1 2 . 8 gm p e r 100 ml o f b lo o d . t o 1 5 .9 gm p e r c e n t . The range was 1 0 . 4 The a v e r a g e hemoglobin v a lu e o f th e 10 c o n t r o l lambs j u s t b e f o r e th e t e r m i n a t i o n of th e e x p e r i ­ ment d e c r e a s e d s l i g h t l y and was 1 2 .4 gm p e r 100 ml of b lo o d . T h is s l i g h t d e c r e a s e o f hemoglobin was not c o n s i d e r e d s i g n i ­ fican t. The c o n t r o l lambs t h e n m a i n ta in e d an approxim*- a t e l y normal hemoglobin l e v e l th r o u g h o u t th e e x p e r i m e n t. In e v e r y v i t a m i n E d e f i c i e n t lamb t h e r e was a g r a d u a l drop of hem oglobin. The a v e r a g e v alu e c a l c u l a t e d from t h e l a s t v a l u e s a v a i l a b l e b e f o r e th e d e f i c i e n t lamb d i e d or was s a c r i f i c e d - 91 - TABLE 11 EFFECT OF COMMERCIAL AND MOLECULAR DISTILLED LARD AND COD LIVER OIL ON TIME REQUIRED TO PRODUCE SYMPTOMS OF VITAMIN E DEFICIENCY No of T r i a l Lambs 5 10 i D iet L a r d Used Cod L i v e r Days to Produce Oil Therapy D eficiency P o s i t i v e Commercial control ---- Commercial --- E low No symptoms 30 - 80 2# P o s i t i v e Commercial 3 ml, d a i l y No symptoms control 3# E low 2 and 3 62 - 74 Commercial 3 ml. d a i l y 7 P ositive control M olecular d istilled 18 E d efi­ cient M olecular d istilled ——— No symptoms 20 - 55 # P r i o r t o a d m i n i s t r a t i o n o f cod l i v e r o i l th e lambs were on commercial l a r d d i e t f o r 40 d ay s . - 92 - was 1 0 . 4 gm p e r 100 ml b lo o d , e x c l u d i n g t h r e e lambs which d e v e lo p e d anemia i n th e f i n a l s t a g e s of d e f i c i e n c y and showed v a l u e s o f 5 . 1 , 5 . 6 and 6 .5 gm of hemoglobin p e r 100 ml of blood. The a v e ra g e l e u k o c y t e count of a l l lambs a t th e b e g i n n i n g o f th e ex p e rim e n t was 8,100 p e r mm3 of blood w ith t h e av e ra g e r a t i o o f n e u t r o p h i l s to lymphocytes 55:4 3 . No s i g n i f i c a n t p a t t e r n was e x h i b i t e d by t h e l e u k o c y t e or d i f ­ f e r e n t i a l counts. However, t h e l e u k o c y t e count and t h e p e r c e n t o f n e u t r o p h i l s were s i g n i f i c a n t l y i n c r e a s e d i f t h e d e f i c i e n c y was c o m p lic a te d w ith pneumonia. The i n i t i a l l e v e l of t o c o p h e r o l s i n the blood plasma was d e t e r m in e d on 23 lam bs. These d e t e r m i n a t i o n s were c a r r i e d o u t on blood drawn from 0-15 days a f t e r t h e lambs were p l a c e d on t h e e x p e r i m e n t a l d i e t s . The v a l u e s ran g ed from 0 .0 4 0 t o 0 .3 7 7 mg o f t o t a l t o c o p h e r o l s p er 100 ml of b lo o d w ith an a v e ra g e v a lu e 0 . 1 7 9 mg. The r e s u l t s of th e b lo o d a n a l y s i s f o r t o t a l t o e o p h e r o l of lambs on the commer­ c i a l and d i s t i l l e d l a r d d i e t s and lambs t r e a t e d w ith cod l i v e r o i l and a l p h a - t o c o p h e r o l a r e shown i n t a b l e s 12 and 13. I n e v e ry c a se th e v a l u e s r e p r e s e n t a t l e a s t two a n a l y s e s f o r each sample of b lo o d . The t o c o p h e r o l l e v e l o f blood plasma of t h e c o n t r o l lambs on th e commercial l a r d d i e t supplemented w i t h 100 mg o f a l p h a - t o c o p h e r o l e v e r y o t h e r day s i g n i f i c a n t l y i n c r e a s e d th r o u g h o u t th e e x p e r i m e n t , w h ile t h e c o n t r o l lambs on t h e m o l e c u l a r d i s t i l l e d l a r d t r e a t e d s i m i l a r l y showed a l e s s p r o - - 93 - TABLE 12 TOCOPHEROL CONTENT OF BLOOD PLASMA IN LAMBS ON COMMERCIAL LARD DIET AND THE EFFECT OF COD LIVER OIL ON THE LEVEL OF PLASMA TOCOPHEROLS No o f Lambs 4 8 2## D iet Cod L i v e r Oil Therapy Positive control V ita m in E — low P ositive control 3 ml d a i l y V ita m in E low 3 ml d a i l y 2**# Da vs on E xperim e nt 0-15 30 - 45 60 - 70 mg/100 ml mg/loo ml mg/100 ml 0 .3 7 2 0 .0 8 4 0.485 0.212 0 .2 6 6 0 .2 6 6 0 .2 2 8 # 0.307# 0 .2 1 6 0.1 1 5 0.3 7 7 0 .2 4 4 0 .1 5 9 0 .1 4 4 0 .1 8 7 0.141 0.075 0 .0 9 5 0 .0 9 7 0.141 0.147 0.141 0 .1 0 6 0 .2 0 6 # 0.118# 0.099# 0.266 0 .2 6 6 0.1 6 9 0.1 5 6 0.266# 0 .1 6 2 # 0.147 0.141 0.0 7 2 0.0 7 2 0.144# 0.072# 0.2 8 1 sacrificed cod l i v e r o i l cod l i v e r o i l 0.281 sacrificed 0 .0 6 9 0 .1 1 9 sacrificed cod l i v e r o i l 0.1 0 9 sacrificed cod l i v e r o i l # Average * # P r i o r to a d m i n i s t r a t i o n of cod l i v e r o i l t h e lambs were on commercial l a r d d i e t f o r 40 days. TOCOPHEROL CONTENT OF BLOOD PLASMA IN LAMBS ON MOLECULAR DISTILLED LARD DIET AND THE EFFECT OF ALPHA-TOCOPHEROL THERAPY ON THE LEVEL OF PLASMA TOCOPHEROLS - 94 - o CO in o o 03 J> 1 ^ rH in t- • • rH ^ i to to 03 • 03 • ooo • O tO 00 05 o to to • o * • • •H o o o o o !•* ©i n 05 £>- 05 05 00 in P in X i 0 3 pH rH W o •tf o o o o >. © Q in 03 in 03 i in • • • • • • * CO C"to ,sJ1 o 'tj* in 05 rH o o o in I o 05 CO to C" to c - rH rH O O O 1 • • • • o o o o o COrHCOrH^OtOOtO to o o• o• o• rH © ooooo O 05C ^05C 0lO O >O O rH H O O O H O H O H • 03 CO 05 • 05 to lO to in rH CO ■sH !> to t - 0 0 OJ CM 0 1 rH t OJ rH l—I M J ) O 05 03 o• o• o• o• rH rH O O O O O • Oo o o o o • > a o • o o o o o o • t - 'tf 05 CO rH to 05 CO to 03 o 03 H OJO H CD tO <3i tOlO COO 1 rHO torH rHr-l •rH o in t- W C*- 03 © o o o o o o o o o 03• O• o• rH• o• O ooO oo * — 105 05 o !>00 to 1 oo CO in 05 o rH• ■—•1 I—•1 o• O• ooooo Xl P •>5 CUi o ©fc * Ol xi ] PC If I—I oc u+ © * CO xJC 0P* OJ oc p<» 1 © © o 13 o © xs a , t> o p o © u o p © > o £ in M © IH M -P © rH l> o £ a © •H J* © p S © s p TS ttO S © O Xi O P rH O ft r-n © + oi PC •r o ., •rr t. P -P •h a O © -H E CO O p X3 Cm 01 O XJ o >» ©© Ih C XI f t — p ©+ x jo ^5 £ © rH bD © E *rl P •r i O 1xs •H © © ❖ 00 rH rH i1 u © rH O O rH in © x> s o oo o 03 to • • • • • • • ❖ tO 05 0 5 0 3 0 5 CO t o 05 r l^ O O O tO C O r l W C O O W O O M to r H C 0 0 3 0 3 0 J OOr Ht H • *to 05 £ W o• o• o• o• *o •o oooooo £ s © p *05 * 03 lO 02 1 ^ W (OH H 03 o o * CO O 05 05 O ^ <75 03 O to tO o CO to co m 05 © in S to CO O 03 )H 03 • • • tO £ 0 - rH 03 • ooo to to p t o CO co LO CO CO -Si1 c- * si* P4 $ rH to in pH CO bD © © t> C - 95 - nounced i n c r e a s e o f blood plasma t o c o p h e r o l . D e f i c i e n t lambs on t h e commercial l a r d d i e t showed a g r a d u a l and c o n s i s t e n t drop i n t o c o p h e r o l s c o n t e n t of blood plasm a o v e r t h e 70 day e x p e r i m e n t a l p e r i o d . On th e d i e t c o n t a i n i n g m o l e c u l a r d i s t i l l e d l a r d , about th e same drop i n t o c o p h e r o l c o n t e n t o f the plasma o c c u r r e d i n 30 t o 35 d a y s . The a v e r a g e t o c o p h e r o l l e v e l o f the d e f i c i e n t lambs which s u r v i v e d over 60 days on t h e m o le c u la r d i s t i l l e d d i e t was 0 .0 4 9 mg p e r 100 ml o f blood w i t h two lambs showing a complete ab sen ce o f t o c o p h e r o l i n the b lo o d . D a il y a d m i n i s t r a t i o n of 3 ml cod l i v e r o i l f o r 25 days to two c o n t r o l and t h r e e d e f i c i e n t lambs low ered th e average to c o p h e r o l c o n t e n t o f blood plasma r e s p e c t i v e l y from 0 .2 6 6 t o 0 .1 6 2 mg and from 0 .1 4 4 t o 0 .0 7 2 mg. Gross P ath o lo g y P o s t mortem e x a m in a tio n o f the c o n t r o l lambs d id not r e v e a l s i g n i f i c a n t c h a n g e s. Three out of 12 c o n t r o l lambs d e v e lo p e d s i g n s o f pneumonia d u r i n g the ex p e rim e n t and were t r e a t e d w ith p r o c a i n e p e n i c i l l i n . The lu n g s of t h e s e t h r e e lambs showed u n i l a t e r a l " c o ld pneumonia" c o n f i n e d t o the r i g h t ap ical lo b e. Gross p a t h o l o g i c a l s t u d i e s o f ev e ry v i t a m i n E d e f i c i e n t lamb r e v e a l e d d e g e n e r a t i o n o f s k e l e t a l and c a r d i a c muscle accompanied by pneumonia or l u n g conges­ tio n . The d e g e n e r a t i o n of s k e l e t a l muscles ran g ed from very m ild t o se v e r e d y s tr o p h y d epend ing on the l e n g t h of time t h e p a r t i c u l a r lamb was on the d e f i c i e n t d i e t . In the e a r ly 96 - s t a g e s o f d e f i c i e n c y th e a f f e c t e d muscles were p a l e and anemic. L a t e r , t h e l e s i o n s i n t h e s k e l e t a l muscles a p p e a r e d on t h e s u r f a c e as g r a y i s h or w h i t i s h a r e a s or s t r e a k s . o n ly a p a r t of t h e muscle was a f f e c t e d . U sually On c r o s s s e c t i o n t h e w h i t i s h or g r a y i s h a r e a s a g a i n i n d i c a t e d t h a t not a l l of th e muscle f i b e r s and bundles were i n v o l v e d . The a f f e c t e d m u sc les were m oist and c h a r a c t e r i s t i c y e llo w g r a y i s h g e l a ­ t i n o u s m a t e r i a l was p r e s e n t between t h e muscles and e s p e c i a l l y a round t h e s c i a t i c and b r a c h i a l n e r v e s . I t was observed t h a t t h e m uscles o f b o t h h in d and f o r e l e g s were a f f e c t e d e a r l i e s t and showed much more se v e r e d e g e n e r a t i o n tha n th e m uscles of th e s h o u l d e r , rump, l o i n or neck. In four severe ca se s , p r a c t i c a l l y a l l of t h e m u s c u la t u r e was a f f e c t e d i n c l u d i n g th e d ia p hra gm , i n t e r c o s t a l m u sc les , m a s s e t e r and t h e to n g u e . E x a m in a tio n s of t h e c a r c a s s e s showed t h a t b o th s i d e s o f t h e body were a b o u t e q u a l l y a f f e c t e d . The most s t r i k i n g f e a t u r e o f the d e f i c i e n t lambs was t h e c h a r a c t e r i s t i c l e s i o n s i n t h e h e a r t which, i n most i n s t a n c e s , were c o n f i n e d or were most pronounced w i t h i n the r i g h t v e n t r i ­ cle (Fig. 1 0 ). A g ain, depending on t h e l e n g t h of time th e a n im a ls were on t h e d e f i c i e n t d i e t , th e d e g r e e o f d e g e n e r a t i o n was v a r i a b l e . The l e s i o n s were u s u a l l y i n th e form of round p a t c h e s r a n g i n g i n s i z e from p i n p o i n t up to 12 mm i n d i a m e t e r ( F i g . 11) and o c c u r r e d s i n g l y , i n groups or were v e r y d i f f u s e and a f f e c t e d most o f t h e v e n t r i c l e . The l e s i o n s were o f y e l l o w i s h o r w h i t i s h c o l o r and t h e l i n e o f d e m a r c a t io n between - 97 - t h e d e g e n e r a t e d and h e a l t h y a r e a was very d i s t i n c t . I n the l a t e r s t a g e s of d e f i c i e n c y under t h e s u p e r f i c i a l a p p e a r i n g l e s ­ i o n s o f th e endocardium , d e g e n e r a t i o n o f t h e myocardium was i n p r o g r e s s and, i n one extreme c a s e , t h e d e g e n e r a t i o n o f t h e c a r d i a c w a l l r e a c h e d t h e e p ic a r d iu m ( F ig . 1 2 ) . In th i s p a r t i ­ c u l a r c a se t h e l e s i o n s were found i n b o th v e n t r i c l e s and a t r i a in c lu d in g the a o r t a . I n f o u r o t h e r i n s t a n c e s th e l e s i o n s were o b se rv e d i n the l e f t v e n t r i c l e and a t r i a . The h e a r t s i n g e n e r a l were f l a b b y and th e amount of f l u i d i n the p e r i c a r d i a l sac was g r e a t l y i n c r e a s e d . D i f f u s e s u b e p i c a r d i a l hemorrhages were o b s e r v e d i n a few. R e s p i r a t o r y d i s t r e s s was one o f t h e e a r l i e s t symptoms o f v i t a m i n E d e f i c i e n c y and l a t e r pneumonia was confirm ed a t necropsy. Twenty f o u r o u t of t h i r t y d e f i c i e n t an im als showed l o c a l i z e d u n i l a t e r a l or b i l a t e r a l pneumonia. I n most i n s t a n c e s pneumonia was u n i l a t e r a l and only the a p i c a l and c a r d i a c l o b e s were i n v o l v e d . I n o t h e r c a s e s the once a f f e c t e d p a r t s of t h e l u n g were c o n s o l i d a t e d and i n t h e p r o c e s s o f h e a l i n g . I n o n l y t h r e e lambs t h e pneumonia was a c t i v e , a f f e c t e d b o th lu n g s and was c o n s i d e r e d a prim ary cause o f d e a t h . In a l l t h r e e lambs th e t h o r a c i c c a v i t y was f i l l e d w ith a l a r g e amount o f f i b r i o u s exuda te which adhe re d to th e w a l l s of t h e th o racic ca v ity . The lu n g s were imbedded i n t h i s e x u d a t e . I n 12 c o n t r o l lambs only two showed pneumonia a t a u t o p s y . The l u n g s o f t h e c o n t r o l and d e f i c i e n t lambs were c u l t u r e d - 98 - on blood a g a r p l a t e s and i n most i n s t a n c e s gram n e g a t i v e ple om orp hic organism s r e s e m b l i n g P a s t e u r e l l a e were i s o l a t e d a a f t e r two to t h r e e day p e r i o d o f i n c u b a t i o n . The changes i n th e l i v e r ranged from very mild f a t t y i n f i l t r a t i o n to f o c a l n e c r o s i s . c u t ap p e ared g r e a s y . f a t t y degeneration. The l i v e r was p a l e and when I n most i n s t a n c e s t h e r e was g e n e r a l I n t h r e e c a s e s f o c a l n e c r o s i s was ob­ served. The k id n e y s o f th e d e f i c i e n t lambs showed g e n e r a l f a t t y d e g e n e r a t i o n i n v o l v i n g both t h e c o r t e x and m e d u lla . In a number o f lambs t h e d e g e n e r a t i o n was pronounced, e s p e c i a l l y i n t h e c o r t e x which was y e l l o w i s h i n c o l o r w ith w hite s t r e a k s r u n n i n g from the c a p s u l e to th e m e d u lla . Most o f t h e a d r e n a l s seemed to be norm al. A few appe ared t o be e n l a r g e d , showed numerous p e t i c h i a l hemorrhages i n t h e c o r t e x and g e n e r a l f a t t y d e g e n e r a t i o n . I n t h r e e lambs which showed a c t i v e bronchopneumonia and s e v e r e muscular d y s t r o p h y th e a d r e n a l s were damaged t o t h e e x t e n t t h a t t h e l i n e o f d e m a r c a t i o n between t h e c o r t e x and medulla had d i s a p p e a r e d . M ic ro s c o p ic P ath o lo g y Gross and m ic r o s c o p ic s t u d i e s of the muscle t i s s u e s from v a r i o u s p a r t s o f th e body i n d i c a t e d t h a t t h e muscle de­ g e n e r a t i o n was p r o g r e s s i n g from th e h i n d p a r t o f th e body toward th e f o r e p a r t . A lso t h e s e v e r i t y of t h e muscle dys­ t r o p h y and i t s e x t e n t o v er v a r i o u s p a r t s o f t h e body was - 99 - d i r e c t l y c o r r e l a t e d w ith the l e n g t h of tim e the lamb was on the d e f ic ie n t diet* However, th e n a t u r e o f the m yodegeneration was ab out t h e same i n a l l c a s e s . The c h a i n o f e v e n t s was v i s u a l i z e d as h y a l i n e d e g e n e r a t i o n of the sa rc o p la s m fo llo w e d by f r a g m e n t a t i o n of th e a f f e c t e d muscle f i b e r s and c o a g u l a t i o n n e c r o s i s accompanied by c e l l u l a r i n f i l t r a t i o n , p r o l i f e r a t i o n o f muscle f i b e r n u c l e i and f i b r o b l a s t s . In th e early stages o f d e g e n e r a t i o n o n ly a few muscle f i b e r s were in v o lv e d w h ile o t h e r s i n th e same a r e a were normal ( F i g . 1 3 ) . The a f f e c t e d muscle f i b e r s f i r s t a p p e a r e d to be s w o lle n and l a t e r became s h r u n k e n i n d i a m e t e r and l e n g t h , assuming a wavy c h a r a c t e r and s t a i n e d d a r k e r w ith Hematoxylin and E o s i n ( F ig . 1 4 ) . The i n t e r s t i c e s o f t h e muscle f i b e r s were f i l l e d w ith c e l l u l a r m a t e r i a l and f l u i d (F ig . 1 5 ) . The muscle f i b e r s l o s t the c r o s s and l a t e r th e l o n g i t u d i n a l s t r i a t i o n , and became g r a n ­ u l a r (Fig. 1 6 ). The g r a n u l a r sa rc o p la sm became a homogeneous mass i n s i d e th e sarcolemma which was s t i l l not a f f e c t e d . F i n a l l y the h y a l i n i z e d sa rc o p la sm fragm ented and v a c u o le s w i t h i n t h e sarcolemma were o b se rv e d ( F ig . 1 7 ) . The n u c l e i o f th e muscle f i b e r s e i t h e r became p y k n o t i c , or had undergone k a r y o r r h e x i s or k a r y o l y s i s . A r a t h e r mild i n f i l t r a t i o n of th e n e c r o t i c a r e a s w ith p o l y m o r p h o n u c l e a r l e u k o c y t e s , lymph­ o c y t e s and macrophages s u g g e s te d t h a t t h e i n j u r e d muscle t i s s u e was i n i t i a l l y in v o lv e d i n an inflam m ato ry p a t t e r n (Fig. 1 8 ). menced. T his was b e f o r e r e p a i r and r e g e n e r a t i o n had com­ At th e same time numerous macrophages and g i a n t c e l l s - 100 - in v a d e d t h e a r e a . The r o l e o f g i a n t c e l l s i n c l e a r i n g th e d e b r i s was e v i d e n t ( F ig . 1 9 ) . As the muscle f i b e r s d i s a p ­ p e a r e d th e y were r e p l a c e d w ith v e r y r a p i d l y p r o l i f e r a t i n g fib ro b lasts. Some a r e a s were c o m p le te ly devoid o f muscle f i b e r s and a t y p i c a l f i b r o s i s w i t h i n th e muscle was o b s e r v e d . However, t h e r e was a l s o ample e v id en ce to show t h a t s i m u l ­ t a n e o u s d e g e n e r a t i o n and r e g e n e r a t i o n of t h e muscle was t a k i n g p lace. New muscle f i b e r s c h a r a c t e r i z e d by f a i n t e r s t a i n and c e n t r a l l y l o c a t e d n u c l e i a r r a n g e d i n rows were o f t e n d e t e c t e d i n t h e m usc les o f b o th th e d e f i c i e n t and t r e a t e d lambs ( F ig . 2 0 ) . The l e s i o n s i n th e c a r d i a c muscle were s i m i l a r t o t h o s e in the s k e l e t a l m usculature. S t a r t i n g w ith the endocardium t h e d e g e n e r a t i o n p r o g r e s s e d i n t o the myocardium ( F ig . 2 1 ) . The l i n e o f d e m a r c a t io n between t h e n e c r o t i c and normal a r e a s was r a t h e r s h a r p . The i n d i v i d u a l muscle f i b e r s showed changes c h a r a c t e r i z e d by h y a l i n e d e g e n e r a t i o n , f r a g m e n t a t i o n and c o a g u l a t i o n n e c r o s i s fo llo w ed by p r o l i f e r a t i o n of f i b r o ­ b l a s t s (Fig. 22). I n c r e a s e d a c t i v i t y of th e muscle n u c l e i and i n v a s i o n of the a r e a s w ith macrophages, n e u t r o p h i l s and lymphocytes were a l s o n o t e d . The g i a n t c e l l s o b se rv e d i n th e d e g e n e r a t e d s k e l e t a l muscle were a b s e n t i n th e c a r d i a c m u sc le. I t seemed t h a t t h e p r o l i f e r a t i o n o f f i b r o b l a s t s i n t h e a r e a s of d e g e n e r a t i o n was more i n t e n s e th a n i n t h e s k e l e t a l m u sc le. Some a r e a s were a lm o st c o m p le t e ly composed of f i b r o b l a s t s and connective t i s s u e (Fig. 23). R e p a i r was a l s o n o t i c e d i n th e lambs w h ile s t i l l on th e d e f i c i e n t d i e t . On many o c c a s i o n s - 101 - s m a l l , young muscle f i b e r s f a i n t l y s t a i n e d were d e t e c t e d i n th e a r e a s o f f i b r o s i s ( F i g . 2 4 ) . I t seemed t h a t th e s e new muscle f i b e r s o r i g i n a t e d from a d j o i n i n g normal muscle and t h a t r e g e n e r a t i o n was p r o g r e s s i n g from the myocardium toward th e endocardium . I t was i n t e r e s t i n g to n o te t h a t the P u r k i n j e f i b e r s a p p e a r e d to be normal d e s p i t e th e f a c t t h a t th e im­ m e d ia t e a r e a s were n e c r o t i c ( F i g . 2 3 ) . The l i v e r i n most i n s t a n c e s was c o n g e s te d and showed changes r a n g i n g from cloudy s w e l l i n g to f o c a l n e c r o s i s . Frozen s e c t i o n s s t a i n e d w ith Sudan IV showed pronounced f a t t y d e g e n e r a t i o n which was r a t h e r d i f f u s e a f f e c t i n g a l l of th e l i v e r . The c e n t r a l p a r t of th e l o b u l e was u s u a l l y f i l l e d w i t h f a t g l o b u l e s to a g r e a t e r e x t e n t t h a n th e p e r i p h e r y . O c c a s i o n a l l y t h e c o n g e s t i o n was v ery se vere i n the c e n t r a l v e i n s and t h e a d j o i n i n g s i n u s o i d s were f i l l e d and d i s t e n d e d w ith blood. I n most a n i m a l s , e s p e c i a l l y i n th o se w i t h secondary pneumonia, t h e k id n e y was c o n g e s te d and th e changes ranged from clo u d y s w e l l i n g to a pronounced f a t t y d e g e n e r a t i o n (F ig . 2 5 ) . The p ro x im al c o n v o l u te d t u b u l e s were d i l a t e d , and f i l l e d to some e x t e n t w i t h h y a l i n e m a t e r i a l . p u s c l e s a p p e a r e d to be more or l e s s normal. The r e n a l c o r ­ The c o n g e s t i o n was e s p e c i a l l y s e v e r e i n t h e m e d u lla and some hemorrhages were a l s o n o te d ( F i g . 2 6 ) . M ic r o s c o p ic s t u d i e s of t h e a d r e n a l s d id n o t show con­ s i s t e n t changes. I n most o f t h e i n s t a n c e s t h e a d r e n a l s were - 102 - normal o r showed s l i g h t f a t t y i n f i l t r a t i o n . I n two o r t h r e e lambs t h e a d r e n a l s were c o m p le t e ly d e g e n e r a t e d . The a r c h i t e c ­ t u r e was c o m p le t e ly l o s t and th e whole a d r e n a l was a mass o f coagulation n ecro sis. T re atm ent and E s t im a te d A l p h a - t o c o p h e r o l Requirement E i g h t lambs from t h e second and t h i r d t r i a l s showing advanced symptoms of d e f i c i e n c y c h a r a c t e r i z e d by i n a b i l i t y to g e t up were t r e a t e d w ith a l p h a - t o c o p h e r o l . The i n i t i a l dose was 500 mg, fo llo w e d by 100 mg ev e ry o t h e r day a d m i n i s t e r e d orally . days. A d e f i n i t e r e s p o n s e was n o t i c e d i n t h r e e to f i v e On t h e f o u r t h and f i f t h day the anim als were a b l e t o g e t up and to move to some e x t e n t . L a t e r th e anim als were a b l e to move f r e e l y and d i d n o t show symptoms of d e f i c i e n c y . The lambs were s a c r i f i c e d a t i n t e r v a l s d u r i n g th e a l p h a t o c o p h e r o l t h e r a p y , namely over a p e r i o d o f 10 t o 45 d a y s . T h is was done to stu d y t h e p r o c e s s of r e c o v e r y g r o s s l y and t h e muscle r e g e n e r a t i o n m i c r o s c o p i c a l l y . a p p e a r e d c o m p le t e ly i n 15 to 20 d a y s . The symptoms d i s ­ P o s t mortem e x a m in a tio n o f t h e lambs t r e a t e d w ith a l p h a - t o c o p h e r o l up to about 25 days r e v e a l e d s t i l l v i s i b l e d e g e n e r a t i o n of the s k e l e t a l and c a r ­ d i a c muscle and some s l i g h t changes i n th e l i v e r and k id n e y . I t was o b s e r v e d , however, t h a t e s p e c i a l l y a f t e r 20 and 25 days of t h e r a p y t h e s k e l e t a l muscle d e g e n e r a t i o n seemed t o be l e s s marked. The h e a r t l e s i o n s were not as e x t e n s i v e and t h e p a t c h e s were s m a l l e r i n s i z e and o f g r a y i s h c o l o r , l e s s w h i t i s h - 103 - and is c h e m i c th a n i n p r o g r e s s i v e d e g e n e r a t i o n . The b o r d e r s between th e normal and a f f e c t e d a r e a s were a l s o l e s s d i s t i n c t . M ic ro s c o p ic ex a m in a tio n o f th e t i s s u e s from th e s k e l e t a l and c a r d i a c muscles r e v e a l e d r e p a i r of the n e c r o t i c a r e a s by v e r y a c t i v e muscle r e g e n e r a t i o n c h a r a c t e r i z e d by i n c r e a s e d p r o l i f e r a t i o n o f muscle f i b e r n u c l e i and f i b r o b l a s t s and by i n c r e a s e d numbers of macrophages and g i a n t c e l l s ( F ig . 2 7 ) , T h is was e s p e c i a l l y t r u e i n the lambs which had been on t h e r a p y 10 and 15 d a y s . The r e g e n e r a t i o n due to t h e r a p y seemed t o be the same a s t h a t o bserved w h ile t h e d e g e n e r a t i o n was s t i l l in progress. The new muscle f i b e r s ap p e ared t o be o r i g i n a t i n g from the sarcolemma of t h e d e g e n e r a te d muscle or from th e muscle f i b e r n u c l e i which p r o l i f e r a t e d e x t e n s i v e l y . The new muscle f i b e r s v a r i e d i n s i z e , s t a i n e d f a i n t l y w ith h e m a t o x y l i n - e o s i n , d i s p l a y e d v a r i o u s d e g r e e s of c r o s s o r l o n g i ­ t u d i n a l s t r i a t i o n and one p o le t e r m i n a t e d i n a round p o i n t which p r o b a b l y was th e a c t i v e c e n t e r of growth ( F ig . 28, 29, 3 0 ) . The n u c le i, always numerous, were a r r a n g e d i n rows and were c e n t r a l l y l o c a t e d ( F ig . 3 1 ) . As th e t h e r a p y c o n t i n u e d , more and more new muscle f i b e r s appeared and i n lambs t r e a t e d f o r 40 and 45 days th e r e g e n e r a t i o n was a lm o st complete e x c e p t t h a t t h e i n c r e a s e d number of muscle n u c l e i was s t i l l noted (Fig. 3 2 ). The same p r o c e s s o f new muscle f i b e r f o r m a tio n was o b se rv e d i n the c a r d i a c muscle. The two lambs t r e a t e d f o r 40 and 45 days d id n o t show h e a r t l e s i o n s g r o s s l y or s i g n i f i c a n t - 104 - changes m i c r o s c o p i c a l l y . T r a n s v e r s e and l o n g i t u d i n a l s e c t i o n of s k e l e t a l muscles from two p o s i t i v e c o n t r o l lambs were shown i n F i g . 33 and 34. U n f o r t u n a t e l y i t was i m p o s s i b l e in t h i s s tu d y to d e t e r ­ mine th e e x a c t q u a n t i t a t i v e r e q u ir e m e n t of the young lamb f o r vitam in E (a lp h a-to co p h ero l). The 100 mg of a l p h a - t o c o p h e r o l a d m i n i s t e r e d o r a l l y ev e ry o t h e r day was s e t a r b i t r a r i l y and a p p e a r e d to be more t h a n a d e q u a t e . Evidence to s u b s t a n t i a t e t h i s was a marked i n c r e a s e of th e blood to c o p h e r o l l e v e l i n th e c o n t r o l lambs over t h e i n i t i a l l e v e l which could be con­ s i d e r e d normal f o r the young lamb. F u r th e r m o r e , a r a t h e r r e m a rk a b le r e s p o n s e to th e t h e r a p y c o n s i s t i n g of 500 mg a l p h a t o c o p h e r o l as an i n i t i a l dose and 100 mg e v e ry o t h e r day s u g g e s te d t h a t 100 mg o f a l p h a - t o c o p h e r o l was enough to a r r e s t t h e d e g e n e r a t i v e changes i n th e m u s c u l a t u r e , to c l e a r up the symptoms, i n i t i a t e muscle r e g e n e r a t i o n and su p p o r t normal b i o l o g i c a l p r o c e s s e s i n the body, " S tif f- L a m b " D is e a s e i n th e F i e l d While t h e s e e x p e r i m e n t a l s t u d i e s were i n p r o g r e s s , many c a s e s of t h e s o - c a l l e d " S tiff-L a m b " d i s e a s e were r e p o r t e d by f a r m e r s i n M ichigan. A number of a f f e c t e d lambs, dead or a l i v e , were b r o u g h t to th e l a b o r a t o r y f o r n e c ro p s y and p r o v i d e d an ample o p p o r t u n i t y to stu d y and compare t h e g r o s s and m ic ro ­ s c o p i c p a t h o l o g y o f bo th f i e l d and e x p e r i m e n ta l c a s e s . Further­ more, th e i n c i d e n c e of " s t i f f - l a m b s " i n th e sheep f l o c k a t - 105 - M ichigan S t a t e C o l le g e p e r m i t t e d o b s e r v a t i o n of the symptoms i n th e f i e l d as w ell a s t h e o p p o r t u n i t y f o r t h e r a p y w ith alpha-tocopherol. The symptoms, g r o s s and m ic ro s c o p ic de­ g e n e r a t i v e l e s i o n s i n the s k e l e t a l and c a r d i a c muscle ( F i g . 35, 36, 37, 38, 3 9 ) , and the re s p o n s e to t h e r a p y were i d e n t i c a l w i t h th o s e p ro duced e x p e r i m e n t a l l y u s i n g t h e p u r i f i e d v i t a m i n E d eficient d ie t. The d e g ree and e x t e n t of muscle d e g e n e r a t i o n was v a r i a b l e b u t i n every case b o th s k e l e t a l and c a r d i a c muscles were i n v o l v e d . I t was n o t i c e d , however, t h a t d e g e n e r a t i o n of s k e l e t a l muscles i n th e lambs from t h e f i e l d was much more s e v e r e and a f f e c t e d l a r g e r a r e a s t h a n i n the lambs on th e experim ental d i e t . I n r e g a r d t o h e a r t l e s i o n s no d i f f e r e n c e was observed between th e e x p e r i m e n ta l and f i e l d c a s e s . Again th e h e a r t l e s i o n s were found u s u a l l y i n th e r i g h t v e n t r i c l e ( F ig . 4 0 ) . However i n one lamb both t h e r i g h t and l e f t h e a r t was i n ­ v o lv e d ( F i g . 4 1 , 4 2 ) . Secondary pneumonia was n o t i c e d i n e v e r y f i e l d case which s u g g e s t e d t h a t r e s p i r a t o r y d i s t r e s s and pneumonia a r e c h a r a c t e r i s t i c f e a t u r e s of n u t r i t i o n a l m uscular d y s t r o p h y o c c u r r i n g under f i e l d c o n d i t i o n s . As none of th e t r e a t e d and r e c o v e r e d " s t i f f lambs" was s a c r i f i c e d , a d i r e c t com parison of th e muscle r e g e n e r a t i o n was n o t a v a i l a b l e . Ju d g in g from t h e d is a p p e a r a n c e of th e symptoms and from t h e complete r e c o v e r y o f t h e lambs i n th e f i e l d , t h e p r o c e s s of muscle r e g e n e r a t i o n was no doubt t h e same. DISCUSSION U n t i l 1928 when Evans and Burr d e v e lo p e d p a r a l y s i s i n young s u c k l i n g r a t s from mothers d e p r i v e d of v i t a m i n E, t h i s v i t a m i n was th o u g h t t o be e n t i r e l y concerned w ith r e p r o d u c t i o n . S in c e t h e n v i t a m i n E d e f i c i e n c y , c h a r a c t e r i z e d g r o s s l y by lo c o m o to r d i s t u r b a n c e s and m i c r o s c o p i c a l l y by d e g e n e r a t i o n of th e s k e l e t a l and c a r d i a c m u sc le s , has been e x p e r i m e n t a l l y pro d u ced and d e s c r i b e d i n guinea p i g s and r a b b i t s (G-oettsch and Pappenheimer 1 931), r a t s .(Pappenheimer 1 9 3 9 ) , dogs ( B r in k house and Warner 1941) and i n mice (Pappenheimer 1 942), V ita­ min E d e f i c i e n c y i n c h i c k s , c h a r a c t e r i z e d by e n c e p h a l o m a l a c i a , was d e s c r i b e d by Pappenheimer (1931) and Ju n g h e rr (1949). D u c k lin g s on th e same d i e t developed a g e n e r a l myopathy (Pappenheimer and G-oettsch 1934) w hile t u r k e y s showed myopathy o f the g i z z a r d ( J u n g h e r r and Pappenheimer 1 9 3 7 ) . D e g e n e r a t i o n o f t h e s k e l e t a l and c a r d i a c muscle i n t h e lamb s i m i l a r to Z e n k e r ' s d e g e n e r a t i o n , produced e x p e r i m e n t a l l y i n l a b o r a t o r y a n i m a l s , was f i r s t d e s c r i b e d by Metzger and Hagan (19 2 7 ). These a u t h o r s r e c o g n i z e d the d e g e n e r a t i v e changes i n the muscles as n o n i n f e c t i o u s and noninflam m atory in n atu re. S ince t h e n many workers i n t h i s c o u n t ry and abroad have d e s c r i b e d a s i m i l a r c o n d i t i o n among lambs and p o s t u l a t e d v a r i o u s c a u s e s r e s p o n s i b l e f o r t h e d i s e a s e and s u g g e s te d a v a r i e t y o f p r e v e n t i v e m easures. - 107 - Willman et^ a l . (1934) produced " s t i f f lambs" from ewes k e p t on a s p e c i f i c n a t u r a l r a t i o n and d e f i n i t e l y proved th e n u t r i t i o n a l n a t u r e o f th e d i s e a s e (Willman et_ al_. 1940). M orgulis and Spencer (1936), S him otori et. al_. (1940), MacKenzie and McCollum (1941) and Willman et. al_. (1941) r e p o r t e d t h a t wheat germ o i l , wheat germ meal, wheat b ran and a l p h a - t o c o p h e r o l p r o t e c t e d guinea p i g s , r a b b i t s and lambs from n u t r i t i o n a l muscular d y s t r o p h y . Willman ert a l . (1945, 1946) p r e v e n t e d and cured " s t i f f lambs" by a d m i n i s t r a t i o n o f pure a l p h a - t o c o p h e r o l or i t s d e r i v a t i v e s to p r e g n a n t ewes or a f f e c t e d lambs im m ediately after b irth . W h i t t i n g a t a l . (1949) a n a ly z e d the blood co lo str u m and m il k f o r t o c o p h e r o l s from ewes on a v i t a m i n E d e f i c i e n t d i e t and blood t o c o p h e r o l l e v e l from a f f e c t e d lam bs. The v a l u e s were up to 48 p er c e n t lower when compared w ith normal c o n t r o l ewes and lambs. I n view of t h e s e f i n d i n g s i t was b e l i e v e d t h a t n u t r i t i o n a l muscular d y s tr o p h y d e s c r i b e d as " S tiff-L a m b " d i s e a s e was due to v i t a m i n E d e f i c i e n c y . The r o l e o f v i t a m i n E i n p r o t e c t i n g th e normal s t r u c t u r e o f th e m uscle, and t h e bioc hem ical changes i n r e g a r d t o t h e c o m p o s itio n and p h y s io l o g y of th e muscle f i b e r due to v i t a m i n E d e f i c i e n c y a r e unknown. There were, however, some f a c t s which p e r m i t t e d s p e c u l a t i o n on t h e p o s s i b l e r o l e of t h i s v i t a m i n i n r e g a r d to muscle d y s t r o p h y . The most s t r i k i n g c h a r a c t e r i s t i c of t o c o p h e r o l s i s t h e i r a n t i o x i d a n t a c t i v i t y . 108 - I f t o c o p h e r o l s would r e t a i n t h e i r a n t i o x i d a n t a c t i v i t y i n v iv o i t could be r i g h t l y th o u g h t t h a t most o f t h e i r b i o l o g i c a l f u n c t i o n s would be c e n t e r e d around t h i s p r o p e r t y i n the m etabolism o f f a t . Dam (1949) i n a s e r i e s o f e x p e rim e n ts d e m o n s tr a te d t h e p r e s e n c e o f p e r o x i d e s i n the body f a t o f c h i c k s and r a t s r e a r e d on a v i t a m i n E low d i e t and supplemented w ith 5 p e r c e n t cod liv e r o il. The c o n t r o l a n i m a l s on t h e same d i e t su pplem en ted w i t h 2 . 5 mg p e r c e n t of a l p h a - t o c o p h e r o l a c e t a t e showed no p e r o x i d e v a l u e and absence o f t h e d e f i c i e n c y symptoms. He s u g g e s t e d t h a t d e g e n e r a t i o n o f the muscle was p r i m a r i l y due to some p r o d u c t which r e s u l t e d from t h e p r o c e s s which th e h i g h l y u n s a t u r a t e d f a t t y a c i d s underwent i n t h e c e l l s w h ile v i t a m i n E was l a c k i n g . F u r t h e r e v id en ce o f a n t i o x i d a n t a c t i v i t y o f t o c o p h e r o l s I n vivo was d e m o n s tr a te d by H a r r i s et al. (1 9 4 4 ). T o c o p h e ro ls e x e r t e d a pronounced s p a r i n g a c t i o n on v i t a m i n A i n r a t s fed a v i t a m i n A low d i e t . W h i t t i n g e_t a l . (1949) s t u d i e d the e f f e c t o f t o c o p h e r o l s on t h e v i t a m i n A mammary and p l a c e n t a l t r a n s f e r i n the sh e e p , g o a t and p i g . The o n ly i n c r e a s e o f v i t a m i n A was noted i n th e l i v e r o f t h e newborn lamb. H a r r i s et_ a l . (1947) i n c r e a s e d b u t t e r f a t p r o d u c t i o n i n d a i r y cows by s u p p le m e n t in g t h e r a t i o n w i t h 5 0 0 -1 ,0 0 0 mg of alp ha-tocopherol• The s p a r i n g or p r o t e c t i v e a c t i o n of t o c o p h e r o l s on f a t i t s e l f was shown by Menschik e t a l . ( 1 9 4 9 ). Mice on a h i g h - 109 - v i t a m i n E d i e t made s u b s t a n t i a l g a i n s i n body weight i n the form o f f a t , w h ile f a t o f t h o s e on th e v i t a m i n E f r e e d i e t was l o s t and r e p l a c e d w i t h u n s a t u r a t e d f a t t y a c i d s , phospho­ l i p i d s and p r o t e i n . Roderuck e t a l . (1949) s t u d i e d r a t h e r e x t e n s i v e l y th e che m ic a l and enzymic a l t e r a t i o n s i n muscles a f f e c t e d by experim ental dystrophy. QOg v a l u e s o f m uscles from v i t a m i n E d e f i c i e n t r a b b i t s , g u in e a p i g s and h a m s te r s were g r e a t l y increased. T h is s u g g e s t e d t h a t a l p h a - t o c o p h e r o l was i n v o l v e d in the o v e r a ll c e l l u l a r o x id a tio n . The n a t u r e of t h e sub­ s t r a t e i n t h e muscle u n d e r g o in g i n c r e a s e d com bustion d u r i n g v i t a m i n E d e f i c i e n c y was unknown. However, d e c r e a s e d c r e a t i n e c o n t e n t of th e m u sc le, c r e a t i n u r i a and i n c r e a s e d c r e a t i n e i n t h e l i v e r , r e p o r t e d a l s o by G o e tt s c h and Brown (1932) i n r a b b i t s , i n d i c a t e d t h a t p r o t e i n m etabolism was i n v o l v e d . A n a ly s e s o f t h e muscle t i s s u e s f o r v a r i o u s amino a c i d s showed g lu ta m in e t o be e s p e c i a l l y abundant i n th e normal muscle a s f r e e amino a c i d . The g lu ta m in e c o n t e n t o f d y s t r o p h i c m uscles was markedly l o w e r e d . However, t h e t o t a l n o n - g lu ta m in e amino a c i d c o n t e n t seemed t o be unchanged. Roderuck p r o v i d e d f u r t h e r e v i d e n c e i n s u p p o r t o f th e i d e a t h a t p r o t e i n r a t h e r t h a n f a t m e tab o lism was i n v o lv e d i n t h e p r o c e s s o f muscle d e g e n e r a t i o n . I t i s a w e ll known f a c t t h a t b i o t i n i s d e s t r o y e d i n t h e p r e ­ se n ce o f a u t o - o x i d i z i n g u n s a t u r a t e d f a t s . I f tocopherol a c t e d a s an a n t i o x i d a n t i n v i v o , i t s absence would promote a u t o - o x i d a t i o n and p r o d u c t i o n o f u n s a t u r a t e d f a t t y a c i d s and - 110 - p e r o x i d e s which would d e s t r o y b i o t i n . However, b o th f r e e and t o t a l b i o t i n v a l u e s o f normal and d y s t r o p h i c m uscles from h a m s t e r s , g u in e a p i g s and r a b b i t s v a r i e d w i t h i n t h e l i m i t s o f error. The a u t h o r s a d m i t t e d t h a t i f f a t t y a c i d p e r o x i d e s were i n v o l v e d i n muscle d y s tr o p h y th e p e r o x i d e s f o r some reaso n did not d e s tro y b i o t i n in vivo. Roderuck, i n t h e c o u r s e o f t h e s e s t u d i e s , a l s o o b se rv e d a marked d e c r e a s e i n th e a c t i v i t y o f t h e s u c c i n i c dehydrogenase system . t i o n was e x p l a i n e d by Ames and R i s l e y ( 1 9 4 9 ). T h is i n h i b i ­ A lpha-tocopherol p r e c i p i t a t e d i o n i c ca lc iu m which i s an a c t i v a t o r o f d ip h o s p h o pyridine n u cleo tid ase. I t was i n t e r e s t i n g t o note t h a t a l l t h r e e t o c o p h e r o l s , a l p h a , gamma and d e l t a p h o s p h a t e s , proved t o have t h e same i n h i b i t o r y e f f e c t and an a d d i t i o n of Ca i o n s i n c r e a s e d th e a c t i v i t y o f t h e system to th e same e x t e n t . The b i o l o g i c a l a c t i v i t y of the v a r i o u s t o c o p h e r o l s , however, was found to be e n t i r e l y d i f f e r e n t . The b lo o d s t u d i e s o f t h e v i t a m i n E d e f i c i e n t lambs showed a d e f i n i t e and c o n s i s t e n t drop of the hem oglobin c o n c e n t r a t i o n th r o u g h o u t t h e e x p e r i m e n t a l p e r i o d . Three d e f i c i e n t lambs d e v e lo p e d anemia toward the end o f the ex p e rim e n t w ith v a l u e s a little o v er 5 gms o f hemoglobin per 100 ml b l o o d . Gyorgy and Rose (1949) s u g g e s t e d and s u b s t a n t i a t e d by many e x p e r i m e n t a l s t u d i e s i n . vivo and in. v i t r o a p r o t e c t i v e a c t i o n o f t o c o p h e r o l a g a in s t hem olysis. R a ts on a t o c o p h e r o l d e f i c i e n t d i e t i n ­ j e c t e d w i t h 160 mg p e r kg of a l l o x a n i n t r a p e r i t o n e a l l y - I ll d e v e lo p e d a s e v e r e h e m o g lo b in u r ia and hemoglobinemia and d i e d due to a l m o s t com plete h e m o l y s i s . c o m p l e t e l y f i l l e d w ith b lo o d . On a u t o p s y t h e k i d n e y s were S u p p l e m e n t a t i o n w ith t o c o p h e r o l d e c r e a s e d t h e m o r t a l i t y and c o m p le t e ly p r o t e c t e d a l l a n i m a l s from h e m o l y s i s . That a l l o x a n a s such and a l o n e was n o t r e s ­ p o n s i b l e f o r t h e h e m o ly sis was e v i d e n t from th e f a c t t h a t i t d i s a p p e a r e d from t h e blood s tr e a m i n two t o t h r e e m in u t e s a f t e r th e i n j e c t i o n . A llo x a n was p o s s i b l y c o n v e r t e d i n ° a number of i n t e r m e d i a t e compounds i n th e ab sen ce o f t o c o p h e r o l s . These compounds t e s t e d on v i t a m i n E d e f i c i e n t r a t s showed more or l e s s t h e same hem olyzing a c t i v i t y . T ocopherol a g a i n f u l l y p r o t e c t e d t h e a n im a ls from h e m o l y s i s . I t1 v i t r o , s t u d i e s showed t h a t u nder a p p r o p r i a t e c o n d i t i o n s blood c e l l s from v i t a m i n £ d e f i c i e n t an im a ls were c o m p le t e ly hem olyzed, w hile t h o s e o f t o c o p h e r o l supplemented a n im a ls were n o t a f f e c t e d . This e x p e rim e n t f u r t h e r d e m o n stra te d t h a t t h e p r o t e c t i o n a g a i n s t h e m o ly s is was a f u n c t i o n o f the c e l l and n o t of t h e body f l u i d . The a u t h o r s s t a t e d t h a t anemia was n o t c h a r a c t e r ­ i s t i c o f v i t a m i n E d e f i c i e n c y u n l e s s under e x tre m e ly abnormal c o n d i t i o n s o f m e tab o lism . From t h e ev id e n c e i t was co n c lu d ed t h a t t o c o p h e r o l was n e c e s s a r y to p r o t e c t th e e r y t h r o c y t e a g a i n s t h e m o ly s is by i t s a n t i o x i d a n t e f f e c t . A r a t h e r s t r i k i n g f e a t u r e of e x p e r i m e n t a l l y produced v i t a m i n E d e f i c i e n c y was th e e x t r e m e ly h i g h i n c i d e n c e of pneumonia. I n p r a c t i c a l l y e v e ry r e p o r t of " S tif f - L a m b " d i s e a s e e n c o u n t e r e d i n t h e f i e l d , pneumonia was mentioned as one o f - 11 2 - t h e symptoms or a s a c o m p l i c a t i o n which r e s u l t e d from th e disease. A l l t h e " s t i f f lambs" b r o u g h t t o t h e l a b o r a t o r y f o r n e c r o p s y showed a c t i v e or p a s s i v e pneumonia. I t was e v i d e n t t h a t a l a c k o f v i t a m i n E i n c r e a s e d the s u s c e p t i b i l i t y f o r r e s p i r a t o r y i n f e c t i o n which f i n a l l y t e r m i n a t e d i n pneu­ monia. I t was s u g g e s t e d i n c o n n e c t i o n w ith r i b o f l a v i n , t h a t th e o r g a n w i t h an e s p e c i a l l y h i g h r e q u ir e m e n t, min was a f f e c t e d f i r s t . fo r the v i t a ­ Evidence s u p p o r t i n g t h i s was a v a i l ­ a b l e i n t h e h e a r t l e s i o n s b u t no s t u d i e s were done i n r e g a r d to th e r e q u i r e m e n t and s t o r a g e o f t o c o p h e r o l s i n t h e l u n g . R e s p i r a t i o n o f b i o p s i e d s k e l e t a l muscle t i s s u e s from v i t a m i n E d e p l e t e d a n i m a l s was e x t e n s i v e l y s t u d i e d by Houchin (1942) and Houchin and M a t t i l l (1942). i n c r e a s e d up to 250 p er c e n t . The oxygen consumption Oral o r i n t r a v e n o u s a d m i n i s ­ t r a t i o n o f a l p h a - t o c o p h e r o l c o n s i d e r a b l y d e c r e a s e d t h e oxygen consumption i n one hour and an a l m o s t normal r e s p i r a t i o n was r e s t o r e d i n 22 h o u r s . The oxygen consum ption was a l s o lo w e r e d by 40 p e r c e n t when a l p h a - t o c o p h e r o l was added t o th e s a l i n e medium w i t h d y s t r o p h i c muscle s l i c e s from r a b b i t s and h a m s t e r s . K au n itz and Pappenheimer (1943) measured t h e t o t a l oxygen consumption o f a group of v i t a m i n E d e f i c i e n t r a t s and a group supplem ented w i t h a l p h a - t o c o p h e r o l . The t o t a l oxygen consump­ t i o n o f th e d e f i c i e n t group was c o n s i d e r a b l y h i g h e r . S in c e s k e l e t a l m uscles a c c o u n t f o r n e a r l y h a l f o f t h e body w e i g h t , i t c o u ld be p o s t u l a t e d t h a t d u r i n g v i t a m i n E d e f i c i e n c y t h e - 113 - oxygen r e q u i r e m e n t i s i n c r e a s e d g r e a t l y . T h i s , no d o u b t , would r e s u l t i n an e x t r a o r d i n a r y s t r e s s on t h e l u n g s and h e a r t and i n c r e a s e th e s u s c e p t i b i l i t y t o a l t e r a t i o n . The c h a r a c t e r i s t i c l e s i o n s i n t h e h e a r t were c o n s i d e r e d pathognomonic o f e x p e r i m e n t a l l y in d u c e d v i t a m i n E d e f i c i e n c y i n th e lamb. T h is was to some e x t e n t i n c o n f l i c t w ith ob­ s e r v a t i o n s and r e p o r t s on the d i s e a s e o c c u r r i n g u n d er f i e l d conditions. A lthough o b se rv e d and d e s c r i b e d , n o t a l l workers c o n s i d e r e d d e g e n e r a t i o n o f h e a r t muscle t y p i c a l of v i t a m i n E deficiency. E i t h e r th e l e s i o n s were n o t r e p o r t e d i n some i n s t a n c e s or were n o t p r e s e n t a t a l l . In the course of t h i s s t u d y i t was o b s e rv e d t h a t lambs from t h e f i e l d a t n e c ro p s y showed much more pronounced d e g e n e r a t i o n of the s k e l e t a l m usc les and few er and l e s s d i s t i n c t h e a r t l e s i o n s t h a n t h e lambs d e p l e t e d on t o c o p h e r o l d e f i c i e n t d i e t . T h is r e v e r s a l was e x p l a i n e d by a l a c k o f e x e r c i s e i n lambs k e p t i n sm all pens u n d er l a b o r a t o r y c o n d i t i o n and t h e e x c e s s i v e e x e r c i s e of lambs r u n n in g w ith ewes. I t was q u i t e p o s s i b l e t h a t t h e s k e l e t a l m usc les i n lambs r u n n in g f r e e l y on p a s t u r e , due t o i n c r e a s e d metabolism, were d e p l e t e d sooner th a n i n th e lambs c o n f i n e d t o sm a ll p e n s . Some e x p l a n a t i o n f o r the delay ed d e g e n e r a t i o n o f th e c a r d i a c muscle as compared w ith s k e l e t a l muscle was r e p o r t e d by Mason ( 1 9 4 2 ). The a n a l y s e s o f t h e h e a r t and v a r i o u s o r g a n s f o r t o c o p h e r o l showed t h a t h e a r t t i s s u e from normal o r d e f i ­ - 114 - c i e n t r a t s c o n t a in s tw ice a s much o f t h e v i t a m i n as any o t h e r t i s s u e i n c l u d i n g th e l i v e r . The a u t h o r f u r t h e r e x p l a i n e d t h e p o s s i b l e s t e p s l e a d i n g to t h e h e a r t l e s i o n s and e v e n t u a l failu re. The o v e r a l l d e f i c i e n c y g r e a t l y i n c r e a s e d the demand f o r oxygen and re d u c e d the c a p a c i t y to work. T h is c o n s e q u e n t l y c aused d i l a t a t i o n and weakness o f t h e o rg an which i n c r e a s e d th e s u s c e p t i b i l i t y f o r p a th o lo g ic a l changes. I t was of i n t e r e s t t o n o t e t h a t l o s s o f a p p e t i t e , r e t a r d e d grow th, d i a r r h e a and e m a c i a t i o n , t h e symptoms of most o f t h e v i t a m i n d e f i c i e n c i e s , were n o t o b s e rv e d i n v i t a m i n E d e f i c i e n c y produced e x p e r i m e n t a l l y or o b se rv e d i n the f i e l d . T h is s t r o n g l y s u g g e s t e d t h a t th e d e f i c i e n c y d e s c r i b e d was due t o l a c k o f v i t a ­ min E and v i t a m i n E a l o n e . SUMMARY AND CONCLUSIONS N u t r i t i o n a l m uscular d y s t r o p h y i n th e lamb was pro d u ced by u s i n g a l i q u i d , p u r i f i e d v i t a m i n E d e f i c i e n t d i e t . The s o u r c e o f f a t i n the d i e t of t h e f i r s t group was commercial l a r d w h ile i n t h e second and t h i r d groups m o le c u l a r d i s t i l l e d l a r d , v e r y low i n v i t a m i n E, was u s e d . The symptoms o f v i t a m i n E d e f i c i e n c y were c h a r a c t e r i z e d by d i s t u r b a n c e s i n l o c o m o t i o n , s t i f f n e s s , p a r a l y s i s , s u s c e p ­ t i b i l i t y t o pneumonia and sudden d e a t h . Blood a n a l y s i s showed lo w e re d hem oglobin c o n c e n t r a t i o n and low t o c o p h e r o l l e v e l s . P o s t mortem e x a m in a tio n s showed d y s tr o p h y o f s k e l e t a l and c a r d i a c muscle and f a t t y d e g e n e r a t i o n of th e l i v e r and k id n e y . The l e s i o n s i n t h e h e a r t were i n v a r i a b l y most p r o ­ nounced i n t h e r i g h t v e n t r i c l e . A d d i t i o n o f 100 mg a l p h a - t o c o p h e r o l to t h e d i e t e v e ry o t h e r day p r o t e c t e d 12 p o s i t i v e c o n t r o l lambs from m u sc u la r d y s t r o p h y and g r e a t l y a l l e v i a t e d th e symptoms o f d e p l e t e d anim als in th re e to f iv e days. M ic ro s c o p ic s t u d i e s showed a l ­ most com plete r e g e n e r a t i o n of t h e muscle a f t e r 30 days o f therapy. The symptoms, g r o s s and m ic r o s c o p i c p a t h o l o g y o f th e m u s c u l a t u r e o f lam bs, a f f e c t e d w ith " S t i f f - L a m b " d i s e a s e u n d e r f i e l d c o n d i t i o n s were a l s o s t u d i e d . 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M o r ris o n and P. O l a f s o n . R e l a t i o n o f F e e d in g and Management to th e Cause o f t h e " S tif f - L a m b " D i s e a s e . New York ( C o r n e l l ) Agr. Exp. S t a . A nnual. R p t. p. 112, 1941. Willman, J . P . , F. B. M o r ris o n , L. A. Maynard and P. O l a f s o n . R e l a t i o n of F eed in g and Management t o the Cause o f th e " S tif f - L a m b " D i s e a s e . New York ( C o r n e l l ) Agr. Exp. S t a . Annual R p t. p . 120, 1939. Willman, J . P . , F. B. M o rriso n and P. O l a f s o n . R e l a t i o n o f F eed in g and Management to t h e Cause of t h e " S tif f - L a m b " D i s e a s e . New York ( C o r n e l l ) Agr. Exp. S t a . Annual R p t . p . 79, 1935. Willman, J . P . , F. B. M o rriso n and P. O l a f s o n . R e l a t i o n o f F e ed in g and Management to th e Cause o f t h e " S tif f - L a m b " D i s e a s e . New York ( C o r n e l l ) Agr. Exp. S t a . Annual R p t. p. 84, 1936. F ig . 8. Lamb N o . 3 9 . A t y p i c a l s t a n c e o f t h e lamb i n e a r l i e r s t a g e s of v ita m in E d e f i c i e n c y . Note t h e a r c h e d b a c k . - 123 - F ig . 9. Lamb N o . 3 4 . L a te r stage of v itam in E d e f ic ie n c y . l o s t e q u i l i b r i u m and f e l l . The lamb The n o r m a l a p p e a r a n c e o f t h e h e a d s u g g e s t s no p h y s i c a l p a i n . - 124 - F ig . 10. Lamb N o. 1 2 . L e s io n s w ith in the r i g h t v e n t r i c l e . N ote t h e c o m p le t e d e g e n e r a t i o n o f t h e w a l l . - 125 - METRIC I F ig . XI. Lamb No. 2 4 . R ight v e n t r i c l e d eg en eratio n . sh o w in g e x t e n s i v e Z e n k e r ' s The d i a m e t e r o f t h e l e s i o n a t t h e a p e x o f t h e h e a r t was a p p r o x i m a t e l y 12 mm. - 126 - F ig . 12, Lamb Mo. 1 2 . The d e g e n e r a t i o n i n v o l v e d t h e whole c a r d i a c w all. N ote t h e w h i t i s h l e s i o n s on t h e surface. - 127 - METRIC I F ig . 13. Lamb No. 1 4 . L o s s o f s t r i a t i o n and e x t e n s i v e c e l l u l a r i n f i l t r a t i o n w i t h i n d e g e n e r a t e d m u sc le fib ers. P a r t s of th e m uscles are i n t a c t . H e m a t o x y l i n and E o s i n s t a i n x 1150 F ig . 14. Lamb N o. 6 . The d i s a p p e a r i n g m u s c le f i b e r s and p r o l i f e r a t i o n of connective t i s s u e . H e m a t o x y l i n and E o s i n s t a i n x 170 - 129 - ♦ V 4 > - • » i J / X W ,/ ' V," A ' 4* \ijv # V U>r 'J— • c r ' • A ' ,V ; ' •*»! , ♦f r ':* , ;f x , ’ - >/ 4 > J • * •> ■ * / x -i « s Y #* * / * t t L s*4 ' A / F ig . 15. Lamb N o . 1 8 . I n t e r s t i t i a l edema i n t h e s k e l e t a l m u s c l e . F r a g m e n t a t i o n o f t h e m uscle f i b e r s i s i n progress. H e m a t o x y l i n and E o s i n s t a i n x 170 - 130 - F i g . 16 * Lamb N o . 2 6 . Beginning of muscle d egen eration. Note the increased g ra n u la r ity o f the muscle f i b e r s . Hematoxylin and Eosin s t a i n x 750 - 131 - 4 » F ig . 17. Lamb N o . 1 1 . V a c u o liz a tio n of the a f f e c t e d muscle f i b e r . Hematoxylin and Eosin s t a i n x 1150 - 132 - F ig . 18. Lamb N o . 1 4 . H i s t i o c y t i c i n f i l t r a t i o n in an area of d egen eration and n e c r o s i s . Hematoxylin and Eosin s t a i n x 750 133 - F ig . 19. Lamb N o . 8 . H i s t i o c y t e s and giant c e l l in the area of d egen eration. Hematoxylin and Eosin s t a i n x 1150 - 13 4 - ri * * * ; < F ig . 20. Lamb N o . 1 7 . Young muscle f i b e r s showing l o n g it u d in a l stria tio n . The n u c le i are lo c a t e d c e n t r a l ly and at the end o f the muscle f i b e r . The lamb was not t r e a t e d . Hematoxylin and Eosin s t a i n x 750 - 135 - F ig . 21. Lamb N o . 1 2 . Degeneration and n ec r o s is extending from the endocardium in to myocardium. Hematoxylin and Eosin s t a i n x 50 - 136 - F ig . 22. Lamb N o . 1 9 . Heart muscle showing fragmentation of the f i b e r s and presence of h i s t i o c y t e s . The a d jo in in g blood v e s s e l i s congested. Hematoxylin and Eosin s t a i n x 750 - 137 - F ig . 23. Lamb N o . 1 2 . Cardiac muscle showing degeneration and n ecrosis. The Purkinje f i b e r s seem to be normal. Hematoxylin and Eosin s t a i n x 170 - i •• \ S ’ i 138 - V •* . . * > ■ • • • I‘ ■. -* | * ,| ^ "V"' * ' ^ r . ; A ' ' */ * ir , ' > *•/ 'J * ] S '4 f * v , . } n •» , i *•• '.Jy !v i l i t - " ■' ,>V -i rj •* 1 iff R a *, * i).-V ’ i . i-V . - / f '* f * # • i • . l ) >< « i / . 'I. /w (j l#/ /. » ]*r. . -I *■ * t #/ ♦ m • I * F ig . 24. Lamb N o . 47. D e g e n e ra tin g c a r d i a c m uscle. T hese young m u s c le s t r a n d s a r e s i m i l a r i n a p p e a r a n c e t o c a p i l l a r i e s b u t t h e p r o t o p l a s m i s con­ t i n u o u s w i t h t h e m u s c le f i b e r s . H e m a t o x y l i n and E o s i n s t a i n x 750 - 139 f •V * I «• \ > * F ig . 25. Lamb N o . 4 3 . F a t t y d e g e n e r a tio n of the k id n e y . C ells o f th e co n v o lu ted tu b u le s a r e f i l l e d w ith f a t d ro p lets. F r o z e n s e c t i o n Sudan IV s t a i n x 170 140 - .4 '»* * . ^ * •‘J r j f i * * H *. n r \ * %S i t — • f y •* ' , * V.V • Utif. NJ v. ' ' ' 1 j ' F ig . 26. Lamb N o. 2 7 . M e d u l l a o f t h e k i d n e y show ing i n t e r s t i t i a l edema and c o n g e s t i o n o f b l o o d v e s s e l s . H e m a t o x y l i n and E o s i n s t a i n x 600 - 141 - 1 1 • V/t /\ ©rV;* * 4 ! * * - > 4 - v T ' * * " » f B •is§jir «*s,$♦•'' i "‘' \ T w 'iV 'Vr * •; %' t #s i r . i .“ » / « . - ' * » * \ \ 4 % F ig . 27. Lamb No. 3 9 . G iant c e l l s w ith en g u lfe d d e b r i s . In ten siv e c e l l u l a r i n f i l t r a t i o n i s also noted. H e m a t o x y l i n and E o s i n s t a i n x 750 « & « f . F ig . 28. Lamb N o . 3 4 . Young m u s c le f i b e r s b e g i n n i n g t o show c r o s s striatio n . H e m a t o x y l i n and E o s i n s t a i n x 750 143 - F ig . 29. Lamb N o . 4 3 . More m a t u r e m u sc le f i b e r s sh o w in g c r o s s stria tio n . Note t h e n u c l e i a t t h e t i p o f t h e y o u n g m u sc le f i b e r s . The n u c l e i a r e c e n tra lly lo cated . H e m z t o x y l i n and E o s i n s t a i n x 750 144 - F ig . 30. Lamb N o . 5 0 . F u lly regenerated muscle f i b e r s are ch aracter­ iz e d by pronounced c r o s s s t r i a t i o n . Hematoxylin and Eosin s t a i n x 750 - 145 - F ig . 31. Lamb N o. 4 2 . Regenerating muscle with numerous n u clei arranged in a row i n the center o f the muscle f i b e r . Hematoxylin and Eosin s t a i n x 1150 146 - ✓ 4 F ig . 32. Lamb N o . 4 2 . Almost f u l l y regenerated muscle from a lamb tr e a te d with alpha-toeopherol for 40 days. Note the in creased number of muscle n u c l e i . Hematoxylin and Eosin s t a i n x 600 - 147 - F ig . 33. Lamb N o. 1 3 . Transversal s e c t i o n of the s k e l e t a l muscle from a p o s i t i v e control lamb. Hematoxylin and Eosin s t a i n x 170 mmm » H 1«1 % a Se* sI 14 1 ♦ t F ig . 34. Lamb N o. 4 0 . Longitudinal s e c t i o n o f the s k e l e t a l muscle from a p o s i t i v e control lamb. Hematoxylin and Eosin s t a i n x 170 - 149 - I * • • j> ' • H t ' 1 i '! >' • V ( ' i 1 ' j. i v • 1 .! F ig . 35. F i e l d Case. Z e n k e r ' s d e g e n e r a t i o n o f th e s k e l e t a l m uscle. The a r e a i s i n f i l t r a t e d w i t h phagocytic c e l l s . H e m a t o x y l i n and E o s i n s t a i n x 170 - 150 - F ig . 36. F ie ld Case. Loss of s t r i a t i o n , f r a g m e n t a t i o n and c e l l u l a r i n f i l t r a t i o n o f t h e m u s c le f i b e r s . Some m uscle f i b e r s i n the a r e a a r e norm al. H e m a t o x y l i n and E o s i n s t a i n x 1150 - 151 - F ig . 37. F i e l d Case. R e g e n e r a t i o n o f m u s c le f i b e r i n t h e a r e a of d eg e n e ra tio n . H e m a t o x y l i n and E o s i n s t a i n x 1150 - 152 - I V' * ■% * i » i 0 F ig . 38. F i e l d Case. H eart m uscle. fib ers. D e g e n e r a t i o n o f t h e m uscle Note i n c r e a s e d c e l l u l a r a c t i v i t y and n o r m a l l y a p p e a r i n g P u r k i n j e f i b e r s . H e m a t o x y l i n and E o s i n s t a i n x 170 153 - S $ k' 1 W ' . F ig . 39. F i e l d Case. C a r d i a c m uscle r e g e n e r a t i o n . New m u sc le f i b e r s a r e g r o w in g i n t h e a r e a o f degeneration. H e m a t o x y l i n and E o s i n s t a i n x 750 - 154 - F ig . 40. F ie ld Case. E x t e n s i v e l e s i o n s i n t h e e n d o c a r d iu m an d myocardium from a lamb a f f e c t e d w i t h "S tiff-L am b" d is e a s e . - 155 - METRIC I F ig . 41. F i e l d Case. W h i t i s h l e s i o n s a f f e c t i n g most o f t h e rig h t v e n tric le . - METRIC I 156 - F ig . 42. F ie ld Case, (same a s F i g . to--). D if fu s e involvem ent o f th e l e f t v e n t r i c l e and a t r i u m c h a r a c t e r i z e d by w h i t i s h a r e a s . Note t h e l e s i o n s t h r o u g h o u t t h e c a r d i a c w all. - 157 - METRIC I