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A biological study of the protein and riboflavin content of British Columbia fishmeals 1942

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 A BIOLOGICAL STUDY OF THE PROTEIN AND RIBOFLAVIN CONTENT OF BRITISH COLUMBIA FISHMEALS by Jean M. P r a t t A T h e s i s submitted i n P a r t i a l F u l f i l m e n t of The Requirements f o r the Degree of MASTER OF SCIENCE IN AGRICULTURE i n the Department of POULTRY HUSBANDRY The U n i v e r s i t y o f B r i t i s h A p r i l , 1942 Columbia ACKNOWLEDGMENTS The w r i t e r wishes to take t h i s o p p o r t u n i t y t o thank Mr. Jacob B i e l y , o f the Department of P o u l t r y Husbandry, f o r h i s guidance and h e l p f u l s u g g e s t i o n s i n c a r r y i n g out t h i s i n v e s t i g a t i o n ; Mr• E. A. L l o y d , Head o f the Depart- ment of P o u l t r y Husbandry, f o r h i s v a l u a b l e a s s i s t a n c e i n the p r e p a r a t i o n of t h i s t h e s i s ; and Mr. C a r l Hedreen, of the Canadian F i s h i n g Company, Vancouver, B eC. , f o r s u p p l y i n g the f i s h m e a l s and by-products used i n the study. t • if ' • . ' • . f j ;j Table of Contents i \ I INTRODUCTION . ... . 1 •U ' •" . . • • • 1 " " " || EISHMEALS (a) Raw Mater i a l s ..«..••••••••»»»•«»••«»««««•• 3 (b) Methods of Manufacture » 5 i (c) P r o t e i n s ..,...»•••••••••©•••.••••••••••••• 7 i (d) V i t a m i n G 1 1 i (©) M i n s r a l s • 13 1 ( f ) E f f e c t s of Method of Manufacture « 17 I (g) Uses of F i sh. Meals • <>•••«•••••••••••••••••• 18 4 • I. SERIES 1 ll (a) The Experiment 1 9 , (b) M a t e r i a l s and Methods • • • 2 0 (c) R e s u l t s 2 3 Experiment 1 ............................ 24* Experiment 2 . . . . . . . . . 2 6 1 Experiment 3 • 2 8 (d) D i s c u s s i o n ^ ..»»»•«•.. • • 2 9 SERIES 2 (a) The Experiment .••..••..«...•.•.*...«•«.>•• 3 1 (b) M a t e r i a l s anCL Methods ••••••••••• ••••••••»• 31 (c) H@sults •«•••••••«•••«•«••«•••»••••••*••••• 32 Exp ©r i meD.t 1 • • • » • • • « • • • • • • • • © • • • • • • • • • • « • 3 2 Exp6riitt©nt 2 • • • • • • « • • • • * • • • • • • • • • • • • • • • • 37 Exp©3?jLTiient 3 • • • • • • • 38. (d) D i s c u s s i o n •••••••••••••••••••••••••••••••« 4-0 STJMMARY •••*«•••••«• • • • • • •©•••• ••••••••• • 4* 2 REFiJJrtEN CES ••••••••••• •»••••• •••••«•••••••••••••• • 44 A BIOLOGICAL STUDY OF THE PROTEIN AND RIBOFLAVIN CONTENT OF BRITISH COLUMBIA FISHMEALS INTRODUCTION The p r o t e i n content o f the common g r a i n s i s d e f i c i e n t i n both q u a n t i t y and q u a l i t y t o meet the n u t r i t i v e r e q u i r e - ments o f growing c h i c k s , l a y i n g b i r d s , and b r e e d i n g s t o c k . Hence, the p r o t e i n c o n t a i n e d i n the g r a i n i s g e n e r a l l y s u p p l e - mented by the a d d i t i o n of p r o t e i n c o n c e n t r a t e s . These may be of animal o r i g i n , such as f i s h m e a l , meat meal or m i l k p r o d u c t s , or to a more l i m i t e d e x t e n t o f v e g e t a b l e s o u r c e , such as soyabean, l i n s e e d or c o t t o n s e e d meals. The purpose o f the p r o t e i n c o n c e n t r a t e s i s t w o f o l d : f i r s t , to i n c r e a s e the p r o t e i n content o f the r a t i o n from t h e 1 0 - 1 2 / - o r d i n a r i l y p r o - v i d e d by t h e g r a i n s or g r a i n by-products t o 1 8 - 2 0 ^ ; and second, t o complete and t o bala n c e the amino a c i d content o f the r a t i o n . The e s t a b l i s h e d p r a c t i c e i n f e e d i n g c h i c k s has been, u n t i l r e c e n t l y , t o r e p l a c e a c e r t a i n amount o f meat meal and f i s h m e a l w i t h d r i e d skim m i l k i n or d e r t o d e r i v e the optimum growth response from the r a t i o n . Asmundson and B i e l y , f o r example ( ? ) , r e p o r t e d t h a t a combination o f d r i e d s k i m m i l k and salmon as the source o f p r o t e i n was more e f f e c t i v e than salmon a l o n e , and at a t o t a l o f 7.5 and lof. seemed to have a supplementary e f f e c t upon one another. Furthermore, B i e l y and Asmundson ( 5 ) a l s o r e p o r t e d t h a t when f i s h m e a l c o n s t i t u t e d 7 , 5 / 0 of t h e ' p r o t e i n of the r a t i o n , the a d d i t i o n of more than 5 / i s k immilk d i d not r e s u l t i n i n c r e a s e d growth. At t h a t time the advantage o f f e e d i n g m i l k i n a d d i t i o n to f i s h m e a l was supposed to be due t o the a d d i t i o n of c e r t a i n amino a c i d s found i n the m i l k and not i n t h e f i s h meal* I t was tho u g h t , moreover, t h a t the 5 f « l e v e l of m i l k p r o v i d e d a l l the e x t r a e s s e n t i a l amino a c i d s . I t seems more l i k e l y now, however, t h a t t h i s e x t r a growth response was due t o the a d d i t i o n a l r i b o f l a v i n s u p p l i e d a t t h a t l e v e l by the m i l k ; and t h a t the amount of v i t a m i n G p r o v i d e d by t h e d r i e d s k i m m i l k at the 51° l e v e l would appear t o be adequate f o r optimum growth. The type o f f i s h m e a l used by B i e l y and Asmundson ( 3 , 5 ) i n t h e i r experiments was a p p a r e n t l y c o n s i d e r a b l y lower i n r i b o - f l a v i n content than d r i e d skimmilk.' More r e c e n t l y such improvements have been made i n t h e method o f manufacture as t o a l l o w f i s h m e a l s to r e t a i n a g r e a t e r percentage o f t h e i r n u t r i - t i v e v a l u e . I t i s known, moreover, t h a t due t o the c o n s t i t u - t i o n of f i s h m e a l s , and more p a r t i c u l a r l y t o t h e organs and t i s s u e s i n c l u d e d i n them, t h e y may v a r y c o n s i d e r a b l y i n v i t a m i n G c o n t e n t . Since l i t t l e i n f o r m a t i o n was a v a i l a b l e as to t h e r e l a t i v e p o t e n c i e s o f d i f f e r e n t f i s h m e a l s and by- products of the f i s h i n g i n d u s t r y i n B r i t i s h Columbia, an experiment was begun i n the summer of 1940 t o i n v e s t i g a t e the va l u e of these p r o d u c t s . A c c o r d i n g l y , the f i r s t s e r i e s i n v o l v e d a t e s t of the e f f e c t s o f some of these supplements on the r a t e o f growth of c h i c k s to f i v e weeks of age. L a t e r a second s e r i e s was undertaken t o i n v e s t i g a t e the r e l a t i v e supplementary v a l u e s of s y n t h e t i c and n a t u r a l r i b o f l a v i n . FISHMEALS (a) Raw M a t e r i a l s The i n c r e a s i n g use o f f i s h m e a l s as a p o u l t r y and l i v e s t o c k feed has been accompanied by a growing r e c o g n i t i o n o f the v a l u e o f marine products as n a t u r a l sources o f p r o t e i n of a v e r y h i g h q u a l i t y . There a r e , however, c o n s i d e r a b l e v a r i a - t i o n s i n the u l t i m a t e v a l u e o f the p r o d u c t , depending upon the nature o f t h e s p e c i e s of f i s h from which i t was prepared, and a l s o upon the method by which i t was manufactured. S i n c e t h e advent and expansion o f the knowledge o f v i t a m i n s i t has been r e c o g n i z e d t h a t heat, l i g h t and o x i d a t i o n may bear a c o n s i d e r a b l e i n f l u e n c e upon th e f i n a l n u t r i t i v e v a l u e o f the p r o d u c t . The raw m a t e r i a l s from which f i s h m e a l s are manufactured f a l l i n t o two d i s t i n c t c l a s s i f i c a t i o n s , "based upon the p h y s i o l o g i c a l c h a r a c t e r i s t i c s o f t h e f i s h from which the pro- duct o r i g i n a t e d ( 1 9 ) • The f i r s t c l a s s , the o i l y f i s h m e a l s , i n c l u d e s the m a j o r i t y o f those manufactured from f i s h (salmon, p i l c h a r d , t u n a , m a c k e r e l , menhaden and h e r r i n g ) which s t o r e the f a t throughout the t i s s u e s o f the body and have com- p a r a t i v e l y s m a l l l i v e r s . The second c l a s s , the n o n - o i l y meals are prepared from f i s h which have l o c a l i z e d f a t - d e p o t s i n the l i v e r and o t h e r v i t a l organs. In t h i s c l a s s are cod, haddock, p o l l o c k , hake and cusk. T h i s l a r g e v a r i a t i o n i n the o i l content of the raw m a t e r i a l s causes a c o r r e s p o n d i n g v a r i a t i o n i n the f i s h m e a l p r o d u c t . Manning p u b l i s h e d a b i b l i o g r a p h y of the comparisons made between meals prepared from d i f f e r e n t s p e c i e s of f i s h u n t i l t h a t t i m e , 1 9 3 0 . In g e n e r a l , the f i s h m e a l s w i t h h i g h o i l content w i l l have r e l a t i v e l y low p r o t e i n c o n t e n t , a l t h o u g h t h e r e are a few e x c e p t i o n s to t h i s statement. However, the o i l has a d e f i n i t e v i t a m i n v a l u e which must be c o n s i d e r e d , s i n c e i t e n t e r s i n t o the r e d u c t i o n phase of manufacture. " A c c o r d i n g t o e x i s t i n g p r a c t i c e , " s t a t e H a r r i s o n et a l , ( 1 9 ) "the dehydrated r e s i d u e a f t e r o i l removal u s u a l l y c o n t a i n s 5 t o 15 percent f a t , depending upon the raw m a t e r i a l used and the e f f i c i e n c y o f the o i l e x t r a c t i o n p r o c e s s . From t h i s i t can be seen t h a t i n the r e d u c t i o n of the two t y p e s of waste, d i f f e r e n t problems are i n v o l v e d . I n t h e case o f the n o n o i l y waste t h e r e i s the r e - quirement of d e h y d r a t i o n a l o n e , and i n t h e case of o i l y waste t h e r e i s the added problem of o i l removal." In a d d i t i o n t o the lower p r o t e i n c o n t e n t , t h e r e i s another o b j e c t i o n t o a h i g h f a t content i n f i s h m e a l s - namely t the f a c t t h a t they t e n d to become r a n c i d through o x i d a t i o n and d e t e r i o r a t i o n upon s t a n d i n g . Ewing ( 1 5 ) s t a t e s t h a t i t has been the e x p e r i e n c e of t e c h n o l o g i s t s t h a t i n o r d e r f o r a f i s h m e a l t o remain a s t a b l e commodity d u r i n g s t o r a g e and - 5 - h a n d l i n g , i t should not c o n t a i n more than 6f° f a t o r 6f. m o i s t u r e . E x c e s s i v e percentages of e i t h e r of the s e f a c t o r s i s l i a b l e t o cause a r a p i d d ecomposition and d e t e r i o r a t i o n of the n u t r i t i v e v a l u e , as w e l l as the danger of o v e r h e a t i n g . A c c o r d i n g t o the Feeding S t u f f s Act of Canada, 1937, (l6) i t i s p r o v i d e d t h a t f i s h m e a l or any o t h e r product (except l i v e r meal) of f i s h o r f i s h waste be guaranteed on a l a b e l as to i t s minimum amount of crude p r o t e i n ; maximum amount o f crude f a t ; maximum amount o f crude f i b r e i f i n excess o f 2 y » ; and maximum amount o f s a l t ( N a C l ) . F i s h l i v e r meals need o n l y show l a b e l s g u a r a n t e e i n g the minimum amounts o f crude p r o t e i n and minimum and maximum amounts o f crude f a t . By d e f i n i t i o n , f i s h m e a l i s "the c l e a n , d r i e d , ground r e s i d u e , c o n t a i n i n g not more than 6 per cent of o i l , from undecomposed whole f i s h and/or f i s h c u t t i n g s " , and O i l y F i s h Meal i s "the c l e a n , d r i e d , ground r e s i d u e , c o n t a i n i n g more t h a n 6 per cent of o i l , from undecomposed whole f i s h and/or f i s h c u t t i n g s " • The f i s h m e a l s of the n o n - o i l y c l a s s a re g e n e r a l l y d e s i g n a t e d c o m m e r c i a l l y as w h i t e - f i s h meals, w h i l e those of the o i l y c l a s s are known by the source from which t h e y were prepared -- as Salmon meal, A l a s k a h e r r i n g meal, e t c . (b) Methods o f Manufacture There are s e v e r a l methods employed i n the p r e p a r a t i o n of f i s h m e a l s , e x p l a i n e d by D a n i e l and McCollum (12) as f o l l o w s : - 6 . - "The raw m a t e r i a l s h o u l d be c l e a n and f r e s h i f i t i s t o be u t i l i z e d f o r s t o c k f e e d i n g . T h i s raw m a t e r i a l i s steam cooked, pressed and d r i e d , or i n the case o f some f i s h meals l o w . i n o i l , such as white f i s h meals, the c o o k i n g and d r y i n g are c a r r i e d out i n one o p e r a t i o n , t h e r e b y o m i t t i n g the p r e s s - i n g as a p a r t o f the p r o c e s s . The methods of d r y i n g a f f o r d the g r e a t e s t d i f f e r e n c e s i n the ma n u f a c t u r i n g p r o c e s s . F o r example, d r y i n g may be accomplished i n r o t a r y d r y e r s which s u b j e c t the meal t o d i r e c t flame or to steam he a t , or the product may be p l a c e d i n s t a t i o n a r y steam-jacketed d r y e r s equipped w i t h a r o t a t i n g s h a f t and bla d e s f o r s t i r r i n g t h e meal. T h i s l a t t e r type may be a form p e r m i t t i n g e v a p o r a t i o n under vacuum. " I n g e n e r a l , the cost o f vacuum d r y i n g i s g r e a t e r t h a n t h a t o f any o t h e r p r o c e s s . However, i n those cases i n which the p r e s s i n g i s e l i m i n a t e d , the vacuum-drying process i s l e s s c o s t l y . Furthermore, t h i s l a t t e r 1-step r e d u c t i o n p r o c e s s has , an a d d i t i o n a l advantage i n t h a t i t e l i m i n a t e s the l a r g e l o s s of p r o t e i n s t h a t are d i s c a r d e d i n t h e pres s l i q u o r s from the wet pr o c e s s . " A l l f i s h m e a l s c o n t a i n a c e r t a i n amount of w a t e r - s o l u b l e p r o t e i n which w i l l form glue o r g l u e - l i k e substances and have a tendency t o cake onto any hot s u r f a c e t o form a h a r d , tough i n s u l a t i n g c o a t i n g . The f a t t y f i s h m e a l s c o n t a i n s u f f i c i e n t o i l t o l u b r i c a t e the hot s u r f a c e encountered i n t h e pr o c e s s of d e h y d r a t i o n , and to prevent s t i c k i n g . Moreover, most of the gluey m a t e r i a l s are removed i n the e x t r a c t i o n of the o i l by cooking and p r e s s i n g , so t h a t t h e r e i s l i t t l e d i f f i c u l t y encountered i n d r y i n g f i s h m e a l s c o n t a i n i n g any a p p r e c i a b l e amount of o i l . However, i f raw n o n - o i l y waste i s p l a c e d i n a r o t a r y h o t - a i r d r y e r , such as t h a t mentioned above, i t e i t h e r cakes on t h e s i d e s or s c o r c h e s and b urns, or e l s e becomes case hardened, which prevents s a t i s f a c t o r y d e h y d r a t i o n . S i m i l a r d i f f i c u l t i e s w i t h n o n - o i l y meals are a l s o encountered i n steam j a c k e t d r y i n g ; and c o n s e q u e n t l y the method g e n e r a l l y adopted u n t i l 1933 was to cook the waste, squeeze out the water- s o l u b l e p r o t e i n s , and dry the cooked r e s i d u e . H a r r i s o n , Anderson and P o t t i n g e r ( 1 9 ) , however, r e p o r t e d t h a t " r e c e n t s t u d i e s by the a u t h ors ...... have demonstrated t h a t raw waste can be d r i e d without a p p r e c i a b l e d i f f i c u l t y from ' c a k i n g ' and ' s t i c k i n g 8 i n s t e a m - j a c k e t e d vacuum d r i e r s , i f steam p r e s s u r e and vacuum are c o n t r o l l e d d u r i n g the p r o g r e s s o f t h e d r y i n g o p e r a t i o n . These s t u d i e s . suggest the f i r s t major d i v i s i o n i n the problems of n o n - o i l y f i s h m e a l manufacture, namely, the r e l a t i v e m e r i t o f wet and d r y p r o c e s s e s o f r e d u c t i o n . " (c) P r o t e i n s The advantages of knowing the p r o t e i n v a l u e o f a supplement are o b v i o u s . Both q u a l i t y and q u a n t i t y o f p r o t e i n i n a meal are important i n s e c u r i n g proper n u t r i t i o n . On t h e o t h e r hand, an excess of p r o t e i n i s n e i t h e r economical nor w i s e , because p r o t e i n i s the most c o s t l y o f f e e d elements, and too h i g h a l e v e l o f i t i n the r a t i o n may have a d e l e t e r i o u s e f f e c t on the animals t o which i t i s b e i n g f e d . There are s e v e r a l f a c t o r s i n f l u e n c i n g the v a l u e of a meal as a source of p r o t e i n . I t must be remembered t h a t p r o t e i n s are complex a g g r e g a t i o n s o f some 2 3 amino acids© Some of these must be n a t u r a l l y p r e s e n t i n the d i e t , o t h e r s can be s y n t h e s i z e d from the o r i g i n a l c h e m i c a l c o n s t i t u e n t s . D i g e s t i o n i n v o l v e s the b r e a k i n g down of these m a t e r i a l s by the d i g e s t i v e agents of t h e stomach, i n t e s t i n e and a c c e s s o r y organs, i n t o , a form which can be absorbed and d i s t r i b u t e d t o the c e l l s of t h e body. The p a r t of the f o o d which cannot be a s s i m i l a t e d i s d i s c a r d e d i n the f e c e s ; and the absorbed amino a c i d s which are not r e q u i r e d f o r replacement, growth or storage are m e t a b o l i z e d f o r energy and d i s c a r d e d i n t h e u r i n e . The d i g e s t i b i l i t y of p r o t e i n s may be^ determined e x p e r i m e n t a l l y by t a k i n g i n t o account the d i f f e r e n c e between n i t r o g e n i n t a k e and the n i t r o g e n l o s s i n t h e f e c e s , a f t e r the l a t t e r has been c o r r e c t e d f o r the n i t r o g e n present as a r e s u l t o f m e t a b o l i c breakdown. T h i s i s determined by e s t i m a t i n g the amount of waste p r o t e i n on a known non-nitrogenous diet,, B e s i d e s a knowledge of the d i g e s t i b i l i t y o f the p r o d u c t , other i n f o r m a t i o n i s o f s t i l l g r e a t e r importance. Even t h a t p o r t i o n of the meal which i s d i g e s t e d and made a v a i l a b l e t o t h e body does not n e c e s s a r i l y s u p p l y a l l the v a r i o u s amino a c i d s i n e x a c t l y the proper p r o p o r t i o n s f o r optimum e f f e c t s . - 9 - I t i s apparent, t h e n , t h a t i t i s of v i t a l importance t o know j u s t what amount o f the d i g e s t e d m a t e r i a l can be u t i l i z e d i n anabolism and the l i f e p r o c e s s e s . The e f f i c i e n c y w i t h which the d i g e s t e d p r o t e i n s u p p l i e s the amino a c i d s r e q u i r e d f o r the c o n s t r u c t i o n o f body t i s s u e s g i v e s a measure o f the " b i o l o g i c a l v a l u e " of the m a t e r i a l . • There are s e v e r a l methods i n use f o r the d e t e r m i n a t i o n o f p r o t e i n q u a l i t y . I n most g e n e r a l use i s the method of measuring the amount of p r o t e i n u t i l i z e d by t h e animal i n metabolism, by d e t e r m i n i n g the d i f f e r e n c e between the amount of n i t r o g e n absorbed and the n i t r o g e n d i s c a r d e d i n the u r i n e , a f t e r the l a t t e r has been c o r r e c t e d f o r endogenous u r i n a r y n i t r o g e n . T h i s i s e s t i m a t e d by d e t e r m i n i n g the amount o f n i t r o g e n d i s c a r d e d on a non-nitrogenous d i e t . Means of d e t e r m i n i n g p r o t e i n q u a l i t y i n c l u d e M i t c h e l l ' s r a t b i o l o g i c a l v a l u e method (32) ; St-*. John and co-workers' b i o l o g i c a l v a l u e method, f o r a v i a n n u t r i t i o n (40) ; the n i t r o g e n - b a l a n c e method o f W i l g u s , N o r r i s and Heuser ( 4 4 ) ; the growth method of d e t e r m i n a t i o n developed by Recor d , Bethke and Wi l d e r ( 3 7 ) ; the s l a u g h t e r method of A c k e r s o n , B l i s h and Mussehl ( l ) ; t h e " g r o s s - p r o t e i n v a l u e " method i n use at Washington S t a t e C o l l e g e (22,38); and more r e c e n t l y , a chemical method, d e v i s e d by A l m q u i s t and co-workers, which g i v e s a " p r o t e i n q u a l i t y i n d e x " (2) . - 1 0 - Since these s e r i e s were i n t e n d e d to demonstrate the r e l a t i v e r i b o f l a v i n p o t e n c i e s o f the supplements, a l l e x p e r i m e n t a l r a t i o n s had t o be b a l a n c e d to c o n t a i n the same amount of p r o t e i n , i • e . , 2 0 $ ( 3 0 ) . The c o n t r o l r a t i o n i n each case c o n t a i n e d 2 0 f ° soyabean meal; and the e x p e r i m e n t a l r a t i o n s were ba l a n c e d by r e p l a c i n g a c e r t a i n percentage of the c o n t r o l r a t i o n c o n c e n t r a t e w i t h an e q u i v a l e n t amount of the t e s t m a t e r i a l * The p r o t e i n content of the supplements i s r e p o r t e d i n the t a b l e s of c o m p o s i t i o n as c a l c u l a t e d by the n i t r o g e n d e t e r m i n a - t i o n method, and c o n v e r t e d t o terms of "crude p r o t e i n " by the f a c t o r 6 . 2 5 , s i n c e most of the p r o t e i n s i s o l a t e d from a n i m a l t i s s u e c o n t a i n s about l 6 / » n i t r o g e n ( 2 j > ) • Throughout the t r i a l s , soyabean meal was used as the r e f e r e n c e source of p r o t e i n , s i n c e e x p e r i m e n t a l data r e p o r t e d by a number of workers had i n d i c a t e d ' t h a t treatment o f soya- beans , e i t h e r by h e a t i n g w i t h moist heat or by a u t o c l a v i n g , produced a meal t h a t had good b i o l o g i c a l v a l u e and was capable of m a i n t a i n i n g normal growth. Re p o r t s by Hayward, Steenbock and Bohstedt ( 2 l ) c o n f i r m e d t h e f a c t t h a t , whereas the com- m e r c i a l meals pr o c e s s e d at low temperatures c o n t a i n e d p r o t e i n s i m i l a r i n v a l u e t o t h a t of t h e raw bean, those prepared by e i t h e r the E x p e l l e r or t h e H y d r a u l i c process at h i g h temperatures c o n t a i n e d p r o t e i n of t w i c e the n u t r i t i v e v a l u e o f the low temperature meal. I t w i l l be n o t i c e d t h a t t h i s i s i n d i r e c t c o n t r a s t w i t h t h e f i s h m e a l s , where the h i g h p r o c e s s i n g - 11 - temperature was s e r i o u s l y d e t r i m e n t a l t o the n u t r i t i v e v a l u e * Hayward and Hafner ( 2 0 ) have p u b l i s h e d a most comprehensive r e p o r t on the f i n d i n g s l e a d i n g up t o the e s t a b l i s h m e n t o f the reason f o r i n c r e a s e d n u t r i t i v e v a l u e o f soyabean processed meal above t h a t of the raw bean. (See a l s o papers by Rose and Womack ( 3 9 , 4 6 ) . ) Heiman, Carver and Cook ( 2 2 ) found t h a t c h i c k s f e d the h i g h e r l e v e l s of p r o t e i n from c a s e i n and from soyabean o i l meal weighed e s s e n t i a l l y the same a t the end of t h e i r e x p e r i m e n t a l p e r i o d * P r e v i o u s experiments conducted at t h i s l a b o r a t o r y ( 3 6 ) u s i n g c o m m e r c i a l l y prepared soyabean o i l meals had confirmed these r e p o r t s , and had shown t h a t when the r e s t of the r a t i o n i s complete i n r i b o f l a v i n and m i n e r a l s , soyabean o i l meal may be employed as an e f f e c t i v e supplement to g r a i n i n the c h i c k r a t i o n . (d) V i t a m i n G As e a r l y as 1 9 3 3 i t had been r e c o g n i z e d by W i l g u s , Ringrose and N o r r i s ( 4 3 ) t h a t the n u t r i t i v e v a l u e o f the more common supplements used i n p o u l t r y r a t i o n s i s connected not o n l y w i t h the q u a l i t y of the p r o t e i n s c o n t a i n e d i n them, but a l s o w i t h t h e i r v i t a m i n G c o n t e n t . A c c o r d i n g l y , t h e y d e v i s e d a method to e v a l u a t e these f a c t o r s s e p a r a t e l y , s i m i l a r t o the one used i n t h i s present paper. Here, the s e r i e s were con- ducted w i t h a view to e v a l u a t i n g the r i b o f l a v i n potency o f the t e s t m a t e r i a l s by the d u a l c r i t e r i a o f growth and the i n c i d e n c e of " c u r l e d - t o e " p a r a l y s i s . The C o r n e l l i n v e s t i g a t o r s made the proper adjustments t o keep the p r o t e i n , f a t and bone ash - 12 - co n s t a n t , so t h a t the o n l y d i f f e r e n c e i n the e x p e r i m e n t a l r a t i o n was caused by v a r i a t i o n s i n t h e v i t a m i n G- content o f the m a t e r i a l s under st u d y . T h i s method was f u r t h e r v i n d i c a t e d , because i n t h e customary b i o l o g i c a l method of assay (Bourquin and Sherman, 7) of t h i s v i t a m i n , growth i s the on l y measure o f e s t i m a t i n g the potency. I t was t h e r e f o r e deduced t h a t , w i t h p r o t e i n content b alanced q u a n t i t a t i v e l y and o t h e r f a c t o r s c o n s t a n t , v i t a m i n G was s o l e l y r e s p o n s i b l e f o r gains i n weight above the c o n t r o l . I n c i d e n c e of c u r l e d - toe p a r a l y s i s was observed as a f u r t h e r i n d i c a t i o n o f the v i t a m i n potency of the supplement* There are at present s e v e r a l methods of c o n d u c t i n g v i t a m i n G a s s a y s . The b i o l o g i c a l method o f Bourquin and Sherman ( 7 ) , e s s e n t i a l l y a r a t growth method, was t h e one f i r s t employed. Another b i o l o g i c a l method i n common use was dev i s e d by i n v e s t i g a t o r s a t C o r n e l l ( 3 4 - ) , u s i n g the c h i c k as the e x p e r i m e n t a l u n i t . Other methods i n c l u d e J u k e s 9 b i o l o g i c a l assay of l a c t o f l a v i n w i t h c h i c k s ( 2 6 ) ; the measurement o f the degree o f f l u o r e s c e n c e g i v e n by f l a v i n i n v i o l e t l i g h t (Supple© et a l . , 4 2 ) ; p h o t o e l e e t r i o f l u o r e s c e n c e measurement as developed by Cohen ( 9 ) and E u l e r (14) and adapted by Hand ( l 8 ) ; the f l u o r o m e t r i c method of Hodson and N o r r i s ( 2 3 ) ; and the m i c r o b i o l o g i c a l method o f S n e l l and Stron g ( 4 1 ) , which measures the i n f l u e n c e o f f l a v i n on both the c e l l growth and the a c i d p r o d u c t i o n o f L a c t o b a c i l l u s e a s e l ( grown on a s y n t h e t i c medium f r e e of r i b o f l a v i n . Emmet and co-workers ( 1 3 ) p u b l i s h e d a r e p o r t on the p r o g r e s s o f r i b o f l a v i n assay, and made a comparison o f t h e s e f o u r methods. Close agreement was found among them. The averages o f the per cent d i f f e r e n c e s , i n c l u d i n g the s i g n , between t h e b i o l o g i c a l and each of the o t h e r t h r e e methods are - 1 0 . 7 , + 5 . 3 , and - 7 . 0 by the m i c r o b i o l o g i c a l , t h e f l u o r o m e t r i c and the F l u o r a y methods r e s p e c t i v e l y . I n the second s e r i e s o f experiments, a f t e r the b a c t e r i o l o g i c a l method had proved t o be v a l u a b l e because o f the advantage o f the c o m p a r a t i v e l y short time r e q u i r e d to conduct an as s a y , m i c r o b i o l o g i c a l d e t e r m i n a t i o n s were c a r r i e d out on the samples i n o r d e r t© study o t h e r supplementary e f f e c t s . The f i r s t s e r i e s was conducted t o e v a l u a t e the r i b o f l a v i n potency o f the c a r r i e r s . A m o d i f i c a t i o n of the method o f W i l g u s , N o r r i s and Heuser ( 4 4 ) and N o r r i s , W i l g u s , R i n g r o s e , Heiman and Heuser ( 3 4 ) was c a r r i e d out. They compared /the average g a i n over c o n t r o l of each r a t i o n w i t h t h e g a i n over c o n t r o l of a standard r e f e r e n c e pork l i v e r , and expressed the v i t a m i n G c a r r i e r i n terms of the pork l i v e r . On d i v i d i n g t h e former v a l u e by the per cent o f t e s t m a t e r i a l and m u l t i p l y i n g by 1 0 0 , the potency was determined i n terms o f pork l i v e r • 1 0 0 . As t h i s product was shown to c o n t a i n 1 0 0 micrograms o f f l a v i n per gram, the C o r n e l l " C h i c k U n i t " was demonstrated t o be a p p r o x i m a t e l y e q u a l to a microgram of f l a v i n . S i n c e the time t h e i r paper was w r i t t e n r i b o f l a v i n has been s y n t h e s i z e d , and t h e potency o f a m a t e r i a l may now be o b t a i n e d d i r e c t l y i n terms o f - 14 - micrograms of r i b o f l a v i n . W i l d e r , Bethke and Record ( 4 3 ) , i n d e t e r m i n i n g t h e r e l a t i v e v a l u e of f i s h m e a l s under d i f f e r e n t methods o f p r e p a r a t i o n , d i s c o v e r e d t h a t c e r t a i n o f the p r o c e s s e s removed "some of the v i t a m i n G complex", and th e y a l s o emphasized the importance o f c o n t r o l l i n g t h e v i t a m i n G content of the r a t i o n " i f t he c o r r e c t c o n c l u s i o n s r e g a r d i n g the p r o t e i n v a l u e s o r the t o t a l n u t r i t i v e e f f e c t o f the product under i n v e s t i g a t i o n are to be reached." T h i s p r i n c i p l e was f o l l o w e d i n t h e present i n v e s t i g a t i o n s , but here a l l f a c t o r s , i n c l u d i n g p r o t e i n , were c a r e f u l l y b a l a n c e d and c o n t r o l l e d i n o r d e r t o study t h e r i b o f l a v i n potency of t h e meals. Day-old S i n g l e - Comb White Leghorn c h i c k s were p l a c e d 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 f o r a p e r i o d o f a week t o t e n days to d e p l e t e them of t h e i r s t o r e of r i b o f l a v i n . At the end of t h i s t i m e they were d i v i d e d i n t o b a l a n c e d l o t s and f e d the e x p e r i m e n t a l r a t i o n f o r f i v e weeks. At the end o f the e x p e r i m e n t a l p e r i o d the average g a i n s i n weight at the t h r e e r e f e r e n c e l e v e l s over the n e g a t i v e c o n t r o l were p l o t t e d a g a i n s t t h e u n i t s of v i t a m i n G (micrograms of r i b o f l a v i n ) per 100 grams of f e e d . From t h i s curve the c o r r e s p o n d i n g potency of t e s t m a t e r i a l s was determined from g a i n over c o n t r o l , and c a l - c u l a t e d from t h e graph i n terms of micrograms of r i b o f l a v i n . The r i b o f l a v i n c o n tent o f t h e b a s a l r a t i o n , e x c l u d i n g the p r o t e i n supplement, was 6 3 micrograms per 100 grams; and of the soyabean meal (20$) was another 50 micrograms, as - 15 - determined i n the second s e r i e s by the method of S n e l l and Strong ( 4 1 ) . Soyabean meal c o n t a i n e d 2 - 3 micrograms; skimmilk, 2 0 ; I m p e r i a l Brand F i s h M e a l , 2 5 ; h e r r i n g meal, 15; meat meal, 7 » 5 ; and c a s e i n , 3 micrograms per gram* N o r r i s et a l 6 ( 3 4 ) r e p o r t e d t h a t i n o r d e r f o r c h i c k s to reach a maximum weight at f o u r weeks of age, t h e y r e q u i r e about 3 2 5 u n i t s per 1 0 0 grams of f e e d ; at s i x weeks, about 3 0 0 u n i t s ; and at e i g h t weeks, 2 9 0 u n i t s . I n o t h e r words, t h e r e i s a d e c r e a s i n g requirement f o r t h i s v i t a m i n w i t h i n - c r e a s i n g age, and r i b o f l a v i n i s i n d i c a t e d as b e i n g much more impor t a n t f o r r a p i d growth than f o r maintenance. These s e r i e s were conducted over a p e r i o d o f f i v e weeks, s i n c e at t h a t time the r i b o f l a v i n r e quirements were b e g i n n i n g to drop o f f , and i n c i d e n c e o f a v i t a m i n o s i s as w e l l as d i f f e r e n c e s i n r a t e of g a i n were becoming l e s s e v i d e n t • C u l t o n and B i r d of the U n i v e r s i t y o f Maryland ( 1 0 ) found t h a t 3 0 0 micrograms o f c r y s t a l l i n e r i b o f l a v i n added to 1 0 0 grams of b a s a l c o n t a i n i n g a p p r o x i m a t e l y 1 7 5 micrograms per 1 0 0 grams was not s u f f i c i e n t t o prevent the symptoms o f a r i b o f l a v i n o s i s . I t was concluded t h a t under c e r t a i n c o n d i t i o n s the r i b o f l a v i n requirement o f c h i c k s may be h i g h e r than the g e n e r a l l y acoepted f i g u r e s . (e) M i n e r a l s Marine products are good examples of p r o t e i n m a t e r i a l s a s s o c i a t e d w i t h m i n e r a l s . F i s h m e a l s are g e n e r a l l y known t o be r i c h i n c a l c i u m and phosphorus (because of t h e i r bone c o n t e n t ) , and a l s o t o have a h i g h e r i o d i n e content than most p r o t e i n - 1 6 foods. D a n i e l and McCollum s t a t e t h a t Orr and Husband ( 3 5 ) p o i n t e d out t h a t the calcium-phosphorus r a t i o i n f i s h m e a l s i s s i m i l a r to t h a t o f cow's or sow's m i l k ; and f u r t h e r m o r e , t h a t o n l y c o m p a r a t i v e l y s m a l l amounts of these p r o d u c t s would be n e c e s s a r y t o supply an adequate q u a n t i t y of the m i n e r a l s i n q u e s t i o n . Because the sea has s m a l l amounts of most of the m i n e r a l elements d i s s o l v e d i n i t , i t i s to be expected t h a t sea foods w i l l prove v a l u a b l e sources of m i n e r a l s i n a d d i t i o n to t h e i r o t h e r n u t r i t i o n a l m e r i t s . I r o n , manganese, potassium and sodium are t o be found i n a l l l i v i n g t i s s u e . N e w e l l and McCollum ( 3 3 ) conducted a s p e c t r o g r a p h i c a n a l y s i s of marine prod u c t s and r e p o r t e d the presence of a l l the above elements. Io d i n e i s not d e t e r m i n a b l e by t h i s method, but was shown t o be present i n r e l a t i v e l y l a r g e amounts i n f i s h m e a l • B e s i d e s t h e s e , t r a c e s o f aluminum, chromium, copper, l e a d , l i t h i u m , manganese, and s t r o n t i u m were found i n a l l o f the meals, w h i l e t r a c e s of f l u o r i n e , n i c k e l , s i l i c o n , s i l v e r , t i n , t i t a n i u m and z i n c were present i n some of the meals* These i n v e s t i g a t o r s i n c l u d e d i n t h e i r paper a d i s c u s s i o n o f the known importance t o animal n u t r i t i o n of the l e s s common m i n e r a l s r e p o r t e d above * The e f f e c t o f the f i s h m e a l m a n u f a c t u r i n g p r o c e s s on m i n e r a l s i s not so pronounced as the e f f e c t on p r o t e i n s or v i t a m i n s , but i t s h o u l d be borne i n mind t h a t i t i s p o s s i b l e to l o s e a c e r t a i n percentage of the m i n e r a l s t h rough e x t r a c - t i o n and s o l u t i o n i n the w a t e r - s o l u b l e p r o t e i n p o r t i o n . - 17 - ( f ) E f f e c t s o f Method of Manufacture As e a r l y as 1929 I n g v a l d s e n (24) had r e p o r t e d t h a t t h e method of p r e p a r a t i o n g r e a t l y a f f e c t e d the n u t r i t i v e v a l u e o f f i s h m e a l s . W i l d e r , Bethke and Record (43) conducted a s e r i e s o f experiments i n which t h e y made comparisons between f i s h - meals prepared under d i f f e r e n t e x p e r i m e n t a l l y c o n t r o l l e d c o n d i t i o n s , and found t h a t the p r o t e i n s o f haddock meals pro- duced by the vacuum-drying method were s u p e r i o r t o those of the f l a m e - d r i e d meals. The p r o t e i n o f vacuum-dried meals was s l i g h t l y more d i g e s t i b l e than t h a t o f the s team-dried haddook p r o d u c t s . Moreover, the method of p r o c e s s i n g p r e v i o u s t o d r y i n g p r o f o u n d l y a f f e c t e d the b i o l o g i c a l v a l u e of the meal*. The absorbed n i t r o g e n from wet-rendered meals was more e f f i c i e n t l y u t i l i z e d t h a n t h a t o f t h e d r y - r e n d e r e d p r o d u c t s . H a r r i s o n , Anderson and P o t t i n g e r (19) i n v e s t i g a t e d the e f f e c t s o f v a r i o u s methods o f manufacture upon haddock meal, a t y p i c a l n o n - o i l y f i s h m e a l , and found t h a t f l a m e - d r y i n g d e f i n i t e l y decreased the amount of f i s h m e a l p r o t e i n t h a t t h e a n i mal c o u l d absorb. Rendering by e i t h e r wet or d r y method d i d not a f f e c t t h e d i g e s t i b i l i t y , which i n d i c a t e d t h a t the w a t e r - s o l u b l e and i n s o l u b l e f r a c t i o n s had p r o t e i n s o f s i m i l a r d i g e s t i b i l i t y . F i n a l l y , the p r o t e i n s of the head and back seemed t o be e q u a l l y d i g e s t i b l e , which seemed to i n d i c a t e t h a t temperature was t h e o n l y f a c t o r i n v o l v e d i n i n f l u e n c i n g t h e p r o t e i n a b s o r p t i o n d u r i n g the process of manufacture. - 1 8 - The b i o l o g i c a l v a l u e , however, was found to be i n f l u e n c e d by s e v e r a l f a c t o r s . The v a l u e f o r the d r y - p r o c e s s meals was d e c i d e d l y i n f e r i o r t o t h a t f o r the wet-process meals, which i n d i c a t e s t h a t the w a t e r - s o l u b l e p r o t e i n s (which would be removed i n the l a t t e r p r o c e s s ) are of low q u a l i t y . The head meal, which was e q u a l l y d i g e s t i b l e as the meal prepared from the backs, d i d not have as h i g h a b i o l o g i c a l v a l u e . High d r y i n g temperatures were shown t o be d e t r i m e n t a l t o the q u a l i t y of the meal as w e l l as to the d i g e s t i b i l i t y , and made i t apparent t h a t f l a m e - d r y i n g had a m u l t i p l e d e t r i m e n t a l e f f e c t . Vacuum-drying, had no p a r t i c u l a r advantage over s t e a m - d r y i n g . D r y - p r o c e s s meals, however, proved to be a b e t t e r source of v i t a m i n G than the wet-process meals; and a l s o the head proved t o be a b e t t e r source o f r i b o f l a v i n than the backbone, which i n d i c a t e d t h a t the w a t e r - s o l u b l e e x t r a c t i v e s removed by the wet p r o c e s s , and the head p r o t e i n s , were the most potent c a r r i e r s . I n c o n c l u s i o n t h e y p o i n t e d out t h a t because p r o t e i n s are g e n e r a l l y q u i t e s t a b l e under normal d r y i n g c o n d i t i o n s , f u t u r e improvements i n t h e method of manufacture of f i s h m e a l s w i l l be dependent upon methods of i n c r e a s i n g the v i t a m i n G content of the p r o d u c t . (g) Uses o f F ishmeals Because o f t h e i r e x c e l l e n t p r o t e i n q u a l i t y , h i g h m i n e r a l content and v i t a m i n potency, f i s h m e a l s are of g r e a t v a l u e i n p o u l t r y and swine f e e d i n g , and may be used i n f e e d i n g beef c a t t l e , d a i r y cows and sheep, or t o s u b s t i t u t e f o r p a r t of the - 1 9 - m i l k i n r a i s i n g d a i r y c a l v e s . When f i s h m e a l s were f i r s t used as supplements i n the r a t i o n s of p o u l t r y and l i v e s t o c k t h e r e were r e p o r t s of t a i n t i n g o f the meat, m i l k and eggs; but i n v e s t i g a t o r s found no such d i f f i c u l t i e s from the f e e d i n g o f marine p r o d u c t s . I t seemed l i k e l y t h a t the u n s t a n d a r d i z e d methods t h a t were used i n the manufacture of f i s h m e a l s f o r f e r t i l i z e r b efore t h e i r great v a l u e f o r f e e d i n g was r e a l i z e d , were l a r g e l y r e s p o n s i b l e f o r the unfounded reports® SERIES 1 (a) The Experiment M i l k and m i l k by-pro ducts have been e x t e n s i v e l y used i n p o u l t r y r a t i o n s as a source of p r o t e i n of a h i g h q u a l i t y . More r e c e n t l y m i l k has a l s o been r e c o g n i z e d as a good source of v i t a m i n G-. I t i s because of the l a t t e r , as a m atter of f a c t , t h a t m i l k has found almost u n i v e r s a l use i n p o u l t r y f e e d i n g , and more p a r t i c u l a r l y i n t h e c h i c k r a t i o n , r i b o f l a v i n b e i n g i n t i m a t e l y concerned i n t h e p r o c e s s of growth. S i n c e i t was known t h a t c a r e f u l l y prepared f i s h m e a l s c o n t a i n p r o t e i n which has a b i o l o g i c a l v a l u e comparable to t h a t of m i l k , i t was undertaken t o f i n d out whether f i s h m e a l s c o u l d be u t i l i z e d as an e q u a l l y good source of r i b o f l a v i n . T h i s study was conducted i n o r d e r t o e s t i m a t e the r e l a t i v e p r o t e i n and v i t a m i n e f f i c i e n c i e s of v a r i o u s w h o l e - f i s h meals and r e s i d u e s and s c r a p s from the canning i n d u s t r y . - 2 0 - •(b) M a t e r i a l s and Methods For the purpose of these experiments a r a t i o n was compiled w i t h a view to d e t e r m i n i n g comparative v a l u e s f o r the r i b o f l a v i n content of v a r i o u s B r i t i s h Columbia f i s h m e a l s and f i s h m e a l b y - p r o d u c t s . The c o m p o s i t i o n of t h e b a s a l r a t i o n i s g i v e n i n Table I . I t w i l l be seen t h a t w i t h the e x c e p t i o n s of the source o f p r o t e i n and the v i t a m i n G content a l l the r a t i o n s are i d e n t i c a l . The soyabean o i l meal was a c o m m e r c i a l l y prepared brand of a r i c h brown c o l o u r and p l e a s a n t odour and t a s t e . The r e p o r t e d a n a l y s i s (Table I I ) showed t h a t i t c o n t a i n e d 4 4 • i f . p r o t e i n , 9 . 6 7 % m o i s t u r e , 5»1lt f a t , and 5 . 7 5 /• ash. A l l o t h e r i n g r e d i e n t s were o f a st a n d a r d type i n common use by the poultrymen i n B r i t i s h Columbia. The f i s h m e a l s o f t h e s e s e r i e s were h i g h i n t h e i r f a t c o n t e n t , c h i e f l y because t h e y were l a b o r a t o r y - p r e p a r e d f o r the purpose of the t e s t . T h i s was o f f s e t by an accompanying h i g h p r o t e i n v a l u e , and was t a k e n i n t o account i n the b a l a n c i n g o f the rations® The p r o t e i n content of the produc t s i n t h e s e s e r i e s was e x c e e d i n g l y h i g h , v a r y i n g from 5 0 f o i n the case of the meal prepared from salmon heads t o 7 8 f » i n t h e whole h e r r i n g meal, and a v e r a g i n g over 6 5 f » . I t w i l l be noted a l s o t h a t the ash content of the f i s h m e a l s v a r i e d c o n s i d e r - a b l y , but was c o n s i s t e n t l y h i g h . I n t h e case of the meals prepared from salmon roe the v a l u e s were l o w e r , because t h e r e was no bone p r e s e n t . Throughout the s e r i e s , w i t h t h e - 21 - : e x c e p t i o n of the " h e a t - t r e a t e d egg meal", the meals used were prepared i n vacuo i n a steam-jacketed d r y e r * A n a l y s i s of the supplementary i n g r e d i e n t s appears i n Table II« No f i g u r e s were a v a i l a b l e f o r the PgO^ and CaO c o n t e n t s . Manganous s u l p h a t e was added d u r i n g the m i x i n g p r o c e s s , s u f f i c i e n t t o ensure a g a i n s t the i n c i d e n c e o f p e r o s i s . The s y n t h e t i c r i b o f l a v i n , o b t a i n e d from Merck and Company, L i m i t e d , was i n c o r p o r a t e d i n the r a t i o n at the l e v e l s i n d i c a t e d . In o r d e r t o secure d i s t r i b u t i o n i n the mash, the r i b o f l a v i n was mixed f i r s t w i t h 5 pounds of f e e d , t h i s t h o r o u g h l y mixed and s i f t e d w i t h 10 pounds, and so on f o r i n c r e a s i n g l y g r e a t e r q u a n t i t i e s . The p r e p a r a t i o n of each 100 pounds of mash r e q u i r e d about h a l f an hour of m i x i n g . The v i t a m i n A and D o i l was i n c o r p o r a t e d w i t h the b a s a l . S u f f i c i e n t b a s a l was prepared at the 'beginning o f t h e experiment t o l a s t throughout the t r i a l . I n o r d e r t o keep r e a s o n a b l y c o n s t a n t a l l f a c t o r s o t h e r than the v i t a m i n G- potency of t h e t e s t m a t e r i a l s , t h e f a t content and t h e m i n e r a l c o n t e n t o f each of t h e t e s t r a t i o n s were c a l c u l a t e d from the l a b o r a t o r y a n a l y s e s , and t h e n balanced by adding t o them Mazola o i l and bone meal. Table I I I summarizes the c o m p o s i t i o n o f the e x p e r i m e n t a l r a t i o n s per 100 pounds, i n c l u d i n g t h e amounts of f a t and ash added to e q u a l i z e the r a t i o n s w i t h i n each s e r i e s . I n each case t h e - 2 2 - p r o t e i n content was balanced t o 2 0 $ , and wheat added t o make the t o t a l up t o 1 0 0 pounds. I n t h i s s e r i e s the l e v e l of p r o t e i n r e f e r s t o the percentage of soyabean o i l meal r e p l a c e d by an e q u i v a l e n t percentage of p r o t e i n from a f i s h m e a l s o u r c e . I n Experiment 1 the r i b o f l a v i n was f e d at t r i a l l e v e l s of 1 0 0 , 2 0 0 and 3 0 0 micrograms per 1 0 0 grams as r e f e r e n c e supplements to t h e c o n t r o l r a t i o n , but t h e r e was found to be no s t a t i s t i c a l d i f f e r e n c e between the 2 0 0 and 3 0 0 microgram l e v e l s . (See T a b l e VI) I t was found p r e f e r a b l e a f t e r the f i r s t experiment to o b t a i n g r e a t e r spreads between t h e g a i n s i n weight of the c h i c k s on t h e r e f e r e n c e r a t i o n s , and subse- q u e n t l y d i f f e r e n t l e v e l s were s u b s t i t u t e d f o r the second and a g a i n f o r the t h i r d assay. E v i d e n t l y t h e l e v e l s f i r s t s e l e c t e d were approaching the r e q u i r e m e n t s o f the c h i c k s f o r r i b o f l a v i n , and the lower l e v e l s o f 7 , 5 , 1 2 5 , and 1 7 5 m i c r o - grams per 1 0 0 grams, and f i n a l l y 5 0 , 1 0 0 and 1 5 0 micrograms, were s u b s t i t u t e d to make d i f f e r e n c e s i n growth due t o t h i s f a c t o r more o b v i o u s . Day-old Single-Comb White/Leghorn c h i c k s were o b t a i n e d from an e s t a b l i s h e d breeder from h i g h q u a l i t y s t o c k known t o have been f e d on s t a n d a r d high-grade r a t i o n s . The c h i c k s were f e d a d e p l e t i o n d i e t f o r a week or t e n days, and then graded on the b a s i s of w e i g h t , and s e l e c t e d so t h a t the primary average weights i n each l o t were comparable — 8 5 t 5 grams. A f t e r b e i n g leg-banded f o r i d e n t i f i c a t i o n , t h e y - 2 3 - were p l a c e d i n se p a r a t e compartments of stand a r d b a t t e r y - brooders i n the P o u l t r y N u t r i t i o n L a b o r a t o r y at the. U n i v e r s i t y of B r i t i s h Columbia, .The c h i c k s were weighed weekly, b e g i n n i n g when t h e y were s e l e c t e d f o r w e i g h t , and at the end o f each week t h e r e a f t e r * Weights were r e c o r d e d f o r each i n d i v i d u a l b i r d a c c o r d i n g t o the i d e n t i f y i n g l e g-band. O b s e r v a t i o n s were made a t t h i s t i m e , as w e l l as s e v e r a l times d u r i n g the week, f o r any s i g n s of a v i t a m i n o s i s or o t h e r a b n o r m a l i t i e s . The b a t t e r y - b r o o d e r s used i n t h i s experiment were so c o n s t r u c t e d t h a t i t was i m p o s s i b l e t o keep a r e c o r d of the feed consumption, but i n order t o encourage the maximum i n t a k e the troughs were always kept f u l l . Lamoreux and Schumacher ( 2 ? ) r e p o r t a lOOf. i n c r e a s e i n r i b o f l a v i n i n the f e c e s when they a re h e l d at room temperature f o r 24 hours, and a 3 0 0 / » i n c r e a s e when they are h e l d f o r a week. Care was t h e r e f o r e t a k e n t o prevent coprophagy because o f t h i s r a p i d s y n t h e s i s of t h e v i t a m i n f o l l o w i n g d e f e c a t i o n . M o r t a l i t y was not a f a c t o r i n e i t h e r s e r i e s o f experiments. The few f a t a l i t i e s t h a t d i d occur were due t o a c c i d e n t a l causes (such as by i n j u r y ) , and were not r e l a t e d w i t h any p a r t i c u l a r r a t i o n . (c) R e s u l t s The r e s u l t s o f t h i s s e r i e s of t h r e e experiments are gi v e n i n Tab l e s IV and V. I n Table VI i s r e p o r t e d a - 24 - s t a t i s t i c a l summary o f the r e s u l t s o b t a i n e d from the " t - t e s t " method. The terms S and N r e f e r t o the s i g n i f i c a n c e o r n o n - s i g n i f i c a n c e of the d i f f e r e n c e s between the means as c a l c u l a t e d by t h i s method, a c c o r d i n g to the formulae: I 2 2 J *1 + n 2 /nj, + n 2 + 2 s s = s 7 (n x+ 1 ) ( h 2 + 1 ) t - (S^ - x 2 ) s_ z where n^ and n 2 r degrees of freedom £T(x - x) - sum of squares x s mean. EXPERIMENT 1 S i n c e r i b o f l a v i n i s w a t e r - s o l u b l e , the p o s s i b i l i t y suggested i t s e l f t h a t s t i c k w a t e r , a waste product from the manufacture o f f i s h m e a l by the wet-process a l r e a d y d i s c u s s e d , might prove a good source of t h e v i t a m i n . S t i c k w a t e r i s the e f f l u e n t r e m a i n i n g when t h e o i l has been s e p a r a t e d from the l i q u i d p r e s s e d from the cooked f i s h ; and s t i c k w a t e r meal i s a product manufactured by e v a p o r a t i o n under vacuum by a patented p r o c e s s ( . ' . 2 8 ) s - 2 5 - In o r d e r t o make comparisons between s t i c k w a t e r meal and other f i s h p r o d u c t s , two o t h e r meals were i n c l u d e d . The f i r s t was a,meal prepared from the whole f i s h ; the o t h e r , one prepared from the waste of the e d i b l e p o r t i o n . H e r r i n g meal was s e l e c t e d f o r the w h o l e - f i s h meal, s i n c e i t i s e x t e n s i v e l y used i n p o u l t r y f e e d i n g i n B r i t i s h Columbia; and salmon meal, a l s o i n common use, was chosen as t h e meal prepared from cannery trimmings,, I n t h i s experiment the c h i c k s f e d the 15% l e v e l of salmon meal reached a weight o f 3 8 9 grams, which r e p r e s e n t e d a g a i n o f 1 5 9 grams over the c h i c k s on the c o n t r o l r a t i o n , and was comparable w i t h the. 2 0 0 and 3 0 0 - u n i t l e v e l s . The f a c t t h a t v e r y s a t i s f a c t o r y growth was o b t a i n e d would suggest t h a t the p r o t e i n from t h i s source was of h i g h b i o l o g i c a l value© The f a c t t h a t t h e r e was a s m a l l i n c i d e n c e of a v i t a m i n o s i s even at the h i g h e r l e v e l , however, would suggest t h a t t h i s meal i s not s u f f i c i e n t l y potent i n v i t a m i n G f o r normal f u n c t i o n i n g and p r e v e n t i o n of symptoms o f d e f i c i e n c y . The s t i c k w a t e r meal of t h i s s e r i e s produced s l i g h t l y i n f e r i o r weights at the 1 0 f. l e v e l , but the c h i c k s showed no symptoms of c u r l e d - t o e p a r a l y s i s ; which would i n d i c a t e t h a t w h i l e i t i s c o m p a r a t i v e l y r i c h i n r i b o f l a v i n , i t s p r o t e i n i s not of p a r t i c u l a r l y h i g h b i o l o g i c a l v a l u e . T h i s i s i n agreement w i t h W i l d e r , Bethke and Record ( 4 3 ) , who showed t h a t the w a t e r - s o l u b l e c o n s t i t u e n t s o f f i s h m e a l s were of e x c e p t i o n a l l y low b i o l o g i c a l v a l u e , a l t h o u g h t h e y d i d not - 2 6 - study s t i c k w a t e r meal as such. L i k e w i s e , W i l g u s , R i n g r o s e and N o r r i s ( 4 5 ) r e p o r t e d t h a t the s t i c k w a t e r from f i s h m e a l manufacture, and the heads, (see Experiment 2 ) add m a t e r i a l l y t o t h e v i t a m i n G- potency. C u r t i s , Hauge and K r a y b i l l ( 1 1 ) found a marked d i f f e r e n c e i n the value o f the h o t - w a t e r - i n s o l u b l e and s o l u b l e f r a c t i o n s of tankages when used as a p r o t e i n supplement to cor n * The s o l u b l e f r a c t i o n s had no supplementary v a l u e t o the p r o t e i n s of c o r n , due t o d e f i c i e n c i e s i n tryp t o p h a n e and c y s t i n e , " S t i c k " (which i s the product r e s u l t i n g from t h e c o n c e n t r a t i o n of the l i q u o r s o b t a i n e d i n the wet r e n d e r i n g o f tankage pr o d u c t s ) i s almost e n t i r e l y s o l u b l e i n b o i l i n g water. T h i s s o l u b l e f r a c t i o n , when f e d t o r a t s as a s o l e source o f p r o t e i n , even at a 15/» l e v e l , was not s u f f i c i e n t f o r maintenance. These f i n d i n g s were f u r t h e r c o r r o b o r a t e d by the f i n d i n g s o f H a r r i s o n , Anderson and P o t t i n g e r (.19) d i s c u s s e d p r e v i o u s l y , who found t h a t water- s o l u b l e p r o t e i n s were o f low q u a l i t y ; ''and t h a t d r y - p r o c e s s meals ( c o n t a i n i n g the w a t e r - s o l u b l e p r o t e i n s ) were a b e t t e r source of r i b o f l a v i n t h a n the wet-process meals. The t h i r d product under t e s t i n t h e f i r s t experiment - h e r r i n g meal - produced much l e s s s i g n i f i c a n t g a i n s i n weight and a l s o a 6of. i n c i d e n c e of a v i t a m i n o s i s even when f e d at the 1 5 / 0 l e v e l , EXPERIMENT 2 ' The r e s u l t s o f the f i r s t experiment l e d t o an e n q u i r y i n t o the n u t r i t i v e v a l u e of another marine by-pr o d u c t , - 2 7 - prepared from salmon r o e . I t was decided t© e v a l u a t e the e f f i c i e n c y o f t h i s meal and to determine whether t h e r e were any changes i n i t s n u t r i t i v e v a l u e induced by d i f f e r e n c e s i n the method of p r e p a r a t i o n . I t was decided at the same time to c a r r y out assays t o accumulate more p r e c i s e data on the n u t r i t i v e v a l u e of meals prepared from salmon heads and v i s c e r a . The r e s u l t s o f t h i s second t e s t are r e p o r t e d i n Tables IV and V. There was no s t a t i s t i c a l d i f f e r e n c e between th e weight o f the c h i c k s on the c o r r e s p o n d i n g l e v e l s of the roe meal under the d i f f e r e n t methods of heat t r e a t m e n t ; and j u d g i n g from t h e growth s t i m u l u s and i n c i d e n c e of a v i t a m i n o s i s , n e i t h e r d i d t h e r e appear t o be any d i f f e r e n c e i n t h e i r r i b o f l a v i n c o n t e n t . T h i s i s not i n accordance w i t h o t h e r f i n d i n g s r e p o r t e d e a r l i e r i n t h i s paper, where h i g h temperatures were shown t o be d e t r i m e n t a l t o b i o l o g i c a l value and even more d e l e t e r i o u s t o r i b o f l a v i n c o n t e n t . The c a l - c u l a t e d number of u n i t s of r i b o f l a v i n per gram of f e e d was the same i n the case of the h e a t - t r e a t e d roe meal, and of the raw egg. The meal appeared t o be of h i g h n u t r i t i v e v a l u e and f a i r v i t a m i n G- c o n t e n t , and to be u n a f f e c t e d by h e a t i n g to the temperatures i n v o l v e d i n i t s preparation© The c h i c k s which were f e d the meal prepared from the salmon heads d i d not make ve r y r a p i d g a i n s i n weight at e i t h e r l e v e l f e d , and a l s o showed s i g n s of d e f i c i e n c y o f - 2 8 - r i b o f l a v i n . H a r r i s o n , Anderson and P o t t i n g e r (19) r e p o r t e d t h a t heads d i d not have so h i g h a n u t r i t i v e value as the backbone, but t h a t they ivere good sources of r i b o f l a v i n . L i k e w i s e W i l g u s , R i n g r o s e and N o r r i s (45) r e p o r t e d t h a t the i n c l u s i o n of heads added m a t e r i a l l y t o the v i t a m i n potency of a meal, and W i l d e r , Bethke and Record (43) showed t h a t the wastes from the e d i b l e p o r t i o n were h i g h e r i n q u a l i t y t h a n the heads or the t a i l s . The present i n v e s t i g a t i o n d i d not i n d i c a t e t h a t salmon-head meal was a good source of r i b o f l a v i n . I n the case of the salmon-gut meal, e x c e p t i o n a l l y f i n e w e ights were o b t a i n e d , even at the 7 . 5 $ l e v e l ; w eights which averaged 1 8 2 grams above the c o n t r o l . The r a t e of growth, i n c o n j u n c t i o n w i t h freedom from any a v i t a m i n o s i s , demonstrated the meal t o have p r o t e i n o f h i g h b i o l o g i c a l v a l u e , and t o c o n t a i n even at t h e 7 * 5 l e v e l an adequate s u p p l y of r i b o f l a v i n . EXPERIMENT 3 I t next seemed of i n t e r e s t , s i n c e t h e meal from salmon v i s c e r a had proved to be o f such v a l u e , to run a t h i r d experiment t o study the r e l a t i v e e f f i c i e n c y of meals prepared from the v i s c e r a of v a r i o u s s p e c i e s o f salmon. I t was d e c i d e d at the same time t o i n c l u d e two l e v e l s of l i v e r mealo I n the p r e p a r a t i o n of salmon meal p r o b a b l y t w o - t h i r d s of the l i v e r remains w i t h the head, s i n c e p a r t of the l i v e r l i e s - 2 9 - i n the s e c t i o n which i s cut o f f w i t h t h e head, Weights of the c h i c k s f e d the t h r e e v i s c e r a meals i n d i c a t e t h a t these products are a l l e x c e p t i o n a l l y f i n e sources of h i g h - q u a l i t y p r o t e i n , and a l l good sources of r i b o f l a v i n . The growth s t i m u l u s accorded by f e e d i n g l i v e r meal was i n d i s p u t a b l y h i g h e r than t h a t of any o t h e r product under t e s t . The l e v e l o f l i v e r meal was comparable w i t h the 5% l e v e l of the v i s c e r a meals of t h i s s e r i e s ; and t h e 5% l e v e l c o n t a i n e d an adequate s u p p l y o f v i t a m i n G- to prevent e n t i r e l y any o c c u r r e n c e o f p a r a l y s i s . T h i s h i g h v a l u e o b t a i n e d f o r l i v e r meal i s i n accordance w i t h t h e f i n d i n g s of B i l l i n g s et a l . ( 6 ) , who l a t e r made m i c r o b i o l o g i c a l assays of s i m i l a r meals; and a l s o w i t h those o f Lunde of Norway ( 2 9 ) , who r e p o r t e d l i v e r and roe t o be e s p e c i a l l y r i c h i n r i b o - f l a v i n , (d) D i s c u s s i o n As a r e s u l t o f the t h r e e experiments o f S e r i e s 1 i t has been shown t h a t the f i s h meals prepared i n o r d i n a r y m a n u f a c t u r i n g p r o c e s s e s o f the canning i n d u s t r y - namely, the h e r r i n g and salmon meals - are not p a r t i c u l a r l y good sources of r i b o f l a v i n , a l t h o u g h t h e y seem t o c o n t a i n p r o t e i n o f good b i o l o g i c a l v a l u e . To be of v a l u e as supplements i n the p o u l t r y r a t i o n they would have t o be f o r t i f i e d w i t h m a t e r i a l s r i c h i n v i t a m i n G-. In the case of the b y - p r o d u c t s , however. - 3 0 - the r e s u l t s suggest t h a t f u r t h e r commercial use should be made of t h e i r v a l u e as v i t a m i n - r i c h p r o t e i n supplements. The value of the, l i v e r meal, i n agreement w i t h the f i n d i n g s of ot h e r i n v e s t i g a t o r s , was p a r t i c u l a r l y h i g h i n r i b o f l a v i n , and the v i s c e r a meals were a l s o c o n s i s t e n t l y r i c h i n t h i s f a c t o r . The two meals prepared from salmon roe were o n l y moderately v a l u a b l e as a source of v i t a m i n G, and the salmon head meal was the lowest o f the s e r i e s . More e x t e n s i v e use c o u l d be made of t h e products r i c h i n r i b o f l a v i n t han i s a t pres e n t the case. I t should prove of b e n e f i t t o use them e i t h e r as independent supplements or i n c o n j u n c t i o n w i t h the o r d i n a r y f i s h m e a l s such as the h e r r i n g or salmon meals. Because of i t s h i g h r i b o f l a v i n c o n t e n t , the s t i c k w a t e r meal s h o u l d prove o f va l u e when used t o r e p l a c e a p o r t i o n of o t h e r supplements w i t h a h i g h b i o l o g i c a l v a l u e . These s t u d i e s i n d i c a t e t h a t the q u a l i t y o f t h e f i s h m e a l s produced i n B r i t i s h Columbia c o u l d be enhanced i n t h e i r v i t a m i n G content by improvements i n t h e method of manufacture (vacuum-drying vs» f l a m e - d r y i n g ) as w e l l as by the i n c l u s i o n of f i s h r e s i d u e s ( l i v e r , v i s c e r a , roe and s t i c k w a t e r ) » g e n e r a l l y d i s c a r d e d i n the f i s h i n g i n d u s t r y . The s o - c a l l e d "waste p r o d u c t s " have a v e r y h i g h n u t r i t i v e v a l u e which would warrant s p e c i a l c a r e b e i n g t a k e n i n t h e i r p r e p a r a t i o n and i n c o r p o r a t i o n i n t o f i s h m e a l s . TABLE I Composition of B a s a l R a t i o n E i s h O i l (100D, 1000A) 1.0 S a l t 1«0 Limestone ^. ..... • l»j> Bone E l o u r ............. . 5 M i d d l i n g s 10.0 Bran , , 10.0 Ground Oats 10.0 Corn Meal ., .. 10.0 Ground Wheat ?6 .0 Soyabean Meal ................a 20.0 100.0* "Manganese s u l p h a t e added, at the r a t e of j£ pound per t o n of feed„ TABLE I I A n a l y s i s of I n g r e d i e n t s • M o i s t u r e Eat P r o t e i n Ash ? 2 ° 5 CaO Salmon Meal 8.91 8 . 7 6 6 0 . 7 5 5 . 8 0 H e r r i n g Meal 4,21 7.84 7 8 . 6 7 9 . 6 ? S t i c k w a t e r Meal 3.10 2 2 . 1 0 67.40 1 2 , 1 5 Heat-Treated Egg 1 8 , 2 0 6 0 , 9 0 3 . 5 6 Raw Egg 11,60 6 2 . 8 0 3.52 Head <> 2 0 . 4 0 5 0 . 2 0 i 5 c 0 3 ' . Gut. 2 0,60 6 2 . 3 0 7 . 8 7 Chum V i s c e r a 9 . 7 3 7 1 . 2 5 7 « 8 9 P i n k V i s c e r a 13,13 72.24 7 * 5 0 > Sockeye V i s c e r a 1 4 * 7 6 6 8 , 7 4 , . 6 . 9 . 6 ' L i v e r Meal 1 7 . 0 9 6 4 . 4 6 5.45. ! Soyabean O i l Meal 9 * 6 ? 5 . 1 7 4 4 . 1 0 5 « 7 5 ; . TABLE I I I Composition of R a t i o n s per Hundred Pounds ^ L e v e l Soyabean Meal Pounds Supplement Pounds Ash Added Grams Eat Added Grams Salmon Meal 7 , 5 0 1 2 , 5 0 5.43 2 5 9 4 1 . 0 1 5 . 0 0 5 , 0 0 1 0 . 8 6 . 3 2 2 0 H e r r i n g Meal 7 . 5 0 1 2 , 5 0 4 . 2 0 2 0 2 1 0 8 . 0 1 5 . 0 0 5 , 0 0 8.40 2 2 1 1 3 5 . 0 S t i c k w a t e r Meal 5 . 0 0 1 5 , 0 0 3 . 2 7 129 5 4 0 , 0 1 0 . 0 0 1 0 , 0 . 0 6.54 6 3 2 9 8 . 0 Heat-Treated Egg 7»50 1 2 , 5 0 5.47 1 2 0 . 0 2 0 5 . 2 1 1 . 2 5 8 , 7 5 .8.21 1 7 8 , 4 7 2 . 6 Raw Egg 7 . 5 0 1 2 . 5 0 5 , 3 0 1 2 1 , 7 3 7 8 . 6 1 1 . 2 . 5 , 8 , 7 5 7 * 9 5 1 8 3 . 9 3 3 5 . 0 Head ; 7.50 1 2 , 5 0 6 . 6 3 0 4 4 . 9 1 1 , 2 5 8 , 7 5 9 . 9 5 0 0 Gut 7 .50 1 2 . 5 0 5 . 3 5 0 1 5 7 . 5 1 1 , 2 5 8 . 7 5 8 . 0 3 0 0 Chum V i s c e r a 5 . 0 0 1 5 , 0 0 3 . 1 0 19 .1 1 6 8 . 7 1 0 . 0 0 1 0 . 0 0 6 . 2 0 " 3 9 , 0 . 1 4 0 . 6 P i n k V i s c e r a 5 , 0 0 1 5 * 0 0 3 . 0 5 2 6 . 3 1 3 2 . 5 1 0 . 0 0 ' 1 0 . 0 0 . 6 . 1 0 5 3 . 0 6 8 . 0 Sockeye V i s c e r a 5 . 0 0 1 5 . 0 0 3 . 2 1 2 9 . 9 9 8 . 4 1 0 . 0 0 1 0 , 0 0 6,42 5 9 . 9 0 L i v e r Meal 2 . 5 0 1 - 7 . 5 0 1.71 1 23*1' 1 2 1 . 6 5 . 0 0 1 5 . 0 0 • 3 . 4 3 4 5 * 4 4 9 * 0 "The " L e v e l " i n d i c a t e s t he number of pounds (per 1 0 0 pounds) of soyabean meal r e p l a c e d by an e q u i v a l e n t amount of p r o t e i n from the source i n d i c a t e d . :• TABLE IT Exp e r i m e n t a l Data : L e v e l *Ave.Wt. of Chi c k s at 5 Wks. (Grams) Number of Chicks C o e f f i c i e n t of V a r i a b i l i t y Gain i n Wt. Over Negative C o n t r o l (Grams) U n i t s of VI tamin per Gram of Eeed, . Normal Showing A v i t . G. Salmon Meal 7 . 5 0 347.5 18 3 1 4 . 2 3 1 1 7 . 2 27.1 15.00 389.4 20 " 2 1 3 » 7 1 1 5 9 . 1 •2.2.4 ' H e r r i n g Meal 7.50 308*9 10 13 14.94 78.6 14 . 3 15.00 324.0 9 12 6.29 93.7 1 1 , 3 S t i c k w a t e r Meal 5.00 3 3 2 . 1 12 6 14.23 101.8 3 3 . 6 ; IO.OO 355.8 21 0 13.71 125.5 2 5 . 2 H e a t - T r e a t e d Egg 7 . 5 0 346.62 16 6 1 3 . 0 7 114.6 21,9 11.25 3 8 2 . 0 5 19 1 13.94 150.1 20.1 Raw Egg 7.50 3 5 6 . 3 3 15 7 1 1 . 1 7 124.5 20.8 1 1 . 2 5 3 8 7 . 1 5 20 0 15.64, 155.2 21.4 Head 7 . 5 0 , 3 2 3 . 3 3 15 6 '12.89 9 1 . 3 14.3 11*25 3 7 0 . 4 2 19 2 1 1 . 0 3 138.4 15.1 :Gut 7.50 413.73 2 3 0 9.58 181.7 37.4 Chum V i s c e r a ' 5.00 270.4 13 8 16.89 40 .4 42.5 jio.oo, 3 0 3 . 6 20 0 1 4 . 0 8 73.6 3 6 . 0 •Pink V i s c e r a 5 . 0 0 275.2 15 3 14.86 45.2 4 8 . 5 1 0 . 0 0 291.9 2 2 0 19 . 5 0 61.9 - 3 1 . 5 •Sockeye V i s c e r a 5.00 289.9 18 2 14.9 3 59.9 . - 10.00 28 2 . 4 1 6 0 14,54 5 2 . 4 5 8 . 3 L i v e r Meal 2 . 5 0 279.8 19 2 1 3 . 8 3 49.8 • 9 3 ? ^ ' 5.00 306.5 2 2 0 1 7 . 50 76.50 •• 6 8 ro % Average 24.8 12.« 29.4 21.0 21.1 14.7 37.4 39.2 40.0 58.3 80,3 Based on Normal Chicks Only. TABLE V Average Weights of C o n t r o l s (Based on Normal Chicks Only) • Wt. of Chicks . at 5 Weeks (Grams) Number of Chi c k s at 5 Weeks Gain- i n Wt-. Over N e g a t i v e C o n t r o l (Grams) Normal Showing A v i t . G Nega t i v e C o n t r o l 2 3 0 „ 3 2 19 0 B a s a l + 1 0 0 r 3 2 6 . 7 2 0 0 9 6.4 B a s a l + 2 0 0 r 3 7 1 . 7 2 3 0 141.4 B a s a l + 3 0 0 ^ 3 8 9 . 3 24 0 • 1 5 9 . 0 N e g a t i v e C o n t r o l 2 3 2 . 0 2 !? 0 B a s a l + 7 5 r 3 0 8 . 4 1 7 3 7 6 . 4 B a s a l + 1 2 5 ^ 3 5 0 . 3 2 3 0 1 1 8 . 3 B a s a l + 1 7 5 r 3 5 9 . 5 21 0 1 2 7 . 5 N e g a t i v e C o n t r o l 2 3 0 . 0 6 1 2 0 B a s a l + 5 0 f 2 3 6 . 4 1 3 6 6 04 B a s a l + 1 0 0 ^ 2 5 8 . 6 1 9 3 2 8 . 6 B a s a l + 1 5 0 / 2 7 5 . I 2 0 0 / : 4 5 » 1 TABLE V l ( a ) S t a t i s t i c a l Summary of S e r i e s 1 A n a l y s i s of S i g n i f i c a n c e of D i f f e r e n c e s O Pt •P o o « rH V o o H cd ra cd © OJ o o CM + > H cd xa cd PQ * o o KN + rH cd 82 cd « - • 6 tt). •rl h © o « rH 60 £ • H JH fH © w e o H cd © U ffl •P aJ O •H -P • © tr- °. o rH H cd © u +5 cd M o •H -P « LTN o H cd CO . e O n o - » ICN rH fl o H cd O rH 1. C o n t r o l s s s s s S S s - s 2. B a s a l + 10G f" ' s - s S s 3 . B a s a l + 200r s s -. N 4. B a s a l + 3 0 0 s s N s. 5 • H e r r i n g 7 . 5 s . 6 . H e r r i n g 15.0 • s s .; H - • S -- - • 7 o S t i c k w a t e r Meal 5.0 s - N 8 . S t i c k w a t e r Meal 10.0 - s •1 N 9 ., Salmon 7 . 5 - K 10. Salmon 15.0 s s — ' • — N _ K s N - TABLE VI(b) S t a t i s t i c a l Summary of S e r i e s 1 A n a l y s i s o f S i g n i f i c a n c e of D i f f e r e n c e s H O u •p Pi o o « H Lf\. + H cd xa cd CM CM H + r-i cd to cd m » V 1 H + • H cd m cd P P » o 1A 9 c— M w « • / E H : e w o LA IA CM . H H W to . « EH * > • i M cd • l A CM « H «> cd CO O C~ cd <D w 9 OS 10.  H ea d 11 ,2 5 j IA « •P 1, C o n t r o l -. . N s . S s S s S s s 2. B a s a l + 75v. N : , s,. ; • 3o B a s a l + 125 < .'• s N • 4. B a s a l + 17 5 f s s N N s 5. H.T. Egg 7.50 s N : 6. H.T. Egg 1 1 . 2 5 s N N. • • • • • • • 7. Raw Egg 7.5 s s 8. Raw Egg 11.25 s S s ?. Head 7.5 s s - S 10. Head 11.25 s , - - •,- s 11. Gut .7.5 s TABLE V l ( c ) S t a t i s t i c a l Summary o f S e r i e s 1 A n a l y s i s of S i g n i f i c a n c e of D i f f e r e n c e s Co nt ro l J V o IA •+' H cd w cd m B as al  +  l OO r o rH *f H cd CO cd o 0 "A •s & O o « o H .3 O o * IA M Pi •H P4 o a O H Ad Pi •H W O • IA CD >, <D AS o o CO S oc ke ye  10 .0  IA CM U <D > •rt o IA <D !=• •H • • • 9 "* . . tr- . CO OX • o « rH H . CM H 1. C o n t r o l - S, S, ; . S. s. . s. s s s 7 s S. s 2. B a s a l + 50< S - s ; S 3. B a s a l + 100^ s s - : N N 4. B a s a l + 150/ s s - S N s s \ 5 o Chum 5•0 3. , s 6 » Chum 10.0 s . s. s . ,7. P i n k 5.0 S . - 8. P i n k 10.0 S H. : N N. . • . 9. Sockeye 5.0 .• - •' N 10. Sockeye 10.0 N N - 11. L i v e r 2.5 s ; f l . . - s 12. L i v e r 5.0 s s - - M I - SERIES 2 (a) The Experiment The f i r s t s e r i e s demonstrated t h a t f i s h m e a l s which were supplemented w i t h s u f f i c i e n t v i t a m i n G- produced s a t i s f a c t o r y growth and freedom from c u r l e d - t o e p a r a l y s i s . T h i s i n t r o - duced the q u e s t i o n o f the r e l a t i v e e f f i c i e n c y of v i t a m i n G o b t a i n e d from v a r i o u s sources -- whether t h e r e were any d i f f e r e n c e i n the e f f i c i e n c y of r i b o f l a v i n as found i n f i s h m e a l s , i n d r i e d s k i m m i l k or i n the s y n t h e t i c v i t a m i n . T h i s second s e r i e s was conducted i n o r d e r t o study the above problem, and a l s o to e s t i m a t e the amount of r i b o f l a v i n r e q u i r e d to o b t a i n optimum growth and freedom from symptoms of a r i b o f l a v i n o s i s . (b) M a t e r i a l s and Methods The m a t e r i a l s and the methods employed i n t h i s second s e r i e s were e s s e n t i a l l y the same as those used i n the f i r s t s e r i e s . There were, however, c e r t a i n minor d i f f e r e n c e s i n the e x p e r i m e n t a l procedure which w i l l be d i s c u s s e d as the p o i n t i n q u e s t i o n a r i s e s . In t h i s second s e r i e s the amounts of r i b o f l a v i n were known from m i c r o b i o l o g i c a l a s s a y . The purpose of t h e experiments was not t o determine the potency o f v a r i o u s c a r r i e r s , as i t was i n the f i r s t s e r i e s , but r a t h e r to study the r e s u l t s when known o p t i m a l amounts o f r i b o f l a v i n were added t o v a r i o u s supplements, and to compare the v i t a m i n e f f i c i e n c y o f d i f f e r e n t m a t e r i a l s w i t h s y n t h e t i c r i b o f l a v i n * Table I i n - e a c h case r e p r e s e n t s the a n a l y s i s of the i n g r e d i e n t s used i n the r a t i o n s of the t e s t ; Table I I g i v e s the c o m p o s i t i o n of t h e b a s a l r a t i o n ; Table I l i a r e p o r t s the c o m p o s i t i o n of each of t h e r a t i o n s per 50 pounds (enough f o r the e x p e r i m e n t ) , and Table I l l b , f o r ready c a l c u l a t i o n , the co m p o s i t i o n of t h e r a t i o n s i n terms of per cent; Table I¥ g i v e s the r i b o f l a v i n content of each of the r a t i o n s as c a l c u l a t e d from m i c r o b i o l o g i c a l d e t e r m i n a t i o n s made f o r each of the i n g r e d i e n t s ; and Table T r e p o r t s the weekly weight averages of the c h i c k s on each of the r a t i o n s , t o g e t h e r w i t h the i n c i d e n c e o f a v i t a m i n o s i s . (c) R e s u l t s The r e s u l t s o f t h i s s e r i e s o f experiments are r e p o r t e d f o r each experiment i n Table V. O b s e r v a t i o n s and i n t e r - p r e t a t i o n s are i n c l u d e d w i t h each i n d i v i d u a l t r i a l , EXPERIMENT I The f i r s t experiment was undertaken to i n v e s t i g a t e the e f f e c t s o f adding 225 micrograms of r i b o f l a v i n per 100 grams of feed to v a r i o u s l e v e l s of d r i e d s k i m m i l k , I m p e r i a l Brand F i s h M e al, h e r r i n g meal, meat meal and c a s e i n . Any Improve- ment i n t h e r a t e o f growth o b t a i n e d upon the a d d i t i o n o f t h e s e supplements would t h e r e f o r e be d i r e c t l y a t t r i b u t a b l e t o the i n f l u e n c e of t h e v i t a m i n , and not t o the n a t u r e of the p r o t e i n . - 33 - C u l t o n and B i r d (10) had r e p o r t e d t h a t the growth - promoting p r o p e r t i e s of d r i e d skimmilk were g r e a t e r than c o u l d be a s c r i b e d to i t s f l a v i n c o n t e n t . The a d d i t i o n of d r i e d skimmilk t o a f l a v i n - d e f i c i e n t d i e t r e s u l t e d i n g r e a t e r growth response per u n i t of f l a v i n added than d i d the a d d i t i o n o f c r y s t a l l i n e r i b o f l a v i n * They a l s o found t h a t w i t h the e x p e r i m e n t a l b i r d s t hey used, 415 micrograms of r i b o f l a v i n i n d r i e d s k i m m i l k or d r i e d whey d i d not prevent c u r l e d - t o e p a r a l y s i s , and n e i t h e r d i d 3 0 0 micrograms of c r y s t a l l i n e r i b o f l a v i n added to 175 micrograms of f l a v i n per 100 grams of feed* Throughout S e r i e s 2, s k i m m i l k was used as a r e f e r e n c e f o r o t h e r e x p e r i m e n t a l m a t e r i a l s , i n o r d e r to determine whether these r e p o r t e d f i n d i n g s were reproduced under the t e c h n i q u e s and e x p e r i m e n t a l procedures employed at t h i s l a b o r a t o r y * In Experiment 1, the c a l c u l a t i o n ' s were based on pounds of supplement; and from t h i s , the amount of p r o t e i n was determined which would r e p l a c e an e q u i v a l e n t amount of soyabean o i l meal p r o t e i n from th e c o n t r o l * That i s , h e r r i n g meal 6$ i n t h i s experiment means 6 pounds of h e r r i n g meal p l u s 10.46 pounds of soyabean o i l meal per 100 pounds of t o t a l r a t i o n . T h i s i s determined from the f a c t t h a t the c o n t r o l r a t i o n , c o n t a i n i n g 20 pounds of soyabean meal, s u p p l i e d 8.73$ p r o t e i n . S i n c e 6$ h e r r i n g meal (69$ p r o t e i n ) s u p p l i e d 4.161$, an amount of soybean meal was r e q u i r e d t o s u p p l y the r e m a i n i n g 4.569$, t o make the t o t a l of 8.73$ — or i n o t h e r words, 10.46 pounds of - 3 4 - soyabean o i l meal. The r e s u l t s of the u n f o r t i f i e d r a t i o n s are due e n t i r e l y , t h e n , to the g r o w t h - s t i m u l a t i n g p r o p e r t i e s of the supplement. At a l e v e l of 2 $ , m i l k ( d r i e d skimmilk) was not s u f f i c i e n t ' t o prevent the o c c u r r e n c e of c u r l e d - t o e p a r a l y s i s ; and even at the h i g h e r l e v e l s of 4 $ and 6 $ d i d not produce c h i c k s e n t i r e l y f r e e from s i g n s of a v i t a m i n o s i s . At t h e ' l a s t l e v e l , however, the growth r e s u l t s were c o n s i d e r a b l y b e t t e r . The a d d i t i o n o f 2 2 5 micrograms of r i b o f l a v i n t o the m i l k supplements d i d not produce any s i g n i f i c a n t g a i n over the u n f o r t i f i e d l e v e l , which would i n d i c a t e t h a t the m i l k i n i t s e l f i s s u f f i c i e n t l y w e l l s u p p l i e d w i t h v i t a m i n G to support good growth. In the case of the I m p e r i a l Brand F i s h Meal l i k e w i s e , the lower l e v e l s o f 2 $ and 4% d i d not s u p p l y s u f f i c i e n t r i b o f l a v i n t o prevent the c h a r a c t e r i s t i c symptoms of v i t a m i n G d e f i c i e n c y . The 6 $ l e v e l a g a i n produced e x c e l l e n t growth, but as i n t h e case o f the m i l k , the weights were not s i g n i f i c a n t l y improved by the a d d i t i o n of r i b o f l a v i n . The use of a c o m b i nation of 2/= I m p e r i a l Brand F i s h Meal w i t h 2% m i l k produced v e r y good growth r e s u l t s , which were not i n f l u e n c e d by a d d i t i o n a l r i b o f l a v i n . T h i s confirmed t h e i n d i c a t i o n t h a t both these p r o d u c t s were good sources of v i t a m i n G. I t w i l l be n o t i c e d t h a t where r i b o f l a v i n was present i n - 5 5 - suboptimal q u a n t i t i e s , the symptoms of d e f i c i e n c y apparent i n the t h i r d week c l e a r e d up by the f i f t h week, due to the d e c r e a s i n g requirement f o r growth. At the 31° l e v e l , h e r r i n g meal was d e f i n i t e l y i n f e r i o r , but t h a t t h i s e f f e c t was due to i t s low v i t a m i n G- content and not t o i n f e r i o r p r o t e i n q u a l i t y was s t r i k i n g l y shown upon the a d d i t i o n o f 2 2 5 micrograms of r i b o f l a v i n . An a d d i t i o n of 6 f « of the product brought about an improvement i n the r a t e of g a i n , which was l i k e w i s e much s u p e r i o r when the v i t a m i n was added. The same s i t u a t i o n e x i s t e d i n t h e case o f the meat meal, where both l e v e l s were g r e a t l y improved by the a d d i t i o n o f r i b o f l a v i n . The poorer v a l u e s o f u n f o r t i f i e d l e v e l s of meat meal are to be expected from r e s u l t s o b t a i n e d by R o b e r t s o n , Carver and Cook ( 3 8 ) . These i n v e s t i g a t o r s r e p o r t e d u n i f o r m l y h i g h v a l u e s from f i s h m e a l s . The average "gross v a l u e " of h e r r i n g f i s h m e a l s was 1 0 1 as compared w i t h c a s e i n , 1 0 0 ; p i l c h a r d meals were v a l u e d at 9 8 ; s a r d i n e meals at 9 5 ; and salmon at 8 6 , 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 the d r i e d s k i m m i l k and b u t t e r m i l k . Both c o n t a i n e d p r o t e i n of h i g h q u a l i t y , but had g r o s s v a l u e s which were lower t h a n those o f the f i s h meals. Meat meals were u n i f o r m l y poorer sources o f p r o t e i n , a v e r a g i n g o n l y 5 5 i n g r o s s v a l u e . T h i s term, "gross v a l u e " i s "a r e l a t i v e n u m e r i c a l e x p r e s s i o n o f the growth response of c h i c k s , o b t a i n e d w i t h p r o t e i n supplements when added to a d i e t b e l i e v e d complete i n a l l r e s p e c t s except - 3 6 - q u a l i t y and q u a n t i t y of p r o t e i n . " The net g a i n per u n i t of supplementary p r o t e i n was compared w i t h the net g a i n per u n i t of supplementary p r o t e i n from c a s e i n , a r b i t r a r i l y a s c r i b e d the v a l u e of 100. The v a l u e s o b t a i n e d w i t h c a s e i n i n t h i s s e r i e s were c o n s i s t e n t l y low, even when supplemented w i t h r i b o f l a v i n . A comparison of the weights o b t a i n e d when 2 0 $ soyabean meal ( c o n t a i n i n g 2 - 3 micrograms per gram, from b a c t e r i o l o g i c a l d e t e r m i n a t i o n ) was f e d , w i t h those when 2 0 $ c a s e i n ( 3 m i c r o - grams) was g i v e n , shows t h a t the soyabean meal produced c h i c k s w e i g h i n g 3 6 6 , 3 8 1 and 4 0 3 grams when 7 5 , 1 2 5 and 1 7 5 micrograms r e s p e c t i v e l y were added, w h i l e the c a s e i n produced s i g n i f i c a n t l y l i g h t e r c h i c k s w e i g h i n g o n l y 3 3 0 , 3 5 7 and 3 3 9 grams at the same l e v e l s . C a s e i n was f o r m e r l y employed as a stand a r d i n most experiments when i t was d e s i r e d t o e v a l u a t e the q u a l i t y o f a p r o t e i n , because i n s p i t e of a low c y s t i n e content i t was b e l i e v e d t o be one of the most complete p r o t e i n s , . a n d c o u l d be o b t a i n e d i n a p u r i f i e d form. However, B r a n i o n et a l 0 ( 8 ) showed t h a t c a s e i n o b t a i n e d from v a r i o u s sources v a r i e s i n q u a l i t y and r i b o f l a v i n c o n t e n t . Moreover, i n r e c e n t i n v e s t i g a t i o n s , A l m q u i s t ( 2 ) has suggested t h a t i t i s b e t t e r t o use a st a n d a r d p i l c h a r d meal supplement as a c r i t e r i o n f o r growth comparisons, because the amino a c i d d e f i c i e n c i e s of c a s e i n f o r c h i c k s i n l o w - p r o t e i n experimental d i e t s may cause c a s e i n t o be a v a r i a b l e , dependent upon t h e amino a c i d content TABLE I A n a l y s i s of I n g r e d i e n t s M o i s t u r e Fat P r o t e i n Ash ; P 2 G 5 . CaO H e r r i n g Meal 8 , 3 0 8.43 6 9 . 3 5 1 1 , 7 5 4 . 9 6 4 . 1 2 Soya Meal 10,12 5 . 0 0 4 3 . 6 5 5 . 5 3 0 . 8 4 0 .40 Wheat 1 2 , 5 6 2 , 2 ? 12.46 1 . 7 3 0.41 0 , 1 5 I m p e r i a l Brand F i s h Meal 8 , 7 5 12.18 6 1 . 4 5 6 . 3 2 2 e 7 7 0 , 3 3 ;Meat Meal 6.3? ? « 0 5 4 8 . 6 5 2 9 . 3 4 1 1 . 7 3 1 3 . 3 3 B a s a l 1 1 , 8 8 3.52 13.85 5 . 9 5 0 . 5 1 1 . 5 8 : S k i m M i l k • 4 . 4 4 - 0 . 0 5 3 4 . 3 4 1*59 2.42 1 . 8 8 C a s e i n 1 0 . 3 ? 0 . 3 1 7 6 , 2 0 4.62 2 , 2 0 1 . 9 5 TABLE I I Composition of B a s a l R a t i o n F i s h O i l (100D, 1000A) . . . . 1.0 S a l t . „..... . ............... • 1.0 Limestone ...... 1.5 Bone F l o u r . 5 M i d d l i n g s . . , , 10.0 Bran . 10.0 Ground Oats 10.0 Corn Meal . . ............» 10.0 Ground Wheat 36.0 Soyabean Meal .,. . 20 .0 100.0 s ^Manganese s u l p h a t e added, at the r a t e of i - pound per t o n o f f e e d . • TABLE, i l l-a Composition, of Rations per F i f t y Pounds Per Cent Supplements Supplement Soyabean Fat Added Ash Added V/heat to Balance PER CENT. PROTEIN Meal Supplement From Supplement From Soyabean Meal Pounds Pounds Grams Grams Poxinds 1 . Control Soya 20/t + 75 r Riboflavin Soya 20^ + 125 / Riboflavin Soya 20$ + 175 * Riboflavin r\ U 1 0 . 0 0 71.41 90.21 10.00 0 8.73 2. 0 1 0 , 0 0 71.41 90.21 10.00 0 8.75 3. 0 y10.OQ 71.41 90,21 10.00 0 8.73 4. 0 1 0 , 0 0 71.41 90.21 10.00 0 8.73 •5. Soya 20$ + 225 ''Riboflavin 0 : 1 0 , 0 0 71,41 90.21 10.00 0 8.73 6. Mile' Z% 1.0 9.21 87.58 75,59 . 9.79 .6868 8.0433 7, Milk 4^ 2.0 8.43 105.56 61,20 9.57 1.3736 7,3564 8. Milk 6% 3 , 0 7.65 136,29 45.97 9.55 2.0604 6.6696 9. Milk 4$ +• 225' Riboflavin 2.0 8.43 105.56 61.20 9.57 1.3736 7.3564 10. Milk 6% + 225 / Riboflavin 3.0 7.65 136,29 45.97 9.35 2.0604 6.6696 1 1 . Imperial Brand Fish Meal 2fo 1.0 8.59 46.70 96,64 10.41 1.2290 7.5010 12. " 4$ 2.0 7.19 23.18 103.49 10.81 2.4580 6.272 15. " 6% 3 . 0 5.77 - 110.32 Ho, 23 3.6870 5.043 14. " Z% + . 225 ^Rib o f l a v i n 1.0 8.59 - 46,70 96.64 10.41 1.2290 7.5010 15. " 4$ + 225 r Riboflavin ,2.0 7.19 23.18 103.49 10.81 2.4580 6.272 16. Herring 3% X o 13 7". 62 66,83 70.08 10.88 2,0805 6.6495 17. Herring 6% 3 , 0 £3 « 23 17.94 0 11.7?'' • 4.1610 4.5690 18. Herring 3$-+ 225. V. " 1.5 7.62 66.83 70.08 10.88 2.0805 6,6495 19. Herring 6% + 225'"' " 3.0 5.23 17.94 0 11.77 4.1610 ' 4,5690 20. Meat 4$ 2.0 7.77 38.32 0 10*23 1.9460 6,7840 21. Meat Q% 4.0 , 5,54 6.73 0 10.46 3.8920 4.8380 22. Meat 4$ + 225 ^ R i b o f l a v i n 2.0 7.77 38,32 0 10.23 1.9460 6.7840 • 23. Meat 8$ + 225^ Riboflavin 4 . 0 : 5.54 6.75 0 10.46 3.8920 4.8380 24. Imperial Brand Fish Meal Z% + Milk 2$ 1.0 +• LO 7.80 64.28 32,37 10.20 2.0158 6.7142 25. Imperial Brand Fish Meal 2$'+ Milk 2$- + ..225T/ Riboflavin 1.0 •+ 1,0 7.80 64,28 32.37 10.20 2.0158 6.7142 26. Casein + 75f Riboflavin 5.73 0 288.81 221.19 14.27 8.73 0 27. Casein + 125< Riboflavin 5.73 0 288.81 221,19 14.27 8.73 0 28. Casein + 175V Riboflavin 5.73 0 • 288.81 221.19 14.27 8.73 0 Fote: Bacteriological assay of supplements showed the following riboflavin..potencyt Soyabean Meal 2-3 * ; Milk 20 — ' — Imperial Brand Fish Meal 25 r ; Herring 15 Y ; Meat 7js Y\ Casein 3 r. TABLE-111b. Composition o f R a t i o n s per 1 0 0 Pounds SUPPLEMENT PROTEIN Lot R a t i o n Supplement Pounds Soyabean Meal Pounds 1. C o n t r o l 0 2 0 2 . Soya 2 0 / » + 7 5 * " R i b o f l a v i n 0 2 0 3 . Soya 2 0 f . + 125^ R i b o f l a v i n 0 2 0 4 . Soya 2 0 / . .+ 1 7 5 ^ R i b o f l a v i n 0 2 0 5 . Soya 2 0 f . + 2 2 5 ^ R i b o f l a v i n 0 2 0 6 . " M i l k 2$ " 2 . 0 1 8 . 4 2 7 . M i l k 4 7 . 4 . 0 1 6 . 8 6 8 . ; M i l k 6 7 . 6 . 0 1 5 . 3 0 9. M i l k 4 $ , + 2 2 5 ^ R i b o f l a v i n 4 . 0 1 6 . 8 6 1 0 * M i l k 6 7 . + 2 2 5 r R i b o f l a v i n 6 . 0 1 5 . 3 0 1 1 . I m p e r i a l Brand F i s h Meal 2% 2 . 0 1 7 . 1 8 1 2 . I m p e r i a l Brand F i s h Meal 4 f . 4 . 0 1 5 . 3 8 1 3 . I m p e r i a l Brand F i s h Meal 6 7 . 6 . 0 11.54 14. Imp.Br.F.M. 2% + 225* R i b o f l a v i n 2 . 0 1 7 . 1 8 1 3 . Imp.Br.F.M. 4 C / . + 2 2 5 * " R i b o f l a v i n H e r r i n g 3"/» 4 . 0 1 5 . 3 8 1 6 . 3 . 0 1 5.24 1 7 . H e r r i n g 6 $ 6 . 0 1 0 . 4 6 I B . H e r r i n g 3 7 ° + 2 2 5 ^ R i b o f l a v i n 3 . 0 1 5.24 19. H e r r i n g 6 f . + 2 2 5 ^ R i b o f l a v i n 6 . 0 1 0 . 4 6 2 0 . • Meat 4% 4 . 0 1 5 > 5 4 2 1 . Meat 8> 8 . 0 1 1 . 0 8 2 2 . Meat 4 7 . + 2 2 5 * R i b o f l a v i n 4.0 1 5 . 5 4 2 3 . Meat 8 f . + 2 2 5 ^ R i b o f l a v i n _ - . . . 8 . 0 1 1 . 0 8 24. Imp.Br.F.M. 2% +• M i l k 2% 2 . 0 + 2 . 0 15.60 2 5 . Imp.Br.F.M. 2 f . + M i l k 2J. + 2 2 5 ^ . R i b o f l a v i n 2 . 0 + 2 . 0 1 5 . 6 0 2 6 . C a s e i n + 7 5 ̂  R i b o f l a v i n , 11.46 0 2 7 . C a s e i n + 1 2 5 * R i b o f l a v i n 1 1 . 4 6 0 2 8 . C a s e i n + 1 7 5 ' ' R i b o f l a v i n 1 1 . 4 6 0". TABLE IY Riboflavin Content of Rations i n f per 100 gm» Lot Ration Basal PROTEIN Crystalline Total Supplement Soyabean Meal Riboflavin 1. Control 63 0 50 0 115 Soya 20% * 75rRiboflavin 65. 0 50 75 188 3. Soya ZQ% + 125 V Riboflavin 63 0 50 125 238 4* Soya 20% + 175 v Riboflavin 63 0 50 175 288 5. Soya 20$ + 225y Riboflavin 63 0 50 225 338 •6. 13.1k 2$ 63 40 46 0 149 7. Milk' 4$ .. 63 80 42 0 185 8, 65 120 38 0 221 9. MilkMfo + 2251- Riboflavin 63 80 42 225 410 10. Milk 6$ + 225{r Riboflavin 63 120 38 225 446 XX » Imperial Brand f i s h Meal 2% 63 50 43 0 156 12. .Imperial Brand Fish Meal 4^ 63 100 35 0 3J99 13. Imperial Brand Fish Heal 6% 63 150 29 0 242 14. Irap.Br.F.M. 2% + 225y Riboflavin 65 50 43 225 . 381 15. Imp.Br.F.M. A% + 225r Riboflavin 63 100 36 225 424 16. Herring 'Sfo 63 45 38 0 146 17. Herring 6% ' 63 90 26 0 179 18. Herring 3% + 225jT Riboflavin 63 45 38 225 371 19. Herring 6^ + 225r Riboflavin 63 90 26 225 404 20. Meat 4$ 63 30 39 0 152 21. Meat 8$ 63 * 60 28 0 151 22. Meat 4$ + 2253- Riboflavin 63 30 39 225 557 23. Meat %$ + 225 V Riboflavin. : 63. 60 28 .225 376 24. Imp.Br.E.H;- 2^ + Milk 2$ 63 50 + 40 39 o 192 25. Imp.Br.F.M. 2$ + Milk 2% + 225V Riboflavin 65 50 +.40. 39 225 417 26. Casein + 75r Riboflavin 63 34 75 172 27. Casein + 125r Riboflavin 65 34 125 222 28. Casein + 175cr Riboflavin 63 54 175 272 . TABLE V 'Summary of Weights-of.-Chicles in Vitamin C-.Ejp'eriment PER CEHT PROTEIH FHOE SUPPLEMSJTS 1. Control 2. Soya 20$ + ^ R i b o f l a v i n 3. Soya 20$ + 125 J-Riboflavin 4. Soya 20$ + 175 V Riboflavin 5. Soya 20$ + 225'J'Riboflavin 6i Milk 2$ 7. Milk 4$ 8. Milk 6$ 9. Milk 4$ + 225 ̂ Riboflavin -10. Milk ff/o* 225 r Riboflavin 11. Imperial Brand Fish Heal Z% 12. Imperial Brand Fish Meal 4$ 13. Imperial Brand Fish Meal 6$ 14. Imperial Brand Pish Heal 2$ + 225VRiboflavin 15. Imperial Brand Pish Meal 4$ •+ 225r Riboflavin 16. Herring 3$ 17. Herring 6$ 16. Herring 3$ + 225» Riboflavin 13. Herring S% + 225!r Riboflavin 20. Heat 4jJ - 21. Meat 8$ -22. Meat 4$ + 225 ̂ Riboflavin 23. -Meat B% * 225 ITRibof lavin 24. Imperial Brand Pish Heal zi .+ Milk 2% 25. Imperial Brand Pish Heal 2$+Milk 2$+ ZZ5* Rioof lavin 26. Casein 20$"+ 75'* Riboflavin 27. -Casein 20$ + 125r Riboflavin - 28. Casein 20$ + 175 j-Riboflavin FIRST WEEK Normal Chicks gt.jgms.) Ho. 80.6 64.8 85.3 81.2 81.6 82.6 81.6 85.5 82.0 86.0 83.5 84.2 84.2 82.3' 83.5 88.9 82.4 83.6 81.9 84.8 82.7 86.7 87.4 79.9 81.1 32.5 83.8 84.7 22 21 20 21 21 22 20 21 22 22 21 20 22 20 21 18 21 19 15 19 2i 20 22 .21 22 21 18 21 SBOOED TfflEff Hormal Chicks! Showing;Avlt. G m. g o . - -m.. . ito 123.2 124.8 .124.9 127.8 141.7 147^8 124.3 ' 136.9 126.8 129.0 112.9 126.9 131.8 128.8 134.7 118; 7 121.5 132.2 132.7 119.4 1-14.3 151.6 136.1 114.4 122.7 12U2 127.4 130.7 28 20 20 21. 21 20 19 20 22 22 -18 15 22 20 21 14 13 19 15 18 19 20 22 18 22 21' 18 21 122.7 116.0-. 121.0 142i0-130.0 131.4 132.4 122.6 122.0: 123.0 111.0 4 1. 0 •0' 0 2 1 1 0 0 ". 3 5 .'•0 0 ' 0 4 8 ;0- 0 1 2 0 0 3 0 . 0 0 0 THIRD-SEEK; Hormal -Chicks;! Showing Avit. S fft. Ho. Wt. Ho. 178.8 166.0 191.4 192.6 223.8 - 186.2 188.3 215.5 192.0 198.8 ' • 205.9 203.0 212.7 200.0 209.9 179.7 183.7 212.7 216.1 168.2 172.3 1-99.7 203.4 •190.0. 203.6 176.9: 189.7 183.8 13 20 20 21 21 16 19 20 22 22 15 14 21 20 21 14 13 19 15 17 15 20 . 22 16 22 21 - 18 21 172i4- 163.0 187;9 207i0 212.0 196.3 196.3 196.0 164.2 169.2 154.5 -162,0 9 1 1 0 0 0 6 " 1 1 0 0 6 6 1 0 - 0 - 4 '8. •0 ,0 '2 6 0 0 4 0 '0 0 0 FOURTH V/EEK " Hormal .Chicks . V»t.. . -Ho. '245.9 5 269.0 21 278.1 20 291.7 ' 21 •287;4 20 270.5 • 20 276.5. 20 314.9 21 274.8 20 314.9 22 289.9 21 297 i'8 : 17 318,5 22 302.1 19 "316.1 21 239.1' 7 264.1 e 305 i 6 19 324.8' 15 .236.7 14 248.3' 14 291.4 '20 299.4 22 285.6 20 301.8 •22 243.4 21 260.1 :18 253.9 21 Showing-Avit. V/t. Ho. 239.3 242.4 216,0 219:0 16 0 0 0 0 2 0 0 0 • 0 0 3 0 0 - 0 11 13 0 0 5 6 0 0 1 0 0 0 0 Sever Shocring Avit. W. Bo. 239.1 269.0 278a 291.7 271.7 ' 274.5 . 314.4 274.8 314.9 291.S 293.4 314.5 - 302.1 316.-1 247.0 - 26S.8 305.6 -.324.8 236.7 . 248.3 29H4 299.4- •290.1 301.8 243.4 260.1 253.9 3 . . 21 •: 2 0 21 0 16 •19" . 20 22 22 15 . 13 21 1C 21 6 - 6 19 15 14 14- 20 22 16 22 21 18 • ' 21 - Hormal Chicks- Ho 365.9 381.1 404.6 369.3 366.5 437.4 394.1 422.8 - .385.3 452.3 400.4 431.3 308.1 -353.0 440.7 469.9 . -312.6- 319.5 399.7 411.8 418.7 424.4 330.4 357.3 339:6 21 20 21 21 20 •21 22 . 22 19 22 19 20 -14 13 13 15 14 -16- 20 . 22 21 22 21 10 21 ; FIFTH YJEEK (F" Shooing Avit. '.'ft. Ho. 390.0 301.0 •326.3 281.6 280.5 Eever Showing 'Avit. 0 . ! <H; •-. ;Ko. - 0 ' 365.9 : 21 0 : 381.1 20 0 -. 404.6 -. 21 1 • 372.2 - 16 0 360.9 . . 19 0 .•;-• 436.0 20 •0 394.1 22 0 422.8 22 1 388.9 13 0 453.3- . 21 6 . 400.4 19 0 431.3 20 .4 295.0 6 8 361.7 . 6 0 44Q.7 19 0 " 469.9 15 5 312.6 14 4 , 321.0 14 0 399.7 20 0 ' 411.8- 22 0 . -418V9 - 16 0 424.4 22 0 330.4- 21 0 357.3 18 0 • 338.6 •21 ~ftie to accident, no valiies for the fi f t h ijoslc are'-available. - 37 - of the r e s t of the r a t i o n , EXPERIMENT 2 A second experiment, s i m i l a r i n p l a n to the f i r s t , was undertaken i n the summer o f 1941; but t h i s time the supplements were c a l c u l a t e d i n terms of per cent p r o t e i n from the v a r i o u s t e s t m a t e r i a l s , i n s t e a d of per cent supplement as p r e v i o u s l y . Here, t h e n , 6f. h e r r i n g meal i n d i c a t e s 6$ p r o t e i n o b t a i n e d from 8.64 pounds of h e r r i n g meal, the balance of the p r o t e i n to be made up from soyabean o i l meal. As b e f o r e , the r i b o f l a v i n content was determined by m i c r o b i o l o g i c a l a s s a y , and r e p o r t e d i n Table IT, The r e s u l t s , however, were not at a l l i n accordance w i t h those t h a t xvould have been expected, and i t would seem t h a t t h e r e had been a d e s t r u c t i o n of the r i b o f l a v i n by some u n e x p l a i n e d means. I t w i l l be seen t h a t the a d d i t i o n o f r i b o f l a v i n had no i mproving e f f e c t , where i t d i d i n Experiment 1. F o r t i f i e d I m p e r i a l Brand F i s h M eal, d r i e d s k i m m i l k , and meat meal were not s i g n i f i c a n t l y improved by the a d d i t i o n of r i b o f l a v i n , above the u n f o r t i f i e d ; and the r e s u l t s o f the o t h e r t e s t m a t e r i a l s cannot be i n t e r p r e t e d . I t i s i n t e r e s t i n g to note the i n c i d e n c e of p a r a l y s i s i n the 6$ h e r r i n g l o t , but not i n the 3$; a l s o i n t h e u n f o r t i f i e d c o n t r o l and the two l e v e l s of meat meal. Hovrever, because of the seeming d e s t r u c t i o n of the r i b o f l a v i n by some means, i t would not be wise t o make any c o n j e c t u r e s as t o the TABLE I A n a l y s i s of I n g r e d i e n t s M o i s t u r e Eat P r o t e i n Ash P 2 0 _ 5 CaO H e r r i n g Meal 8 * 3 0 8 . 4 3 6 9 . 3 5 1 1 * 7 5 4 , 9 6 4 . 1 2 Soyabean Meal 1 0 * 1 2 3 . 0 0 4 3 . 6 5 5 . 5 3 0.84 0.40 Wheat 1 2 . 3 6 . 2 . 2 ? 1 2 * 4 6 1 . 7 3 0.41 0 . 1 5 I m p e r i a l Brand P i s h Meal , 8 . 7 3 1 2 . 1 8 61.45 6 , 3 2 2 . 7 7 0 . 3 3 Meat Meal , 6 . 3 ? 9 . 0 5 4 8 * 6 5 2 9 . 3 4 1 1 . 7 3 1 3 . 3 3 B a s a l t 1 1 , 8 8 3 . 5 2 1 3 . 8 5 5 . 9 5 0.51 1 . 5 8 Skim M i l k 4 .44 . - 0 . 0 5 3 4 . 3 4 7 . 5 9 2.42 1.88 TABLE I I Composition of B a s a l R a t i o n F i s h O i l (100D, 1000A) 1.0 S a l t 1,0 Limestone . 1.5 Bone F l o u r .. , 5 M i d d l i n g s 10.0 Bran . . .. 10 ,0 Ground Oats 10.0 Corn. Meal 10.0 Ground Wheat . „ 36.0 Soyabean Meal . 20.0 100.0* 3E Manganese s u l p h a t e added, at the r a t e of \ pound per t o n o f feed© TABLE I l i a C o m p o s i t i o n of Rations per F i f t y Pounds Supplement Pounds Soyabean Meal Pounds Fat Added Grams Ash Added Grams 1 , C o n t r o l 0 1 0 . 0 0 5 6 . 7 5 1 2 7 . 5 7 4 2 . Soya 8 . • 7 - 5 % + 2 25* R i b o f l a v i n 0 1 0 . 0 0 5 6 . 7 5 1 2 7 . 5 7 4 3 . B a s a l + 3 7 . H e r r i n g Meal 2.16 6 . 5 6 5 2 . 2 1 9 8 . 9 7 2 4 . B a s a l + 3 $ H e r r i n g M eal + 2 2 5 ^ R i b o f l a v i n 2.16 • 6 . 5 6 5 2 . 2 1 9 8 . 9 7 2 5 . B a s a l + 6 $ H e r r i n g Meal 4 . 3 2 3 . 1 3 4 7 . 6 7 7 0.143 6 s B a s a l + 6 $ H e r r i n g Meal + 2 2 5 ^ R i b o f l a v i n 4 , 3 2 3 . 1 3 4 7 . 6 7 7 0.143 7 . B a s a l l M M i l k • 2 . 1 8 8 . 2 8 9 5 . 3 4 9 5 . 7 9 4 8 e B a s a l + i f f . M i l k + 2 2 5 f R i b o f l a v i n 2.18 8 . 2 8 9 5 . 3 4 9 5 . 7 9 4 B a s a l + 3 > M i l k 4 . 3 6 6 . 5 6 1 3 3 . 9 4 64.014 1 0 . B a s a l + 3 $ M i l k + 2 2 5 r R i b o f l a v i n 4 , 3 6 6 . 5 6 1 3 3 . 9 4 6 4.014 1 1 . B a s a l + 3 / 1 I m p e r i a l Brand F i s h Meal 2 . 4 4 6 . 5 6 0 1 4 4 . 7 7 2 12. B a s a l + 3 > I m p e r i a l Brand F i s h Meal + 2 2 5 < R i b o f l a v i n 2 . 4 4 6 . 5 6 0 1 4 4 . 7 7 2 1 3 » B a s a l -J" •67 . I m p e r i a l Brand F i s h Meal ..• 4 . 8 8 3 . 1 3 0 160.716 14. B a s a l + 6 $ I m p e r i a l Brand F i s h Meal + 2 2 5 ^ R i b o f l a v i n 4 . 8 8 3 . 1 3 0 1 6 0.716 15. B a s a l + 3 7 . Meat 3.08 6 . 5 6 8.40 0 16. B a s a l + 3 $ Meat + 2 2 5 r R i b o f l a v i n 3 . 0 8 6 . 5 6 8.40 0 1 7 . B a s a l + 6f. Meat / 6.16 3 . 1 3 0 0 1 8 . B a s a l + 6f. Meat + 2 2 5 * R i b o f l a v i n 6.16 3 . 1 3 0 0 1 9 . B a s a l + 4-g-fo I m p e r i a l Brand F i s h Meal 3 . 6 6 4 . 8 4 - 0 1 5 2 . 5 4 4 2 0 . B a s a l + 4-|fo I m p e r i a l Brand F i s h Meal + - 225< Riboflavin 3 . 6 6 4 . 8 4 0 1 5 2 . 5 4 4 2 1 . B a s a l 2±% M i l k 3 . 2 7 7 . 4 2 1 0 8 . 0 5 8 0 . 1 3 1 2 2 . B a s a l + 2-J7I M i l k + 2 2 5 * R i b o f l a v i n 3 . 2 7 7.42 1 0 8 . 0 5 8 0 . 1 3 1 Note: B a c t e r i o l o g i c a l Assay of•Supplements showed the f o l l o w i n g r i b o f l a v i n potency: Soyabean Meal, 2 - 3 / ; H e r r i n g , 15/'; M i l k , 2 0 ^ ; I»P. B ^ n d F i s h Meal,- 25 ; Meat, 7 .5 * . TABLE IHb Composition of Rations per Hundred Pounds PRO TEIN;. Supplement Supplement Pounds Soyabean Meal Pounds 1. C o n t r o l Meal 8.73f» + 2 2 5 ^ R i b o f l a v i n 0 10.00 2. Soyabean 0 10.00 3* B a s a l + 3i H e r r i n g Meal 4 . 3 2 13.12 4, B a s a l + 3% H e r r i n g Meal + 22$r R i b o f l a v i n 4 . 3 2 13.12 5 . B a s a l 6% H e r r i n g Meal 8.64 6 . 2 5 6. B a s a l + 6B/. H e r r i n g Meal + 225^ R i b o f l a v i n 8.64 6 . 2 5 7. B a s a l + l-lfo M i l k lii M i l k +•225^ R i b o f l a v i n 4.36 16.56 8« B a s a l + 4.36 16.56 9. B a s a l + 3 7 » M i l k 8.72 13.12 10. B a s a l + 3t M i l k + 2 2 5 ^ R i b o f l a v i n 8.72 13.12 IX© B a s a l + 3% I m p e r i a l Brand F i s h Meal 4.88 13.12 12. B a s a l + 3% I m p e r i a l Brand F i s h Meal + 223< R i b o f l a v i n 4.88 13.12 1?. B a s a l + 6 7 . I m p e r i a l Brand Eish. Meal - 9«76 6.25 14. Basal" 6 / 0 I m p e r i a l Brand: F i s h Meal + 225r R i b o f l a v i n 9.7-6 6 . 2 5 1 5 . B a s a l + 3% Meat 6.16 1 3.12 16. B a s a l + 3% Meat + 2 2 5 r R i b o f l a v i n 6.16 1 3.12 17. B a s a l + 6f. Meat -• 1 2 . 3 2 6,25 18. B a s a l + -6,f. Meat + ' • • 2 2 5 * ' R i b o f l a v i n 12 . 3 2 6.25 19. B a s a l + 4-J5-/0 I m p e r i a l Brand F i s h Meal 7.32 9.68 2 0 . B a s a l A-ii« I m p e r i a l Brand F i s h Meal + 225< R i b o f l a v i n 7 . 3 2 9.68 21. B a s a l + 2-ifo M i l k 6.54 14.84 22. B a s a l + 2±f M i l k + 2 2 5 r R i b o f l a v i n 6.54 14.84 per Hundred Grams Lot R a t i o n B a s a l : PROTEIN C r y s t a l l i n e R i b o f l a v i n T o t a l Supplement Soyabean Meal .1. C o n t r o l Meal 8 . 7 3 % + 2 2 5 v R i b o f l a v i n 6 3 0 5 0 1 1 3 - • 2 . Soyabean 6 3 0 5 0 225 3 3 8 -v • 3 . B a s a l + 3 % H e r r i n g Meal : 6 3 6 5 3 3 161 , 4. B a s a l + 3 > H e r r i n g Meal + 2 2 5 * " R i b o f l a v i n 6 3 6 5 3 3 2 2 5 3 8 6 3 . Ba s a-1 6 > H e r r i n g Meal • 6 3 1 3 0 16 2 0 9 ' " 6 . B a s a l + b% H e r r i n g Meal + 2 2 5 * R i b o f l a v i n 6 3 1 3 0 16 2 2 5 4 3 4 i « B a s a l ItJo M i l k 6 3 8 7 41 191 B a s a l + l-lfo M i l k + 2 2 5 * R i b o f l a v i n 6 3 8 7 41 2 2 5 416 9 . B a s a l + 37»- M i l k 6 3 174 3 3 2 7 0 1 0 . B a s a l + 3 % M i l k + 2 2 5 1 " R i b o f l a v i n 6 3 174 3 3 2 2 5 4 9 5 ' 11. B a s a l + 3 % I m p e r i a l Brand F i s h Meal 6 3 1 2 2 3 3 2 1 8 . 12. B a s a l + 3 ' / . I m p e r i a l Brand F i s h Meal + 2 2 5 * " R i b o f l a v i n 6 3 1 2 2 3 3 2 2 5 4 4 3 l j . B a s a l + 6 > I m p e r i a l Brand F i s h Meal - 6 3 244 16 3 2 3 "14. B a s a l + 6 f . I m p e r i a l Brand F i s h Meal * -2 2 5 * R i b o f l a v i n 6 3 244 16 2 2 5 5 4 8 -15 . B a s a l + 3 > Meat 6 3 46 3 3 142 . • 1 6 . B a s a l + 3 % Meat + 225*" R i b o f l a v i n 6 3 46 3 3 2 2 5 3 6 7 1 7 . B a s a l + 6yo Meat - - 6 3 92 16 1 7 1 1 8 . B a s a l + 6f Meat + 2 2 5 * R i b o f l a v i n 6 3 92 16 2 2 5 3 9 6 19. B a s a l + 4i-f° I m p e r i a l Brand F i s h Meal 6 3 9 2 1 2 1 6 7 2 0 . B a s a l + 4 - | % I m p e r i a l Brand F i s h Meal + 2 2 5 * R i b o f l a v i n 6 3 92 1 2 2 2 5 3 9 2 21. B a s a l + 2 j 7 o M i l k • 6 3 6 5 1 8 146 2 2 , B a s a l + 2 i % M i l k + 2 2 5 r R i b o f l a v i n 6 3 6 5 1 8 2 2 5 3 7 1 TABLE V Summary of Weights of Chicks in Vitamin August, 1941 FIRST WEEK Hormal Chicks Wt. •Ho. 66.7 1 8 66.2 18 66.1 19 67.0 ' 8 66. J 18 65.8 19 65.8 19 66.5 19 65. J 1 7 67.5 19 66.2 8 .66.4 1 8 , 66.4 1 8 , 66.0 18 66.5 19 66.5 19 69.6 20 66.4 19 58.6 20 58.4 19 : 57.8 20 ..  57.2 17 SECOND WEEK Normal Chicks Wt. No. 101.9 18 101.8 1 8 107.5 19 104.7 18 109 .1  117.8 19 0 9 .  19 107.8 19 105.? 17 111.6 19 112 .1 18 112.8 1 1 5 . J 1 8 116.6 1 8 104.4 19 •  1 1 1 . 9 19 101.2 20 108..7 19 I O 5 . I 20 100.8.." 19 9 5 . 7 , 20 9 5 . 1 n I, . Soyabean -Meal 8 . 7 5 / . -.2, "Soyabean Meal 8 . 7 3"/.'+ 2 2 5 f Riboflavin -"• ••.3'. Herring Meal ?f •• -"•'4v Herring Meal-3% + 2 2.5>' Riboflavin "'"5, Herring Meal,6f. • • • 6 . Herring Meal bf * 225*" Riboflavin 7. Milk lH-8 . ' Milk + 2 2 5 ^ Riboflavin 9. Milk 37. " 10; Bilk'37. +• 2 2 5 r Riboflavin .". •-',-II. Imperial Brand Fish Meal 3°/=' ' '-v . -; 1 2 ; ,Imperial Brand Fish Meal 3*'. • 2 2 5 ' H!bi.rl<ivlr. -.13; Imperial' Brand' F i B h ' Meal: 6-jl .14.- Ini.oriul -irun-l Fish V.o>\X t", ' 2 2 5 ' ' Riboflavin -15. Meat 3f. • IS-," .-Meat- 3'/-:'•+. 2 2 5 ^ Riboflavin'. 17. .Meat 6 f . - <r8' . Meat 6.7.' + ' . 2 2 5 * ' Riboflavin -~ 19. Imperial Brand Fish Meal 4 V / . . ^ 2 0 . Imperial .Brand. Fishs-Heal + 225*«' Riboflavin' •2i; Milk 2-H • 2 2 . Milk 2-Jy. -+ 2 2 5 f Riboflavin G Experiment THIRD 1 t WEEK FOURTH WEEK = =^==============^ \ FIFTH WEEK - ' . . .....u.^"Chicks with'Avitaminosis 8 Chicks With Avitaminosis G uoriuaj-'̂ isî -̂ Y : » , Normal Wt. Chicks ! Ho. ! Normal • Wt. \J 11 -L v «  0 Ho. 1 1 .Wt. Mo. 217-0 7 3 2 0 0- U 18 312.7  6 0 • ' 156.8 18 232.9 1 8 0 284;4 I T ' 0 157.8 1 8 . 1 2 . 0 0 415.6 LI.:. - > 0 18 3.0 19 2 8 9 . 4 17 18 0 404.2 •S3 3 2 6 . 2 9 1 6 5 . 6 1 8 .274.2 235.9' 1 2 3 5 9 . 2 19 ; 0 1 7 0 . 0 18 2 5 8 .3 0 4 5 ° i 2 , 0 • : . . " 1 9 4 . 7 19 3 O 9 . 0 19 i <i 0 369.8 19 19; 0 1 7 6 . 7 19 1 2 6 4 . 8 j - 7 0 366.3 0 1 5 9 . 5 2 5 7 .8 19 11 0 3 3 4 . 7 17 • • 19 0 164.3 17 242.2 J- / 0 3 5 1 . 6 1 a ' 0 176.3 1 9 245.1 19 1 8 0 416.8 lo . • 18 - . " . '.- - - ' 0 - - 187.2 1 8 2 8 6 , 2 1 8 0 4 26;3 18 0 192.3 1 8 29.4.1 1 8 0 3 9 7 .7 v l 8 0 1 9 6 . 8 1 8 244.4 0 430.7 " 3 1 0 . 5 ' -'. * : 197.5 18 3P3.1 1 8 1 8 2 3 0 . 0 3 345.7 . 15 19 p 0 3 0 2 . 2 - 162.0 19 • 24-8 • A 0 3 9 8 . 4 1 8 2 . 7 19 279.1 19 229.7 3 342.8 ;16 ' 0 1 5 8 . 2 2 0 235.1 7 1 8 0 371.7 n 20 • , 0 0 160.1 19 255 • £ 2 0 • 0 420 • 0 19 180.7 2 0 29.2*. 19 0 •401.0 2 0 • ' 0 170,7 19 2 7 . 1 * . 0 333.2 i ̂  5 .1 ' 149.5' 2 0 17 2 3 4 . 2 2 6 . L 2 0 3 17 0 3 2 8 . 1 ... 1.1 1 : — reason why none of the o t h e r l e v e l s were a f f e c t e d , and no c o n c l u s i o n s were drawn from t h i s p a r t i c u l a r experiment, (Not c o n s i d e r e d i n c a l c u l a t i o n s . ) EXPERIMENT 3 A s l i g h t l y d i f f e r e n t p l a n was d e v i s e d i n the next experiment. Because a c e r t a i n amount of v a r i a t i o n was found i n Experiment 1, f u r t h e r work was c o n t i n u e d on t h i s p r o j e c t i n o r d e r t o t e s t the v a r i o u s supplements to determine whether the s y n t h e t i c v i t a m i n d i f f e r e d i n a v a i l a b i l i t y from the combined form. Once ag a i n t h e r a t i o n s were a l l c a r e f u l l y balanced to c o n t a i n the same amount of p r o t e i n . As i n the second experiment, 6 / . f i s h m e a l i n d i c a t e s 6 f » p r o t e i n o b t a i n e d from f i s h m e a l . Here, however, the r i b o f l a v i n content was a l s o s t a n d a r d i z e d to c o n t a i n , i n the f o r t i f i e d l e v e l , 3 6 8 . 5 1 1 4 . 1 u n i t s per 100 grams • T h i s ' amount wa-s known from p r e v i o u s i n v e s t i g a t i o n to be more than adequate f o r normal f u n c t i o n i n g , so t h a t any d i f f e r e n c e s here c o u l d be a t t r i b u t e d to d i f f e r - ences i n a v a i l a b i l i t y between the s y n t h e t i c and n a t u r a l l y o c c u r r i n g (combined) forms. P r e v i o u s l y t h e r i b o f l a v i n had been added i n c o n s i d e r a b l e e x c e s s . Here i t was c a r e f u l l y c a l c u l a t e d and added i n amounts t h a t made the v i t a m i n G- v a l u e of each r a t i o n the same. The u n f o r t i f i e d r a t i o n s would serve at the same time as a f u r t h e r i n d i c a t i o n of the n u t r i t i v e v a l u e s of the supplements. - 3 ? - The 4 $ d r i e d skimmilk produced, as would have been expected from i t s r i b o f l a v i n v a l u e of 3 2 3 micrograms, weights s l i g h t l y s u p e r i o r t o the weights of c h i c k s r e c e i v i n g o n l y 2 $ ( 2 l 8 micrograms), and d i d not cause the s m a l l o c c u r r e n c e of a v i t a m i n o s i s encountered i n the lower l e v e l . The a d d i t i o n of r i b o f l a v i n , however, e q u a l i z e d the r a t e s of g a i n from the two l e v e l s . The same s i t u a t i o n was encountered i n the case of the f i s h m e a l and the meat meal, where the 4 $ l e v e l was e i t h e r not s i g n i f i c a n t l y d i f f e r e n t from the 2 7° l e v e l , o r was o n l y v e r y s l i g h t l y s u p e r i o r t o i t . A g a i n , when the s y n t h e t i c r i b o f l a v i n was added t o a l l the l e v e l s to make the t o t a l v i t a m i n G v a l u e i n each e q u a l , t h e r e was no d i f f e r e n c e between them. The m i l k proved t o he s u p e r i o r to the f i s h m e a l and the meat meal of t h i s experiment, because of i t s r i b o f l a v i n c o n t e n t . At the 2 f . and 4 $ l e v e l s f o r each of these m a t e r i a l s , the v i t a m i n G content was: M i l k 2 1 8 and 3 2 3 F i s h m e a l ...... 145 and 1 7 6 Meat meal , 4 . . , 1 3 2 and 1 5 2 . The reason f o r t h i s v e r y h i g h v i t a m i n v a l u e , however, was, i n p a r t , i t s low p r o t e i n content ( 3 4 . 3 4 $ ) . At the 4 $ l e v e l s i t was n e c e s s a r y t o add 1 1 . 6 pounds of s k i m m i l k , 5 . 7 pounds of f i s h m e a l , and 8 . 2 pounds of meat meal. TABLE I A n a l y s i s o f I n g r e d i e n t s M o i s t u r e Fat P r o t e i n Ash p 2 o 5 CaO F i s h Meal 8 * 3 0 8 . 4 3 6 9 . 3 5 1 1 . 7 5 4.96 4 . 1 2 Soya Meal 1 © > 1 2 5 . 0 0 43.65 5.53 , 0.84 0 . 4 0 Whe at 1 2 . 5 6 2 . 2 9 1 2 . 4 6 1 . 7 3 . 0.41 0 . 1 5 C.F.C. Sp. F i s h Meal 8 .75 1 2 e 18 6 1 . 4 5 6 . 3 2 2.77 0 . 3 3 Meat Meal 6.39 9 . 0 5 48 .65 29.34 1 1 . 7 3 1 3 . 3 3 B a s a l l i e 88 3 .52 13«85 5 . 9 5 0 .51 1 . 5 8 Skim M i l k 4.44 0 . 0 5 3 4 . 3 4 7.59 2 . 4 2 l e 8 8 T A B L E I I C o m p o s i t i o n o f B a s a l R a t i o n F i s h O i l (100D, 1000A) . . . , . 1.0 S a l t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 L i m e s t o n e 1 . 5 B o n e F l o u r . 5 M i d d l i n g s . . 10.0 B r a n 1 0 . 0 G r o u n d O a t s 10.0 C o r n M e a l . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 G r o u n d W h e a t . 5 6 . 0 S o y a b e a n M e a l .' . 20.0 100.0 s ^ M a n g a n e s e s u l p h a t e a d d e d , a t t h e r a t e o f 4 p o u n d p e r t o n o f f e e d . TABLE I I I Composition of D i e t s Supplements 1. 4. ' , 5 . 5 a . 6 . 6a. 7 . 8 . . ?. 9a. 1 0 . 1 0 a . 1 1 . 12. 1 5 . 14. 1 5 . 1 5 a . 16. 1 6 a . 17 . 1 8 . Soya Soya Soya Soya M i l k M i l k M i l k M i l k M i l k M i l k F i s h F i s h F i s h F i s h F i s h F i s h F i s h F i s h Meat Meat Meat Meat Meat Meat 8.75$ 8 . 7 5 $ + 65J R i b o f l a v i n 8,73$ + 130^ R i b o f l a v i n 8.73$ + 260^ R i b o f l a v i n 2$ 2$ 2$ + 160?" R i b o f l a v i n 2$ + 1 6 0 / R i b o f l a v i n 4$ 4$ + 6 0 ^ R i b o f l a v i n 2$ 2$ 2$ + 21$fRiboflavin 2$ + 215^ R i b o f l a v i n 4$ 4$ + 1 7 0 y R i b o f l a v i n 6$ 6$ 2$ 2$ + 125 if R i b o f l a v i n 2 $ + 23Qf R i b o f l a v i n 2 $ + 2 3 0 c T ^ R i b o f l a v i n 4$ 4$ + 2 0 0 ? r R i b o f l a v i n PROTEIN Supplement Soyabean Meal Pounds Pounds 1 0 1 0 1 0 1 0 2.912. 7 .705 2.912 7.705 2.912 7 .705 2.912 7 .705 5.824 5.415 5 0 8 24 5.415 1.442 7.705 1.442 • " 7.705 1.442 7.705 1.442 7 .705 2.883 5.415 2.883 5.415 4/325 3.126 4 .325 3.126 2 .055 7.705 2 .055 7 .705 2.055 7 .705 2 .055 7 .705 4.110 5.415 4.110 5.415 FAT Grams 65.0712 65.0712 65.0712 65.0712 116.2240 116.2240 116.2240 116.2240 167.5487 167.5487 61.7213 61.7213 61.7213 61.7213 58.5433 58.5433 49.8492 49.8492 32.4610 32.4610 32.4610 32.4610 0 0 ASH Grams 85.3974 85.3974 85.3974 85.3974 42.7668) ) 42.7668) 42.7668) ) 42.7668) 0 0 66.1478) ) 66.1478) 66.1478) ) 66.1478) 46.8982) ) 46.8?82) 21.2245) ) 21.2245) 0 0 0 0 0 0 ) ) WHEAT TO BALANCE SUPPLEMENT Pounds 9 . 3 8 3 9 . 3 8 3 8 . 7 6 I 10.855 10.855 11.705 12.545 9.760 9.760 9.525 ! Note; B a c t e r i o l o g i c a l assay o f supplements showed potency: Soyabean M e a l , 2-3 r ; M i l k , 20 f ; the f o l l o w i n g r i b o f l a v i n F i s h , 15 r ; Meat 7^ ;r . I l i a . TABLE I l i a . C omposition of R a t i o n s per 100 Pounds SUPPLEMENT PROTEIN Lot R a t i o n Supplement Pounds Soyabean Meal Pounds 1. Soya 8 . 7 3 % 0 20 2. Soya 8 . 7 3 % + 65 * R i b o f l a v i n 0 20 3 . Soya 8 . 7 3 % + 1 3 0 * R i b o f l a v i n 0 20 4. Soya 8 . 7 3 % + 2 6 0 r R i b o f l a v i n 0 20 5 . M i l k H 5.824 15.410 5 a . M i l k 2i 5.824 15.410 6. M i l k 2i + 160 *" R i b o f l a v i n 5.824 15.410 6 a . M i l k 2i + l 6 0 * R i b o f l a v i n 5 . 8 2 4 15.410 7 . M i l k . 4% 11. 648 1 0 . 8 3 0 8 . M i l k 4% + 6 0 * R i b o f l a v i n 11 .648 1 0 . 8 3 0 ?.. F i s h 2% 2% 2 .884 15.410 9 a . F i s h 2 .884 15.410 1 0 . F i s h 2% + 215 r R i b o f l a v i n 2 .884 15.410 1 0 a . F i s h 2% + 215 Y R i b o f l a v i n " ' 2 .884 15.410 11. F i s h 4% 5.766 1 0 . 8 3 0 '12. F i s h Ai + 170 * R i b o f l a v i n 5.766 10 .830 13. F i s h 6i 8.650 6 .252 14. F i s h 6 % + 125 r R i b o f l a v i n 8 . 6 5 0 6 . 2 5 2 15. Meat 2% 4 . 1 1 0 15.410 15a. Meat 2% 4 . 1 1 0 15.410 1 6 . Meat 2i + 2 3 0 r- R i b o f l a v i n 4 . 110 15.410 16a. Meat 2% + 2 3 0 > - R i b o f l a v i n 4 . 1 1 0 15.410 17. Meat Ai 8 . 2 2 0 1 0 . 8 3 0 18. Meat 4% + 200 r R i b o f l a v i n 8.220 IO .83O TABLE IT Riboflavin Content of Rations In r per 100 gm. Lot Ration Basal PROTEIN Crystalline Riboflavin Total Supplement Soyabean Meal.. 1. Soya 8.73$' 63 0 50 0 113 2. Soya 8.73$ + 65 *r Riboflavin 63 0 50 65 178 3. Soya 8.73$ + 130rRiboflavin 63 0 50 130 243 4. Soya 8.73? y + 260*" Riboflavin 63 0 50 260 373 5. Milk tC/o 63 116.5 38.5 0 218 5a. Ifilk 63 116.5 38.5 0 218 6. Ifilk d/o + 160V Riboflavin 63 116.5 38.5 160 378 6a. Milk 2% 4- 160V Riboflavin 63 116.5 160 378 7. Milk. 4% 63 235 27 0 525 8. Milk 4$ + 60er Riboflavin 63 235 27 60 383 9. Fish 2% 63 43 38.5 0 145 9a. Fish 2% 63 45 ' 58.5 0 145 10. Pish 2% + 2T5r Riboflavin . 63 45 38.5 215 360 10a. Fish 2% -f 215s- Riboflavin 63 45 38.5 \ - 215 360 11* Fish 4$ 63 86 27 0 176 12. Fish 4$ + 170-r Riboflavin 63 86 27 170 346 13. Fish 6$ 63 > 193 15 0. 271 14. Fish erf O/O 63 193 15 125 396 15. Meat 2% 63 31 38.5, 0 132 15a. Meat 2% 63 31 38.5 0 132 16. Meat 2% + 230v Riboflavin 65 31 " .. .58.5 230 362 -,. 16a« Meat 2% + 230r Riboflavin 63 31 58.5 230 562 17. Meat 4$ 63 62 27 0 152 18. Meat A% + 200V Riboflavin 63 62 27' 200 552 TABLE T Summary of Weights of Chicks in Vitamin G Experiment October, 1941. • TEH DAYS. THIRD -'.VEESI FOURTH WEEK SOEKLEIEHTS Initial wt. Ho. of Chicks : Hormal Chicks --Showing Avit.-G Hormal Chicks Hever'Showing Avit.G Showing Avit Wt. Ho. , 7ft. Ho. Wt. Ho. Wt. - Ho. Wt. Ho 1. Soya 8.73$- •< - , - 75.4 21 143.0 13 143.0 7 • 186*8. 12 191.5 11 : 196.2 6 2. Soya 8.73$ + 65 K Riboflavin : ,- -77.0 . 22 163.1 18- 151.0 - 4 232.2 19 233.7 17 201.3 3 3. Soya 8.73$+ 130r Riboflavin 76.4 .22 -160.9 22 - - . ' .0 241.3 20 241.3 20 - 0 4. Soya- 8.73$ +2S0ir Riboflavin.! 75.3 - 22 .174.6 21 , , - .:•-. 0 249.0 22 249.0 - 22 - 0 5. Milk 2$ . 75.7,: 21 .166.2. 18" 175.3 ' 3. 249.2 21 245.3 16 - 0 5a. Eilk 2$ SUlk -2$ + 160r Bibof lavin- V .77.9 - 21 .155.4 11 159. .3-: 3 - 254.8 17 254.2 15 - 0 6. , 78.9 , 22 176.8 21 , - 0 259.6 21 259.6 21 - 0 6a. Milk ,Z% * 1600iboflavin - 82.8 20 174.9 19 • - . 0 . 258.6 20 258.6 20 - 0 ' 7. Hilk 4$ - 73.5 - 21 174.2 21 - 0 259.1 21 259.1 21 - - . 0 0. Milk 4$ + eo/Riboflavin 75.6 - '21 169.1 21 0 254.6 20 254.6 20 - 0 9. Pish 2$ . • • 74.1 21 146.7 11 137.3 9 202.7 3 202.7 3 189.1 16 9a. Pish 2$ -. -78.0 ,22 - 139*9 10 •135.5 12 181.8 4 181.8 4 192.9 17 10. Pish 2$-+ 215if-Riboflavin • .' 78.1 22 .189.3 22 ' :• -. • o - 283.4 23 283.4 23 • - 0 10a. Pish 2$ +215/ Riboflavin - 77.4 -21 177.0 21 - . 0 270.3 20 270.3 20 - 0 11. Pish 4$ ' ' . 74.8 22 -143;7 6 144.5 16 203.0 5 209.8 4 169.5 15 12. Pish 4$ + 17orRiboflavin 76.4 21 183.0 21- 0 274.1 17 274.1 17 - 0 13. Fiah 6$ • -78.3 20 144*6 7 - 131.4 13 203.1 6 209.8 4 179.6 14 14. Fish'6$.+ 125»' Riboflavin .' 76.8 20 , 169.8 19 ' 174.0 1 262.9 20 261.8 19 - .. 0 IB. Heat -2$ - . -. - 75.0 22 146.3 - 12 140,3 9 200.3 12 195,0 9 189.2 9 15a. Kent 2$ - 78.6. 21 157.9 17 • 147.0 - 4 239.1 15 239.0 14 ; 212*0 4 16. -Meat 2$ + 230><t Riboflavin .-: - - '75*0 ,22 '174.3 22 0 254.7 22 254.7 22 -0 ;l6a. Moat 2$ + 230yRibofiavin 82.2 22 174.0 22 0 259.3 22 259.3 22 - 0 17. Moat 4$ .' . 79.1-, "22 146.9 -16 146.5 ' 6 178.6 20 178.8 16 - 0 18. Meat 4$ + 200r"Ribof lavin 76.3 22 175.5 22 0 . 259.5 22 . 259.5 22 - 0 FIFTH --TCEEK-. Normal Ghicks Kales ••"-Females .'.Average wt. Wt. 242.5 218.4 301.4 303.0 297.1 285.6 307.6 335.0 337.1 342.3 326.6 349.3 346.6 •364.8 350.7 366.3 348.4 378.9 359,5 - 368.0 264.2 i 279.5 271.5 - 254.0 392.8 402.7 371.6 - 387.1 286.0 290.0 368.6 390.6 277.8 272.0 362.5 377.6 270.2 241.0 292.3 • 281.1 337.7 363*7 347.4 378.9 245.3 259.4 348.7 373.4 Hever Showing Avit. G Males Females -Average Wt. Wt. Showing Avit. G - Males Females Average Wt. Wt. Wt. Hever Afflicted Males Females -Ho. - .Ho. - - Afflicted Hales. Female Ko. Ho.- . - Recovered - Hales .Feaale Ho. - Ho* 232.0 299.-9 285.6 335.0 351.9 :349.3 ,364.8 - 366.3 378.9 "368.0 275.3 254.0 402.7 387.1 284.0. 390.6 . 282.0 377.6 241.0 275.1 . 363.7 : 378.9. 259.4 • 373.4 253.0 302.4 -308.5 • • 290.0' 324*0 301,6 326.6 -335*1 314.8 •' 352.6 .259.4 280.0 •370.0 338.0 285.8 -349.1 . 276.0 •347*4 272.7 •316.9- 327.9 325.5 235.9 319*0. 253.0 305.5 308*5 290.0 324.0 301.6 326.6 ' -335.1 314.8 352.6 266.0 266.7 370.0 338.0 300.0 349.1 292.7 347.4 278.5 316.9 327.9 325.5 235.9 319.0 237.3 304.5 297.1 307.6 332.1 326.6 346.'6 350.7 348.4 359.5 272.5 263.6 392.8 .371.6 295.0 368.6 287.5 362.5 274.3 297.8 337.7 347.4 245.3 348.7 231.7 230.0 231.3 262.4 248.7 255.9 190.3 248.6 283.0 234.0 207.3 262.0 254.4 245.6 251.7 240.4 257*9 225.9 247.1 283.0 5 17 20 9 11 1-1- 11 •10 --11 9 2 2 16 13 - 3 13 3 •10 1 10 6 6 12 11 14 10 10 10 -10 -10 11 2 .'4 7 • 6/ 4 8 7 10 12 7 16 IS 12 10 3 2 0 ' 0 0 0 0 0 -0 0 7 12 0 : 0 8 ' 0 4 '0 5 3 0 0 0 0 - .1 2 0 0 3 0 0 0 0 0 . 1 1 0 0 ' 2 , 0 2 0 ' 0 2 0 0 0 0 (d) B j s cuss i o n From the t h r e e experiments c a r r i e d out i n S e r i e s 2 i t has been shown t h a t p r o t e i n supplements f o r t i f i e d w i t h r i b o f l a v i n are e q u a l l y as good as supplements n a t u r a l l y r i c h i n t h i s f a c t o r . The f i r s t experiment was c a r r i e d out by adding s y n t h e t i c r i b o f l a v i n i n excess o f the known re q u i r e m e n t s , supplementing one member of each p a i r of r a t i o n s at a g i v e n l e v e l w i t h 2 2 5 u n i t s of r i b o f l a v i n . Here the p r o t e i n v a l u e s were g i v e n i n terms of per cent supplement. R e g a r d l e s s of the f i n a l r e s u l t s on t h e u n f o r t i f i e d r a t i o n s , the c h i c k s a t t a i n e d a p p r o x i m a t e l y the same weights upon the a d d i t i o n of an excess of the v i t a m i n . Skimmilk i n c l u d e d i n t h e t e s t was no more e f f i c i e n t i n i t s g r o w t h - s t i m u l a t i n g p r o p e r t i e s t h a n o t h e r r a t i o n s e qual i n p r o t e i n and supplemented w i t h t h e s y n t h e t i c r i b o f l a v i n , c o n t r a r y t o r e s u l t s r e p o r t e d by some o t h e r investigators© The second experiment was c a r r i e d out by supplementing the r a t i o n s w i t h s y n t h e t i c r i b o f l a v i n as i n t h e p r e v i o u s one, but here the p r o t e i n v a l u e s were c a l c u l a t e d i n terms of per cent p r o t e i n , f o r ease o f comparison on a p r o t e i n b a s i s . There was some u n e x p l a i n e d d e s t r u c t i o n of r i b o f l a v i n , however, which rendered the r e s u l t s o f t h i s experiment beyond i n t e r p r e t a t i o n . The t h i r d experiment was d e v i s e d so t h a t one r a t i o n of each p a i r of r a t i o n s at a g i v e n l e v e l c o n t a i n e d a c e r t a i n d e f i n i t e amount of r i b o f l a v i n , 368+14.1 micrograms. In o t h e r words, i n s t e a d of adding 2 2 5 micrograms to each l e v e l , - 41 - i r r e s p e c t i v e of i t s o r i g i n a l v i t a m i n c o n t e n t , an in d e p e n d e n t l y c a l c u l a t e d q u a n t i t y was added to each i n d i v i d u a l r a t i o n . The p r o t e i n was ; a g a i n c a l c u l a t e d i n terms of per cent p r o t e i n from a g i v e n source. As a r e s u l t o f t h i s experiment i t was shown t h a t v i t a m i n G was e q u a l l y a v a i l a b l e whether o b t a i n e d from n a t u r a l sources or from the s y n t h e t i c p roduct. No improvement through the f e e d i n g of d r i e d skimmilk was noted, which con- f i r m e d the c o n c l u s i o n s drawn from the r e s u l t s of Experiment 1, Moreover, 3 7 0 micrograms per lOO grams of feed was more than adequate f o r normal growth and maintenance, and the minimum requirements f o r s a t i s f a c t o r y growth were 2 2 5 micrograms. O b s e r v a t i o n s and comments on the i n d i v i d u a l supplements used i n t h i s s e r i e s are i n c l u d e d w i t h each experiment, and the r e s u l t s summarized i n the t a b l e s . F i g u r e 1 r e p r e s e n t s the " s c a t t e r diagram" o b t a i n e d by p l o t t i n g , f r o m the data o f Experiments 1 and 3, the weights of c h i c k s i n grams a g a i n s t the number of u n i t s of r i b o f l a v i n f e d . The mean of the c h i c k weights was c a l c u l a t e d , from 49 independ- ent v a r i a b l e s , as b e i n g 357.14+56.42 ( c o e f f i c i e n t of v a r i a b i l - i t y = 15.80%); and the mean of the c o r r e s p o n d i n g l e v e l s of r i b o f l a v i n as 267 .45+.102.23 ( c o e f f i c i e n t of v a r i a b i l i t y - 38.22%). The c o r r e l a t i o n c o e f f i c i e n t , r , was c a l c u l a t e d from t h e e q u a t i o n : , o ft 2SO 26 O 300 5 6 o •too o O O ' o X \ X t X • N . oo X X X X V> M « ft o °\ X X o X X "I o X o o .1 s X In o j'o I* id o o : 2 e o o o \ X X o o X X *> o \ \ x X IS X X 1 X F i g u r e 1 - 42 - r x y = 5 ( * " S ) . ( y ^ - y ) A = +0,4843 <r x . <r y Th i s i n d i c a t e d a h i g h l y s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between g a i n i n weight and micrograms of r i b o f l a v i n f e d . With t h i s i n f o r m a t i o n , the r e g r e s s i o n c o e f f i c i e n t was d e t e r - mined from the formula: y X ' £(x - x K b x y = S l l L . - y) (* - £). = G.S776 E(y - y ) Z For each i n c r e a s e i n 1 u n i t r i b o f l a v i n t h e r e i s a correspond- i n g i n c r e a s e of .2673 grams i n weight; and c o n v e r s e l y , each gram g a i n i n weight would r e q u i r e the a d d i t i o n of micrograms o f r i b o f l a v i n . The r e l a t i o n between the v a r i a b l e s , b y X , i s r e p r e s e n t e d by the r e g r e s s i o n l i n e i n the f i g u r e . SUMMARY Two s e r i e s of f e e d i n g experiments were conducted i n ord e r t o determine the n u t r i t i v e v a l u e of B r i t i s h Columbia f i s h m e a l s i n c h i c k f e e d i n g . I n the f i r s t s e r i e s , which c o n s i s t e d o f t h r e e experiments i n v o l v i n g t w e n t y - t h r e e l o t s - of twenty-two c h i c k s each, t h e n u t r i t i v e v a l u e of eleven f i s h m e a l s and by-products of the f i s h i n g i n d u s t r y was determined. The h e r r i n g and - 4 3 - salmon meals under i n v e s t i g a t i o n were not p a r t i c u l a r l y good sources o f r i b o f l a v i n , but c o n t a i n e d p r o t e i n of high, q u a l i t y . L i v e r meal was p a r t i c u l a r l y r i c h i n the v i t a m i n G f a c t o r , the t h r e e meals prepared from salmon v i s c e r a being o n l y s l i g h t l y l e s s so. Two samples of salmon roe meals were o n l y moderately good sources of r i b o f l a v i n . The meal prepared from salmon heads had the lowest v i t a m i n G potency of the s e r i e s . I t was suggested, because of t h e i r h i g h r i b o f l a v i n c o n t e n t , t h a t s t i c k w a t e r meal and c e r t a i n of the o t h e r by-products under i n v e s t i g a t i o n should be u t i l i z e d i n the manufacture of f i s h m e a l s . S e r i e s 2 , a l s o c o n s i s t i n g o f t h r e e experiments, i n v o l v e d s e v e n t y - f o u r l o t s o f twenty-two c h i c k s each. I t was shown as a r e s u l t of t h i s s e r i e s t h a t s y n t h e t i c r i b o f l a v i n was e q u a l l y as e f f e c t i v e i n promoting growth as was the r i b o f l a v i n from n a t u r a l s o u r c e s . C o n t r a r y t o p r e v i o u s p r a c t i c e , i t was not found necessary t o add d r i e d s k i m m i l k when f i s h m e a l s were supplemented w i t h s u f f i c i e n t v i t a m i n G. I t was found t h a t 3 7 0 micrograms of r i b o f l a v i n per 1 0 0 grams of f e e d were adequate f o r o p t i m a l growth and f o r the p r e v e n t i o n of c u r l e d - t o e p a r a l y s i s . The minimum r i b o f l a v i n r e q u i r e m e n t s f o r s a t i s f a c t o r y growth were much lower - namely, 2 2 5 micrograms per 1 0 0 grams. - 44 - REFERENCES (1) A c k e r s o n , C.W. , M.J. B l i s h and F.E. Mussehl 1935 The u t i l i z a t i o n of n i t r o g e n , c a l c i u m , and phosphorus by the growing c h i c k . Nebraska E x p t . S t a . Res. B u i , 80, (2) A l m q u i s t , H.J. 1941 Chemical e s t i m a t i o n of q u a l i t y i n animal p r o t e i n c o n c e n t r a t e s . J . N u t r i t i o n , v o l , 21 , No. 4: 3 4 7 - 3 5 0 . (3) Asmundson, V.S., and Jacob B i e l y 1932 F i s h meal supplements f o r c h i c k s . I . Comparative r a t e of growth- t o - e i g h t -weeks on r a t i o n s supplemented w i t h p i l c h a r d , salmon, h a l i b u t , d r i e d skim-milkj o r w i t h salmon meal and d r i e d skim m i l k . S c i . Agr., v o l . 13, No. 4: 236-248. (4) B e a l l , Desmond 1933 Loss i n the e f f l u e n t of p i l c h a r d r e d u c t i o n p l a n t s i n B r i t i s h Columbia. B u i . No. 3 5 , B i o l , Board of Canada, Ottawa. (5) B i e l y , Jacob,and V.S. Asmundson 1933 F i s h meal supplements f o r c h i c k s . I I . Comparative r a t e of growth- t o - e i g h t - w e e k s on r a t i o n s supplemented w i t h v a r i o u s p r o p o r t i o n s o f f i s h meal and d r i e d skim m i l k . S c i . Agr. v o l , 13, No, 1435-438. (6) B i l l i n g s , F.L., Jacob B i e l y , H e r b e r t F i s h e r and C a r l Hedreen 1941 The r i b o f l a v i n content of f i s h p r o d u c t s , J . 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McCollum 1951 S t u d i e s on the n u t r i t i v e v a l u e of f i s h meals. U.S. Dept. of Commerce, Bur. of F i s h e r i e s , Wash., D . C . , I n v e s t . Report No. 2 . ( 1 5 ) Emmet, A.D., O.D. B i r d , R.A. Brown, G a i l Peacock and J.M. Vandenbelt 1941 D e t e r m i n a t i o n of v i t a m i n B.,, ( r i b o f l a v i n ) . Comparison of b i o a s s a y , m i c r o b i o l o g i c a l and f l u o r o m e t r i c methods. Ind. and Eng. Chem. 13: 219 . (14) E u l e r , H, von, and E.Z. A d l e r 1934 Uber das VorKommen von F i a v i n e n i n T i e r i s c h e n Geweben. Z, P h y s i o l . Chem. 2 2 3 : 105. (15) Ewing, W. Ray 1941 Handbook of P o u l t r y N u t r i t i o n . F i r s t p r i n t e d e d i t i o n . P r i n t e d i n U.S.A. by J . J . L i t t l e & Co., New York. ( 1 6 ) The'"Feeding S t u f f s A c t , 1937 and R e g u l a t i o n s . Dom. Dept. o f Agr. Prod'n S e r v i c e , P l a n t P r o d u c t s . 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Bohstedt 1936 The e f f e c t - o f heat as used i n the e x t r a c t i o n of soybean o i l upon the n u t r i t i v e v a l u e o f the p r o t e i n of soybean o i l meal. J o u r . N u t r i t i o n 11: 2 1 9 - 2 3 4 . (22) Heiman, V i c t o r , J.S. Carver and J.W. Cook 1939 A method f o r d e t e r m i n i n g the gross v a l u e of p r o t e i n concentrates. P o u l t r y S c i . 18, No. 6: 4 6 4 . - 46 - (23) Hodson, A.Z., and L.C. N o r r i s 193© A f l u o r o m e t r i c method f o r d e t e r m i n i n g the r i b o f l a v i n content of f e e d s t u f f s . J . B i o l . Chem. 131: 6 2 I - 6 3 O . (24) I n g v a l d s e n , T. 1929 F i s h Meals: P a r t I - The e f f e c t of the h i g h temperature employed f o r d r y i n g , on the n i t r o g e n p a r t i t i o n i n f i s h meals. Can. Chem. and M e t a l l . 13: 9 7 - 9 9 . (25) Jones, D. 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' (31) Manning, J.R. 1939 Importance of f i s h e r y by-products i n p o u l t r y n u t r i t i o n . Proc® World's P o u l t r y Congress, C l e v e l a n d , Ohio. S e c t . 2: 2 1 2 . - (32) M i t c h e l l , H.H. 1926 B u i . Nat. Res. C o u n c i l , P a r t I , No. 35 ( a b s t r . ) . (33) N e w e l l , John M.t and E.V. McCollum 1931 S p e c t r o g r a p h i c a n a l y s i s of marine p r o d u c t s . U.S. Dept. of Commerce, Bur. of F i s h e r i e s , Wash. D.C., I n v e s t . Report.No. 5. (34) N o r r i s , L.C., H.S. W i l g u s , J r . , A.T. R i n g r o s e , V i c t o r Heiman and G.F. Heuser. 1936 The v i t a m i n - G r e q u i r e - ments of p o u l t r y . B u i . 660, C o r n e l l U n i v . Agr. E x p t . S t a . , I t h a c a , New York. (35) O r r , J.B. and A.D. Husband 19 22 The importance o f m i n e r a l matter f o r growing a n i m a l s . S c o t t i s h J o u r , of Agr. v o l . 5, No. 3: 244 - 2 5 0 . - 47 - ( 3 6 ) Pratt,.J".M e 1940 A study of the soyabean and i t s " n u t r i t i v e v alue i n the c h i c k r a t i o n . Unpublished data - U n i v e r s i t y of B.C. ( 3 7 ) Record, P.R., R.M. Bethke and O.H.M. W i l d e r 1934 E f f e c t of method of manufacture on the n u t r i t i v e value of f i s h meals as determined by growth s t u d i e s w i t h c h i c k s . J o u r . Agr. Res. 49: 7 1 3 . ( 3 8 ) Robert son, E . I . , J.S. Carver and J.W. Cook 1940 Gross va l u e of p r o t e i n supplements f o r p o u l t r y . Washington • E x p t . S t a . B u i . 3 8 8 . ( 3 9 ) Rose, Wm. C. 1 9 3 7 The n u t r i t i v e s i g n i f i c a n c e of the amino a c i d s and c e r t a i n r e l a t e d compounds. S c i e n c e , v o l . 8 6 , No. 2 2 3 1 : 2 9 8 - 3 0 0 . (40) S t . John, J.L. , 0. Johnson, J.S. Carver and S.A. Moore 1931 A method of de t e r m i n i n g the b i o l o g i c a l v a l u e o f p r o t e i n i n t h e study o f a v i a n n u t r i t i o n . J o u r . N u t r . 3* 2 6 7 . (41) S n e l l , E.E., and F.M. Stro n g 1939 A m i c r o b i o l o g i c a l assay f o r r i b o f l a v i n . I n d . Eng. and Chem. v o l . 11: 3 4 6 . (42) Supplee, G.C. , S. Ansbacher, E.E. F l a n i g a n and Z J , Hanford 19 3 6 The f l u o r o m e t r i c e s t i m a t i o n of l a c t o f l a v i n . I n d . Eng. Chem. A n a l . Ed,, 11: 495. ( 4 3 ) W i l d e r , O.H.M., R.M, Bethke and P.R. Record 1934 E f f e c t o f method of manufacture on the n u t r i t i v e v a l u e of f i s h m e a l s as determined by growth s t u d i e s w i t h r a t s . J o u r . Agr. Res, 49, No. 8 : 7 1 5 - 7 3 0 . (44) W i l g u s , H.S., J r . , L.C. N o r r i s and G.F. Heuser 1935 The r e l a t i v e p r o t e i n e f f i c i e n c y and the r e l a t i v e v i t a m i n G content of common p r o t e i n supplements used i n p o u l t r y r a t i o n s . J o u r . Agr. Res. 5 1 : 383-399. (45) W i l g u s , H.S., J r . , : R.C. RinRro.se and L.C. N o r r i s 1933 S t u d i e s of th e e s s e n t i a l n u t r i t i v e p r o p e r t i e s of common pr o t e i n - s u p p l e m e n t s used i n p o u l t r y r a t i o n s . A t t i d e l v Congress© Mondiale d i P o l l i t i c o l t u r a N.44, 2 a S e z i o n e . ( 4 6 ) Womack, Madelyn, Kenneth S. Kemmerer and Wm. C, Rose 1937 The r e l a t i o n o f c y s t i n e and methionine to growth. J o u r . B i o l . Chem. 121: No. 2: 403-410. - 4 8 - A i m q u i s t , H.J., E.L.R. S t o k s t a d and E.L. Halbrook 1935 Values of animal p r o t e i n c o n c e n t r a t e s i n c h i c k r a t i o n s . J o u r . N u t r . 1 0 : 19 3. The c o m p o s i t i o n and n u t r i t i v e p r o p e r t i e s of soybeans and soybean o i l meal. - A L i t e r a t u r e Review. 1939 Soybean N u t r i t i o n a l Research C o u n c i l , 3 8 1 8 Board of Trade - B u i l d i n g , Chicago, 111. Csonka, F.A. and D.B. Jones 1934 The c y s t i n e , tryptophane and t y r o s i n e content o f the soybean. J o u r . Agr. Res. 49 : " 2 7 9 . Csonka, F.A., and D.B. Jones 1 9 3 3 D i f f e r e n c e s i n the amino a c i d content of the c h i e f p r o t e i n ( g l y c i n i n ) from seeds of s e v e r a l v a r i e t i e s of soybean. J o u r . Agr. Res. 4.6: 51. C u l t o n , T.G., and H.R, B i r d 1940 The r i b o f l a v i n content o p o u l t r y f e e d s t u f f s . P o u l t r y S c i . 20: No. 1: 3-6. Hayward, J.¥., J . G. H a l p i n , C.E. Holmes, 0. Bohstedt and E.B. Hart 19 3 7 Soybean o i l meal prepared at d i f f e r e n t temperatures as a f e e d f o r p o u l t r y . P o u l t r y S c i . 16:3-14 Hayward, J.W., H. Steenbock and G, Bohstedt 1 9 3 6 The e f f e c t of c y s t i n e and c a s e i n supplements upon the n u t r i t i v e v a l u e of the p r o t e i n of raw and heated soybeans J o u r . N u t r . 1 2 : 2 7 5 - 2 8 3 . Henry, W.A., and F.B. M o r r i s o n Feeds and Feeding. 20th ed. The M o r r i s o n P u b l i s h i n g Co,, I t h a c a , N.Y. L l o y d , E.A., and Jacob B i e l y 1941 P r a c t i c a l P o u l t r y - F e e d i n g . Prov. of B.C. Dept• o f Agr. B u i . 1 0 7 , V i c t o r i a , B.C. M i l l e r , David 1 9 3 8 The v i t a m i n s of the B-G complex. P o u l t r y S c i . 17, No e 6 : 5 2 3 - 5 3 8 . R i b o f l a v i n . Annotated B i b l i o g r a p h y . 1941 Merck &„Co. I n c . Rahway, N.J. Rob e r t s o n , E . I . , J.S. Carver and J.W. Cook 1 9 3 8 Gross p r o t e i n v a l u e s of some c o n c e n t r a t e s . P o u l t r y S c i . 1 7 : 4 3 3 . R o b e r t s o n , E . I . , J.S. C a r v e r , B.B. Bohren and J.W. Cook 19 3 9 Gross v a l u e s of combinations of some p r o t e i n c o n c e n t r a t e s . P o u l t r y S c i . 18: 410» - 49 - Rose, • Wm. C., and E l d o n E. R i c e 1939 The u t i l i z a t i o n of c e r t a i n s u l p h u r - c o n t a i n i n g compounds f o r growth purposes. J o u r . B i o l . Chem. 1 3 0 : No. 1 : 3 0 3 - 3 2 3 . Sherwood, F.W., and J.O. H a l v e r s o n 1939 The v i t a m i n G content of some o i l press-cake meals and r e l a t e d p r o d u c t s . . J o u r . Agr. Res. 58, No. 10: 787-794. W i l g u s , H.S. , J r . , L.C. N o r r i s , and G.F. Heuser 1935 Haddock meal. E f f e c t of manufacturing process upon • n u t r i t i v e v a l u e s . Ind. and Eng. Chem. 21'- 419-422.

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