UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The biological assay of fish oils for vitamin A Miller, John Peat 1937

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1937_A4_M5_B5.pdf [ 28.62MB ]
Metadata
JSON: 831-1.0105547.json
JSON-LD: 831-1.0105547-ld.json
RDF/XML (Pretty): 831-1.0105547-rdf.xml
RDF/JSON: 831-1.0105547-rdf.json
Turtle: 831-1.0105547-turtle.txt
N-Triples: 831-1.0105547-rdf-ntriples.txt
Original Record: 831-1.0105547-source.json
Full Text
831-1.0105547-fulltext.txt
Citation
831-1.0105547.ris

Full Text

THE BIOLOGICAL ASSAY 03* FISH OILS FOR VITAMIN A by John peat M i l l e r  A Thesis Submitted i n P a r t i a l F u l f i l l m e n t 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 of B r i t i s h Columbia A p r i l 1937.  AMOwXEDGEMMT The w r i t e r wishes to acknowledge h i s indebtedness to Professor E. A. Lloyd and Mr. J* B i e l y of the Department of Poultry Husbandry f o r t h e i r keen i n t e r e s t and invaluable c r i t i c i s m i n the preparation o f t h i s manuscript; also to Messrs. J . B. O'Heil and C. W. Wood f o r t h e i r u n t i r i n g assistance during the experimental work.  THE BIOLOGICAL ASSAY OE FISH OILS FOR VITAMIN A TABLE OF CONTENTS I.  Introduction--Vitamins i n N u t r i t i o n  II.  Discovery of Vitamin A  III.  Vitamin A i n the Plant Kingdom  .Page "  1 . Vitamin A Content of Plants . ..  [V.  .  4 4  "  2. Synthesis of Vitamin A i n Plants .....  "  9  3. PIaxit Carotinoids and Vitamin A ......  "  12  4. E x t r a c t a b i l i t y of Carotinoids from p l a n t s  14  Vitamin A i n the Animal Kingdom  15  n  1 . Synthesis of Vitamin A i n F i s h . . .  "  15  2. The Vitamin A Content of Animal Products  »  19  3. Storage of Vitamin A i n the Body  "  25  "  27  "  28  "  32  ^  32  4. Transmission of Vitamin A from Parents to Young 5. Conversion of Carotinoids i n t o Vitamin A r  1  Vitamin A Therapy 1#  _Cn GTo3.cl s  • • • • • • • • • o « » » * « * a « «  « • • • « • « > * • « •  2. In the Dressing of Wounds  "  34  3. Antagonism between Vitamins A and C, and Vitamin A and Thyroxin  "  38  TABLE OF C.OFIBNT S ( Cont' d ) VI.  B i o l o g i c a l Assay of Vitamin A Using the Rat as the Test Animal  ............Page 41  1. Feeding Technique  "  2 . Results o f Vitamin A D e f i c i e n c y xn Kelts • • » « * > * * « » 4 * « * > « » > « « * « « « « * o 3. A s s i m i l a t i o n of Vitamin A i n the Presence of Mineral O i l s . " 4. Influence of Vitamin A on Fat  VII.  Chemical and P h y s i c a l P r o p e r t i e s of Vitamin A  49  53  and P h y s i c a l  Agents on Vitamin A 3. Oolorimetric Reaction  c  "  57  .. "  63  4. Absorption Spectrum  "  69  5. Solvents f o r Carotene 6. Vitamin A. and Anti  "  73  7. Concentrati on of Vitamin A ........ „ 8. The Measurement of Vitamin A {International Units) . .. . VIII.  45  53  1. The Chemical Structure of Vitamin A.. " 2. The E f f e c t of Chemical  41  Vitamin A i n P o u l t r y ..  76  u  "  78  "  83  "  83  "  89  1. Symptoms and Lesions due to Lack Gi* \Pxtctii2X.Q .A.  e t * > « « « « * . > « « « « « > « f t 6 * > 0 o  2. Requirements o f Chicks f o r Growth 3. Requirements of Hens f o r Egg !Px*o^.liGtxon  « » « « « o e * « » * * » » » » » e * » * «  ^ X03  TABLE PIT CONTENTS (Cont'd) IX.  Experimental  . .Page  No,  1. Experiment No. 1 (a) Introduction......  ..  "  109  (t>) M a t e r i a l and Methods ........  "  109  (c) Results ..  u  111  (d) Discussion  u  117  2. Experiment No. 2 (a) Introduction. (t>) M a t e r i a l and Methods  ....  (c) Results . ... (d) Discussion ..  "  121  «  122  "  124  "  149  . ..  3. Experiment No. 3. (a) Introduction ... ..  . . "  154  ("b) M a t e r i a l and Methods ........  "  154  (c) Results  "  155  "  159  "  165  .  (d) Discussion .. X.  Summary  XI.  References ...........  -- - -  168 11  I. TEE. BIOLOGICAL ASSAY OF FISH OILS FOR VITAMIN A.  Introduction. Domestic animals must be furnished w i t h a r a t i o n that i s complete i n carbohydrates,  f a t s , p r o t e i n s , vitamins, a,nd  minerals i f normal h e a l t h , reproduction, and growth are to be maintained.  A d e f i c i e n c y i n any n u t r i e n t or vitamin may  cause  r e t a r d a t i o n of growth i n young animals, or decreased output i n mature animals of such products as milk or eggs.  Furthermore  d e f i c i e n c i e s i n diet may r e s u l t i n diminished h e a l t h or vigor as evidenced by such diseases as xerophthalmia*  nutritional  roup, r i c k e t s , p e l l a g r a , a,naimia, or g o i t e r . The causes of the most common of these d e f i c i e n c y diseases have been tra.ced, i n recent years, to a lack of some s p e c i f i c vitamin.  Of the many d i f f e r e n t vitamins known to  e x i s t today, Vitamin A has received considerable a t t e n t i o n . Many i n v e s t i g a t o r s have shown that t h i s vitamin i s indispensable f o r the normal growth of animals although the actual amount needed i s very small.  Of t h i s small t o t a l r e l a t i v e l y moderate  q u a n t i t i e s are required f o r maintenance, somewhat l a r g e r q u a n t i t i e s f o r growth, and r e l a t i v e l y large q u a n t i t i e s for the production of eggs or milk. Since Beach (12) i n 19 23 showed that Vitamin A was e s s e n t i a l f o r normal p o u l t r y n u t r i t i o n , a great deal of work has be.n done by other i n v e s t i g a t o r s to determine the  II. q u a n t i t a t i v e requirements of chicks f o r t h i s v i t a m i n . O r d i n a r i l y s Vitamin A i s supplied through the feeding of yellow corn, dehydrated a l f a l f a , or green feed.  These feeds  .as a r u l e , must be supplied i n comparatively large amounts to meet the Vitamin A requirements of growing chicks and p a r t i c u l a r l y of l a y i n g b i r d s .  In recent years f i s h o i l s have become  widely u t i l i z e d as sources of Vitamin A f o r p o u l t r y because they are very potent sources of t h i s v i t a m i n .  According to  Praps and T r e i c h l e r (83) a good grade of Cod L i v e r O i l contains 600-1000 units of Vitamin A per gram, while a good grade of yellow corn contains only 5. units, of Vitamin A per gram and dry a l f a l f a 12 u n i t s per gram.  I t w i l l be r e a d i l y seen, there-  fore, that the Vitamin A content of a r a t i o n can be r e a d i l y f o r t i f i e d and c o n t r o l l e d by•the mere a d d i t i o n of a small quantity of a Vitamin A potent f i s h o i l . Amongst the f i s h o i l s , Cod L i v e r O i l has h i t h e r t o occupied a pre-eminent p o s i t i o n a.s a source of Vitamin A i n both human and animal n u t r i t i o n .  In recent years i t has been  shown, however, that many other f i s h o i l s a.re j u s t a.s r i c h sources of Vitamin A. Amongst the various f i s h -oils, BRITISH COLUMBIA PILCHARD OIL i s already e x t e n s i v e l y used as a source of Vitamin D.  On account of t h i s , i t was deemed advisable to  carry out at The U n i v e r s i t y of B r i t i s h Columbia i n v e s t i g a t i o n s regarding the Vitamin A potency of t h i s o i l . The purpose of these i n v e s t i g a t i o n s were'not only to throw l i g h t on the Vitamin A content of P i l c h a r d O i l , but to  III. determine the Vitamin A requirements of growing chicks as w e l l . At the same time, i t appeared that a s a t i s f a c t o r y method should he developed, i f p o s s i b l e , f o r the b i o l o g i c a l and chemical assay of Vitamin A i n f i s h o i l s . The present i n v e s t i g a t i o n c o n s i s t s of three d i s t i n c t experiments, (a) Estimating the Vitamin A content of B r i t i s h Columbia p i l c h a r d O i l i n terms of I n t e r n a t i o n a l U n i t s . (b) Developing a s u i t a b l e basal r a t i o n free from Vitamin A f o r the b i o l o g i c a l assay of Vitamin A i n f i s h o i l s , using the chick as the test animal, (c-^)  Comparing the b i o l o g i c a l and chemical  methods of assaying B r i t i s h Columbia P i l c h a r d O i l .  (c ) 9  Determining the Vitamin A content of P i l c h a r d O i l s produced at d i f f e r e n t periods of the f i s h i n g  season.  l o reference i s made i n t h i s i n v e s t i g a t i o n to the storage of Vitamin A i n the l i v e r s of chicks fed varying amounts of Vitamin A. in progress.  Work on t h i s phase of the problem i s  DISCOVERY Off VITAMIN A. The s p e c i f i c growth-promo t i n g property which i s now "associated withihe occurrence of Vitamin A was by McCollum and Davis (142) i n 1913, who  f i r s t observed  found that an ether  extract of butter or egg yolk i n a synthetic r a t i o n had a stimu l a t i n g action on growth which v/as not possessed by other f a t s such as l a r d or o l i v e o i l . These workers pointed out that c e r t a i n mixtures  of  f a t s of animal o r i g i n , as butter f a t , egg f a t , and the f a t s ext r a c t e d from the i n t e r n a l organs, eg. the kidney, l i v e r , e t c . , contain something which i s absolutely indispensable for e i t h e r maintenance or growth, and that t h i s substance i s not found i n vegetable o i l s or f a t s and i n but very small and inadequate amounts i n the bodyfats of animals. They showed that when the d i e t i s inadequate i n i t s content of t h i s substance which they designated  as fa,t-soluble  A, the animals become emaciated and suffer from edema, of the • eyes. . Blindness r e s u l t s i f the animals are permitted to go without t h i s d i e t a r y e s s e n t i a l or w i t h an inadequate supply f o r a s u f f i c i e n t time. Very s h o r t l y thereafter Osborne and Mendel (171) publ i s h e d experiments i n which i t v/as pointed out that t h e i r milk rations had s p e c i a l d i e t a r y properties not found i n t h e i r milkfree r a t i o n s and that t h i s c h a r a c t e r i s t i c seemed to be true of r a t i o n s carrying an equivalent amount of butter as w e l l . Later  2. (172) they obtained uniform success by s u b s t i t u t i n g cod l i v e r o i l f o r a p o r t i o n of the. l a r d i n t h e i r standard d i e t s . Stephenson (238) i n 1920 found that a crude a l c o h o l light-petroleum extract of dried carrot when added to a f a t not containing vitamin confers upon i t the growth promoting property and the power of p r o t e c t i n g the animal from or...curing i t of keratomalacia.  His work showed that the substance of  substances i n the e x t r a c t , responsible f o r these properties was not carotene.  Other experiments that he conducted demon-  s t r a t e d that the colouring matter of butter f a t may be completely removed or destroyed by f i l t r a t i o n through charcoal without i n the l e a s t a f f e c t i n g the vitamin content of the b u t t e r . Z i l v a (267) i n 1919 found that the f a t soluble A f a c t or i n butter became i n a c t i v a t e d when the b u t t e r was exposed to u l t r a v i o l e t l i g h t f o r eight hours. Drummond and Coward (60) i n 1920 decided that no hard and f a s t l i n e could be drawn between the animal and the vegetable o i l s and f a t s when t h e i r value as a source of Vitamin A was being considered.  These i n v e s t i g a t o r s also found that the  animal f a t s , taken as a c l a s s , possessed a growth-promoting power superior to that of the vegetable o i l s .  This superior  growth promoting power of the animal o i l or f a t appeared to be influenced considerably by the diet of the animal. In 1919, Haring, Bea.ch and J a f f a (26) reported a, study of several outbreaks of a disease occuring i n f l o c k s of p u l l e t s i n C a l i f o r n i a , which, so f a r as could be determined by a search  3. of the l i t e r a t u r e , had not been p r e v i o u s l y described.  The symp-  toms resembled those of "roup" more than of any other disease of fowls known at that time, but d i f f e r e d enough so that a d i f f e r e n t i a l diagnosis was r e a d i l y made.  Later on Beach (12) d e f i n -  i t e l y showed that the disease w i t h which he was dealing i n poult r y was due to a lack of Vitamin A.  4.  VITAMIN A IN THE PLANT KINGDOM Vitamin A Content of Plants* Steenbock, Kent and Gross (226) i n 1918,  discovered  that barley contained an abundance of the wa.ter soluble v i t a •min but was  d e f i c i e n t i n the f a t soluble vitamin.  Osborne and Mendel (173) i n 1919, were the f i r s t i n v e s t i g a t o r s to note that green vegetables supplied an  import-  ant addition to the d i e t of man because the staples such a.s c e r e a l s , meats, potatoes, f a t s and sugar furnished too small an amount of the vitamin to meet f u l l y the requirements of an adequate d i e t a r y . As e a r l y as 1919  Steenbock and Gross (228) demonstrat-  ed the high f a t - s o l u b l e vitamin content of c a r r o t s and yellow sweet potatoes as compared with other r o o t s . Steenbock and Boutwell  A year l a t e r ,  (229) showed that yellow corn  contained  enough of the f a t - s o l u b l e vitamin to allow growth at the norma l r a t e to take place i n the r a t .  White corn on the other  hand did not contain any demonstrated amount of the f a t - s o l u b l e vitamin. In 1920  Steenbock and Gross (230) found that 5 % of  clover or a l f a l f a as the sole source of f a t - s o l u b l e vitamin i n a r a t i o n , when other d i e t a r y requirements were met,  allowed  normal growth and the r e a r i n g of some young. Steenbock, S e l l and Boutwell (234) worked w i t h peas to f i n d t h e i r f a t - s o l u b l e vitamin content i n r e l a t i o n to t h e i r pigmentation.  The r e s u l t s of t h i s i n v e s t i g a t i o n demonstrated  that i n r i p e peas, those of a green colour, also carrying cons i d e r a b l e yellow pigment, were f a r r i c h e r i n t h e i r f a t - s o l u b l e vitamin content than yellow peas which contained much l e s s yellow pigment. About the same time as thes.e i n v e s t i g a t i o n s were going on Coward and Drummond (35) were i n v e s t i g a t i n g nuts as a source of Vitamin A. They found that t h e various nuts contained a large percentage of f a t but possessed l i t t l e or no vitamin A. Their r e s u l t s f u r t h e r substantiate the theory that Vitamin A i s formed i n the green part of the l i v i n g plant and i s not stored to any appreciable extent as such i n the seed and other r e s t i n g tissues. Willimont contained  (259) showed that 5 c c . of navel orange j u i c e  s u f f i c i e n t Vitamin A f o r growth and well-being i n the  ra.t. The Vitamin A content of barley was i n t e n s i v e l y studied by Hughes (124).  He noted that when growth was used as a measure  of the Vitamin A content of barley yellow corn, white corn and barley, the i n d i c a t i o n s were that the Vitamin A content of barley l a y somewhere between that of yellow corn and vtfiite corn. When growth and v a g i n a l smears were used as a measure o f the Vitamin A content of b a r l e y , i t was shown that the quantity i n barley was low, i n f a c t , much lower than that of yellow but higher than that of white corn.  The experiment  corn  definitely  showed that barley, as the only source of Vitamin A i n the d i e t did not produce normal growth i n r a t s .  In conclusion he stated  that barley contained l e s s than one s i x t h as much Vitamin A as  6. did yellow corn. Extensive tests of the Vitamin A potency of a l f a l f a were c a r r i e d out by M i l l e r and Bearse (155a.). They found that a sample of commercial dehydrated a l f a l f a contained twice as much Vitamin A as a. sample of commercial sun-cured a l f a l f a . A f t e r a year's storage the potency of the two samples was  still  the same. They also found that the dehydrated a l f a l f a s they used were t h i r t y times more potent than samples of yellow corn. Hauge (106) found that the Vitamin A a c t i v i t y of freshl y cut young a l f a l f a was preserved when the enzymes were destroyed immediately before drying.  A d i r e c t c o r r e l a t i o n was  found between the e f f e c t of temperature on the Vitamin A and on the enzyme a c t i v i t y .  The influence of the sun's rays on the  destruction of the Vitamin A was thought to be due to the produ c t i o n of temperature which accelerated enzyme a c t i v i t y . Guilbert (98) observed no l o s s on drying but considerable l o s s on autoclaving and during sun-drying, most of which was due to the photochemical action of the sun, though i n slow drying, enzyme and b a c t e r i a l action also played a p a r t .  Temp-  erature was the most important f a c t o r i n f l u e n c i n g l o s s i n storo age; below 5 c. no l o s s was detected i n 6 months but the rate of l o s s increased very r a p i d l y with r i s e of temperature. Recent work on a l f a l f a , hay by V a i l , Tobisha and Dougl a s s (252) showed how Vitamin A may be l o s t or conserved according to methods of handling. 1. Loss or i n a c t i v a t i o n of Vitamin A i n a l f a l f a hay  r e s u l t s from:  7.  (a) Usual p r a c t i c e of curing and s t o c k i n g . (b) Exposure to u l t r a - v i o l e t l i g h t i n presence of moisture. (c) Storage of a l f a l f a meal i n c l o t h sacks. 2. Conservation of Vitamin A i n a l f a l f a - hay r e s u l t s f r om: (a) Curing indoors. (b) Curing by r a p i d a r t i f i c i a l drying. (c) Curing by crushing and r a p i d drying. (d) Storage i n the b a l e . (e) Storage of a l f a l f a meal i n paper sacks. The Vitamin A i n a l f a l f a hay tended to become more i n a c t i v e the longer i t was stored. Woods e t . a l . (266 a ) found that t h i r d c u t t i n g a l f a l f a hay, cock cured and i n f r e s h c o n d i t i o n , contained 308± 13 Sherman u n i t s of Vitamin A a c t i v i t y per gram (leaves 48 3-t 34, stems 121 dr. 7 u n i t s ) .  A sample of the same hay ground and  stored i n d i f f u s e d l i g h t f o r 4 months, contained only 233 _ 20 units.  Hay cured i n the swathe f o r 3 days, then cock cured  and sweated i n the stack contained 116dr 9 u n i t s per gram compared w i t h 144±10 u n i t s f o r s i m i l a r hay which had been exposed i n the swathe only one day before cocking.  Sweating i n the  stack caused a s i g n i f i c a n t reduction i n the Vitamin A a c t i v i t y from 233+ 20 Sherman r a t u n i t s f o r cock cured, 2-g- months o l d unsta.cked hay to 1 4 4 i l 0 u n i t s f o r similar* hay sweated i n the stack 49 days.  8, Praps, T r e i c h l e r and Kemmerer (84 a ) showed that a l f a l f a products containing from 7.3 to 63.5 micrograms of carotene had a Vitamin A potency of from 13 to 77 Sherman-Munsell u n i t s (15.6 to 92.4 i n t e r n a t i o n a l u n i t s ) per gram, w i t h an average value of 1.4 u n i t s (1.6 i n t e r n a t i o n a l u n i t s ) per microgram of carotene.  They also observed that one microgram of  carotene i n the i n t e r n a t i o n a l standard had a value of 1.4 Sherman-Munsell u n i t s , and 1 microgram of p u r i f i e d carotene had the same value.  9. Synthesis of Vitamin A i n P l a n t s . Wilson (260) i n 1922 found that e i t h e r e t i o l a t e d or green wheat sprouts furnished an adequate amount of Vitamin A when the dried sprouts made up 5 % of the d i e t of white r a t s . He drew the conclusion that Vitamin A i s produced i n the growing plant w i t h or without any accompanying photosynthesis. Coward (37) i n 1923 pointed out that l i g h t i s necessary f o r the formation of Vitamin A i n plant t i s s u e s .  This  process, however, can "be c a r r i e d out i n the absence of carbon dioxide and of oxygen i n the surrounding atmosphere.  The  production i s also independent of the u l t r a - v i o l e t rays of the spectrum (or such of those as are held back by 2 p l a t e s of window g l a s s ) and can be c a r r i e d on under the i n f l u e n c e of e l e c t r i c l i g h t i n the absence of s u n l i g h t .  On the other hand  the presence of chloroform i n the atmosphere prevents the formation of the Vitamin. In 1925 Coward (39) f u r t h e r showed that Vitamin A i s not used i n any process c a r r i e d on by l i v i n g plant t i s s u e i n the dark.  The Vitamin A appears to increase when a l e a f loses  i t s green color and becomes yellov/; but i s completely destroyed when the l e a f d r i e s up and d i e s . The same i n v e s t i g a t o r (40) l a t e r presented data to show that the l i g h t from a quartz mercury vapor lamp was eff e c t i v e i n a c c e l e r a t i n g the formation of Vitamin A i n l i v i n g plant t i s s u e s .  These short u l t r a - v i o l e t rays from such a  lamp, used i n conjunction w i t h the v i s i b l e rays, did not have any i n f l u e n c e on the u l t i m a t e amounts of Vitamin A contained  10. i n the t i s s u e s . The i n v e s t i g a t i o n also showed that the amount of Vitamin A i n e t i o l a t e d shoots i s an inverse function of the temperature at which they have been grown, t h i s having  influ-  enced markedly the rate of growth. Moore (159) corroborated the work of both (260) and (40).  He f u r t h e r showed that r a t s fed f o r some months on  e t i o l a t e d wheat shoots appeared lean and rough-coated when compared w i t h s i m i l a r r a t s r e c e i v i n g a l i b e r a l d i e t . The year a f t e r the above evidence had been published Moore (160) came out w i t h the opposite idea.  He fed e t i o -  l a t e d wheat shoots to r a t s under conditions i n v o l v i n g the minimum of red l i g h t i l l u m i n a t i o n consistent with the feeding and handling of the animals.  They were e f f e c t i v e as a source  of Vitamin A, thus supporting h i s conclusion that l i g h t i s not e s s e n t i a l during any stage of the formation of the V i t a min from seed. Dye, Mediock and C r i s t (72) observed that l e a f l e t tuce exceeded head l e t t u c e i n the promotion of growth i n r a t s that had ceased to gain on a d i e t d e f i c i e n t i n Vitamin A. The outside green leaves of head l e t t u c e were f a r superior to the i n s i d e yellow leaves i n f u r n i s h i n g the Vitamin. H e l l e r and S t . J u l i a n (113) found that Vitamin A was formed and stored c h i e f l y i n that p o r t i o n of the plant exposed to l i g h t and that i t d i d not migrate to other portions of the plant* Luce and MacLean (141) stated that Vitamin A i s synthesized by the yeast c e l l and the synthesis may be brought  11. about i n the absence of s u n l i g h t . According to Goode (91) the Vitamin A content of white corn sprouts was increased by i r r a d i a t i o n and the Vitamin was transferred i n part from the sprouts to the g r a i n during the i r r a d i a t i o n of the growing process.  12. Plant Carotinoids And Vitamin A. Palmer and Kennedy (174) i n 1921 showed that c a r o t i n oids and f a t - s o l u b l e Vitamin are not even q u a n t i t a t i v e l y associated i n the plant tissues i n which both are presumably synthesized. Two years l a t e r Coward (38) stated that the c h i e f point i n the formation of the Vitamin which was apparent i n her experiments was that some lipochrome (generally carotene) was always associated with the Vitamin i n plant t i s s u e s ; and that where carotene was found, p a r t i c u l a r l y carotene exposed to s u n l i g h t , there the Vitamin may be expected to be present also. Smith and Morgan (223) l a t e r found that c h l o r o p h y l l was not a necessary intermediary i n s i t u f o r the formation of carotene, lycopene, or any other p o s s i b l e precursors of V i t min A.  F r u i t s which develop carotene and Vitamin A a c t i v i t y  under normal l i g h t conditions do so also under glass and i n the dark although u s u a l l y i n s l i g h t l y smaller amounts.  They  also noted that a constant r e l a t i o n e x i s t s between the l e v e l of d a i l y intake of aarotene by Vitamin A free r a t s and t h e i r growth per unit of t o t a l carotene ingested.  This r e l a t i o n  i l l u s t r a t e s the "Law of Diminishing Returns." Norris (169) discovered that the exposure of e t i o l a t e d plants to a r t i f i c i a l l i g h t r e s u l t e d i n the formation of chlorophyll and xanthophyll i n constant proportions. creased at f i r s t , but a f t e r 4 — 5  houES  than the c h l o r o p h y l l andxanthophyll.  Carotene de-  increased more r a p i d l y With i n c r e a s i n g oxygen  13. i n the atmosphere carotene developed s t i l l more r a p i d l y , hut formation of c h l o r o p h y l l andxanthophyll reached a maximum w i t h 20% oxygen.  The proportion of a l l pigments increased w i t h  r i s i n g carbon dioxide i n the atmosphere up to 3-5^, hut subsequently declined. Indications were obtained by Macv/alter and Drummond (149) that the lipochrome content of young f i s h may be increased when they are fed on a d i e t containing a t y p i c a l green alga. Examination of the lipochromes of Pucus Vesiculosus by Heilbron and Phipers (111) showed that wheras the dead material contained b e t a carotene a.nd zeaxanthine, the l i v i n g plant contained beta carotene and fucoxanthine. No trace of xanthophylls u s u a l l y associated w i t h t h e higher p l a n t s could be found and zeaxanthine was probably a post mortem product of fucoxanthine. Heilbron, Parry and Phipers (112) found that i n the unsaponifiable matter from extracts of cladophora, s a u t e r i , n i t e l l a opaca, oedogonium and rlodymenia palmata, various s t e r o l s could be i d e n t i f i e d and also the lipochrome pigments, l u t e i n , taroxanthine and b e t a carotene. only i n oedogonium.  A l f a carotene was observed  14. I n t r a c t a b i l i t y of Garotinoids from P l a n t s . In 1920 Steenbock and Boutwell (232) cound that when carrots were saturated with l a r d or corn o i l and then e x t r a c t ed w i t h ether, none or l i t t l e of the f a t - s o l u b l e vitamin was removed.  The l a r d preparation gave no evidence of containing  the vitamin, but the corn o i l preparation contained i t i n small but p e r s i s t e n t amounts.  They also noted that ether had l i t t l e  solvent p r o p e r t i e s f o r the f a t - s o l u b l e vitamin as found i n carr o t s ; chloroform and carbon d i s u l p h i d e removed some of i t ; while a l c o h o l and benzene removed considerable amounts of i t . While the vitamin i s not extracted from forn by ether, alcohol removed i t q u a n t i t a t i v e l y and w i t h l i t t l e i f any, d e s t r u c t i o n . They give t h e i r procedure f o r f r a c t i o n a t i n g an extract from alfalfa. These same two i n v e s t i g a t o r s (229) l a t e r demonstrated that the f a t - s o l u b l e vitamin as found i n the plant kingdom i n a g r a i n , , i n l e a f and stem t i s s u e , i n f l e s h y  roots and i n a  cucurbitous vegetable was comparatively stable at a high temperature.  15. VITAMIN A IN THE ANIMAL KINGDOM. Synthesis of Vitamin A i n Fish.. In 1922 Jameson, Drummond and Goward.(125) demonstrated that pure cultures of a common marine diatom grown i n Miguels' s o l u t i o n or s t e r i l i s e d sea water synthesised large amounts of Vitamin A. These i n v e s t i g a t o r s draw a p a r a l l e l between the dependence of land animals on f r e s h green leaves and that of marine animals on the synthetic a c t i v i t y o f the marine f l o r a f o r t h e i r supplies of Vitamin A. Working w i t h brown t r o u t , Coward and Drummond (36) found that the ova of these trout normally contain r e l a t i v e l y large amounts of Vitamin A.  I f i n the p o s t - l a r v a l period or  even before the yolk sac i s completely absorbed, the f i s h are given food r i c h i n Vitamin A t h e i r growth and development are s a t i s f a c t o r y and they appear able to store that factor i n t h e i r tissues. Drummond and Z i l v a (63) concluded from t h e i r work that the ultimate o r i g i n of the Vitamin A found i n the o i l s derived from f i s h , and p a r t i c u l a r l y f i s h l i v e r o i l s , to be c h i e f l y the u n i c e l l u l a r marine p l a n t s .  Except very o c c a s i o n a l l y these org-  anisms are not consumed, d i r e c t l y by the f i s h . These i n v e s t i g a t o r s also noted that the extraordinary r i s e i n the number of marine plants which begins as soon as the i n t e n s i t y and duration of sunlight increase, e a r l y i n the year, i s followed by a r a p i d r i s e i n organisms l a r g e l y copepods and l a r v a l decopods and molluscs, whose growth and development are  dependent on t h e i r food supply which consists of minute p l a n t s . These minute animals, which form a la,rge proportion of plankton contain r e l a t i v e l y large q u a n t i t i e s of Vitamin A presumably der i v e d from the deatoms on which they have t h r i v e n . The plankton forms the staple food of innumerable species of marine animals from small f i s h to some whales. This no doubt accounts f o r the presence of Vitamin A i n the tissues or f a t depots of these animals. Two years la.ter Z i l v a , Drummond and Graham (268) demonstrated that the sexual condition and age of the cod d i d not influence the Vitamin A potency of the l i v e r o i l . B a i l e y (6) l a t e r corroborated t h i s work while working with Ling Cod of the p a c i f i c Coast. Finn (82) i n 1931 published data to show that p i l c h a r d o i l contained a substance which promoted the growth of r a t s which had f a i l e d t o grow on a Vitamin A d e f i c i e n t d i e t .  Biely  and Chalmers (16) working w i t h chicks further demonstrated the Vitamin A potency of p i l c h a r d o i l . T r u e s d a i l and Boynton (246) undertook a study of the Vitamin A content of body o i l s of 5 species of P a c i f i c Coast Salmon.  They found a l l 5 were decidedly i n f e r i o r to the sample  of high-grade medicinal cod l i v e r o i l w i t h which they were compared.  The Vitamin A potency was found t o be proportional  to the i n t e n s i t y of the n a t u r a l yellow colour of the o i l . While working w i t h h a l i b u t l i v e r o i l , Lavern, Edisbury and Morton (139) obtained very i n t e r e s t i n g r e s u l t s ,  ilo p a r a l l -  ism could be traced between the Vitamin A and Vitamin D potencies.  In c e r t a i n species, the Vitamin A content of the l i v e r  o i l was found to increase w i t h the age and or s i z e of the f i s h , the t o t a l Vitamin A reserve i n c r e a s i n g more r a p i d l y than the o i l potency.  They b e l i e v e that h a l i b u t l i v e r o i l i s by f a r the  r i c h e s t known n a t u r a l source of Vitamin A a v a i l a b l e i n quantity. I t has been found, however, that the o i l v a r i e s i n potency a wider range than any other source.  over  Eo c o r r e l a t i o n has emerged  between the immediate d i e t of the h a l i b u t and the o i l potency. Their work also showed that h a l i b u t l i v e r o i l s e x h i b i t w e l l marked seasonal f l u c t u a t i o n s i n Vitamin A concentration which cannot be a t t r i b u t e d to changes i n the o i l content of the l i v e r occasioned by spawning.  The best o i l s from the standpoint of  Vitamin A content are obtained from large halibut caught i n northern waters i n the l a t e spring or e a r l y summer, and i n the autumn.  Very r i c h o i l s at other times of the year are except-  ional . Working along similar- l i n e s Lavern and Sharp (140) found that the diet of the h a l i b u t was  of a general nature,  w i t h no outstanding r i c h source of Vitamin A to account for the high potency of h a l i b u t l i v e r o i l . Taking the glycogen content of the l i v e r as a c r i t e r i o n of i n t e n s i t y of feeding, they found that no c o r r e l a t i o n could be established between i n t e n s i t y of feeding and the Vitamin A potency of the o i l . They obtained further evidence that i n general the l i v e r s of older f i s h a f f orded a more potent o i l than those of younger f i s h .  18O  Weekly records were taken of the f a t , Vitamin A and Vitamin D contents of the l i v e r s o f halibuts(caught near Seattle by B i l l s , Imboden and Wallenmeyer (18).  The o i l content i n -  creased slowly from January (12%) to June, there was then a sudden r i s e to a majcimum value [25%) i n August, followed by a slow d e c l i n e during the rest of the year.  The Vitamin A content  (January 240,000, August 35,000 i n t e r n a t i o n a l units per g.) and Vitamin D content (January 1,400, August 900 i n t e r n a t i o n a l u n i t s per g.) moved i n v e r s e l y w i t h the f a t content although i n the case of Vitamin A the f l u c t u a t i o n was wider than i n the case of Vitamin I). B i l l s et. al.(19) conducted a toxonomic-study  of the  d i s t r i b u t i o n of the Vitamins A and D i n many species of f i s h . They found that f o r a given species, no r e l a t i o n s h i p could be established between the Vitamin A and D potencies of the l i v e r or body o i l s .  In general, but w i t h many exceptions, l i v e r o i l s  r i c h i n the other, and the potency tended to vary i n v e r s e l y w i t h the o i l content of the l i v e r .  19. The Vitamin A Content of Animal Products. In 1917 McCollum, Simmonds and Steeribock (143) stated that fat f r e e milk, when included i n a d i e t c o n s i s t i n g otherwise of p u r i f i e d food substances, promoted growth and prevented decline of animals i n a manner which i n d i c a t e d i t s t i l l contained the f a t - s o l u b l e e s s e n t i a l i n appreciable amounts.  This led  them to suspect that the f a c t o r was appreciably soluble i n water.  They demonstrated that when butter f a t was melted and  thoroughly  agitated w i t h twenty successive portions of water i t  no longer contained the f a t - s o l u b l e f a c t o r . Continuing along t h i s l i n e of thought, Steenbock, Boutwell and Kent (227) found that while the vitamin was removed from the washed b u t t e r f a t the washing s did not contain i t . They were convinced  that somewhere i n the course of the man-  i p u l a t i o n s to which the butter f a t had been subjected, fa.ctors had been introduced which were responsible f o r the vitamin destruction.  They then r e a l i z e d that heat alone i n the absence of  water or i n the absence of conditions designed to bring about intimate contact w i t h a i r was responsible f o r the vitamin des t r u c t i o n observed i n the early experiment. Hopkins (122) found that the f a t - s o l u b l e A substance i n b u t t e r , which d i s p l a y i n g marked r e s i s t a n c e to heat alone at o temperatures up to 120  was r e a d i l y destroyed by simultaneous  aeration of the f a t , presumably because i t i s a substance prone to oxidation by atmospheric nitrogen. Working along the same l i n e s as Hopkins (122), were  20. Drummond and Coward (61).  They noted that the destruction of  the Vitamin present i n "butter f a t occured on heating i n the presence of a i r . They concluded that the loss was due to chang es' of an o x i d a t i v e nature.  They also noted that the destruct-  ion took place r a p i d l y at high temperatures hut provided the exposure to a i r i s extensive, considerable l o s s of n u t r i t i v e value takes pla.ce at temperatures as low as 37o. Steenbock, S e l l and B u e l l (233) found that butter f a t showed a seasonal v a r i a t i o n i n the f a t - s o l u b l e vitamin content when obtained from' s t a l l fed cows during the winter and pastured i n the summer. The note that the f a t - s o l u b l e vitamin content of b u t t e r f a t does not run c l o s e l y p a r a l l e l to the yellow pigment; yet i n general, due to determination by t h e i r content i n thle feed, butter highly pigmented are r i c h i n the vitamin; b u t t e r low i n pigment should be looked upon w i t h suspicion. In beef f a t s the r e l a t i o n s are somewhat s i m i l a r ; those most pigmented are also generally r i c h e s t i n t h e i r f a t - s o l u b l e vit° amin content.  These i n v e s t i g a t o r s demonstrated that the f a t -  soluble vitamin withstood  severe methods of s a p o n i f i c a t i o n .  This indicated that i t was not a fat and probably not an ester. The i n a c t i v a t i n g action of some f a t s on Vitamin -A i n other f a t s was studied by F r i d e r i c i a (85) i n •1925.  He reported  that r a t s d i d not grow on an apparently adequate diet when the butter f a t y i e l d i n g the Vitamin A of the diet was mixed ( a f t e r melting) w i t h a brand of hydrogenated whale o i l .  This hydro-  genated whale o i l , had no t o x i c action on the oh the: growth, of ;  21. r a t s but had an i n a c t i v a t i n g a c t i o n on the Vitamin A of butter f a t when the two f a t s were mixed a f t e r melting at a low temperature.  Two hydrogenated vegetable oils(hydrogenerated coc-  panut o i l , s-nd hydrogenated hemp-seed o i l ) and a non hydrogenated vegetable o i l (cocoanut o i l ) showed neither any t o x i c e f f e c t on the growth of r a t s nor any i n a c t i v a t i n g action on the Vitamin A of b u t t e r f a t , t h i s action accordingly not being regu l a r l y connected w i t h the process of hydrogenating.  Untreated  abdominal p i g fat did not i n a c t i v a t e the Vitamin A of butter f a t . o A f t e r being heated i n t h i n layers to 1G2 to 105 6. f o r 24 hours on exposure to a i r , the same p i g fat acquired an i n a c t i v a t i n g action on the Vitamin A of b u t t e r f a t when the two f a t s were mixed a f t e r melting©  This i n a c t i v a t i n g action i s supposed to  depend on the generation of peroxides i n the aerated heated f a t , the Vitamin A of b u t t e r fat being destroyed by these peroxides through oxidation© S j o r s l e n (219) showed that b u t t e r f a t gave a reaction w i t h sulphuric a c i d but not i n such low concentrations  as cod  l i v e r o i l and colour i n d i c e s do not a t t a i n so high a value© Lard did not give a r e a c t i o n w i t h sulphuric acid©  A mixture of  equal parts butter f a t and non-heated l a r d gave a reaction with sulphuric a c i d .  When b u t t e r f a t Y/as heated 4 hours i n the a i r  i t s a b i l i t y for a t y p i c a l sulphuric a,cid r e a c t i o n  disappeared.  G i l l a m (88) found that on the average, the r a t i o of carotene to xanthophyll i n butter i s 14 to 1 by weight. Many i n v e s t i g a t o r s have studied the vitamin content of  21 cow's milk.  Q Ci  Kennedy and Butcher (131) noted that the presence  of Vitamin A i n cow's milk i s e n t i r e l y dependent upon i t s occur ance i n the r a t i o n .  S t a l l fed cows w i l l produce a milk r i c h  i n vitamins provided t h e i r r a t i o n c o n s i s t s of a proper combination of grains and l e a f y foods.  Bussel et. a l . , (195) found  that i n f r e s h milk the vitamin content may vary a.s a r e s u l t of change i n l a c t a t i o n period and of v a r i a t i o n s of provitamin A and the Vitamin A content  of the r a t i o n when the assay extends  over a period of months.  They show that the percentage output  . of vitamin expresses the r e l a t i o n s h i p between intake and output but does not take into consideration the p o s s i b i l i t y that some of the Vitamin A i n the milk may  come from the body stores. The  percentage of the f a c t o r which appears in.the milk decreases as the amount of i t i n the r a t i o n i s increased and that the i n crease i n Vitamin A content of the milk i s not p r o p o r t i o n a l to the increased consumption.  In a comparison of Ayshire and  Guernsey b u t t e r s , Wilbur* H i l t o n and Hauge (255) found that, although the Ayshire b u t t e r contained 1.8mg  c a r o t i n per 100 gr.  of b u t t e r and the Guernsey 4.0mg. per lOOgr. of b u t t e r , they were i d e n t i c a l i n Vitamin A a c t i v i t y ,  Hathaway and Davis  demonstrated that the Vitamin A content ociated w i t h the b u t t e r f a t content.  (103)  of milk i s c l o s e l y ass-  They show that skim milk  or separated milk containing only a small quantity of b u t t e r f at contains also only a small amount of Vitamin A.  They also noted  that H o l s t e i n cream i s mire potent i n Vitamin A than i s Jersey cream.  As the percentage of f a t i n the H o l s t e i n and Jersey  22. milk approach each other the d i f f e r e n c e i n Vitamin A content of the cream separated from them w i l l disappear.  In f u r t h e r tests  Hathaway and Davis (104) shov/ed that there i s l i t t l e difference i f any i n sour and sweet cream b u t t e r . Margarines on the other hand are poor sources of Vitamin A. I n v e s t i g a t i n g the Vitamin A content of b u t t e r , Praps, Copeland and H r e i c h l e r (84) demonstrated that cows receiving feeds which were low i n Vitamin A content produced butter low in Vitamin A potency.  The Vitamin A content of butter depends  both upon the Vitamin A potency of the feed and the length of time the cow has been fed upon i t .  When a cow i s on a. feed  supplying i n s u f f i c i e n t q u a n t i t i e s of Vitamin A, the Vitamin. A i n the butter f a t decreases w i t h the period of time the cow has been on the feed or the stage of l a c t a t i o n on account of the depletion of the reserve of Vitamin A stored by the cow at the beginning of lactation,,  In the cow i t requires approximately  eleven u n i t s of.Vitamin A to give one unit o f Vitamin A i n the butter.  The cow uses Vitamin A much less e f f i c i e n t l y than  poultry. According to Moore (163) carotene when supplied to cows undergoes conversion to Vitamin A. Guilbert and Hart (96) found that carotene being the p r i n c i p l e pigment of beef f a t , could be withdrawn from the adipose t i s s u e during Vitamin A p r i v a t i o n without a coincident reduction of f a t reserve.  They showed that the carotene i n the  f a t of c a t t l e c o n s t i t u t e d a s i g n i f i c a n t part of the t o t a l V i t -  23. amin A reserve. These same i n v e s t i g a t o r s noted that the storage of Vitamin A i n the l i v e r of newborn calves i s r e l a t i v e l y low r e gardless of the storage i n the dam. The Vitamin A content of the l i v e r s of normally fed oxen, guineapigs, r a b b i t s , r a t s and dogs was tested b i o l o g i c a l l y by Simonnet, Busson and A s s e l i n (218) and found to vary considerably.  The l i v e r of the ox was the most potent while  that of the guinea p i g was the least potent.  Using a dog. these  same i n v e s t i g a t o r s (217) f e d l i b e r a l d a i l y doses o f carotene f o r a, month.  I t wa.s then k i l l e d and the lungs, l i v e r , kidneys,  b r a i n and part of the body f a t were tested f o r Vitamin A. They found that only the l i v e r and kidneys contained Vitamin A and these i n approximately equal amounts. Ahmad and Malik (5) showed that animals d i f f e r i n their a b i l i t y to synthesize Vitamin A from carotene, as judged by the Vitamin A content of the l i v e r .  With the a b i l i t y of rats to  synthesize Vitamin A rated at 100, chickens give a r a t i n g of only 24, while r a b b i t s give a r a t i n g of only 16 and cats zero. They conclude that the Vitamin A potency of feeds may vary f o r d i f f e r e n t animals according to the r e l a t i v e proportion of the Vitamin A and carotene present. L o c a l i s a t i o n of Vitamin A i n tissues by a fluorescent microscope was noted by Querner (178).  In the c e l l s of the  l i v e r and other organs a fluorescent material was present which was ra,pidly destroyed by exposure to u l t r a v i o l e t l i g h t • This  24. "Leuchtstoff" was found to correspond to the amount of Vitamin A present. The n u t r i t i v e value of l a r d was extensively studied by Drummond e t . a l . (62) i n 1920.  They concluded that the p i g  was able to store up supplies of Vitamin A i n the body f a t when fed a diet containing ample supplies of the f a c t o r .  When the  diet was d e f i c i e n t i n Vitamin A no appreciable amounts of the d i e t a r y f a c t o r could be found i n the body f a t . The process employed i n the manufacture of l a r d causes a very marked dest r u c t i o n of the vitamin present i n p i g f a t . I n v e s t i g a t i n g t h i s problem of the Vitamin A content of l a r d f u r t h e r , Mallon and Clark (151) concluded that l a r d made from leaves and back f a t of hogs whose d i e t contained Vitamin A, d i d not contain an adequate supply of t h i s vitamin to prevent xerophthalmia even when fed i n large amounts.  25. Storage of Vitamin A i n the Body. The storage of Vitamin A i n the body was reported by Goldblatt and Soames (90) i n England and Steenbock, S e l l and Nelson (235) i n America i n 1923.  Both groups of investigators  cagie to the same conclusion that Vitamin A i s stored i n the l i v e r and that i t i s stored i n rough proportion to the amount contained i n the d i e t . Continuing t h i s work f u r t h e r , Sherman and Cammack(207) showed that by feeding diets graded i n t h e i r content of Vitamin A, the r i c h e r the d i e t i n t h i s vitamin, the greater i s the ount stored i n the body.  am-  The attainment of the maximum store  of Vitamin A i s a process of gradual accumulation which i s r e l a t i v e l y r a p i d i n i t s e a r l i e r stages and becomes slower as the maximum i s approached. amount i s not  A r a p i d storage of the e n t i r e maximum  possible.  McGoord and Clausen (145) demonstrated that when 1 drop of h a l i b u t l i v e r o i l was added to the d i e t of the rat and the animal k i l l e d 24 hours l a t e r , there was a d e f i n i t e increase of Vitamin A i n the l i v e r and body f a t . When 4 drops were so added, marked increase was found i n a number of other organs and t i s s u e s , p a r t i c u l a r l y the adrenals. Working on the problem q u a n t i t a t i v e l y Baumann, R i i s i n g and Steenbock (10) found that 95 % of the Vitamin A i n the body was stored i n the l i v e r . and kidney t i s s u e .  The remainder was located i n lung  The minimum d a i l y dose of Vitamin A nece-  ssary to produce storage i n the l i v e r was between 25 and 50 blue u n i t s .  When Vitamin A was fed i n the form of halibut  26. l i v e r o i l , the amount stored i n the l i v e r was found to p a r a l l e l the amount administered, hut only 10 to 20 % of the Vitamin could be accounted f o r . When equal amounts of Vitamin A were fed  to normal and to Vitamin A depleted r a t s , the l i v e r stor-  age was greatest i n the normal animals.  When equal amounts  of Vitamin A were fed to animals i n various stages of deplet i o n , the amount stored was i n v e r s e l y p r o p o r t i o n a l to the s t a t e of d e p l e t i o n . The absorption and storage of Vitamin A to a large extent takes place w i t h i n 6 hours a f t e r ingestion of the Vitamin. The e l i m i n a t i o n of Vitamin A from the l i v e r s of rats was studied by Davies and Moore (49).  Female r a t s 18 months  o l d , which had f i n i s h e d breeding and had been fed a d i e t w e l l supplied with Vitamin A, received f o r 12 weeks, i n addition, a large amount of a Vitamin A concentrate.  The mean Vitamin  A concentration i n the l i v e r at the end of t h i s time was 18,000 blue u n i t s per gram.  The s u r v i v i n g animals then r e -  ceived a diet d e f i c i e n t i n Vitamin A and the concentration i n the l i v e r f e l l r a p i d l y , being reduced i n 4 weeks to an average of 2,700 b l u e . u n i t s per gram; after 12 weeks i t f e l l further to about 400 u n i t s and  v<ras  s t i l l at that l e v e l a f t e r 24 weeks.  The r a t e of depletion of these enormous reserves of Vitamin A was f a r more r a p i d than a r a t e representing the r a t s ' d a i l y requirement.  27. Transmission of Vitamin A Prom Parents to Young. The problem of Vitamin A transmission from the mother to the foetus has "been extensively studied by Dann (44) and '(46).  He showed that Vitamin A i s normally found i n the  l i v e r of the r a t and rabbit at b i r t h , but only i n small amounts which cannot be increased by g i v i n g the mother a diet r i c h i n carotene during g e s t a t i o n . The store of Vitamin A i n the l i v e r of the young r a t increases 2 or 3 f o l d during s u c k l i n g , but the increase may be greater when the l a c t a t i n g parent receives a d i e t r i c h i n the f a c t o r .  There i s a l i m i t  to the amount of t h i s increase, due i n turn to a l i m i t a t i o n of the amount of Vitamin A which can pass into the milk. Working w i t h c h i c k s , however, Bearse and M i l l e r (13) have shown that the hen's d i e t markedly controls the amount of Vitamin A found i n baby chicks. The work of Hale (101) i s i n t e r e s t i n g i n that he produced 3 l i t t e r s of pigs born without eyeballs or with very serious eye defects when the mother had received a diet def i c i e n t i n Vitamin A before mating and during the f i r s t 30 days of g e s t a t i o n .  28 © Conversion of Carotinoids Into Vitamin A. The transformation of carotene into Vitamin A was studied by Copper (30) i n 1930.  He used the absorption specu u  t r a of r a t l i v e r o i l s .  He found that the band at 325  generally a t t r i b u t e d to Vitamin A was absent from the absorpt i o n spectra of the l i v e r o i l s of rats s u f f e r i n g from Vitamin A d e f i c i e n c y , but was shown by the l i v e r o i l s of s i m i l a r rats which had been subsequently cured by massive doses of carotene, u u The 325 / / band was absent from the absorption spectrum of carotene, therefore, he concluded, the substance responsible for the e x h i b i t i o n of t h i s band by the l i v e r o i l s of carotenetreated r a t s , had been synthesised i n vivo from the carotene, Moore (161) noted that the conversion of carotene into Vitamin A took place i n the l i v e r , but the e f f i c i e n c y of the conversion was by no means q u a n t i t a t i v e . The absorption of carotene from the i n t e s t i n a l tract was i n v e s t i g a t e d by Ahmad (3) v/ho observed that i t may be considerably a f f e c t e d by the composition of the d i e t . Studying the isomers of carotene K a r r e r , Euler and H e l l stroza (128) showed that the blue values given by alpha and beta isomerides i n the Sb.Clg r e a c t i o n were i d e n t i c a l . Rat growth tests f o r Vitamin A a c t i v i t y however, demonstrated that the beta i s more active than the alpha isomeride. The c u r a t i v e e f f e c t s of 5 to 10 y of pure carotene on r a t s s u f f e r i n g from symptoms of Vitamin A deficiency was d e f i n i t e l y established by Glanzmann (89).  He found that the  blood p l a t e l e t s of the r a t s f a l l i n numbers r a p i d l y when  29..  diets d e f i c i e n t i n Vitamin A are given, and r i s e r a p i d l y when carotene i s supplied. The Vitamin A a c t i v i t y of palm o i l s was found to be "in accord w i t h t h e i r carotene content by Ahmad (4).  After  hydrogenation, however, a l l the a c t i v i t y was l o s t . Sea and Drummond (180) i n v e s t i g a t e d the formation of Vitamin A from carotene i n the animal organism.  In the incu-  b a t i o n of the l i v e r of r a t s and cats w i t h s o l u t i o n s of carotene, they f a i l e d to demonstrate the formation of Vitamin A. The intravenous i n j e c t i o n of c o l l o i d a l suspensions of carotene into the l i v e r s o f cats which had received a Vitamin A d e f i cient d i e t , produced no detectable increase i n the Vitamin A content of the l i v e r s as shown by spectrographic examination of the non-saponifiable f r a c t i o n s before i n j e c t i o n , and 4 to 72 hours a f t e r i n j e c t i o n . Conversion of carotene into Vitamin A i n the animal body was studied by Skarzynski (220).  He made spectroscopic  and c o l o r i m e t r i c examinations of e x t r a c t s from the caecum, spleen, l i v e r and serum of young r a t s which had developed xerophthalmia on ©• Vitamin A d e f i c i e n t diet and which had died or been cured w i t h carotene ( 4 mg. i n 36 days). I n the extracts from the d e f i c i e n t group which had died, no trace of Vitamin A or carotene could be detected.  In the  group which received carotene, Vitamin A was found i n the l i v e r and serum, and carotene i n the caecum and probably i n the l i v e r ; the spleen contained n e i t h e r . The author suggests that transformation of carotene does not take place i n  5 0 .  the gut as supposed "by Ahmad (4), but i n the l i v e r from which i t passes into the blood. The Vitamin A content of r a t l i v e r a f t e r feediing .alpha, beta and gamma carotene was reported by Brockmann and Tecklenburg (22). They examined the unsaponifiable f r a c t i o n of the l i v e r o i l w i t h S b C l and found that beta carotene produced 3  2 or 3 times as much Vitamin A as the other two. Moore (164) reported i n 1933 that carotene i s u t i l i s e d i n the body as e f f i c i e n t l y  as preformed Vitamin A at l e v e l s  approaching the minimum dose. Using a spectrophotometer method, Drummond and MacWa.lter (68) observed that a f t e r i n j e c t i n g carotene into the p o r t a l c i r c u l a t i o n of r a b b i t s no increase i n the Vitamin A i n the l i v e r takes place u n t i l eight days after the experiment. They suggest the p o s s i b i l i t y  that formation of the vitamin  occurred w i t h i n much shorter periods but either i t was u t i l ised and converted into another substance or that the amounts were too small to be detected by t h e i r technique. The fats of carotene introduced into the c i r c u l a t i o n was investigated by Drummond, G i l d i n g and MacWalter (69). These i n v e s t i g a t o r s i n j e c t e d intravenously into anaesthetised cats a c o l l o i d a l suspension of carotene i n i s o t o n i c glucose s o l u t i o n . Blood samples were taken i n the ensuing 15 minutes and after death, the remaining blood and organs were examined for carotene Wo carotene was. found i n any of the blood samples and, of the organs, only i n the lungs, l i v e r and spleen.  In the lungs the  31. carotene appeared to be trapped i n the c a p i l l a r i e s .  The  greater part was absorbed s e l e c t i v e l y by the l i v e r and was c h i e f l y located i n the Kupffer c e l l s . Working w i t h r a t s , Drummond and MacWalter (70) r e ported that Vitamin A and carotene were evenly d i s t r i b u t e d through the three lobes of the animals l i v e r , Pure c r y s t a l l i n e xanthophyll was shown not to be i d e n t i c a l w i t h Vitamin A by W i l l i m o t t and Moore (257) i n 1927. A few years l a t e r Kuhn, e t . a l . (134) showed that the xanthop h y l l s , l u t e i n , zeaxanthin and v i o l a x a n t h i n were negative as w e l l as a z a f r i n methylester, dihydrocrocetin dimethylester and chlorophyll. Certain microorganisms  were found by Baumann, Steen-  bock and Ingraham (9) to synthesize carotene.  Whenever an  organism showed Vitamin A a c t i v i t y as determined by feeding experiments, enough carotene was found to be present to account f o r that a c t i v i t y .  The Vitamin A a c t i v i t y of microorgan-  isms d i d not appear to be a f f e c t e d by the presence of yellow pigments other than carotene.  They concluded that since  spectrographic determination f a i l e d to reveal any absorption band at 328 M  i t i s exceedingly improbable that Vitamin  as such i s g e n e r a l l y present i n b a c t e r i a .  Attempts, however,  to e f f e c t the transformation of carotene into Vitamin A by microorganisms  failed.  A  32» VITAMIN A 'THERAPY III Ool&B  I t i s generally recognized that a l a c k or deficiency of Vitamin A i n the Ration r e s u l t s i n the greater s u s c e p t i b i l i t y of animals as w e l l as man to various Respiratory i n f e c t i o n s . In view of the wide prevalence of simple colds i n man,  con-  siderable emphasis has been l a i d on the i n c l u s i o n of optimum amounts of Vitamin A i n the d i e t .  As a matter of f a c t , d i r e c t  evidence that Vitamin A plays the role of an a n t i - i n f e c t i v e agent, as has been commonly accepted, Is l a c k i n g . In t h i s connection i t i s i n t e r e s t i n g to note Cameron's work (27), (28) and (29), on Vitamin A Therapy i n colds.  In an  experiment (27) conducted at the U n i v e r s i t y of West V i r g i n i a , he used f i v e groups of women students.  The members of one group  took a tablespoonful of cod l i v e r o i l d a i l y from November to March 1932-33, and the other groups an equivalent dose of a Vitamin A concentrate, an i n e r t t a b l e t , a cod l i v e r o i l residue i n t a b l e t , form, and no medication.  Records were made twice a  week during the experimental period on the presence and duration of colds.  At the end of the experiment i t was found  that the number of days with colds reported from the group taking no medication was considerably higher than from any the other groups.  of  The Vitamin A concentrate and the eod l i v e r  o i l residue t a b l e t s appeared to give s l i g h t l y better r e s u l t s than the cod l i v e r o i l .  The f o l l o w i n g i s a quotation from the r e s u l t of the above experiment. "Ho conclusion i s as yet j u s t i f i e d from these r e s u l t s , but the volunteers p a r t i c i p a t i n g i n the experiment were unanimous i n r e p o r t i n g subjective improvement from cod l i v e r o i l and i t s concentrates.  This improvement took the form of  increased a p p e t i t e , weight, or endurance, or r e l i e f from s k i n eruptions or from sinus t r o u b l e , as w e l l as i n reduction i n number and duration of colds.  These changes can probably be  a t t r i b u t e d to the improved n u t f i t i o n r e s u l t i n g from an abundance of Yitamin A, and t h i s improved n u t r i t i o n i s worth while i n i t s e l f whatever f i n a l conclusions are reached with reference t o Yitamin A Therapy against common Golds". In h i s second experiment (28) the e f f e c t of increasing the Yitamin A intake of men and women students, e i t h e r by increasing the amount of foods r i c h i n Yitamin A, of by direct dosing with cod l i v e r o i l , h a l i v e r o i l , cod l i v e r o i l t a b l e t s or carotene s o l u t i o n , was studied i n regard t o the number and s e v e r i t y of colds during a period of two years.  The control  groups received no medication or a lactose supplement. S t a t i s t i c a l examination of the r e s u l t s i n d i c a t e d that increase i n the Vitamin A intake d i d not decrease the number of colds, but d i d tend to l e s s e n the duration and s e v e r i t y of the infection.  Approximately  f i f t y percent of the subjects of the  t e s t reported improvement i n powers of endurance, i n appetite and i n decrease of s k i n eruptions.  He concludes, however,  34. that Vitamin A Therapy should not be considered as a s p e c i f i c against oolds. From h i s t h i r d i n v e s t i g a t i o n (29) he gave the f o l l o w ing  conclusion: "Whether the e f f e c t of Vitamin A upon colds, therefore,  r e s t s upon i t s a b i l i t y to maintain i n t a c t a b a r r i e r of healthy mucous membrane to act as a l o c a l defense against invasion by v i r u s or b a c t e r i a , or whether i t s e f f e c t i s due also to i t s influence on c i r c u l a t i o n and nervous mechanisms which weather and d i e t a r y imbalance undoubtedly play a p a r t , i t has been shown to be a f a c t o r i n the duration of colds i n adults and worth a t r i a l i n a l l susceptible i n d i v i d u a l s . " In the Dressing of Wounds. The l o o a l use of Vitamin A i n the treatment of wounds has been observed by many workers.  Horn and Sandor  observed that dressings of a concentrate  of Vitamin  (123) A,  containing two thousand i n t e r n a t i o n a l u n i t s i n 1 ml. of an o i l y medium, influenced favorably the healing of f l e s h wounds and of i n f e c t e d t i s s u e s .  Zoltan (269) obtained good r e s u l t s  when purulent wounds were treated with an ointment containing cod l i v e r o i l , carotene and i r r a d i a t e d e r g o s t e r o l .  Direct  a p p l i c a t i o n of crude cod l i v e r o i l was found by Steel  (225)  to be most successful i n the treatment of severe burns and ulcers.  The use of the cod l i v e r o i l prevented the  of scar t i s s u e and pigmentation f o l l o w i n g healing.  formation  D r i g a l s k i (55) made wounds of a measured size with s c i s s o r s i n the s k i n of guinea pigs and dressed them with a cod l i v e r o i l ointment "Norguentolan ', or with the same 1  ointment through which a i r had been bubbled at 150° c. f o r t h i r t y - s i x hours.  B i o l o g i c a l t e s t s on r a t s showed the  d e s t r u c t i o n of Yitamin A by t h i s procedure to be complete. Healing was considered t o be more favorable and more r a p i d i n wounds t r e a t e d with unoxidized "Unguentolan", whether on the same or on d i f f e r e n t animals, even when the wounds were experimentally i n f e c t e d with stophylococci or s t r e p t o c o c c i . Shortly a f t e r the above observations had been made "Strauss (259) reported that over one hundred twenty cases of wounds, burns, e t c . , were s a t i s f a c t o r i l y treated with "Unguentolan". He stated that pain was reduced since the wound seldom needed redressing and drains were unnecessary.  Using t h i s same  ointment "Unguentolan", Dreyfus ( 5 3 ) observed that i s a p p l i c a t i o n t o burns of the second and t h i r d degrees r e s u l t e d i n r a p i d and complete h e a l i n g .  S i m i l a r r e s u l t s were obtained  i n purulent and gangrenous u l c e r s , but l i t t l e improvement was obtained i n erythrodermia ichthyosiforma.  He suggests  that the h e a l i n g might be due t o Vitamin A or D, or the Phosphorus or Iodine content, or the protective e f f e c t s of the preparation or a combination of these f a c t o r s . An extensive study of the e f f e c t of ointments containing Vitamins on the healing of wounds was conducted by Lauber  and Rooholl (135).  Surface wounds on white mice were treated  with various ointments containing Vitamins "H", "B", "C", "D", and with c o n t r o l ointments without Vitamins.  "A",  The wounds  treated with vogan (Vitamin A) i n a base containing Cholesterol healed best; vogan i n a v a s e l i n e - l a n o l i n e base was considerably l e s s e f f e c t i v e , and i n large doses even tended to i n h i b i t healing.  Vitamin "H" had some b e n e f i c i a l e f f e c t and Vitamin  "B", very l i t t l e , while Vitamins "C" and "D" appeared to hinder the healing process.  D r i g a l s k i (56) disagreed with the  above r e s u l t s and stated.that they would go no further than the authors" own f i n d i n g s . The e f f e c t of Vitamins on the growth of t i s s u e s and transplanted tumors was studied by G-ordonoff and Ludwig (92). They noted that the growth of f i b r o b l a s t s from embryo chicken heart, or of small transplants of mouse tumors, was very much l e s s ' i n plasma from r a t s which had been kept on d i e t s d e f i c i e n t i n Vitamin A, than i n plasma from r a t s which had received large doses of vogan.  The growth of normal and path-  o l o g i c a l t i s s u e s seemed also to be i n h i b i t e d i n plasma from r a t s deprived of the Vitamin "B" complex, but not i n plasma from r a t s r e c e i v i n g doses of yeast.  There was no difference  i n plasma from r a t s on MoCollum's rachitogenic diet 3143, whether the animals received Vitamin "D" or not.  The presence  of Vitamin "0" seemed to have an i n h i b i t i n g e f f e c t , as shown by cultures i n plasma from scorbutic and normal guinea pigs.  57. The work of Turner and Loew (249), shows that carotene i s p r o t e c t i v e against "bacterial invasion of the upper r e s p i r a t ory t r a c t and the development of the pathologic systemic conditions accompanying Vitamin A d e f i c i e n c y i n r a t s .  Carotene  therapy reduces the number of spontaneous supperative l e s i o n s i n the upper r e s p i r a t o r y t r a c t occuring i n animals deprived of Vitamin A; Xerophthalmia  i s cured i n one hundred percent,  and normal health regained i n seventy-four percent. percentage  The  incidence of b a c t e r i a i n the nasal c a v i t i e s and  middle ear i n animals fed active carotene i s noticeably l e s s than that encountered  i n animals given faded carotene or that  i n animals r e c e i v i n g no source of Vitamin A.  In another  i n v e s t i g a t i o n (247), they noted that the withdrawl of Vitamin A from the monkey does not develop a c h a r a c t e r i s t i c suscepti b i l i t y toward i n f e c t i o n of the upper r e s p i r a t o r y t r a c t .  They  also observed while working with r a t s (248), that the age of the animal, the previous storage of Vitamin A, the season, had no marked e f f e c t on the b a c t e r i a l f l o r a . In reviewing the researches on the value of ood l i v e r o i l p l a s t u s i n the h e a l i n g of burns and wounds, Gortzen (92), points out that ood l i v e r o i l does not i n h i b i t the growth of bacteria i n cultures.  He claims i t s . healing e f f e c t cannot,  therefore, be explained by any b a c t e r i c i d o l a c t i o n on infected tissue.  Hayaski (107) suggests that i t i s the l i p o i d content  of cod l i v e r o i l that has the b e n e f i c i a l e f f e c t on infected  38. wounds and not Vitamins "A" and "D". Antagonism Between Vitamins "A" and "C" and Vitamin "A" and Thyroxin Wendt and Schroeder (253) studied the antagonism between Vitamins "A" and "C". When guinea pigs were given l a r g e simultaneous, d a i l y doses of Vitamin "A" (1.5 ml. vogan) and Vitamin "C" (60 mg, Cebain Merck), they showed no signs of hypervitaminosis "A". I f only 0.25 t o 0.5 ml. d a i l y of vogan was given together w i t h 10 mg. Cebain Merck, there was not even r e t a r d a t i o n of growth.  The Vitamin "A" content of the  l i v e r s of the guinea pigs r e c e i v i n g both Vitamins was lower than that of others having Vitamin "A" only, but the Vitamin "G" content of the organs was not a f f e c t e d by the presence or absence of Vitamin "A", While studying the problem of hyperthyroidism, Schneider and Windmann (201) noted that when r a t s were f u l l y depleted of Vitamin "A", o r a l a d m i n i s t r a t i o n of carotene f o r ten days d i d not cause deposition of Vitamin "A" i n t h e i r l i v e r s j although a d m i n i s t r a t i o n of the Vitamin i t s e l f caused storage i n large amounts.  When the l i v e r s were depleted of glycogen  by i n j e c t i n g the animals with the t h y r o t r o p i c p r i n c i p l e of the p i t u i t a r y , f a i l u r e of deposition of Vitamin "A" a f t e r a d m i n i s t r a t i o n of carotene a l s o occured.  This f a i l u r e i s  a t t r i b u t e d t o an increased demand f o r Vitamin "A" rather than to i n a b i l i t y of the l i v e r t-o convert carotene i n t o the  39. Vitamin.  They suggest that the metabolism of glycogen i s c l o s e l y  bound up w i t h that of Vitamin  "A",  Wendt (254) obseryed that an excess of thyroxine increases the rate at which Vitamin "A" i s metabolised.  In  hypothyroidism the capacity to convert carotene to Vitamin "A" and to store carotene and Vitamin "A" i n the l i v e r are reduced. Further studies by Sherwood and Luchner (216) dealt with the h i s t o l o g i c a l e f f e c t of ood l i v e r o i l on the t h y r o i d gland.  They found that excessive d a i l y doses of carotene,  " h a l i v e r " o i l or cod l i v e r o i l , over a period of several weeks, r e s u l t e d i n a;depletion of the o o l l o i d and an increase i n the height of the e p i t h e l l o l c e l l s i n the t h y r o i d glands of young albino rats.  Carotene and " h a l i v e r " o i l produced i n a d d i t i o n  a marked increase i n stroma.  Administration of potassium  i o d i d e , i n amount approximately equivalent t o the iodine i n 0.3 ml. d a i l y of ood l i v e r o i l , had no e f f e c t up to the eighteenth day.  There was then an increase i n c o l l o i d and the  a c i n i were distended, with extremely low epitheluim.  They  concluded that the iodine of ood l i v e r o i l was l e s s a v a i l a b l e f o r metabolic a c t i v i t y than that of potassium iodide, possibly being l o s t by excretion of the o i l , and that such changes i n the gland as were produced by cod l i v e r o i l were due t o Vitamin "A", rather than to iodine* The work of A b e l i n ( l ) noted that the ingestion of a Vitamin "A" preparation by r a t s , diminished the r i s e i n basal  40. metabolism r e s u l t i n g from t h y r o i d administration.  The action  of excess of doses of t h y r o i d i n lowering the growth rate was a l s o p a r t i a l l y counteracted  by administration of Yitamin  "A",  which was shown to increase the glycogen content of muscles. He suggests that Thyroxine and Vitamin "A" are antagonistic because of t h e i r opposite influence on l i p o i d and carbohydrate metabolism. In s p i t e of the w e l l recognized importance of Vitamins i n n u t r i t i o n , p r a c t i c a l l y no work has been done on the a s s o c i a t i v e a c t i o n of the various Vitamins.  While i n the case  of p o u l t r y and other domestic animals the minimum and optimum amounts have been f a i r l y accurately determined, no information i s a v a i l a b l e as to the most s a t i s f a c t o r y combina t i o n of any two or more Vitamins to promote normal growth, h e a l t h and  reproduction.  BIOLOGICAL ASSAY OF YITAMIN A USING .THE RAT AS THE TEST ANIMAL. .. Feeding Technique. The technique used i n carrying out the Vitamin A assays i s described i n great d e t a i l by the United Pharmacopoeia (see appendix 1 ) .  States  These recommendations have  arisen as the r e s u l t of very c a r e f u l work conducted during the past decade by several i n v e s t i g a t o r s of whom Coward of Great B r i t a i n has been the leading i n v e s t i g a t o r . In 1920 Drummond and Coward (59) stated that i n t e s t ing foodstuffs f o r the presence of the fa,t-solub"le vitamin the greatest care- should be devoted to ensuring that the basal d i e t a r y i s rendered as free from that vitamin as p o s s i b l e . They give d e t a i l s f o r the preparation of a highly p u r i f i e d ration.  They conclude that the f a i l u r e to work with a suff-  i c i e n t l y pure d i e t may lead to c o n f l i c t i n g and misleading results. St eenbock,;: Nelson, and; Black.-(236) noted that the absence of growth on s u i t a b l y constituted r a t i o n s cannot be taken as an i n d i c a t i o n of the absence of Vitamin A unless the a n t i r a c h i t i c f a c t o r i s supplied. Steenbock and Coward (237) advocated the use of the incidence of ophthalmia as a, sign of exhaustion store of Vitamin A i n preference  of the animals'  to cessation of growth. The  two are often simultaneous, but the use of the former c r i t e r i o n prevents l o s s of animals through the very sudden and r a p i d dec l i n e that may ensue while waiting to become c e r t a i n that growth  42. has r e a l l y ceased.  Growth ceases during the worst stages of  ophthalmia and i s only resumed when d e f i n i t e improvement i n the animals' condition i s observable. In the b i o l o g i c a l assay f o r Vitamin A according to J a v i l l i e r and Emerique (126) i t i s necessary to observe certain precautions with regard to: an adequate supply of Vitamin D i n the d i e t of the experimental animals; 2. The period of the test proper which should begin only a f t e r there has been a loss i n weight of 10%; 3. The large number of animals (10, 20, 30) to be used i n the tests of each dose; 4. The s i m i l a r sex of a l l animals compared; 5. The manner of administration of the substance t e s t e d . The J a v i l l i e r unit of Vitamin A i s defined as "the q u a l i t y of the vitamin which, when added to the minimal dose of maintenance (per lOOg. of r a t ' s body weight under the s p e c i f i e d conditions) w i l l induce a growthresponse l a s t i n g at o least 30 days, the angle of growth being 30  and the gain i n  weight about 30%." Sherman and Batchelder (208) claim on s t a t i s t i c a l grounds that w i t h l e v e l s of Vitamin A feeding which induce a gain i n weight of about 3 grams a week, a decrease of about 25% or an increase of 33% i n the value of the d a i l y intake of Vitamin A can undoubtedly be measured by this method. According to Honeywell, Butcher and E l y (121) the response to administration of Vitamin A of r a t s depleted of t h i s f a c t o r i s influenced by the type of yeast used i n the diet.  This influence i s thought to be a function of the  Vitamin B complex.  The authors Suggest that Vitamin A'  consists of two f a c t o r s , one possessing anti-xerophthalmic p r o p e r t i e s , concerned i n maintaining the normality of the cornea and other e p i t h e l i a l t i s s u e s , and another with a purely growth-promoting f u n c t i o n . Nelson, Walker and Jones (167) noted that when single doses of m a t e r i a l containing Vitamin A were given to r a t s deprived of the f a c t o r , the growth response and subsequent duration of l i f e of the animal were d i r e c t l y p r o p o r t i o n a l to the amount of Yitamin A given. The i n v e s t i g a t o r s Polak and Stokvis (175) found that d a i l y doses of 0.5 to 1.0 gamma of carotene were s u f f i c i e n t to prevent and cure xerophthalmia i n r a t s on a diet d e f i c i e n t i n Yitamin A. Working along s i m i l a r l i n e s too(1.75) were Baumann, R i i s i n g and Steenbock (11).  A s o l u t i o n of carotene i n cotton-  seed o i l i n which no d e t e r i o r a t i o n could be detected spectr os c o p i c a l l y throughout the experiment, was administered f o r 8 weeks to r a t s on d i e t s d e f i c i e n t i n Yitamin A. A weekly dosage of 3 to 5 gamma of carotene d i d not cure or prevent xerophthalmia but 10 gamma d i d so and also promoted moderate growth; amounts l a r g e r than 20 gamma weekly d i d not enhance growth. They found i t impossible to maintain r a t s at a growth rate of 3 grams per week, and also emphasise the danger of using a standard of animal response rather than the i n t e r n a t i o n a l standard substance f o r reference. Coward and Key (42) found that the mean weight i n crease of groups of r a t s r e c e i v i n g 7 mg. of cod l i v e r o i l  44. weekly, after depletion of Vitamin A, was the same whether 1 mg. was administered d a i l y , or 4 mg. on Thursday and 3 mg. pn Monday. The high degree of s t a t i s t i c a l p r o b a b i l i t y that the r e s u l t i s v a l i d i s worked out. The determination of Vitamin A values by a method of s i n g l e feedings was reported by Sherman and Todhunter (209). V/hen male or female r a t s , depleted of Vitamin A received single large doses of 28, 56 or 112 gamma of carotene, the weight i n crease and s u r v i v a l time were p r o p o r t i o n a l to the dose f o r each sex.  The value best expressing t h i s r e l a t i o n s h i p was obtained  by p l o t t i n g weight increase and subsequent decline against the -time i n days and estimating the area, bounded on the upper side by t h i s curve and on the lower side by a base l i n e drawn paral l e l to the abscissae through the point representing the average weight loss of a group of untreated c o n t r o l s , at the end of the f i r s t week from beginning of dosage.  This method i s  s p e c i a l l y recommended f o r perishable or unstable materials.  45. Results of Vitamin A Deficiency i n Bats. In studying avitaminosis A of r a t s , McCollum, Simmonds and Becker  (144) found that the feeding of excessive amounts  of any one element or i o n did not induce ophthalmia. The h i s t o l o g i c a l changes are very w e l l described by Tyson and Smith (250) who  say "The c h a r a c t e r i s t i c h i s t o l o g i c a l  changes found i n r a t s fed a d i e t l a c k i n g Vitamin A are the s u b s t i t u t i o n of s t r a t i f i e d h e r a t i n i z i n g epithelium f o r normal epithelium i n various parts of the r e s p i r a t o r y t r a c t , alimentary t r a c t , eyes, paraocular glands and the genito-urinary tract i " Replacement of the normal epithelium of . the body organs w i t h k e r a t i n i z e d c e l l s was observed i n mice maintained on V i t amin A free diet by Wolfe and Salter (265).  Xerophthalmia dev-  eloped i n from 25 to 120 days although metaplastic changes i n the r e s p i r a t o r y t r a c t were found p r e v i o u s l y to t h i s . Sure and Smith (242) noted that Vitamin A deficiency i n r a t s d i d not a l t e r the true blood sugar, the a l k a l i n e reserve of the blood or the glycogen content of the l i v e r .  The reducing  non-sugars, however were frequently increased, thus r a i s i n g the apparent blood sugars. Pathologic changes i n the t i s s u e s during early stages of Vitamin A d e f i c i e n c y were i n v e s t i g a t e d by Thatcher and Sure(244) Their r e s u l t s showed metaplasia i n the p o s t e r i o r p o r t i o n of the tongue, i n the s a l i v a r y glands, and i n the r e s p i r a t o r y and urinary t r a c t s i n the majority of cases of early Vitamin A defi c i e n c y , a l t ho ugh i n many i n d i v i d u a l s there were as yet no ext e r n a l symptoms such as cessation of growth, persistence of the  c o r n i f i e d c e l l stage of the oestrous cycle or i n c i p i e n t ophthalmia. The e f f e c t of Vitamin A on metabolism was recorded by C h e v a l l i e r and Baert (33). Young r a t s with t y p i c a l signs of Vitamin A depletion showed a basal metabolic rate 13 per cent higher than that of s i m i l a r normal r a t s .  Rats r e c e i v i n g d a i l y  2G00 U.S.P. u n i t s of Vitamin A and a guineapig receiving d a i l y 10,000 such u n i t s , showed a r a t e lowered by 10 to 15 percent. According to Malmberg (152) increase i n weight and i n t a i l length siakens simultaneously i n young r a t s i n process of depletion of Vitamin A. Prom Tornblom's (245) experiments no s i g n i f i c a n t d i f f erence could be detected i n the oxygen consumption of r a t s developing w e l l maxked symptoms of deficiency, on a diet deprived of Vitamin A, and r a t s maintained i n h e a l t h on the same d i e t , by the d a i l y a d d i t i o n of 10 gamma of carotene. Hematopo&ietic function i n d e f i c i e n c y diseases was i n vestigated by Sure, et. a l . (241). Prom t h e i r work they concluded that a f t e r Vitamin A d e f i c i e n c y has progressed to t he ophthalmic stage i n a n i t i o n complicates the blood p i c t u r e , so that the high figures of hemoglobin and erythrocytes may be an expression of anhydremia, i n d i c a t e d by the concentration of t o t a l blood s o l i d s of the p a t h o l o g i c a l animals as compared with normal animals of the same age and weight.  In the pre-  ophthalmic stage they found a suggestion o f an anemia characterized by reduction i n e i t h e r hemoglobin or erythroytes. Ho  47. connection could toe established between aisritaminosis and perni c i o u s anemia. Batchelder (7) found that u r i n a r y c a l c u l i were present i n r a t s r e c e i v i n g small q u a n t i t i e s of Yitamin A hut were not present i n r a t s r e c e i v i n g no Yitamin A.  He also noted that the  general "breakdown r e s u l t i n g i n death, though accompanied "by simi l a r symptoms, was postponed to successively l a t e r stages as the Yitamin A content was increased. Sutton, S u t t e r f i e l d and Krauss (243) demonstrated that nerve degeneration occurred about the same time as  ophthalmia  and became extensive before external symptoms of p a r a l y s i s appear.  Working w i t h a p o l a r i z i n g microscope on formalinr.fixed  frozen p e r i p h e r a l t i s s u e s , they showed that after external symptoms of p a r a l y s i s appear, adequate amounts of Vitamin A do not r e l i e v e them but nerve degeneration i s arrested. The data published by Evans (80), demonstrated that i n adequate Vitamin A i n j u r e s the female reproductive system so that f e r t i l i z a t i o n and implantation often f a i l .  In t h i s respect  i t d i f f e r s r a d i c a l l y from the reproductive impairment due to low Vitamin E content, where, t y p i c a l l y the eggs are always healthy and implantation takes place but r e s o r p t i o n f o l l o w s . A study of the e f f e c t s on the male g e n i t a l organs was c a r r i e d out by Sampson and Korenchevsky (196).  They observed  that i n most r a t s on a Vitamin A d e f i c i e n t diet the weights of the testes and e s p e c i a l l y of the penis and of the prostate w i t h seminal v e s i c l e s were greater than those of r a t s kept on a  48. paired complete d i e t . were oedematous.  The testes of r a t s on the d e f i c i e n t diet  The decreased food intake produced no  not-  iceable e f f e c t on the penis, decreased the weight of the prostate with seminal v e s i c l e s and i n some cases s l i g h t l y the weight of the t e s t e s .  increased  49. A s s i m i l a t i o n of Vitamin A i n the Presence of_Mineral O i l . Rowntree (191) i n 1931 undertook a study of the effect of the use of mineral o i l upon the absorption of Vitamin A. V(hen' r a t s p r e v i o u s l y deprived of Vitamin A were given small doses of cod l i v e r o i l s u f f i c i e n t only to allow of slow growths the a d d i t i o n of mineral o i l to the diet i n amounts equivalent to the human therapeutic dose cause the anijjials to lose weight and d i e .  When, however, the dose of cod was an adequate one, t  the i n g e s t i o n of the mineral o i l d i d not appear to i n t e r f e r e with the absorption or u t i l i z a t i o n of the vitamin. The a s s i m i l a t i o n of carotene and Vitamin A i n the presence of mineral o i l was studied by Dutcher e t . a l . (71) i n 1934 Rats depleted of Vitamin A recovered when r e c e i v i n g 40 mg. of b u t t e r " d a i l y , but-not when they also received 100 mg. of mine r a l o i l , w i t h or without hydroquinone.  A comparable r e s u l t  was obtained w i t h carotene but the adverse e f f e c t was overcome i f the carotene was considerably increased r e l a t i v e to the mineral o i l . Mineral o i l exercised scarecly any unfavorable e f f e c t when administered w i t h cod l i v e r o i l or a Vitamin A concentrate.  When carotene was fed w i t h mineral o i l , i t could be  recovered from the faeces i n an am&unt p r o p o r t i o n a l to that fed, i n d i c a t i n g that the a c t i o n of the o i l was to i n h i b i t the absorpt i o n of carotene from the gut. The influence of Vitamin A on f a t metabdlism was f i r s t investigated by Drummond (58) i n 1919.  He found that rats were  able to absorb large amounts of f a t t y acids, and presumably synthesise these into f a t s , i n the absence of Vitamin A i n the  50-. diet. lines.  Several years l a t e r , Green (95) worked along s i m i l a r 1 From h i s work no evidence was obtained that a diet  • very r i c h i n f a t accelerates the u t i l i z a t i o n of Vitamin A i n the body.  The diminuation i n the t o t a l crude f a t of the whole  body i s not s i g n i f i c a n t l y , d i f f e r e n t i n . the Vitamin A d e f i c i e n t r a t from that i n the r a t on a. complete diet whose food intake is, r e s t r i c t e d to that of the d e f i c i e n t animal.  A r i s e i n the  iodine value of the f a t t y a c i d s of the l i v e r , coincident with a f a l l i n the percentage amount of f a t i n the l i v e r , occurs i n the end stages of Vitamin A d e f i c i e n c y .  The r i s e i s of a sim-  i l a r loss of l i v e r f a t has been produced by a r e s t r i c t i o n of food intake or by i n f e c t i o n . Working on the esterase content of the blood-serum Green (94) reported that Vitamin A d e f i c i e n c y i n the rat"produced a large and progressive decrease of t h i s enzyme. R e l a t i v e l y large amounts of cod l i v e r o i l i n the d i e t , however, produce a r i s e i n the serum esterase content w e l l above normal.  51. Influence of Vitamin'.A on Fat  Metabolism.  The t o x i c e f f e c t s due to large doses of Vitamin A was studied by D r i g a l s k i (54) i n 1935.  He noted that rats given  bread, oats and water together w i t h 20,000 or 40,000 r a t u n i t s of Vitamin A d a i l y i n the form of a concentrated commercial preparation died i n 5 to 19 days w i t h loss of weight and the development  of c o n j u n c t i v i t i s and hemorragic r h i n i t i s . Control  r a t s , given the same concentrates a f t e r the destruction of the Vitamin A by u l t r a v i o l e t i r r a d i a t i o n , remained i n normal health. Tn<the same year Bomagk and Dobeneck (52) made a detailed h i s t o l o g i c a l examination of r a t s of 80 gram weight, which had received 100,000 "units" of Vitamin A i n I c e . of sesame o i l d a i l y f o r a week. Kupffer's star c e l l s of the l i v e r , the pulp c e l l s of the spleen, e n d o t h e l i a l c e l l s i n the. kidney and epithe l i a l c e l l s of the stomach were f i l l e d w i t h f a t , as compared with those of c o n t r o l s , r e c e i v i n g sesame o i l only.  The c e l l s of the  g a s t r i c epithelium showed abnormally rapid multiplies, t i o n . A year l a t e r Davies and Moore (48) fed massive doses of a Vitamin A d i s t i l l a t e to young r a t s .  They observed that t h e i r  doses proved t o x i c , causing emaciation, haemorrhagic r h i n i t i s , l o s s of h a i r round the mouth and i n one r a t gross lung l e s i o n s . Their attempts to induce hypervitaminosis A by feeding carotene f a i l e d on account of i t s l i m i t e d s o l u b i l i t y i n f a t s . Lewis and R e t i (136) studied the effects, of large doses of carotene when fed to r a t s .  They noted that when young r a t s  were; given large amounts of a Vitamin A concentrate (vogan) they declined i n weight, and died w i t h i n 10-14 days, w i t h the  52  a  usual symptoms of hypervitaminosis A (emaciation, c o n j u n c t i v i t i s skin l e s i o n s on the face and paresis of the hind l e g s ) .  Yi/hen -  they fed 40 mg. of pure carotene dissolved i n an innocuous o i l there were no i l l  effects.  P r a c t i c a l l y no work has been reported on the t o x i c e f f e c t i f any, of Vitamin A i n domestic animals.  The majority of i n -  v e s t i g a t i o n s to date have been concerned w i t h determining the minimum and optimum requirements A.  of domestic animals f o r Vitamin  ;CHEMICAL AND PHYSICAL PROPERTIES Off VITAMIN A. The Chemical Structure of Vitamin A. A great deal of work has "been done on the Struct - and Chemical composition of Vitamin A and carotene* Smith (222) interpreted h i s observations that caroten contained 9 e a s i l y saturated double bonds i n a conjugated s e r i e s , f u r t h e r conjugated w i t h 2 other unsaturated linkage e i t h e r double bonds or cyclopropane linkages.  He suggested  that t h i s system was responsible for the colour of the carotene molecule. B r u i n s , Overhoff and Wolff (23) calculated the r a t i o of the molecular weights of carotene and Vitamin A and from t h i s they deduced a molecular weight of about 330 for Vitam: A.  Erom t h i s value they concluded that the assumption of a  simple chemical r e l a t i o n between Vitamin A and carotene to appear improbable. In 1931 K a r r e r , Morf and Schopp(129) suggested the f o l l o w i n g formula f o r Vitamin  A,(C20H30O)»  which i s now the  " p r o v i s i o n a l formula."  - CH- OH- Cr CH-. CH= CH-Ci CH- CHgOH :-CH*  A year l a t e r , H e i l l b r o n , Morton and Webster ( 1 1 0 )  5 4 . '•  suggested the previous formula, and also the f o l l o w i CH H H C  GH  Hq  G-CH  2  2  N  C  C  Hg  3 93 H  •^3^'  C'H=dH-C=CH-CHQH 2  The next year Karrer, Morf and Schopp (130) synthesised perhydro-vitamin  A, ( C H  0) w i t h the f o l l o w i n g  ?Q  structure: H  H,  /  R  \  C ( G H  <? HCH  CH-CH  ^  3  )  - C H  CH  2  2  - C H - C H  - C H  (L<, P  P  - C H  - C H - G H  d Q  » C H  2 0  2 o  0 H  The i d e n t i t y of the substance thus synthesised with that prepared by the reduction of the natural Vitamin A was establ i s h e d by comparing the b o i l i n g p o i n t , density and molecular refraction. According to Drummond (67) the formulae f o r a l f a and beta carotene are as f o l l o w s :  55.  ©j .o  CM  W  c\j  O — O  O.  Oi  o  ,0  - o  0 = t  w  to  0 11  o  to w o  w  1  W  to  0  0 8  — -O H  K  CD  >  0 1  W  O K  w  •H  tio  0 -*->  <  CD fH CD  •rt  0  o CD  9  O II 1  +> O  w w  0  K  n3  +» CD pq  «•  O  o -  11  o  —  o  X  © c  CD O  M  i - o .  (—4  o3 O  O a 0  to  CQ . -M •H rH  0 SI 0 1  W o  4-»  •O I  K  '3  Cd  0  CD  a  r4  CES  jp CD  w  •H i> 0  CO 0) rH 3 O CD rH O g  56. He also summarized and compared the properties of Vitamin A and carotene. *  Carotene ,  Vitamin A.  s  Synthesised i n plant  Stored i n animal  Orange red  Almost colourless  Antimony t r i c h l o r i d e gives s t e e l blue colour, o hand at 5900 A.  o  Band i n U.V. at 3280 A Royal blue colour with antimony t r i c h l o r i d e , o band at 5720 and 6060 A.  57 The E f f e c t of Ghemioal and P h y s i c a l Agents on Yitamin A Observations upon the chemical nature and properties of Yitamin A were reported by Drummond (57) i n 1919.  He noted  that the f a t soluble accessory food f a c t o r A was r e a d i l y destroyed by short exposures (one hour to a'tempature of 100°), Destruction was l e s s r a p i d at temperatures of 50° to 100°  He  #  believed at t h i s time that the destruction was not due to oxidation or h y d r o l y s i s .  The f a c t o r was not extracted from  o i l s by water or d i l u t e a c i d but was soluble i n a l c o h o l .  The  f a t soluble A faotor could not be i d e n t i f i e d with any of the recognized components of f a t s , such as g l y c e r o l , saturated or unsaturated  f a t t y a c i d s , c h o l e s t e r o l , l e c i t h i n , phosphatides  or the lipochromes.  In conclusion he suggested that i n view  of the low temperature at which destruction occurs, f a t soluble A may  be a l i a b l e substance of i l l - d e f i n e d c o n s t i t u t i o n .  Working with whale o i l s Delf (50) found that the temperature used f o r e x t r a c t i n g the o i l played an important part i n the potency of the o i l .  In h i s work he also found that the  sperm whale gave from i t s head an o i l r i c h e r i n Yitamin A than from the  blubber. Emmett and Luros (77) showed d e f i n i t e l y that benzine  or acetone d i d not extract from the pancreas, thymus and suprarenal glands a f a t that contained the f a t soluble A Yitamin.  58 Brummond, Channon and Coward (64) prepared a concentrate of Yitamin A from Cod L i v e r O i l and examined i t a chemical properties.  They found i t contained no detectable traces of  iodine or nitrogen, so that these elements are not r e l a t e d to the p h y s i o l o g i c a l a c t i o n of the o i l i n promoting growth.  Their  r e s u l t s showed that approximately 50% of the unsaponifiable matter from Cod L i v e r O i l i s c h o l e s t e r o l , which may be removed q u a n t i t a t i v e l y without l o s s of vitamin a c t i v i t y .  Yitamin A  was found on d i s t i l l a t i o n to pass over a t 180-220° at 2-3 m.m. A chemical examination of the active d i s t i l l a t e indicated the presence of (a) a saturated  s o l i d a l c o h o l , (b) the unsaturated  hydrocarbon spinaeene, (c) one or more than one unsaturated a l c o h o l , b o i l i n g about 200° a t 2-3 m.m.  Spinaeene and the  s o l i d alcohol were without Yitamin A a c t i o n . Cody and Luck (25) studied the e f f e c t of d i f f e r e n t gases upon Yitamin A.  They discovered that SOg r a p i d l y  destroyed the a c t i v e p r i n c i p l e of Cod L i v e r O i l . A l f a l f a and spinach, when sulphured i n the dry and green conditions, experienced no l o s s of Vitamin A a c t i v i t y .  Phosphorous penta-  c h l o r i d e , c h l o r i n e , a c e t y l c h l o r i d e , n i t r o u s fumes and Benedicts' a l k a l i n e copper reagent destroyed the active p r i n c i p l e i n Cod L i v e r O i l . Prolonged treatment with sodium b i s u l p h i t e had the same e f f e c t .  Hydrogen sulphide, ethylene, ammonia  and Benedicts' reagent a f t e r n e u t r a l i z a t i o n exhibited no destructive a c t i o n .  Formaldehyde had l i t t l e e f f e c t .  59 Hydrogen peroxide brought about a p a r t i a l l o s s .  They concluded  that Vitamin A a c t i v i t y i s the property of a s p e c i f i c atomic grouping rather than of a s p e c i f i c molecule, and that the active p r i n c i p l e of Cod L i v e r O i l possesses aldehyde properties, The destruction of Vitamin A by u l t r a - v i o l e t radiations was studied by N o r r i s (168),  He found that the curve of  destruction of Vitamin A i n Cod L i v e r O i l by u l t r a - v i o l e t i r r a d i a t i o n , determined by means of b i o l o g i c a l experiments, d i f f e r e d i n several p a r t i c u l a r s from that obtained by use of the c o l o r t e s t .  The f i r s t showed no disappearance of the  Vitamin up to one hour's exposure, t h e r e a f t e r there was a r a p i d destruction, only 1.5$ of the o r i g i n a l content remaining a f t e r four hours i r r a d i a t i o n .  The color t e s t , on the other  hand, i n d i c a t e d that destruction started at once and proceeded slowly, the curve f o l l o w i n g that of a bimolecular  reaction.  Forty-four percent of the o r i g i n a l ohromogenio value s t i l l remained a f t e r sixteen hours and t h i r t y percent a f t e r t h i r t y two hours i r r a d i a t i o n s . This suggested that Vitamin A and the ohromogenio substance were separate e n t i t i e s . Marcus' (153) found the storage of the ether-soluble p o r t i o n of the unsaponifiable  f r a c t i o n of Cod L i v e r O i l with  f i n e l y divided s o l i d s , such as lactose granulate,  ferric  sulphate etc., i n the presence of e i t h e r a i r or carbon dioxide caused a progressive i n f i f t e e n days.  destruction of Vitamin A, often complete  Destruction also occured when the concentrate  60 was stored w i t h "nuchar" charcoal from which a l l traces of a i r had "been removed, showing that i n t h i s instance oxidation was not the d e s t r u c t i v e agent*  The presence of hydroquinone or  water as ten percent of the t o t a l m a t e r i a l delayed, but d i d not prevent, the d e s t r u c t i o n of the Yitamin. From the work of Monaghan and Schmitt (158), carotene, the precussor of Yitamin A i n the animal body, g r e a t l y i n h i b i t s the oxygen uptake of l i n o l e i c a c i d .  Oxidized carotene,  on the other hand, s l i g h t l y accelerates the oxygen uptake of this acid.  Yitamin A i n small concentration may completely  i n h i b i t the oxygen uptake of l i n o l e i c a c i d f o r some hours. This i n h i b i t i o n wears o f f , as i n the oase of carotene, when the vitamin i s destroyed by oxidation.  They suggest the  p o s s i b i l i t y that Yitamin A may be concerned with phospholipid metabolism. Measurements made i n a Warburg apparatus by Euler and Ahlstrom (79) showed that the rate of oxygen uptake of a s e r i e s of f i s h - l i v e r o i l s and Yitamin A concentrates increased with increasing "blue value" and growth-promoting power. L i v e r sections from r a t s showed a higher rate of oxygen uptake i n blood than i n Ringer's s o l u t i o n .  They suggest that Yitamin A  plays an important part i n oxidation processes i n the body. Davies (47) i n h i s work w i t h the absorption spectrum observed that at room temperature, Yitamin A deteriorated l e s s r a p i d l y i n l i v e r specimens treated w i t h potash than i n untreated  61  tissues kept without preservative treatment.  In the case of  specimens of l i v e r t r a n s f e r r e d immediately post mortem to potash s o l u t i o n and then stored at room temperature, no serious decrease i n Vitamin A content was.to be anticipated i f the assay was c a r r i e d out w i t h i n fourteen days a f t e r death. The production of high grade feeding o i l from pilohards and s i m i l a r f i s h was investigated by Brocklesby and B a i l e y ( 2 1 ) . A dry, acid-free p i l c h a r d o i l stored i n a cool dark place and having a minimum access to a i r w i l l remain unoxidized f o r a very long period.  Samples kept i n t i g h t l y  stoppered g l a s s and t i n containers and stored i n a dark cupboard f o r f i v e years showed but a trace of oxidation. with  0.001$  An o i l  maleio a c i d dissolved i n i t w i l l r e s i s t oxidatine  r a n c i d i t y twice as long as an untreated o i l . G-utteridge ( 1 0 0 ) at the C e n t r a l Experimental Farm, Ottawa, showed from an examination of the free f a t t y a c i d and nitrogen content of several v a r i e t i e s of Cod L i v e r O i l , that, i n general, o i l s with a high f a t t y a c i d content also had a high nitrogen content.  Such o i l s abnormally high i n these  constituents were e i t h e r manufaoture.d from s t a l e l i v e r s or improperly processed.  One sample of Cod L i v e r O i l was  deaminised and compared w i t h the untreated o i l by feeding experiments.  The deaminised o i l was superior to the untreated  o i l i n that i t had the e f f e c t of equalising the rate of growth and lowering the m o r t a l i t y i n growing chicks, while  62 also rendering more e f f i o i e n t u t i l i s a t i o n of food f o r egg production. E e t i (184) proved the i n v a r i a b l e existence of Yitamin A i n e s t e r i f l e d form i n the l i v e r . Aocording to M i l l e r (156) the Yitamin A potency of Cod L i v e r O i l i n a feed mixture was preserved by mixing the o i l w i t h cottonseed meal "before incorporating i t i n the feed. The chemical and p h y s i c a l constants of God L i v e r O i l was extensively studied by Lindholm (138),  F i f t e e n samples  of Cod L i v e r O i l showed remarkable constancy i n c o l o r , v i s c o s i t y , a c i d i t y , s a p o n i f i c a t i o n value, iodine value, unsaponifiable matter, sulphuric a c i d test of the U, S, Pharmacopoeia, Carr and P r i c e t e s t , i n a few samples, i n the Yitamin A value, generally as determined spectrographically but i n a few samples b i o l o g i c a l l y .  The Yitamin A content  was e a s i l y diminished by o x i d a t i o n without a f f e c t i n g any of the other constants, but i f any of these became seriously altered,; the Yitamin A was also found to be completely destroyed.  63 Oolorimetrlo  Reaction  Among the f i r s t to use the c o l o r l m e t r i c method of assaying the Vitamin A content of God L i v e r O i l were Rosenheim and Drummond (186). one drop of o i l .  They used arsenic t r i c h l o r i d e , 1 o.c. to  They found that the o i l dissolved r a p i d l y  and gave a blue s o l u t i o n which i n a few seconds changed to purple and gradually faded.  Complete agreement was found to  occur between the c o l o r i n t e n s i t y and growth-promoting a c t i v i t y as tested b i o l o g i c a l l y . Drummond, Coward and Hardy (65) demonstrated the s e n s i t i v i t y of c o l o r reactions with t r i c h l o r o a c e t i c a c i d or dimethyl sulphate, and found that they seemed to be of about the same order as the animal feeding t e s t .  They stated, how-  ever that the r e a c t i o n with arsenic t r i c h l o r i d e was decidely more d e l i c a t e . Fearon (81) found that phosphorus pentoxide formed a deep v i o l e t c o l o r on a d d i t i o n to o i l s containing Vitamin A, Using a 12% s o l u t i o n of t r i c h l o r o a c e t i c a c i d i n dry l i g h t petroleum as a condensing agent, p y r o g a l l o l and other polyphenols intereacted with o i l s containing Vitamin A to give stable pigments which were s u i t a b l e f o r colorimetry. Continuing t h i s work were W i l l i m o t t , Moore and Wokes (256).  They noted that concentrated  sulphuric acid and  phosphorous pentoxide were l e s s s e n s i t i v e tests for Vitamin A than were arsenic t r i c h l o r i d e or antimony t r i c h l o r i d e .  They  64 concluded, "In view of the t r a n s i e n t nature of the colors obtained with both these reagents, i t i s suggested that readings be taken not more than t h i r t y seconds a f t e r mixing. Using the antimony t r i c h l o r i d e t e s t Wilson (261) observed that the f a t t y extract from the human l i v e r gave the same color r e a c t i o n as Vitamin A found i n Cod L i v e r O i l , In a study of e f f e c t of heat and o x i d a t i o n on Cod L i v e r O i l , Wokes and W i l l i m o t t (263) used four color tests--coneent r a t e d sulphuric a c i d and phosphorus pentoxide being q u a l i t a t ive only and arsenic and antimony t r i c h l o r i d e s being quantitative as w e l l as q u a l i t a t i v e .  They obtained r e s u l t s i n agreement  w i t h those of workers using animals, Drummond and Morton (66) measured the color reactions with a Lovibond tintometer and found t h e i r r e s u l t s were i n agreement w i t h those obtained by Rosenheim and Drummond (186) and a l s o those obtained by the absorption bands.  They found  t h a t c o l o r i m e t r i c a n a l y s i s was i n agreement with the spectroscopic methods and recommended e i t h e r f o r g i v i n g a r a p i d , r e l i a b l e q u a n t i t a t i v e measurement of the Vitamin A content of God L i v e r O i l s . Wokes (264) conducted a spectroscopic study of the colors produced by the"Vitamin Reagents" (arsenic and antimony t r i c h l o r i d e s ) on a s e r i e s of Cod L i v e r O i l s and concentrates whose Vitamin A content had been ascertained by feeding t e s t s . In each case he found two absorption bands which appeared to  65 be c h a r a c t e r i s t i c of the chromogen.  Arsenic t r i c h l o r i d e gave  bands at about 587 and 475 J j and antimony t r i c h l o r i d e gave 1  a  hands at about 614 and 530/  He observed that the chromogen  1  on standing i n contact with e i t h e r reagent gradually passed from the stage g i v i n g the i n i t i a l band (at about 587 or 615?" to the stage g i v i n g the second band (at about 475 or 530 z  1  J) 1  J ). 1  This change was accompanied by a gradual l o s s i n blue color and gain i n red c o l o r , which can be measured by means of the t i n t ometer."-? He also stressed the time e f f e c t i n reading the r e s u l t s as (256) had done previously. According to Smith and Hazley (221) the unsaponifiable f r a c t i o n of God L i v e r O i l gives with antimony t r i c h l o r i d e i n chloroform a blue c o l o r p r o p o r t i o n a l to i t s concentration. The l i n e representing the d i l l u t i o n e f f e c t f o r the t o t a l unsaponifiable f r a c t i o n i s t a n g e n t i a l at the o r i g i n to the d i l u t i o n curve f o r the corresponding God L i v e r O i l .  They  describe a method f o r oarrying out the color t e s t on the unsaponifiable f r a c t i o n extracted with chloroform, Emmerie, Eekelen, and Wolff (76) found that by t r e a t i n g a Yitamin A preparation from Cod L i v e r O i l with drops of furan, methylfuran, p y r r o l , i n d o l or s k a t o l p r i o r to use of the antimony t r i c h l o r i d e reagent, a purple color i n place of the usual blue was given.  The 610'  J  absorption band was found  to be suppressed, but the 572// band was unaltered. u  They  conclude that the v a r i a t i o n i n d i f f e r e n t specimens of l i v e r  66 o i l may  perhaps be due to v a r i a t i o n s i n t h e i r content of indol-  l i k e substances, Gillam and Morton (87) observed from t h e i r work that l i v e r o i l s contained two chromogens which with antimony t r i chloride gave colored substances with absorption maxima at 606  u  f and 57 2f F r e s p e c t i v e l y .  In concentrates these maxima  were displaced to 620v F and 583/ F u  U  ,  A comparison of u l t r a -  v i o l e t absorption spectra w i t h a spectroscopic data on the color t e s t disclosed, " ( l ) that the p a r a l l e l i s m between the i n t e n s i t y of the 6G6)  1  J band and the i n t e n s i t y of the 328, u  u  u  band breaks down so s e r i o u s l y i n extreme cases or to render i t improbable that the 606 u u the 572; 328 f-  J  1  chromogen i s Vitamin A; (11) that  / chromogen and the substance responsible f o r the  P- band are probably i d e n t i c a l ; ( i l l ) that the blue  color f o r r i c h o i l and concentrates i s often much deeper than would be expected on the basis of c o r r e l a t i o n between blue color and u l t r a - v i o l e t absorption,  "Hence they concluded that  the matching of blue c o l o r s w i t h Lovibond glasses, though i t may act as a rough guide to Vitamin A potency was  theoretically  unsound. The i d e a l conditions f o r accurate c o l o r i m e t r i c determinations are stated and reviewed by Heilbron, Gillam and Morton (109).  They observed that i n a considerable number of  o i l s characterised i n the c o l o r t e s t by predominance of the 572f J band over the 606 f 1  F band, a large increase i n the  67 i n t e n s i t y of the l a t t e r band could be obtained by t r e a t i n g the o i l beforehand w i t h ozonised oxygen, hydrogen peroxide or benzoyl peroxide.  O i l s which i n i t i a l l y showed an excess of  the 572 / J - chromogen over the 606 u  3  U  7  J - chromogen underwent a 3  slow spontaneous ageing which r e s u l t e d i n a marked increase i n the i n t e n s i t y of the 606 JP-. J - band. 3  Increases i n the 606 ,u u  absorption were not a t the expense of the 572 J - J chromogen 3  1  and were not accompanied by s i m i l a r increases i n the l a t t e r , and the absorption at 328 m / remained p r a c t i c a l l y u  constant  throughout. Rosenthal and E r d e l y i (187) (188) found that a modifi c a t i o n of the antimony t r i c h l o r i d e c o l o r r e a c t i o n f o r Yitamin A by the a d d i t i o n of pyrocatechol d i s t i n g u i s h e d between Yitamin A and the known carotenoids.  The v i o l e t - r e d color  resembling that of a d i l u t e s o l u t i o n of potassium permanganate was more stable than the blue of the Carr-Price r e a c t i o n . A short time l a t e r these same two Investigators (189) noted that a bfo guaiaool s o l u t i o n produced with Yitamin A the same r e d v i o l e t c o l o r as &fo catechol.  The s t a b i l i t y of the  c o l o r rendered the guaiaool t e s t s u i t a b l e f o r quantitative purposes. Continuing t h i s work Rosenthol and Weltner (190) found that the p u r p l i s h red c o l o r of the antimony t r i o h l o r i d e catechol t e s t f o r Yitamin A, when examined speotroscopically  68 w i t h i n the f i r s t ten minutes, showed maxima at 545 and 475 /  u  on longer standing two other maxima appeared.  /  U  When catechol  was replaced by guaiacol the purple c o l o r remained unchanged f o r hours, with maxima at 545 and 478 f- / , U  Anderson and Levine (5 a) observed that on heating the r e a c t i o n mixture to 60° C. the blue color given by Vitamin A with the antimony t r i c h l o r i d e reagent was changed to red, whereas the c o l o r given by carotene remained blue.  They con-  cluded that the use of pyrocutechol, as recommended by (187), was not necessary to b r i n g about the change of c o l o r i n the case of Vitamin A* According to Przeydziecka (177) i f the mixture i s heated i n the presence of guaiacol f o r two minutes on a water bath at 60° 0, a rose or red color i s produced which i s stable and may be compared w i t h a standard s o l u t i o n of suelan three. Holmes and Bromund (119) found that the orthodox method of matching against potassium diohrornate was s a t i s f a c t o r y f o r estimating carotene when d i s s o l v e d i n petroleumether.  Solvents  of higher r e f r a c t i v e index, such as benzene and chloroform, however, caused a change i n the c o l o r towards the red end of the spectrum, and accurate matching against a dichrornate s o l u t i o n was impossible.  Accurate r e s u l t s could be obtained by  using s o l u t i o n s of o r y s t a l l i n e bixen as a standard.  69' Absorption Spectrum The absorption spectra of Yitamin A was observed  first  by Schultz and Morse (199) and Schultz and Z e i g l e r (200). Shortly a f t e r these i n v e s t i g a t o r s were Heibron, Kamm and Morton (108). In 1928 Morton and Heilbron (165) claimed that Vitamin A was characterised by an absorption band w i t h a maximum at 328  u  u  .  They also suggested that one of the  decomposition  products of Vitamin A had an absorption band near 275 to 285/  u  Morton, Heilbron and Thomson (166). found that a crude God L i v e r O i l of high potency gave a d d i t i o n a l s e l e c t i v e absorption between 565 and 585 J  1  J i n the blue s o l u t i o n . 1  Dann (45) showed that Vitamin A was more rapidly o destroyed than carotene by r a d i a t i o n of wave length 2650 A. He d e f i n i t e l y denied the p o s s i b i l i t y that Vitamin A was the end product of the photochemical r e a c t i o n of carotene with this radiation, C h e v a l l i e r and Ghabre (32) applied a spectrophotometrie method, which permitted a very accurate measurement of the u l t r a - v i o l e t absorption given by a substance to the measurement of i n t e n s i t y of absorption at 3280 1 of d i f f e r e n t samples of Cod L i v e r O i l .  The examination of a great number of o i l s of  d i f f e r e n t o r i g i n s showed, t h a t , besides the presence of Vitamin A, the free a c i d i t y of the o i l and i n c e r t a i n cases i t s pigment ( i f very concentrated) must be taken into account  70 i n consideration of the absorption i n the neighborhood of o 3280 A.  They concluded that when the free a c i d content of the  o i l was low, the r e s u l t s of the p h y s i c a l and b i o l o g i c a l t e s t s agreed f a i r l y w e l l , the differences not exceeding the usual experimental errors inherent i n b i o l o g i c a l methods. B i l l s (17) demonstrated that the spectrograph gave two adjaoent spectrograms, and an o p t i c a l wedge i n front of the c o l l i m a t o r provides means of r e v e a l i n g d i r e c t l y the i n t e n s i t y of the banded absorption due to Vitamin A, Macwalter (150) found that the aeration of God L i v e r O i l f o r a short time reduced i t s absorption at 328 / y but u  u  aeration f o r a long time increased i t s absorption g r e a t l y . He devised a method which uses a curve r e l a t i n g absorption at 328  u  u  to duration of a e r a t i o n , whereby an accurate measure  of the absorption due to Vitamin A may be obtained. When t h i s u u method was impracticable, the absorption of 328 7 / of the unsaponifiable f r a c t i o n of an o i l was a t r u e r measure of Vitamin A than the absorption of the o i l i t s e l f . In Hotevarp's (170) experiments the H i l y e r Vitameter A was equipped w i t h a simple photographic possible to determine E. 328 J  1  device which made i t  J w i t h an accuracy approaching 1  that a t t a i n e d by conventional spectrophotometry methods. The values thus obtained f o r pure and crude God L i v e r O i l s , h a l i b u t l i v e r o i l s , concentrates of herring o i l , etc., agreed  71 w e l l with the blue values obtained by the antimony t r i c h l o r i d e method, when the l a t t e r were c a l c u l a t e d according to a special formula.  With Cod L i v e r O i l s the absorption of the unsapon-  i f i a b l e matter was 85 to 90$ of that of the o r i g i n a l o i l , the reduction being a t t r i b u t e d c h i e f l y to losses i n the preparation of the concentrate.  He a l s o noted that the value f o r E 1 cm,  328  U  u w a s  reduced from 0,52 to 0.027 i n a God L i v e r O i l  exposed f o r s i x months on a roof i n a white glass b o t t l e . Shrum and How (210) found that the r e s u l t s of b i o l o g i o a l estimations of Yitamin A i n a Cod L i v e r O i l and i n a concentrate, agreed w e l l with the value found by m u l t i p l y i n g the e x t i n c t i o n c o e f f i c i e n t s (S  1  / G m  at 328/ J ) by 1600. In  two samples of p i l c h a r d o i l , however, the p h y s i c a l method gave values more than three times the b i o l o g i c a l values f o r the o i l s and 2,5 times those f o r the unsaponifiable f r a c t i o n s . Removal of the pigment by absorption on diatomaceous l a r c h reduced the absorption only s l i g h t l y .  To measure the absorption  due to substances other than Vitamin A, the l a t t e r was destroyed.by  o x i d a t i o n , and the absorption of the treated  m a t e r i a l measured.  A f t e r s u b t r a c t i o n of t h i s value from the  t o t a l f o r the untreated m a t e r i a l , the remainder, due presumably to Vitamin A, y i e l d e d a value only 30% greater than the b i o l o g i c a l value.  They b e l i e v e d that the high values obtained f o r  p i l c h a r d o i l were caused by other substances absorbing i n the  region of 328 y  >.  u  u  They concluded that the spectroscopic  method can he a p p l i e d t o p i l c h a r d o i l only when the Vitamin A i s removed completely from the o i l without otherwise modifying or a f f e c t i n g i t . Sample  Vitamin A Content Biological  Physical  12,500  11 7 0 0  Standard S o l u t i o n Cod Liver O i l P i l c h a r d O i l No. 1 P i l c h a r d O i l No. 2  '650 120 175  '  Q 380 590 6 2  The f i n d i n g s of Shrum and How. According to McFarlane and Rudolph ( 1 4 8 ) , at the University of A l b e r t a , there i s need f o r further i n v e s t i g a t i o n of p i l c h a r d o i l t o b r i n g i n t o closer agreement the r e s u l t s of the b i o l o g i c a l assay and p h y s i c a l measurements of t h i s o i l f o r Vitamin A. Milne ( 1 5 6 ) found that spectrophotometrie measurements of the Vitamin A content of p i l c h a r d o i l d i d not equal the b i o l o g i c a l value of the o i l .  I t was also found that the  carotenoid pigment of t h i s o i l was too small t o account f o r the high b i o l o g i c a l value.  73 Solvents f o r Carotene Dann (43) reported that Yitamin A was r a p i d l y oxidized by a i r when dissolved i n some solvents, and slowly or not at a l l i n others.  He found i t to be p a r t i c u l a r l y stable i n ethyl  a l c o h o l , a l c o h o l i c potash and e t h y l acetate. i t was stable toward hydrogen peroxide.  In e t h y l alcohol  The solvent (or'  impurities associated with the vitamin or solvent) appears to play a leading part i n determining the s t a b i l i t y of Vitamin A. McDonald (146) found that carotene dissolved i n e t h y l butyrate, l a u r a t e , or palmitate, or i n peanut o i l , and stored with access of a i r , was destroyed to the extent of 80% i n four weeks, even a t 5°C., while with the same conditions, only 8% was destroyed i n Cod L i v e r , Wesson or Maize o i l .  Higher  temperatures accelerated, and storage i n vacuo retarded destruction. Baumann and Steenbock (8) examined the s t a b i l i t y of carotene i n a number of solvents, quantitative estimation being made speotrophotometrically by observing the i n t e n s i t y of absorption at 485 and 460 m J . 1  Refined cottonseed o i l (Wesson  o i l brand) was most s a t i s f a c t o r y ; cocoanut o i l , among others, was l e s s good. studied.  The e f f e c t of various conditions of storage was  I t was c a l c u l a t e d that when the solutions were kept  i n the conditions exacted by t h e i r use i n a feeding t e s t , 95% of carotene was l o s t a f t e r a month when dissolved i n o l i v e or  74 coooanut o i l , "but only 26$ when cottonseed o i l was used.  The  a d d i t i o n of hydroquione increased the s t a b i l i t y of carotene i n c e r t a i n solvents ( e t h y l laurate, ethyl sebosate) but not i n others. An elaborate study of the-;,-stability of carotene i n e t h y l esters of f a t t y acids and i n l i v e r and vegetable o i l s was conducted by McDonald (147).  A 0.05$ s o l u t i o n of mixed a l f a  and beta carotene i n e t h y l butyrate, l a u r a t e , and palmitate, i n peanut o i l , ood l i v e r o i l , maize o i l and Wesson o i l was stored i n p a r t l y f i l l e d stoppered b o t t l e s at 27°, 24°, and 5° C and i n evacuated sealed tubes at 37°C. and the carotene content observed spectrographically (462 mj l i n e ) from time to time. 1  The oarotene was conserved best i n the Wesson and maize o i l s and worst i n the e s t e r s . Dyer and Key (73) found the b i o l o g i c a l value of the carotene, used as i n t e r n a t i o n a l standard f o r Yitamin A, was greater i n s o l u t i o n of oocoanut or arachis o i l than i n hardened cottonseed o i l or e t h y l l a u r a t e .  Three samples of cocoa-  nut o i l , with and without the a d d i t i o n of q u i n o l , gave approximately the same r e s u l t .  God l i v e r o i l gave a higher  b i o l o g i c a l value f o r Yitamin A, when d i l u t e d w i t h coooanut o i l , than with hardened cottonseed or o l i v e o i l .  "Vitamin A and Antioxidants Jones and C h r i s t i a n s e n (127) found that hydroquione i n a concentration of 0.03% retarded the absorption of oxygen and the d e t e r i o r a t i o n of the "blue value" of two samples of r e f i n e d halibut l i v e r o i l .  I t a l s o retarded without wholly preventing  the d e t e r i o r a t i o n of the Yitamin A value of one sample as estimated b i o l o g i c a l l y .  Maleic a c i d d i d not act as an a n t i -  oxidant i n h a l i b u t l i v e r o i l . L e c i t h i n and hydroquinone as antioxidants f o r Yitamin A was studied by Holmes, Corbet and H a r t z l e r (120).  The e f f e c t  of l e c i t h i n and of hydroquinone i n delaying the oxidation of Yitamin A, as measured by the S b C l  g  reaction, i n halibut l i v e r  o i l and cod l i v e r o i l was studied both at room temperature and i n an atmosphere of a i r , and w i t h the a i d of s p e c i a l l y apparatus, a t 96° C., i n an atmosphere of oxygen.  designed  The a n t i -  oxidant a c t i o n of both substances was confirmed under a l l conditions and at a l l concentrations.  The, a c t i o n of l e c i t h i n  i n supplementing the a c t i o n of hydroquinone was much greater than would have been expected from i t s a c t i o n alone, and the best r e s u l t s were obtained with a combination  of both  substances.  In some instances, and notably with hydroquinone i n cod l i v e r o i l , there appears to be an optimum concentration f o r the a n t i oxidant, and l e s s s a t i s f a c t o r y r e s u l t s are obtained i f t h i s concentration i s exceeded.  At 96° C, the presence of water  s l i g h t l y retarded the rate of oxidation.  Concentration of Vitamin A W i l l i m i t and Wokes (258), working w i t h spinach, prepared an e x t r a c t that was two hundred times as concentrated as f r e s h leaves.  This e x t r a c t , they found, was potent source of  Vitamin A, i n that 25 mg. d a i l y gave complete freedom from xerophthalmia and induced s a t i s f a c t o r y growth. Moore (163) prepared concentrates from the l i v e r o i l s of r a t s and pigs which had received d i e t s containing large amounts of carotene as red palm o i l and compared them with concentrates derived from turbot and s o l - l i v e r o i l s .  Although the  i n i t i a l "blue values of the o i l s v a r i e d widely, l i t t l e difference could he detected i n the a c t i v i t i e s of the f i n a l concentrates, which approached an average value of 2400 B.TJ. per mg. (Pharmacopoeia color value 45,000) i n the Shcl, t e s t , corresponding to a minimal dose of ahout 0.001 mg. i n r a t growth experiments.  He b e l i e v e d that the concentrates must have been  consisted s u b s t a n t i a l l y of actual Vitamin A, or a l t e r n a t i v e l y that the vitamin must have been associated with other substances of s i m i l a r s o l u b i l i t y p r o p e r t i e s i n remarkably constant proportion. The preparation of a potent Vitamin A concentrate was described i n 1935 by Holmes et a l . (118).  They prepared  concentrates of halibut l i v e r o i l having blue values up to 140,000, and claimed them to be 40$ more potent than any other previously prepared samples.  77 Karrer et a l (129) suggest the f o l l o w i n g process f o r concentrating Vitamin A, HALIBUT LIVER OIL saponify UN" SAPON IE I ABLE RESIDUE \ ' cool i n MeOH to -15° to -60° (to remove s t e r o l s ) SOLUTION \ F r a c t i o n a l l y adsorb on F u l l e r ' s larth. VISCOUS OIL CONTAINING OVER 50$ OF VITAMIN A  •'"V • Esterify VITAMIN A ACETATE (Crude) \ saponify and d i s t i l i n vacuo. NEARLY PURE VITAMIN A (•10,00.0 times as active as o r i g i n a l material) The Vitamin A concentrate obtained from the above process represents about one part i n two thousand of the o r i g i n a l o i l . The dose needed to cure a r a t i s as l i t t l e as l/lOOO of a m i l l igram (  s  1/30,000,000 of an ounce).  78 The Measurement of Yitamin A ( I n t e r n a t i o n a l Units) The need f o r a unit measurement of Yitamin A has been recognized by i n v e s t i g a t o r s throughout the world.  To avoid  confusion the so c a l l e d "International U n i t " has been evolved a f t e r several years of intensive i n v e s t i g a t i o n .  U n t i l recently  the Sherman and Munsell u n i t (206) has been extensively used i n the Yitamin A determinations of foods, Eexelen, Emmerie and Wolff (73 a ) i n 1934 converted the various " u n i t s " used to express the r e s u l t s of Vitamin A estimations by the antimony t r i c h l o r i d e method, into International u n i t s .  Their findings were as f o l l o w s :  1 Cod L i v e r  O i l unit (Rosenheim and Webster) equals 208 i n t e r n a t i o n a l u n i t s , 1 blue value (Drummond and H i l d i t c h ) equals 20.8 i n t e r n a t i o n a l u n i t s , 1 lovibond u n i t (Wolff) equals 4.2 i n t e r n a t i o n a l u n i t s , 1 blue u n i t (Moore) equals 0.39 i n t e r n a t i o n a l units* showed that by s i m i l a r c a l c u l a t i o n s an o i l having 328 u  They also Q j £  c  e  n  ^  u equals 1 i s assumed to contain 5 Cod L i v e r O i l u n i t s ,  50 blue values, 250 lovibond units (Wolff), 2750 blue units (Moore) and 625 gamma Vitamin A or 1042 i n t e r n a t i o n a l units per gram. The f i r s t I n t e r n a t i o n a l Conference f o r the Standardisation of Vitamins was held i n 1931 (183.) i n London England. p r o v i s i o n a l u n i t of Vitamin A was established  i n terms of the  A  79 b i o l o g i c a l a c t i v i t y of a sample of carotene.  The second Confer-  ence was held i n 1934 at which a s o l u t i o n of pure beta carotene was adopted as the permanent standard i n terras of which u n i t s of Vitamin A were i n future to be expressed (154). The Pharmacopoeia of the United States has evolved a standard procedure f o r the b i o l o g i c a l t e s t i n g of Vitamins A and D.  Their standards are Reference Cod L i v e r O i l s of known  standard potency.  See Appendix No. 1 f o r the procedure.  The above two standards are f o r the use of r a t s .  Up  to the present there i s no standard procedure f o r Vitamin A determinations w i t h chicks. Many investigators have used chicks as a t e s t animal but a l l express t h e i r r e s u l t s d i f f e r e n t l y and therefore i t i s very d i f f i c u l t to compare the r e s u l t s of one i n v e s t i g a t o r w i t h another.  A standard procedure using chicks  as the t e s t animal i s needed i n order that the findings of d i f f e r e n t i n v e s t i g a t o r s may be comparable. The d e f i n i t i o n of a unit of Yitamin A as outlined by the "Permanent Standards Commission of the Health Organization of the League of Nations" i s as follows: (a) I n t e r n a t i o n a l Standard The Conference recommends that pure B—carotene be adopted as the I n t e r n a t i o n a l Standard f o r Vitamin A,  The  Standard Preparation s h a l l conform to the requirements stated i n Note 1 i n regard to i t s chemical and p h y s i c a l constants.  8D  (b) D e f i n i t i o n of Unit The Conference reoommends that the u n i t f o r Yitamin A p r o v i s i o n a l l y adopted at the 1931 Conference s h a l l be maintained. I t has been e s t a b l i s h e d that one such u n i t i s contained i n 0 6 a  microgram (0.6 gamma) of pure B--carotene. The I n t e r n a t i o n a l Unit f o r Yitamin A recommended f o r adoption s h a l l be defined as the Yitamin A a c t i v i t y of 0.6 microgram (0.6 gamma) of the I n t e r n a t i o n a l Standard Preparation. Daily doses of two to four I n t e r n a t i o n a l Units of Yitamin A, when administered t o young r a t s s u i t a b l y prepared on a Yitamin A d e f i c i e n t d i e t , have been found adequate to restore growth; somewhat l a r g e r doses are required f o r the cure of xerophthalmia. (c) Mode of Preparation I t i s recommended that the Health Organization of the League of Nations s h a l l be requested to obtain a sample of B—carotene as defined by the Conference (see Note 1), and that the National I n s t i t u t e f o r Medical Research, London, a c t i n g for t h i s purpose as c e n t r a l laboratory on behalf of the Health Organization of the League of Nations, s h a l l undertake the care, storage and d i s t r i b u t i o n of the I n t e r n a t i o n a l Vitamin A Standard so obtained. (d) Mode of D i s t r i b u t i o n The Conference recommends that the International Standard  81 Preparation s h a l l be issued i n the form of a standard solution i n o i l , the strength of the s o l u t i o n being such that 1 gramme .contains 500 I n t e r n a t i o n a l Units, or 300 micrograms (300 gamma) of B--carotene. (e) Adoption of Subsidiary Standard of Reference The Conference recommends that a sample of God Liver O i l , the potency of which has been accurately determined i n terms of the I n t e r n a t i o n a l Standard Preparation of B—carotene, s h a l l be provided as a Subsidiary Standard of Reference, In view of the f a c t that the Reference Cod Liver O i l of the United States Pharmacopoeia, which has been accurately assayed i n terms of the p r o v i s i o n a l I n t e r n a t i o n a l Standard adopted i n 1931, has been i n e f f e c t i v e use i n the United States of America f o r some time, the Conference recommends that the Board of Trustees of the United States Pharmacopoeia be approached and i n v i t e d to place a quantity of t h e i r Reference God L i v e r O i l at the disposal of the Health Organization of the League of Nations, w i t h a view to i t s adoption f o r i n t e r n a t i o n a l use as a Subsidiary Standard f o r Vitamin A. In the event of the Reference God L i v e r O i l of the United States Pharmacopoeia not being available f o r i n t e r n a t i o n a l adoption, the Conference recommends that another sample of God L i v e r O i l be selected, i t s potency i n terms of the Intern a t i o n a l Standard Preparation of B—carotene accurately determined by b i o l o g i c a l comparison and independently by  82 spectrophotometries measurements, and that t h i s selected sample be then adopted as a Subsidiary International Standard f o r Yitamin A.  83 YITAMIH A.IN POULTRY Symptoms and Lesions Due to Lack of Vitamin A . Beach (12) i n 1924, was the f i r s t i n v e s t i g a t o r to describe avitaminosis A i n p o u l t r y . He noted that the characteri s t i c symptoms of t h i s disease were confined t o the head L . > i n v o l v i n g the nasal passages, the mouth, pharynx, esophagus and the eyes.  According to h i s early work avitaminosis A was  associated with the f o l l o w i n g symptopathology: "A discharge from the n o s t r i l s , of a watery or v i s c i d f l u i d i s nearly always present.  Later t h i s may c o l l e c t i n the  i n f r a o r b i t a l sinuses, become transformed into a  Gaseous  mass  and cause a s w e l l i n g of the face. "The l e s i o n s i n the mouth, pharynx, esophagus and crop consist of c o l l e c t i o n s of white caseous material i n the mucous glands. "The l e s i o n s i n the eye consist of an ophthalnia which produces p u f f i n e s s of the e y e l i d s , reddening of the conj u n c t i v a , a profuse watery secretion which soon becomes v i s c i d and may glue the e y e l i d s together. "The kidneys are u s u a l l y pale and marked by a network of fine white l i n e s which are u r a t e - f i l l e d tubules.  Occasion-  a l l y there i s a deposit of urates on the heart, pericardium, l i v e r , omentum and i n t e s t i n e s .  In some cases the uraters are  greatly distended with urates. "The p o s s i b i l i t y that i t i s Vitamin D rather than  84 Vitamin A i n the ood l i v e r o i l that prevents the development of t h i s disease would seem to "be controverted by the following: 1. "The s i m i l a r i t y of the ophthalmia to that occuring i n r a t s fed a r a t i o n d e f i c i e n t i n Vitamin A and the t o t a l lack of any symptom of r i c k e t s suggestive of Vitamin D deficiency. 2. "Exposure of the fowls t o an abundance of d i r e c t sunl i g h t d i d not prevent the development of t h i s disease, although i t d i d prevent r i c k e t s . 3. " I t would seem, therefore, that t h i s disease which the w r i t e r has previously designated as a n u t r i t i o n a l disease resembling roup should now be designated a n u t r i t i o n a l disease caused by Vitamin A d e f i c i e n c y although the name n u t r i t i o n a l roup might be more s u i t a b l e f o r use by the poultrymen". In 1930 S e i f r i e d (203) described the e s s e n t i a l h i s t o p a t h o l o g i c a l changes i n the r e s p i r a t o r y t r a c t of chickens caused by avitaminosis A.  He s a i d , "There i s f i r s t an atrophy  and degeneration of the l i n i n g mucous membrane epithelium as w e l l as of the epithelium of the mucous membrane glands. process i s followed or accompanied by a replacement  This  or s u b s t i t -  u t i o n of the degenerating o r i g i n a l epithelium of these parts by a squamous s t r a t i f i e d k e r a t i n i z i n g epithelium.  This newly  formed epithelium develops from the p r i m i t i v e columnas epithelium and d i v i d e s and grows very r a p i d l y .  The process  appears t o be one of s u b s t i t u t i o n rather than a metaplosia, and resembles the normal k e r a t i n i z a t i o n of the skin or even more  85 c l o s e l y the incomplete k e r a t i n i z a t i o n of the mucous membranes. A l l parts of the r e s p i r a t o r y t r a c t are about equally involved i n the process, and the o l f a c t o r y region as w e l l , so that the sense of smell may be l o s t .  The l e s i o n s , which f i r s t take place  on the surface epithelium and then i n the glands, show only minor differences ". "The p r o t e c t i v e mechanism inherent i n the mucous membranes of the e n t i r e r e s p i r a t o r y t r a c t i s s e r i o u s l y damaged or even e n t i r e l y destroyed by the degeneration of the c i l i a t e d c e l l s at the surface and the lack of secretion with bacteri c i d a l properties.  Secondary i n f e c t i o n s are frequently found,  and nasal discharge and various kinds of inflammatory  processes  are common, i n c l u d i n g purulent ones, e s p e c i a l l y i n the upper r e s p i r a t o r y t r a c t , communicating sinuses, eyes and trachea. The development of the c h a r a c t e r i s t i c h i s t o l o g i c a l process i s not dependent upon the presence of these i n f e c t i o n s , since i t also takes place i n the absence of i n f e c t i o n .  The s p e c i f i c  h i s t o l o g i c a l l e s i o n s make i t possible to d i f f e r e n t i a t e between A avitaminosis and some i n f e c t i o u s diseases of the r e s p i r a t o r y tract". Continuing t h i s work f u r t h e r S e i f r i e d (204) s a i d "When fowls are placed on a diet l a c k i n g i n Vitamin A l e s i o n s appear i n the upper alimentary t r a c t which are confined l a r g e l y to the mucous glands and t h e i r ducts.  H i s t o l o g i c a l l y i t i s shown  that the o r i g i n a l epithelium becomes replaced by a s t r a t i f i e d  86 squamous k e r a t i n i z i n g epithelium and that secondary i n f e c t i o n s are r e l a t i v e l y common.  The ducts of the glands may be blocked  leading .to d i s t e n t i o n with secretions and nurotic  materials.  These l e s i o n s macroscopically resemble very c l o s e l y c e r t a i n stages of fowl pox and the two conditions can be separated only by h i s t o l o g i c a l examination.  These l e s i o n s produced by a lack  of Vitamin A may enable b a c t e r i a and other viruses to enter the body". Ackert, Mcllvaine and Crawford (2) found that the r e s i s t ance of growing chickens to the i n t e s t i n a l roundworm, A s c a r i d i a Lineata, was lowered when the fowls, four to seven weeks of age, were kept on a d i e t d e f i c i e n t i n Vitamin A.  The l a r g e r  number of the roundworms remaining i n the chickens on the Vitamin A d e f i c i e n t diet were a t t r i b u t e d i n part to evidences of weakened p e r i s t a l s i s , thus making i t l e s s d i f f i c u l t f o r the young worms to withstand the r i g o r s of p e r i s t a l s i s . According to i n v e s t i g a t i o n s conducted at the University of Idaho, (251) the symptoms and l e s i o n s of chicks fed a Vitamin A free r a t i o n are as follows: "A c h a r a c t e r i s t i c wobbly g a i t , sore eyes, swelling under the throat, excessive mucous i n the mouth, r u f f l e d feathers, general i n a c t i v i t y and i n a b i l i t y to maintain normal equilibrium, and extreme paleness of s k i n , beak, and shanks.  The i n t e r n a l  l e s i o n s observed consisted of extreme paleness of the kidneys, followed by a c h a r a c t e r i s t i c white network of urate deposit i n  8 7  the kidney t i s s u e , enlargment of the ureters with an accumulation of urates, and enlargment of the g a l l bladder and .riculus.  provent-  A white deposit often appeared over the surface of  the i n t e r n a l organs and w a l l s of the body c a v i t y . A gelatinous substance- was frequently found around the heart or over the breast muscles.  In those chicks which survived to or beyond s i x  Yi/eeks of age, t y p i c a l white abscesses (pustules) developed on the mucous membrane of the throat, and cankerous growths appeared i n the mouth.  In some instances the small i n t e s t i n e s  e x h i b i t e d extreme inflammation, with d e f i n i t e hemorrhagic spots. As previously stated, these l e s i o n s did not appear uniformly i n a l l the c h i c k s .  The urate deposits i n the kidneys and the  enlargement of the u r e t e r s , g a l l bladder, and proventriulus, were the l e s i o n s which appeared more c o n s i s t e n t l y . The above symptoms occur i n chicks at about four weeks of age and become more severe before the chicks f i n a l l y die". Hinshaw and Lloyd (114) report that poults fed a Vitamin A d e f i c i e n t r a t i o n from the time of hatching developed symptoms of avitaminosis A i n twenty-five days.  Chicks kept  as penmates to the poults showed s i m i l a r symptoms i n twentyseven days.  The disease was much more acute among the poults  than among the chicks.  Lesions i n the poults were confined to  mucous membranes of the head, the upper digestive t r a c t , the r e s p i r a t o r y t r a c t and the Bursa of Fabricus.  Deposits of  urates i n the kidneys and the ureters seldom occured i n the  88 poults.  They a l s o found that turkeys required a r a t i o n contain-  ing eight percent of dehydrated a l f a l f a l e a f meal (containing approximately 130 gamma of carotene per gram), f o r normal growth to t h i r t y weeks of age.  Chicks kept as penmates to the  turkeys made normal gains and showed no evidence of avitaminosis A on a l e v e l of four percent a l f a l f a meal.  Prom t h e i r  i n v e s t i g a t i o n they prepared tables showing the d i s t r i b u t i o n of l e s i o n s i n the turkeys and the chickens. S e i f r i e d (205) i n l a t e r work showed that chicks developed the f i r s t macroscopic e p i t h e l i a l changes a f t e r t h i r t y - t h r e e days on a d i e t d e f i c i e n t i n Yitamin A. M i l l e r and Bearse (155 a ) noted that the f i r s t symptom observed i n chicks i n a Yitamin A d e f i c i e n t d i e t was a staggering g a i t .  This c o n d i t i o n became progressively worse u n t i l  death occurred.  Staggering g a i t , u n t h r i f t y appearance, and  s o i l e d f l u f f were the only e x t e r n a l symptoms of deficiency i n chicks dying early i n the t r i a l .  Those dying l a t e r frequently  showed a watery eye c o n d i t i o n which was often accompanied by pus.  They a l s o found that the post mortem examination of  chicks on a Yitamin A d e f i c i e n t d i e t revealed enlarged g a l l bladders, enlarged gray kidneys (due to accumulation of urates), ureters distended with urates, enlarged p r o v e n t r i c u l i and gray hearts.  G u l l e t l e s i o n s were observed i n only a few birds*  89 Requirements of Chioks For Growth Sugiura and Benedict (240) i n 1923 diets f o r the n u t r i t i o n of pigeons.  studied synthetic  In t h e i r work they used  two d i f f e r e n t r a t i o n s and obtained normal growth and uction.  reprod-  Ho. 1 d i e t consisted of casein 22, cane sugar 10,  starch 27, agar 2, s a l t mixture 3, butter f a t 30 and yeast 6$. Ho. 2 d i e t had the starch increased to 37$ and the butter fat replaced by l a r d 20$.  They concluded from t h e i r work,  "Pigeons on.a d i e t of s u f f i c i e n t c a l o r i c value, even though the d i e t l a c k s f a t and f a t soluble vitamins, may maintain excellent c o n d i t i o n , and may produce f e r t i l e eggs and rear healthy squabs. Hence f a t - s o l u b l e vitamin i s not e s s e n t i a l i n any stage of avain n u t r i t i o n " . A few months l a t e r Emmett and Peacock (78) showed that young chicks require the f a t soluble Vitamin A.  They noted that  i n the absence of Vitamin A, the onset of the symptoms of ophthalmia appeared and unless the diet was properly reinforced, or an o r a l treatment r i c h i n the Vitamin was given, death eventually ensued.  They claimed that t h i s eye condition  r e s u l t i n g from the l a c k of Vitamin A was the same as the poultry n u t r i t i o n a l roup described by Beach (12.),  The presence  of urates i n the u r e t e r s , kidneys and at times on the surface of the heart, l i v e r and spleen were observed and believed r e l a t e d to the deficiency of the f a t soluble Vitamin A.  They  concluded that, "Young mature pigeons require very l i t t l e i f  90 any, f a t soluble A.  Yitamin A does however play a r o l e i n the  n u t r i t i o n of some species of avian". The f o l l o w i n g year, Hart, Steenbock, Lephovsky and Haplin, (102) agreed with Emrnett and Peacock (78) and disagreed with Suyiura and Benedict (240), Hauge, Canick and Prange (105) found that when they used 25$ of yellow corn i n t h e i r basal r a t i o n , the f a t soluble A requirements  of growing chicks were met f o r the f i r s t ten  weeks of t h e i r l i v e s . met the requirements l a y i n g age.  On the other hand 50$ of yellow corn f o r the development of p u l l e t s , up to the  Chicks f e d on r a t i o n s d e f i c i e n t i n f a t soluble A  usually r e f l e c t e d such a d e f i c i e n c y i n t h e i r growth responses at about four weeks of age. R u s s e l l and Weber (192) i n v e s t i g a t e d the r o l e played by plant pigments i n the n u t r i t i o n of chickens,  Pour groups  of chicks were placed on a Yitamin A d e f i c i e n t d i e t .  After  four weeks on t h i s d i e t alone, d a i l y supplements of 0.03 mg, of carotene, xanthophyll and c h l o r o p h y l l were fed t o the chicks i n each of three of the four groups r e s p e c t i v e l y , the fourth serving as c o n t r o l s . The group r e c e i v i n g the carotene throve, but the chicks i n the c o n t r o l group and i n the two groups r e c e i v i n g the other pigments f a i l e d and died i n two weeks.  The u r i c a c i d i n the blood of the Yitamin A deficient  chicks was much increased but s t a r v i n g chicks y i e l d e d s i m i l a r values.  The conversion of carotene into Vitamin A was proven by Capper, McKibben and Prentice (31).  definitely  They also  observed that the beaks and shanks of chickens, which had become c o l o r l e s s through the absence of carotenoids from the d i e t , d i d not become more yellow when carotene was added to the ration.  They showed that the Vitamin A content of hen l i v e r  o i l s was very high and that the Vitamin A requirements of fowl were l a r g e . Elvenjem and New  (75) used chicks instead of r a t s i n  t h e i r study of avitaminosis A.  They evolved a basal diet  c o n s i s t i n g of : 58 parts ground white corn, 25 parts wheat middlings, 12 parts crude casein, 1 part common s a l t , 1 part p r e c i p i t a t e d calcium carbonate, 1 part p r e c i p i t a t e d calcium phosphate, 2 parts d r i e d yeast. They observed that the chicks grew normally to three weeks when they began to e x h i b i t general i n c o o r d i n a t i o n , became drowsy and crouched on t h e i r haunches.  The feathers  were very r u f f l e d and there was some soreness around the eyes, but there was no t y p i c a l ophtholmia.  The beaks and shanks  became c o l o r l e s s and most of the b i r d s were dead by the end of the f i f t h week.  They a l s o found that the u r i c a c i d content of  the blood of normal chicks was approximately 5 mg. per 100 c c . of whole blood, w h i l e that of Vitamin A d e f i c i e n t chicks went as high as 44 mg. per 100 c c . of blood.  The amount of u r i c  a c i d i n the blood was independent of the p r o t e i n intake.  Yitamin A deficiency d i d not d i s t u r b the u r i c a c i d metaboils Lsra but injured the structure of the kidney s u f f i c i e n t l y t o prevent normal e l i m i n a t i o n of u r i c a c i d .  The amount of u r i c a c i d i n  the blood was dependent upon the degree of kidney damage. The degree of incoordination, however, was independent of the u r i c a c i d content of the blood. K l i n e , Schultze and Hart (132) found that Xanthophyll, m.p. 174^ prepared from spinach d i d not serve as a source of Yitamin A f o r the chick.  There were, however no t o x i c e f f e c t s ,  even when i t was f e d at l e v e l s of 0.25 mg. d a i l y per chick. Carotene, m.p, 172,5° prepared from spinach, when fed i n adequate amounts served as a source of Vitamin A f o r chicks. When the chicks reached the age of seven to eight weeks, 0.03 mg. of carotene d a i l y were not s u f f i c i e n t when i t was the sole source of Yitamin A.  Chicks that had been depleted of  Yitamin A required more than 0.05 mg. of carotene d a i l y i n order to grow to maturity. In determining the minimum amount of yellow corn necessary i n a growing r a t i o n Smith {224) used a basal r a t i o n which contained:  35 pounds ground corn meal, 5 pounds oat meal,  20 pounds wheat middlings, 7 pounds meat scrap, 3 pounds f i s h meal, 5 pounds dried skim milk, 1^ pounds oyster s h e l l , •g- pound s a l t . When white corn was used i n the basal r a t i o n one hundred percent m o r t a l i t y r e s u l t e d by the end of the eighth  week. Normal growth and v i a b i l i t y r e s u l t e d when twenty-five percent and f o r t y - f i v e and one h a l f percent of the t o t a l r a t i o n ,was made up of yellow corn.  He concluded that the minimum  amount of yellow corn required i n a growing r a t i o n l a y between twelve percent and twenty-five percent when other sources of Vitamin A were l a c k i n g . The Idaho A g r i c u l t u r a l Experiment Station (251) used the f o l l o w i n g basal r a t i o n f o r t h e i r work on avitaminosis A: 43 pounds ground-wheat, 15 pounds ground oats, 20 pounds bran, 10 pounds d r i e d milk, 7 pounds meat and bone meal, 4 pounds oyster s h e l l , 1 pound s a l t .  Vitamin D supplied by i r r a d i a t i o n .  They observed that the f i r s t i n d i c a t i o n of Vitamin A deficiency occured at about three weeks.  A high death rate  r e s u l t e d a f t e r four weeks up to one hundred percent by eight weeks. Studying avitaminosis A i n turkeys Hinshaw and Lloyd (114) fed the f o l l o w i n g r a t i o n :  25 pounds white corn, 25 pounds  barley, 25 pounds ground wheat, 10 pounds f i s h scrap, 10 pounds dried milk, 3 pounds bone meal, 2 pounds ground limestone and •§- pound s a l t .  They kept chicks as penmates to the poults and  t h e i r observations are discussed l a t e r under pathology. Gutteridge (99) found that the a d d i t i o n of p i l c h a r d 011 or God L i v e r O i l to a r a t i o n otherwise d e f i c i e n t i n Vitamin A, increased growth and prevented the development of deficiency synptoms i n c h i c k s .  He also observed that neither p i l c h a r d o i l  9 4  nor God L i v e r O i l , when fed with a Yitamin A d e f i c i e n t r a t i o n at l e v e l s of one percent and two percent of the t o t a l feed consumed "brought about as r a p i d growth as was attained by feeding a w e l l balanced r a t i o n .  In conclusion he claimed that  p i l c h a r d o i l and God L i v e r O i l were of equal value i n so f a r as Yitamin A content i s concerned, and that p i l c h a r d o i l was s l i g h t l y more e f f i c i e n t i n t h i s respect. In a study of the carotene and Vitamin A requirements f o r white leghorn.chicks I r o h r i n g and Wyeno (86) used the following ration:  52-§- pounds ground white corn, 10 pounds  wheat bran, 15 pounds wheat middlings, 10 pounds meat scrap, 10 pounds skimmed milk powder, 2 pounds calcium carbonate, •J- pound sodium c h l o r i d e .  100 A.D.M.A. u n i t s of Vitamin D per  chick per day. They found that the minimum requirements of Yitamin A f o r a chick at the age of about eight weeks was 65 A.D.M.A. u n i t s per day.  approximately  P r a c t i c a l l y a l l chicks depleted i n  Vitamin A showed marked a t a x i a three to fourteen days before complete depletion and death, even though given an adequate amount of Vitamin D.  There was a wide v a r i a t i o n i n the number  of days chicks l i v e d on the Vitamin A free d i e t , no doubt due to v a r i a t i o n i n storage from the egg from which the chicks hatched, which i n t u r n was dependent on the storage or r a t i o n of the parent fowl.  The a d d i t i o n of carotene or Vitamin A to  the carotene, and Vitamin A f r e e r a t i o n delayed the appearance  96 of deficiency symptoms and prolonged l i f e i n a rather d e f i n i t e r e l a t i o n to the amount of carotene or Vitamin A added to the diet. Sherwood and Praps (214) fed rations containing 50, 100, 150 and 300 Sherman-Munsell (206) u n i t s per 100 grams of feed to chicks hatched from eggs produced by hens r e c e i v i n g rations containing 310, 440 and 560 u n i t s of Vitamin A per 100 grams of feed.  During the f i r s t few weeks of the experiment the  m o r t a l i t y of the chicks from the hens on the lowest Vitamin A l e v e l was so high that the d i f f e r e n t l e v e l s of Vitamin A i n the chick feeds studied d i d not show the r e s u l t s on chick mortality that they d i d on the chicks from the hens on the higher l e v e l s . The m o r t a l i t y was lower i n the case of the chicks from the hens r e c e i v i n g 440 and 560 u n i t s of Vitamin A per 100 grams of feed as the amount of Vitamin A i n the r a t i o n increased.  Ho  advantage was shown i n the weight of the chicks f o r the 300 u n i t s over the 150 u n i t s per 100 gms., i n the chick r a t i o n s . In another study with chicks hatched from hens receiving an adequate supply of Vitamin A these same two investigators found that the m o r t a l i t y i n twelve weeks was as follows: i n s i g n i f i c a n t amount of Vitamin A, 100$ m o r t a l i t y ; 42 units per 100 gms. 41%; 84 u n i t s per 100 gms, 15%; and 126 units per 100 gms. 12%. The percentage of healthy chicks remaining at the close of the experiment was 0, 24, 72, 83 f o r the respected lots.  Record, Bethe and Wilder (181) used a r a t i o n c o n s i s t i n g of:  58 pounds white corn, 25 pounds wheat middlings, 12 pounds  domestic casein, l i - pounds d r i e d yeast, % pound I r r a d i a t e d yeast (200 D), 1 pound steamed bone meal, 1 pound ground lime stone, 1 pound s a l t . They found In p r o p h y l a c t i c t r i a l s that from f i f t y to one hundred gamma of carotene per 100 gms. of feed were required t o produce normal growth and prevent symptoms of Yitamin A d e f i c i e n c y during the f i r s t eight weeks of a c h i c k s life.  8  I n c u r a t i v e t r i a l s chicks were fed the basal r a t i o n f o r  twenty-six days, at which time a large percentage of the birds showed Yitamin A symptoms.  They then divided the chicks into  groups and fed d i f f e r e n t amounts of carotene or God L i v e r O i l and found that i t required approximately f i f t y gamma of carotene or 60 to 100 i n t e r n a t i o n a l u n i t s d a i l y of God L i v e r O i l t o produce normal chicks f o r seven or nine weeks of supplemental  feeding.  In 1935, Schroeder, Higgins and Wilson (202) reported that chicks up to nine weeks of age required 6000 i n t e r n a t i o n a l u n i t s of Vitamin A per pound of feed (about 1320 u n i t s per 100 gm) t o prevent c l i n i c a l and p a t h o l o g i c a l symptoms of hypovitaminosis A.  They also reported that 1200 u n i t s of  Vitamin A per pound of feed appeared to be adequate to promote f a i r growth; but that there was a tendency f o r body weights to increase as the Yitamin A l e v e l was increased.  The f o l l o w i n g  97  year these same i n v e s t i g a t o r s (262) used the same basal r a t i i .on and found 1200 i n t e r n a t i o n a l u n i t s of Vitamin A per pound of feed were adequate to promote s a t i s f a c t o r y growth.  They  suggested that t h e i r previous high requirements were due to the severe mixed i n f e c t i o n that occured amongst the experimental b i r d s They f u r t h e r noted that the storage of Vitamin A was cumulative and bore a marked r e l a t i o n s h i p to the amount of the factor i n the d i e t .  Unit f o r u n i t , carotene and Vitamin A obtained from  a f i s h o i l concentrate were found to be u t i l i z e d by the chick with equal e f f i c i e n c y . In a study of the Vitamin A storage of growing chicks, Holmes, Tripp and Campbell (116) used the f o l l o w i n g r a t i o n : 52 pounds corn-meal a t t r i t i o n , 15 pounds wheat bran, 15 pounds wheat f l o u r middlings, 12 pounds ground oat groats, 8 pounds dry skim milk, 5 pounds a l f a l f a l e a f meal, 5 pounds meat scraps, 5 pounds f i s h meal, •§• pound d i calcium phosphate, 1 pound oyster s h e l l meal, 1 pound s a l t . When 0.5% sardine o i l was added to the above basal r a t i o n and fed to young chicks the l i v e r s of these chicks contained s i g n i f i c a n t l y more Vitamin A than when 0.25% was added.  The amount stored was approximately four times more f o r  the higher l e v e l .  The Vitamin A reserve i n the l i v e r increased  i n the p e r i o d from eight to twelve weeks of age.  Older birds  showed wide v a r i a t i o n s both i n egg producing power and i n the Vitamin A content of t h e i r l i v e r s .  9'8 Working at C o r n e l l , Ringrose and IJTorris (185) used the following ration:  55 pounds white corn-meal, 25 pounds wheat  f l o u r middlings, 10 pounds commercial casein, 5 pounds dried yeast, 2£ pounds p u l v e r i z e d limestone, 1 pound steamed bone meal, 1 pound cottonseed o i l , -g pound i o d i z e d s a l t ,  Yitamin D  was supplied by i r r a d i a t i o n . They e s t a b l i s h e d the growth response curve f o r the chick to Yitamin A feeding, a b i o l o g i c a l assay expressing r e s u l t s i n terms of Yitamin A per gram of m a t e r i a l thus became possible.  The assay was conducted by feeding the t e s t material  at such a percentage of the r a t i o n as to induce a growth response which was s l i g h t l y , but d e f i n i t e l y , subnormal.  The  growth r e s u l t s obtained at eight weeks of age were then applied to the. growth response curve and the u n i t s of Yitamin A per 100 gms. of feed determined.  By d i v i d i n g the number of u n i t s  per 100 gms. of feed by the percent of the test material i n the r a t i o n , the potency of the test m a t e r i a l i n u n i t s per gm, was obtained.  They found that the minimum Vitamin A  requirement  of the chick during the f i r s t eight weeks of l i f e was about 150 U.S.P.X. Revised 1954 u n i t s per 100 gms. of feed. Holmes, Tripp and Campbell (117) obtained embryos and young chicks from d i f f e r e n t sources and determined the Vitamin A stores i n t h e i r l i v e r s and unabsorbed egg yolks by means of the antimony t r i c h l o r i d e r e a c t i o n . From the eighteenth day of Incubation to the f i f t h day a f t e r hatching the Vitamin A  99 content of the l i v e r rose ana that of the unabsorbed yolk f e l l , the combined t o t a l from both sources f a l l i n g  slowly.  At the U n i v e r s i t y of B r i t i s h Columbia B i e l y  and  Chalmers (16) fed chicks the d i e t of Elvehjem and Neu which i r r a d i a t e d yeast was U.S.  added.  (76)  to  Once a week a dose of the  Pharmacopoeia Reference Cod L i v e r O i l was  fed d i r e c t l y  i n t o the crop of each b i r d i n an amount to supply 0, 25,  50,  75 and 100 i n t e r n a t i o n a l units of Vitamin A d a i l y to the various groups.  The group reoeiving no o i l showed symptoms of  Vitamin A deficiency a f t e r three weeks; these became marked at f i v e weeks and a f t e r eight weeks a l l the birds were dead.  Of  the remainder those r e c e i v i n g 25 i n t e r n a t i o n a l units d a i l y , began to l a g i n growth and to show some symptoms a f t e r four weeks.  The remainder grew w e l l and showed no symptoms.  They  concluded that 75 i n t e r n a t i o n a l units d a i l y were s u f f i c i e n t to ensure normal growth and protect chicks up to eight weeks against any symptoms of Vitamin A deficiency, 100  units  appeared to be above the immediate normal requirements of the chicks up to t h i s age. the white corn (59$)  When yellow corn was  substituted for  i n the r a t i o n with the omission of  Cod  L i v e r . O i l , they found approximately the same performance as the dose of 75 i n t e r n a t i o n a l u n i t s d a i l y had given. Record, Bethke and Wilder (182) Elvehjem and Neu  used the basal diet of  (75) i n which i r r a d i a t e d yeast supplied 4,000  i n t e r n a t i o n a l units of Vitamin D per 100 grams of feed.  They  IQO conducted both prophylactic type and curative type of feeding. The prophylactic t r i a l s showed that, under the experimental conditions employed, i t required a minimum of approximately 50 t o 100 micrograms of carotene or 80 to 160 i n t e r n a t i o n a l u n i t s of Yitamin A from Ood L i v e r O i l per 100 gms. of r a t i o n f o r normal growth and the prevention of external and i n t e r n a l symptoms of Yitamin A deficiency i n chicks to about eight weeks of age.  The curative experiments showed that about 100 micro-  grams of carotene or 120 to 200 i n t e r n a t i o n a l u n i t s of Yitamin A from Ood L i v e r O i l were required every other day to cure and prevent symptoms of Yitamin A deficiency and restore good growth i n chicks depleted of t h e i r Yitamin A reserves to ten to twelve weeks of age. Both types of experiments showed that the chick u t i l i z e d carotene as a source of Yitamin A, The response of the chicks to carotene or Vitamin A was approximately s i m i l a r when equivalent r a t u n i t s were fed; i n d i c a t i n g that the chick and r a t u t i l i z e carotene as a source of Yitamin A i n the same order.  They presented data to show that  the Yitamin A requirements of chicks inoreased with age. I n conclusion they pointed out that these experiments showed no s i g n i f i c a n t storage of Vitamin A i n the l i v e r s of the chicks u n t i l several times the minimum l e v e l , as determined by growth and external and i n t e r n a l symptoms was f e d . At the Western Washington Experiment Station Bearse and M i l l e r (13) used a basal r a t i o n c o n s i s t i n g of: 4 4 i pounds  101 ground white corn, 15 pounds ground wheat, 15 pounds ground oat 10 pounds m i l l r u n , 7 pounds meat scrap, 7 pounds powdered skim milk, 1 pound oyster s h e l l f l o u r , £ pound s a l t .  Yitamin  D was supplied by i r r a d i a t i o n . They supplemented t h i s r a t i o n with varying l e v e l s of dehydrated a l f a l f a f o r a twenty-four week growing period. These l e v e l s furnished approximately 87.5, 175, 350, 700 and 1400 Sherman-Munsell (206) Yitamin A u n i t s per 100 gms. of ration.  The r e s u l t s based on avitaminosis A m o r t a l i t y , growth  and l i v e r storage of Yitamin A showed that 175 Sherman-Munsell Yitamin A u n i t s per 100 gm. of r a t i o n met the Yitamin A requirements  of the chicks.  Feeding efficiency-was greater i n  the l o t s r e o e i v i n g the highest l e v e l s of Yitamin A during the f i r s t eight weeks of the t r i a l .  This difference was not so  great f o r the e n t i r e twenty-four week period.  There was a  close c o r r e l a t i o n between mature body weight and t o t a l feed consumption. In summarizing the f i n d i n g s of many i n v e s t i g a t o r s Dr. Parkhurst (174 a) stated:  "When feeds are mixed previous  to the feeding period, the minimum Yitamin A requirements of chickens to eight weeks of age would appear to be i n the v i c i n i t y of 150 U.S.P. Yitamin A u n i t s per 100 grams of feed (680 u n i t s per pound) and the p r a c t i c a l requirement  for satis-  factory growth and l i v e r storage about 300 units per 100 grams  s  102 of feed (1362 u n i t s per pound. For chickens t o t h i r t y weeks age, the requirement may be double.  Turkeys require at least  double the amount of Vitamin A i n t h e i r r a t i o n as do chickens  103 Requirements of Hens For Egg  Production  Bethke, Kennard and Sassaman (15) reported i n 1927  that  -the f a t soluble vitamin content of hen's yolk was greatly influenced by the amount of these substances present i n the r a t i o n and by environment of the l a y i n g hen.  The yolks of  eggs l a i d by hens which had access to a blue-grass range were approximately  f i v e times as potent i n Yitamin k  t  and ten times  as a c t i v e .antirachitfeally as the yolks of eggs l a i d by hens which received the same basal d i e t but were confined indoors. When two parts of Ood L i v e r O i l were added to the mash there was a f i v e f o l d increase i n the a n t i r a c h i t i c and f a t soluble A vitamin content of the egg yolks. Holmes, D o o l i t t l e and Moore (115) found that the a d d i t i o n of f a t soluble vitamins to the poultry rations d e f i n i t e l y stimulated egg production.  The average weight of eggs,  produced by the o i l fed b i r d s was s l i g h t l y greater than that of the eggs produced by the c o n t r o l b i r d s .  In t h i s experiment  they observed that the percentage of eggs containing blood c l o t s decreased c o n s i s t e n t l y w i t h the increase of Ood L i v e r O i l i n the experimental r a t i o n .  The number of eggs discarded  during incubation e i t h e r from being i n f e r t i l e or on account of containing a weak germ was l e s s f o r the o i l fed group.  The  number of chicks obtained and the v i a b i l i t y of these chicks was greater i n the b i r d s fed the supplement.  Contrary to  104 previously observed conditions, the body weight of the high producing b i r d s d i d not decrease but t h e i r weight at the end of experiment exceeded that of the c o n t r o l s .  As the experiment  progressed the f a t soluble vitamin potency of the eggs from the experimental decreased.  b i r d s increased while those from the controls No detectable f l a v o r was imparted to either the egg  or the f l e s h of the birds fed the God L i v e r O i l . They d e f i n i t e l y concluded that supplementary f a t soluble vitamin feeding increased the reproductive performance of domestic fowl. From the work of Sherwood and Fraps (211) i n 1932, i t was found that yellow corn alone was not s u f f i c i e n t l y high to supply enough Vitamin A to p u l l e t s f o r egg production.  They  estimated that p u l l e t s required f o r maintenance alone, about 105 u n i t s of Vitamin A potency per day, or 33 units per pound per day, ( t h i s i s eight times the estimate of 4 u n i t s per day per pound f o r maintenance of growing r a t s ) .  Ordinary  alfalfa  was found not to supply enough Vitamin A to produce eggs of high potency i n t h i s vitamin, even i f f e d at Q$ of the mash. They c a l c u l a t e d that one u n i t of Vitamin A i n the egg required 6.3 u n i t s i n the feed at 270 u n i t s , 5,7 u n i t s at 336 u n i t s , and 4,0 u n i t s at 44-4 u n i t s i n the feed. The f o l l o w i n g year Sherwood and Fraps (212) published f u r t h e r data on the subject. were used:  In t h i s experiment three rations  105 No. 1  HO*: 2 50 pounds yellow oorn 50 " white com  20 pounds yellow corn 20 " wheat shorts 20 " wheat bran 20 " ground oats 20 " meat and bone scrap No.  3  A l l White Corn These r a t i o n s were fed to groups of p u l l e t s that had been normally fed. At the close of the experiment the yellow corn group (No, 1) averaged 25% more i n weight than the white corn group (No. 3) and the mixed oorn group (No. 2) weighed 17% more than No. 3.  These r a t i o n s had a d e f i n i t e effect on  the number of eggs l a i d a f t e r the experiment had been i n progress two months.  During the l a s t four months of the experiment  the yellow corn group l a i d approximately 65% more eggs than did the white corn group and the mixed corn group l a i d 55% more eggs than did the white corn group.  The Vitamin A content of  the eggs showed d e f i n i t e l y , as the experiment advanced, that none of the r a t i o n s contained enough Vitamin A f o r high egg production.  The Vitamin A content of the eggs from a l l of the  groups became loiver as the experiment progressed.  They con-  cluded that i t required approximately 1,365 units of Vitamin A (Sherman-Munsell u n i t s ) per day f o r a l a y i n g p u l l e t f o r maintenance and production of eggs at the rate of twenty eggs per month.. •  106 R u s s e l l (193) also observed that the Yitamin A content of eggs from hens r e c e i v i n g yellow corn was greater than those .from hens r e c e i v i n g white corn.  He found that 47 to 48% of  the Yitamin A fed was contained i n the eggs. In 1934 Sherwood and Fraps (213) stated,  "The percent-  age of vitamin recovered i n the eggs i s greatest at the lowest l e v e l and l e a s t at the highest l e v e l of feeding.  This  apparent percentage recovery i s not correct because the Vitamin A stored by the hen i s not taken into account and some of the Yitamin A or carotene fed i s used f o r maintenance while some of i t i s probably not digested".  They found that on the  average 1 unit of Vitamin A i n the egg required 4.7 u n i t s i n the feed.  The u n i t s of Vitamin A required i n the feed for  1 u n i t i n the eggs decreased as the average number of u n i t s fed increases. Bishey, Appleby, V/eis and Cover (20) conducted an . i n v e s t i g a t i o n to c o r r e l a t e the Vitamin A a c t i v i t y of egg yolks w i t h the amounts of c a r o t i n o i d pigments they contained.  They  observed that there was a d i s t i n c t gradation of the color i n the egg yolks from the hens on the d i f f e r e n t rations and that the c o l o r of the yolks on a given r a t i o n was uniform.  They  concluded that while there seemed to be some r e l a t i o n s h i p between color and growth, the Vitamin A a c t i v i t y of egg yolks could not be explained on the basis of the c a r o t i n o i d pigments they contained.  They also noted that the Vitamin A  107 a c t i v i t y of the egg yolks was d i r e c t l y dependent upon the rations of the hens, Mohler (157) claimed that the optimto l e v e l of Cod L i v e r O i l feeding i n the diet of chickens was between 1 and 2%.  I f t h i s l e v e l was exceeded, impairment i n egg production  and h a t c h a b i l i t y was l i k e l y to occur. The work of DeVaney, L i t u s and Nestler (51) showed that extra Vitamin D, as 0.5$ v i o s t e r o l (160 D), i n the diet of chickens r e c e i v i n g graded amounts of Cod L i v e r O i l had no e f f e c t on the t r a n s f e r of Vitamin A to the egg. Eggs from p u l l e t s r e c e i v i n g 8$ of Cod L i v e r O i l were several times r i c h e r i n Vitamin A than those from p u l l e t s r e c e i v i n g lower doses. R u s s e l l and Taylor (194) determined the Vitamin A content of the egg yolk and the diet "by the method of Sherman and Munsell w i t h a reference o i l as a standard.  The r e s u l t s  were expressed i n U.S.P. 1934 ( i n t e r n a t i o n a l ) u n i t s by r e f e r ence to a curve of response.  They found that with varying  egg production and intake of Vitamin A, the output of Vitamin A i n the eggs, expressed as a percentage of intake, varied from 11 to 32$. In 1935 Sherwood ana Praps (215) estimated that hens l a y i n g per year one hundred and f i f t y eggs, high i n Vitamin A potency required approximately 600 Sherman-Munsell b i o l o g i c a l u n i t s of Vitamin A per day, or 7.5 u n i t s per gram of feed.  108 Koeriig, Kramer and Payne (133) found that the eggs of p u l l e t s l a i d i n the winter about the fourth month of egg t  production, and tested by the method of Sherman and Munsell, contained about 25 Sherman-Munsell u n i t s of Vitamin A per gram, whether the b i r d s were of high or low p r o d u c t i v i t y .  Autumn  eggs produced near the end of the f i r s t year contained 20 and 33 Sherman-Munsell u n i t s per gram, when l a i d by b i r d s of high and low p r o d u c t i v i t y , r e s p e c t i v e l y .  They conclude that egg  production made pronounced n u t r i t i v e demands upon the hen and that the recognized demand f o r Vitamin A was more pronounced the greater the number of eggs produced and the longer the l a y i n g period. Working at the Western Washington Experiment Station, Bearse and M i l l e r (14) found that eggs from hens r e c e i v i n g d i f f e r e n t q u a n t i t i e s of Vitamin A i n t h e i r r a t i o n s contained d i f f e r e n t q u a n t i t i e s of Vitamin A i n t h e i r egg yolks i n proportion to the amount of Vitamin i n the r a t i o n .  Chicks  hatched from such eggs l i v e d and grew on Vitamin A d e f i c i e n t r a t i o n i n proportion to the amount of Vitamin A i n the breeding hen r a t i o n .  They concluded that 500 Sherman-Munsell u n i t s  of Vitamin A per 100 gm. of feed i n the breeding hen r a t i o n supplied s u f f i c i e n t Vitamin A f o r maximum h a t c h a b i l i t y .  109. EXPERIMENTAL Experiment No. 1. Preliminary experiments conducted at The U n i v e r s i t y of B r i t i s h Columbia by Wood (266) indicated that P i l c h a r d O i l , as produced i n B r i t i s h Columbia, contained an appreciable amount of Vitamin A.  In f a c t , Wood's experiments showed that  one-half on one percent of a sample of commercial P i l c h a r d O i l when fed i n combination with a basal r a t i o n free from Vitamin A, promoted normal growth and development of chicks to eight weeks of age. The purpose of the present experiment was t o determine the Vitamin A potency of a sample of commercial P i l c h a r d O i l i n terms of I n t e r n a t i o n a l Units, using the chick as the test animal. The r e s u l t s of t h i s test were' compared w i t h those of B i e l y and Chalmers (16), who determined the Vitamin A requirements of growing chicks up to eight weeks of age by feeding them gradecd doses of Reference Cod L i v e r Oil(251a). The method followed by B i e l y and Chalmers was to feed ©, 25, 50, 75, and 100 units of the Reference Cod L i v e r O i l once a week with a p i p e t t e per orem.  The chicks i n the  experiment described herein, were fed t h e i r Vitamin A supplement i n the mash, but were brooded under s i m i l a r conditions. Consequently, the data may be considered as comparable. Experimental Methods: Day-old s i n g l e comb White Leghorn cockerels were  110. placed i n battery brooders under the same conditions as described by B i e l y and Chalmers (16).  These cockerels had been  separated-from the p u l l e t s by the Japanese method of "chick sexing".  The b a s a l r a t i o n fed to;the chicks consisted of 59  pounds ground white corn, 25 pounds wheat middlings, 12 pounds crude casein, 1 pound calcium carbonate, 1 pound calcium phosphate, 1 pound s a l t , and 1 pound i r r a d i a t e d yeast (70 D). . chemical composition  The  of t h i s ra.tion was as follows: Protein  ,20.84%, Calcium 1.32%, Phosphorus  .54%.  This basal r a t i o n i s s i m i l a r to that used by Elvehjem and Hew  (75) i n t h e i r vitamin determinations, except that i r r -  adiated yeast (70 D) served as the source of Vitamin D i n the present experiment i n place of u l t r a - v i o l e t l i g h t as used by the above i n v e s t i g a t o r s . I t d i f f e r s from the basal r a t i o n used by Hart, K l i n e , and Keenan (101a) f o r the production of r i c k e t s i n chicks, i n that, the yellow corn i s replaced by white corn, and the yeast i s changed t o i r r a d i a t e d yeast. The basal r a t i o n was supplemented with P i l c h a r d O i l a. s f o l l o w s : .Lot I  1/8%  Pilchard O i l  Ho. 1  Lot I I  1/4%  Pilchard O i l  Ho. 1  Lot  1/2%  Pilchard-0111  Ho.  Lot IV  1/4%  Pilchard O i l  No. 2  Lot V  1/2%  God L i v e r O i l  Lot VI  Control  III  1  Wesson O i l was used to d i l u t e the l / 8 % and 1/4% of o i l up to 1/2% by weight while the controls received  lots 1/2%  111.  Wesson O i l only. Results: The average weekly weights of the various l o t s of chicks are given i n Table No. 1, and the growth curves are shown i n Figure. 1,. It w i l l he r e a d i l y seen from Table No. 1 that the c o n t r o l chicks began to l a g i n growth between the second and . t h i r d week.  From then on the. difference i n the weight of the  c o n t r o l chicks, and those which received Vitamih A as contained i n P i l c h a r d O i l ( l / 4 % or 1/2%) or God L i v e r O i l (1/2%), became p r o g r e s s i v e l y greater as the experiment neared completion.  This i s s t r i k i n g l y shown i n Figure No. 1 .  Furthermore,  the c o n t r o l chicks showed d i s t i n c t symptoms of avitaminosis A at two weeks of age, and c h a r a c t e r i s t i c l e s i o n s at the time of death which u s u a l l y followed three t o four days a f t e r the f i r s t appearance of symptoms.  In every instance, the c o n t r o l chicks  showed considerable loss of weight before death. The c o n t r o l chicks were a l l dead by the end of the eighth week. From Table No. 1, i t w i l l be seen that the groups of chicks which received various amounts of P i l c h a r d O i l or God L i v e r O i l , grew at a f a i r l y uniform rate during the f i r s t four weeks of the experiment. . A f t e r the fourth week, the group which received 1/2% P i l c h a r d O i l No. 1, and the group which received 1/2% Cod L i v e r O i l , grew f a s t e r than the groups which received e i t h e r 1/8% or 1/4% P i l c h a r d OilNo. 1.  At eight weeks  of age, the average weight of the chicks which were fed 1/2% P i l c h a r d O i l No. 1 exceeded the average weight of the chicks  112. o  o o  co o  o>  OJ CO  OJ  CO IO  o>  0 0 .crj  m  IO  CO  CO to  IO  OJ  co  «  9 CO  £>  co  rH rH  IO  o  CO r H T j • rH CO  e  to  CQ  M  CD CD |  to  CC CO rH  CQ  co co «  CD  CO  to to  rH  CO  co  O O  CO CO  OJ OJ  to co  rH  to  e  ' 0  CO C-  OJ  02  OJ  tO  Oi  co  CO  D  o  "I  r-  rH  a  to to «  o  to o  rH  co «  tO  o  © ©  OJ  « to o oj  CO  to  rH O  co  CO  to  - 0  to  to  •a  to to «  ^  o>  Oi  to  CO  o  £> rH  rH  to to  to  co to  0  «  •  o  « or <o  o  to  CO  «  CO  to  IO  o>  to  z>  o  OJ  CO  to  rH •H O  o  rH •H  •H O  •cs  t».  •CS  ©  n3 ,3  •H  o  rH •H  PM  & o  rH •H A*  ^  O rH •H rH  CO  CO « to  OJ  %  n  o  rH •H f4  >  H  nd  o O  V*. ^. ^  ^  02  Oi  CO  9 CO  e  o  ?H Co  o  rH  rH CO  rH •H  r4 Co  Oi  o to,  rH  CO  P  Oi  H  ts  P-  rH  rH  •rt  ©  O  rH  O  rH  IO IO  co «  *te **.  ©  OJ CO  Oi  «  4J  rH  CO  0  H i  co  OJ  Oi rH  rH  tO  rH tO rH  ^<  O  IO  O  c  ^'  Oi  rH  to to  0  rH  « to  e  CO  OJ  rH  OJ  00  to  Oi  to  Oi o  •  rH  to  CO  4  to to  OJ  CO  0  9  9  ©  o o>  t>  OJ  ^'  OS tO to  \  rH  CQ  .y » o & o  OJ  CO  o  .  to  o  co to  o CO CO CO  IO  OJ  ©J  co to  LO  rH  l[  OJ  Oi CO  OJ  ©  © ©  Oi  ©  rH O  u a o a +»  X X *5 $  114. which were fed 1/2% Cod L i v e r O i l . I t should be noted that the average w eight of the chicks which received 1/4% of P i l c h a r d O i l Ho. 2 were only 9 grams less than the average v^eight of the chicks which received 1/2% Cod L i v e r O i l and also, only 20 grams below the average weight of the chicks which were fed 1/2% P i l c h a r d O i l No. 1* With the exception of a few chicks which received 1/8% of P i l c h a r d O i l No. 1, none of the chicks showed any evidence of avitaminosis A.  As judged e x t e r n a l l y , the chicks appeared  ' to be quite normal and healthy i n e v e r y r e s p e c t .  Post-mortem  examination of a few. chicks i n each l o t d i d not reveal any i n d i c a t i o n s of Vitamin A deficiency i n the i n t e r n a l organs. The s t a t i s t i c a l analyses of the data are given i n Tables No. 2 and 3.  Prom these r e s u l t s , i t w i l l be seen that  the difference between the average.weight of the chicks which received the basal r a t i o n supplemented with either 1/2% P i l c h a r d O i l No. 1 or 1/2% Cod L i v e r O i l and the chicks which received 1/8% of P i l c h a r d O i l No. 1, was s t a t i s t i c a l l y ficant.  signi-  Furthermore, i t i s important to note that the  d i f f e r e n c e between the average weight of the chicks which were fed 1/4% P i l c h a r d O i l No. 2 and. the chicks which were fed 1/8% P i l c h a r d O i l No. 1, was s t a t i s t i c a l l y s i g n i f i c a n t .  As previous-  l y stated, i t i s of considerable importance to note that the average weight of the chicks which received 1/4% of P i l c h a r d O i l No. 2 compared very favorably with the average weight of the chicks which received either 1/2% P i l c h a r d O i l No. 1, or 1/2% Cod L i v e r O i l . The r e s u l t s of the s t a t i s t i c a l analysis  115. O -p  4Jl  m  +1  -hi  O ,Q  o> to  0  o o  o to  CVJ  rH  OJ OJ  o o  CO  •  o  o •H +>  ro  o  > CD  HI 41  A  -I-l  .H  OJ  o  O OV  . *  r-l rH  t l  o>  CO OJ rH  o o  o  ro  rH H t  l  LO  o>  CO  •  e  to  LO CO  to to  LO rH - t l  9  £>  «  (Jl  +J  to  •H CD  Hi  (  t l O  I"-  CO H  I  o ^' CO  rH •H o  ro o  rH rH t l  rH rH  9  co o  A •H OJ  +1 o o  «  to  LO  LO  rH •H  rH •H  O  o  *Si (H  *}  .3  A' O  ro o  fH CO  to o  «  rH  rH  +i Oi  + 1 to  co  ©  co  LO LO  rH •H O  H  CD fc> •H H"  rH «H  rH •H  ^  ^  ^  fes.  OJ  OJ  Pi  E-  o  to  rH •H  A,  0  00  .  r-i  »  CQ  OJ  +1  OJ  CO  CO  OJ  -I-l  t—i  «  •H  *  -fi  to  ra i>  o  6 02  ®  0 r-t" •H  OJ OJ  o  HI  •tf O  \ rH  LO  rH •H O  id  M co-  &  o rH •H fH  ^  ^  \ rH  116. 4J •a  +»  +>•  s o  CO  o  •H  •H  CH  •H  •H  •H  SO  £sQ  a  S3  •H  CO  bO  taO  S3  •H  •rH  CO  •ri  •rH tH •H  +»  •S  o J25  rH  to  00  CM CO  »  rH  to  to  CO  to to  to  O CM  £>  to  CM  "tl  CM  t l CM  O CO  *  en  o o>  CM  O co  oo  o>  . «  rH •H O  rH •H  "d  •a  0  CO  •ri  o  rH  rH  ro  CM o  CM LO  o o  o>  to  CM  CO »  e  rH £>  CT>  LO 9  o  CM  CM  o -il rH i>  O to  o "f 1 CM LO  * rH  o LO  HI  -d  rH •H O  •ri O  rH CD  XS H  t>  fH cd  •H rH  CO  CM  r-i  T 3  rH •H O  rH •H O  rH •H  rH  •d  •d  -d  CO  ra  m  H  CO  ^3  co  o  ft ^ co  r-l •H ft  iH 'H ft  ^  ^  \  rH  TH  O •H  •H  HI  o  o  rH •H ft  ^  o  ft ^  CO  &  o iH •H ft  rH  CO  Hi  ^1 o  -d o  •H •H ft  ^  rH  Oi  o>  rH  ft-  o>  "CM  ^ rH *H O  •d  fH  CO  & o  i—!  •ri  ft  •t-i  -d  rH =8  •ri  o  O  •d  •d H  fH CO  ^3 o  rH •H ft ¥ L  CO  r-i  43  o  o rH  rH  •ri •ri ft  ft  rH •H O fH CD f>  •ri t-H  •d  o ©  ^  fe?.  CM •  e  CM  o fH  fH  •H  rH •H O  O  •H O  o  rH •H  -d o  rH  •H  c3  rH  rH  H  <=8  \  ft  o rH •H  to  *  o  =3  CM  A)  ^3  £> CM,  to  e  ««  O  O  rH  c-  CO  rH  rH  •d o  A'  rH  + 1 Hrl- +1  -=8  *d  rH •H ft  -d  rH  C7>  CO  to CM  eg  -d  o  rH •rH O  CO  ©  CM  rH "H O rH  o  o  <  CM \ rH  o  co  o  rH  ft  o  rH  O  co  t 1  o  o  o  + 1 to  +i  o  rH  CM  CM  CO  CO to  CM  CM  EJO  •H  CO  S2i  CQ  to  S3  3  to  *  o  Jzj  CM \ rH  rH •H  ft  •H  bQ •ri  o  CM \ rH  rH •H  Hi •H  rH  •H <H ' (H —«  o  4->  rH •iH  o  Hi  s>  •rH <H •H  CO  .S3 o rH •H  ^o3  .d  \  fH  •H «H •H £3  O  CM rH •H O  Co  CM  e  S3  S3 cO  o  w +»  co  is;  o  •r+ «H •rH •(30 •H  •H  -!-> O  ra  CO  c  a  03  4-»  o  •H  •H  CO  4->  •H «H  4J  c  co o  o  0  •H  CO  +•>  a a  S3 CD O  •H <H  •rH  <M  EI  a ro o  o  CH  -l->  •M  S3 CO •  CM \  rH  CM  117. (Table No. 3) show ahat the d i f f e r e n c e i n the weights of the chicks which were fed 1/2% P i l c h a r d O i l No. 1 or 1/4% P i l c h a r d O i l No, 2, or 1/2% God L i v e r O i l were d e f i n i t e l y of no . s t a t i s t i c a l significance. Discussion: At the end of eight weeks, the average weight of the chicks that received 1/2% P i l c h a r d O i l No. 1 or 1/4% P i l c h a r d O i l No. 2 or 1/2% Cod L i v e r O i l compared very favorably v/ith those reported by Buckner e t . al.(24a), Rlngrose and Norris (185) and Record e t . a l .  (182).  According to Record e t . a l , (182), at the end o f eight weeks the average, w eights of chicks which were f e d various amounts of Vitamin A supplement per 100 grams of feed were as follows: Supplement  -  40 units.as C.L.O. 80  11  Aver. Weight 'Qm. 504 9.4  " C.L.O.  532 8.6  160  «'  " C.L.O.  240  "  " C.L.O.  581 13.0  320  "  " C.L.O.  542 9.9  1% poultry  -  C.L.O. (700 u n i t s )  564 13.8  564 7.7  Records e t . a l . data show that the chicks which received 160 u n i t s of Vitamin A per 100 grams of basal r a t i o n grew as w e l l as the. chicks which received 240 and 320 u n i t s respectively.  Furthermore, the average weight of the chicks which  received 160 u n i t s was equal to those which received 1% Cod L i v e r ©il (700 u n i t s ) .  Since the chicks which were fed 40 or  118. 80 u n i t s of. Vitamin A showed symptoms of avitaminosis A, they concluded that 160 units of Vitamin A per 100 grams of feed may he considered  as the minimum requirements of chicks up to  eight weeks of age. The average weight of the chicks which were fed 1/2% P i l c h a r d O i l No. 1, or 1/4% P i l c h a r d O i l No. 2, or 1/2% Cod L i v e r O i l (Table No. 1) were almost i d e n t i c a l with the average weights of the chicks of the above mentioned i n v e s t i g a t o r s . I t may be concluded, therefore, that the chicks which were fed 1/4% P i l c h a r d O i l Np, 2 or 1/2% P i l c h a r d O i l $o. 1 received at l e a s t 160 units of Vitamin A per 100 grams of feed from t h e i r respective q u a n t i t i e s of P i l c h a r d O i l . Since 1/4% P i l c h a r d O i l No. 2 produced a r a t e of growth equally as good as 1/2% P i l c h a r d O i l No. 1, i t would appear that the Vitamin A. potency of P i l c h a r d O i l No. 2 was greater than that of P i l c h a r d O i l No. 1. Since 160 u n i t s of Vitamin A per 100 grams of feed i s considered by several i n v e s t i g a t o r s to be the minimum requirement of chicks up to eight weeks of age, i t would appear that l / 4 % of a f i s h o i l containing a minimum of 600 units of Vitamin A per gram of o i l (minimum requirements of U.S. Pharmacopea) would meet the minimum requirements of growing chicks to eight weeks of age. The r e s u l t s of t h i s experiment show that 1/4% of P i l c h a r d O i l No.2, and 1/2% of P i l c h a r d O i l No. 1 amply supplied the above requirements.  I t may be concluded, there-  f o r e , that the Vitamin A content of the two samples of P i l c h a r d O i l used i n t h i s i n v e s t i g a t i o n ranged between 300 and 600 units  119. of Vitamin A per gram of o i l . Possibly another way of estimating the Vitamin A potency of the P i l c h a r d O i l used i n t h i s experiment i s through estimating the t o t a l feed consumption and the amount of P i l c h ard O i l contained t h e r e i n . Unfortunately no record was kept of the amount of feed consumed by each group of chicks. However, the data reported by several i n v e s t i g a t o r s (126a), (127a), and (128a) show that normally developing White leghorn chicks w i l l consume 3.6 pounds or 1,634.4 grams feed up to eight weeks of age.  This amount of feed would contain 4.086  grams of P i l c h a r d O i l when 1/4% i s added or 8.172 1/2% i s added.  grams when  Since the basal r a t i o n was pra.ctica.lly free  from Vitamin A, the chicks must have of necessity derived t h e i r Vitamin A requirements from t he r, bove amounts of o i l i n the 1/4% and 1/2% groups.  Since, as has been shown before, a  r a t i o n must contain 160 u n i t s of Vitamin A per 100 grams of feed to promote normal growth of s i n g l e comb White Leghorn chicks to eight weeks of age, the t o t a l number of Vitamin A units supplied i n the r a t i o n to t h i s age would have to be approximately  2615.04 u n i t s .  In view of the normal rate of  growth attained by the chicks which received e i t h e r 1/4% P i l c h a r d O i l No. 2(4.086 gm. o i l ) or l / 2 % P i l c h a r d O i l No. 1 (8,172 gm. o i l ) i t may be concluded  that the above number of  units were supplied i n the respective amounts of o i l ,  If  4.086 grams supplied 2615.04 u n i t s of Vitamin A, then 1 gram of P i l c h a r d Oil,.No. 1 would contain 640 u n i t s of Vitamin A. S i m i l a r l y the 1/2% P i l c h a r d O i l No. 1 would contain at least  120. 320 units of Vitamin A per gram of o i l . B i e l y and Chalmers (16) have shown that the minimum requirements of growing chicks up t o eight weeks of age are 50 units of Vitamin A per day.  On t h i s b a s i s the chicks i n  the present experiment must have received during the entire period(50x56) 2,800 units of Vitamin A.  In accordance with  the method of c a l c u l a t i o n shown above,.the sample of Pilchard O i l Ho. 1.would have contained 343 units of Vitamin'A, while the sample No. 2 would have contained 685 units of Vitamin A. Whether the Vitamin A potency of P i l c h a r d O i l i s estimated on the b a s i s of Record e t . a l . (182).minimum requires ment; per 100.grams of feed or B i e l y and Chalmers (16) minimum requirements of 50 units per day, the r e s u l t s are i n close agreement, i . e . the Vitamin A content of P i l c h a r d O i l Ho. 1 i s over 300 units and P i l c h a r d O i l Ho. 2 over 600 u n i t s . Both methods of c a l c u l a t i o n show that P i l c h a r d O i l Ho. 2 would meet the minimum requirements f o r Vitamin A as s p e c i f i e d by the United States Pharmacopoeia f o r Cod l i v e r O i l .  121.  Experiment 2-  The basal r a t i o n used i n the tests? i n Experiment 1 was s i m i l a r to that employed by Elvehjem and Hen (75) i n t h e i r studies of 'Vitamin A.  I t consisted of 59 pounds of white corn,  25 pounds of wheat middlings, 12 pounds of casein, 1 pound calcium carbonate, 1 pound calcium phosphate, 1 pound s a l t , and 1 pound i r r a d i a t e d yeast.  Because of the fact that white corn  i s very expensive and not obtainable i n Canada, i t was deemed necessary to d evelop a r a t i o n c o n s i s t i n g of commonly a v a i l a b l e Vitamin A-free g r a i n s . Eor t h i s purpose, a r a t i o n s i m i l a r to that used by Erohring and Wyeno (86) was l i m i n a r y test conducted. the experimental  compounded and a pre-  Unfortunately a large percentage of  chicks developed slipped tendons.  l y , the r a t i o n had to be  Consequent-  discarded.  At the. time the present experiment was the cause of s l i p p e d tendons was not known.  i n progress  I t was generally  believed then that an improper balance of calcium and phosphorous was mainly responsible for the occurrence of slipped tendons.  However, several i n v e s t i g a t o r s have since shown that 10  to 20% of ground oats, added to a r a t i o n which o r d i n a r i l y i n duced slipped tendons, tends to prevent or diminish their occurrence.  Eor t h i s reason, i t was  decided to. include 16% oats  i n the r a t i o n s used i n t h i s experiment and modify the calciumphosphorous r a t i o by using various amounts of milk and meat. The l a t t e r also provided the animal p r o t e i n i n the r a t i o n , instead of casein which was used i n the r a t i o n of the f i r s t experiment.  Casein, unfortunately, contains a v a r i a b l e , but  appreciable, amount of Yitamin A, and therefore , i s not quite s u i t a b l e f o r use i n a basal r a t i o n f o r Yitamin A -experiments. To obtain f u r t h e r information on the r e s i d u a l amount of V i t a .min A i n casein, i t was decided to compare a basal r a t i o n containing casein with other r a t i o n s containing varying amounts of milk and meat. Thus the r a t i o n s used i n the present experiment varied i n t h e i r calcium and phosphorus content, and also i n the source of animal p r o t e i n .  The percentage of  Calcium and Phosphorus i n the various r a t i o n s was as follows: Ration  Calcium %  Phosphorus %  No. I  1.426  0.628  No. I I  1.579  0,699  Ho. I l l  1.865  0.853  No. IV  0.978  0.608  A l l r a t i o n s contained oats f o r the prevention of slipped tendons, while none contained white corn. TAble No. 4 shows the various r a t i o n s that were used i n t h i s experiment.  I t w i l l be seen that rations I , I I , and  I I I contained 17-g- pounds of animal p r o t e i n by weight..  Ration  No. I contained l2|-% powdered skim milk, and 5% meat scrap. Ration N0.II!  contained 10% skim milk, and  Ration No. I l l contained  meat scrap.  7t% milk, and 10% meat scrap.  Ration  No. IV contained 12% commercial casein only. Experimental  Methods:  Day-old s i n g l e comb White Leghorn cockerels were obtained from a l o c a l hatchery 25 chicks i n each.  and divided i n t o 16 groups of  At 24 hours of age, the chicks were placed  125. co  co  O  CQ  o  Oi  O rH  02  O rH  IrH  Oi rH  H O O  CQ  © H fH CO pq  CO  H-» CO CD  •H r-l  CQ  •CJ «H  cS  o  CD CO o3  COrn pq  m  o  +J a as •rl  T J  CD  CD  O  o3 co O O  £  •H  CD H-» 03 •H  4-> cti  A  O CO rH O 05 . 3 O ft  +»  crj co fH 03 fH CD H >H  H CO CO  CQ Q rH  CO  r  rH O Oi  co Oi  O  rH  o  £>  O rH  rH  co* rH O CO  W)  CD rH  u  CTJ CD  c\3 O  CO pq  CO  CO +>  a  •H rH Tj Id •H  CO fH pq  rH •H  rO rH  o  CO Oi  Oi  +3 CO CD  pq  CO  CD +•»  CO  o3  fH CD -P CO  >J o  •H  CO CO  CD S-i fH H  CQ  «5  CD J>H  CQ O rH  o rH  O rH  O rH  CO  ra  «3 CD  rH  rH rH CD  CO  rQ  CD rH fH  4->  o o  Oi  O.  i-k  CO  rH  CO H->  ra o  •H  J>  CO fH o  •H rH  CO 4-» 03  s fn  CD  3  pq  rH  r-IJOJ  Oi  rH  o  rH rH CD  T3 n3  fH CD H->  4-»  O  CO  •H  CO CO fH 03 fH CD rH  CO  CO  rH  o Oi  CO Oi  o rH  o rH  O rH  w  pq  CO  CO  o  Oi  CD fH O  ti0 co ©  rH  •H rH  co CD fH pq  O  rH  co  rH  IrH  ©  co  CQ >>  O  rH  +> o3 ©  o  rH rH ©  rH  ffl  rS  H-»  CO •rH  co  iH ©  +» CQ >> O  •d H J  •a'  CO  03 fH fH H  CQ  CO © f>H  124. i n f o u r - t i e r battery brooders, each t i e r being 24 x 60 inches and divided into two equal sized •compartments.  One compartment  i n each t i e r contained a heating unit of three carbon filament lamps» The four basal rations used are shown i n Table l b . 4. Each basal r a t i o n was divided into four l o t s and supplemented w i t h various amounts of Yitamin A„  The amount of supplement  added to Lot I was 1/4% of P i l c h a r d O i l ; Lot 2, 1/2% Pilchard O i l ; Lot 3, 1% P i l c h a r d O i l , and Lot 4 served as the c o n t r o l . Wesson O i l was used to dilute the 1/4% p i l c h a r d O i l up to 1/2% by weight i n order to assure thorough mixing of the o i l i n the mash.  One h a l f percent Wesson O i l was added to the  rations.  control  The respective amounts of o i l were mixed into the  mash.and run through a fine-meshed sieve. Mash and wa|er were constantly available to the chicks. A time clock c o n t r o l l e d the number of hours of a r t i f i c i a l l i g h t that- the chicks received.  I t was set f o r a twelve-hour day,  from 6:00 A.M. t o 6:00 P.M. At the end of the experiment, several chickswere examined to determine the percentage of ash i n t he t i b i a f i b u l a . The average ash analysis proved to be over 45%, thus i n d i c a t i n g normal bone c a l c i f i c a t i o n . Results: The average weekly weights of the chicks i n the various groups and r a t i o n s are given i n Tables l o . 5 and Ho. 6, and the growth fiurves, i n Figures 2 to 9, i n c l u s i v e .  The  m o r t a l i t y of the control groups i s shown i n Table H'o. 7.  125. COS  OJ  Ml col  o co  to  ©I  j> co to  CO  C-  02  Ml  «  KM  CO  to  CO CO to o CO  LO CO a co LO CO  CO  o LO  LO LO CO  H  LO 03  OJ  © ©  rH  •  ' •  LO to  tO CO OJ  OJ  CO  ©  o  o>  ©I  to e  1  LO to rH  £> £> rH  Ml  Oi rH  to  to  ©1 ©  OJ  B  e  LO  J> O rH  OJ  M  © ©  •  o LO  rH OJ  CO OJ  CO  J> to to  to  o> ^1  to OJ  to  rH  o o  o LO  tO LO  o LO OJ  OJ  CO OJ  o OJ  CO o tO  CO  CO o OJ  OJ to  rH  o rH  OJ . to OJ  CO to OJ  to to rH  O o>  LO  o  CO  rH  CO  rH  to  OJ  OJ  o rH  OJ o rH to  ©  to  LO  OJ r-  co  OJ  CO CO  to £>  H  4  rH  OJ  rH  OJ  to  ©  CO  CO  Oi  £> LO  O OJ'  to CO  z> o  o o rH  to o rH  OJ 'rH rH  CO LO  O LO  Z> OJ  CO  to  to to  LO to  o to  to CO  to CO  o o  o  O  o  rH  rH  «  o .rH CO  ©  »  LO  co to  co to  rH  ft  S3 O •H 4-> CO pH  I'- .. °  «  O •H 4J CO £H  iH •H o  rH •H O  rH •H O  H  t) U  •d  43 d rH •H  43 o rH •rH  43 o rH •H  ft  \  rH  CO  ft ^  •  a  o  to  ft  rH  »  9  •  o OJ  o o  u  •4-9  rH  CO  Oi  o> ©  9  9  «  «  •r-i •H  b 8  OJ  \  „  £> rH  to iH  CO  9  OJ to rH  o  *  9  rH  O  rHl  to  9  «  J> rH  e  CQ  «  »  e  k  CQ 1  o  «  o o -?p  rH  OJ  •  iH LO  OJ  o  rH  OJ  •d rH O  1  0  «  o  LO  to LO »  OJ  OJ  9  »  o o  "H<  o rH LO  to  ©I ©  rH to OJ  o to  to  to  LO CO o  OJ  «  S3 o  «  to to c-  rH £>  e  o o  «  LO o 2>  CO LO  ©  o o  •  «  OJ  to  OJ  M  r~  OJ  to  CO iH to  to  rH  ©I  to to £>  «  LO Ml •<D|  «  Oi  o to to  •  e  CO  to  ©  to  to  *  $3 o • *H +3  d  fH CO 43 O  r-i  ft  •d fH  cO 43 o rH •H  ft ^  OJ  \  O  •d fH cO .s3 o rH  •r-i ft  rH O fH  S3 o  126. CQ  <o to  © ©  to to  CQ M © © It  to o  CO  CD .'  o  . ©  to o H  u  IH bO  rH CO  ©  «  C  •H  P CO rH  M  O  M  >1  f3  LO CQ  bO  •H CD  •© ©'  ce  ©  CQ  © ©  tO o H  to  o oQ  to  CD  - OJ  o  o  CO  o  00 OJ  co  o o-  CO 02  CO CO OJ  £-  OJ o  CO o  CO to OJ  e  »  r> o  IO to  to to OJ  CO CO OJ  to 00 rH  to OS  OJ  CO  O  CO  H  to  to  e  rH  2>  rH  rH  r> o  Z>  rH rH to  CO  CO 6  Cr-  CO  •  ca  to  rH PH  o 125  £ i  O; •H 4-» crj M l  co OJ  to co OJ  OJ  OJ  H  9  o CD rH  rH  O O  rH OJ  CO  o 02  to to  OJ  rH  IO rH rH  rH OJ rH  to rH rH  rH rH rH  to to  rH CO  OJ OH  OJ  IO •c-  to' to * to z>  •xH O  CD  CO o CO E>  o to  o o  o o  O o o  OJ  •  o  ©  tO  e  Q  0  ©  CO OJ  ..  «  o  CD CO  o  rH •H  H •H  -d fH CO  Tj  •d fH CO  -d fH cd  -d fH CO  o rH •H  o rH •H  o rH •H PH  o rH •H  -d f-r n3 rCi u rH •H PH  ••rt  01  «  rH •H  \ rH  CO  Pi  OJ  > —  to rH  o o OJ  .rH •rH O  &  CD rH  CD CO rH  rH <H O  o  ct5  ©  rH •H o  C  -P  ©  to CO rH  CD  «  &  to a  to  to rOJ  *  «  & ©  «  to CO  e  8  rH  «  CO  •d rH O  2  ©  IO  OJ  »  H  0  CO z> OJ  to  o  ©  C0 rH  CO  to o>  OJ  to to  «  OJ to  to OJ  ©  to  CD C-  H H  CO  CO  o  ,£  •ss. rH  ©  rH Z> o r> to to  CO  H to  rH rH  r-i  o  OJ  •e  o to  to IO  ©  © ©  CD IO  o o  o r~ to  to  o to  CM  CO OJ  to  CO  ©I ©  O 00 c» OJ to to  to  trH  CQ  M  o  . «  to  OJ CD  #  to  OJ  M  o o  to  •  CD bp  to  o to  CQ  o o  rH t>-  o  to  *  tO  to zr-  CD to Z>  a  o o  ©  co  rH to to  OJ  « oi  o  ©  to to to  i>  to to  o o  o  CO CO  CO  © © CQ  <o  CO  CQ  •w  to to  OJ  «  R  rH O  m  125 o  M l  ^!  o rH •H PH  \  rH  OJ  ^.  rH  rH*.  \  £>  o  SK.  •H  O  o  0  o  r-i  r-l O fH  a o  •&  .  127, 800 -760  Figure No. 2  -720  Growth curves of chicks which received Ration #1, plus various supplements of Vitamin A.  .680  1. \$ P i l c h a r d O i l .  :640  2. i% P i l c h a r d O i l .  .600  3. 1% P i l c h a r d O i l . 4. C o n t r o l .  -560 -520 CQ  -480  fH  J440 -S 4400 J360  i M CO  fH ©  4320 |j .280 -240 .200 .60 ,20  /  300  Figure No, 5. .760  Growth Curves of Chicks which received Ration No, 2, plus various Supplements of Vitamin A.  130.  800  Figure No. 5  760  Growth Curves of Chicks which received Ration No* 4, plus various Supplements of Yitamin A,  |720 680  1, \% P i l c h a r d O i l .  640  2, i $ P i l c h a r d O i l . 3. 1$ P i l c h a r d O i l .  1600  4. C o n t r o l ,  560  CO  S 480 440 -S 400 -H © 360  S FH  ©  320 «{  / / /  / /  1 / k /  V  1st.  800 Figure Ho: 6,  -.760 • 720  Growth Curves of Chicks which received ^ P i l c h a r d O i l ,  .680  1,  Ration No,'. 1,  2,  Ration No. 2,  -640  Ration No. 5, 4.  .500  Ration No, 4.  .560 .520  4440  -5  1 Week Age i n Weeks — — i —  ;  i •  -•800  :  Figure No. 7  .760  Growth Curves of Chicks which received %fo P i l c h a r d O i l .  .720  1.  Ration No, 1»  -.660  2.  Ration No. 2.  -£40  3.  Ration No, 3.  4.  Ration No. 4.  / 1  I i 1  /  I  -.600  /  /  .560  /  1520  /  CQ  |480g  -1440*5  s HOC"0 1360 0 k  0  -L320-S;  .580 540 -200 • 160 .120 80  1 Week Age i n Weeks -—i  800 Figure Ho, 8  .760  Growth Curves of Chicks which received ifa P i l c h a r d O i l . 1.  Ration No. 1.  2.  Ration No. 2.  3.  Ration No. 3.  4.  Ration No. 4.  134.  340  Xo5 © I  rH O  43  cO © •d  t>>  ©. 43  © rH  fH  CQ  O  o CQ  fH  CO  ©  cO G  a  ©  42  CQ  W  ©  •d ©  PH  O 41 CQ  «  m  G  col  4-> •H r  d  oj  M o rH O  o  m  o o O rH  EH  H  a: EH  4J  -1-  G  CQ  •H  © G  H->  +'  ~|-  •H  S.  =  CM  CM  o  o  rH  4H  +>  o  M  P3  © fH  CQ !>  ^ © 3 0'43 C Q  43 4->  CmQ - H rH  © 43  © o  -h +> •H  -d  o  o 43 01  rH  •sF CM  -f i-  4->  -t'  •ri  CO  + =  =  rH  rH  o  -f  T  ~f* r  -t-» •H  s-  CM  r-  co  ~t"  CM  +  -+  + +  +  -f—  ©  is  +  +  cO G  xi © © rH 43 02 o  ^3  fH o CQ  fH  H co © 43  -+ 43  43  ©  CQ  4->  43  •d ©  G  43 CQ  -t  •H Xi •d  rH O >>  fH o C oQ <H 43 © CO Si  •+  CQ  O U •H ©  ©  id  •d -d  N  •H © H 4:) CO •H H-> <H -S-> 4-> O CO O fH O ft © © CQ rH O 4d a X< -H EJ © fH is +> " d *H o G © rH  CQ  CQ  -W • rH 4 3 O H-> >> ro ©  43  H  P H CO ©  43  LO •  !  ft PH H-> © o G « CQ •ri H-» * d © O © o  <H «H O  rH  CO  o  43 T  43-+  •rH =  rH <tf .CM rH CO CM rH rH H-  i-  '  +  "t 43  i-  :  CO  LO  CO  rH  CT> <vH CM CO CO CM CO rH  CQ  •M  o bO •H G  CM CM  -ri  CM  o> >»  LO  CM  o>  rH  • fH O  CQ  M O A4  i  CM  ofij^d  CO  G  o  &\  •& CQ O •H  43 4-> 4-S  O  1-  r~  O i+H : o  43  1  CO fH  O  4-3  S3  CO  o G fH O H-» - H  G +>'  o ro o P3  o> CM  CJ> CM  CO CM  CM  CO  t.O CM  136. The growth rates of the control chicks are i n accord w i t h those of Blvehj em and Neu (75), and B i e l y and Chalmers(16) By the end of the t h i r d week avitaminosis A sygiptoms . were observed amongst the control chicks fed Rations No* I , No. IT, and No. I l l * The chicks fed Ration No. IV d i d not show symptoms u n t i l the fourth week.  By the f i f t h week, a l l control  chicks showed d e f i n i t e symptoms and were unsteady o n t h e i r f e e t , and crouched on t h e i r haunches.  In advanced stages, the  chicks were found l y i n g on one side w i t h t h e i r heads f a l l e n • forward.-  The feathers were very r u f f l e d , end the beaks and  shanks were without any pigmentation.  In s p i t e of being i n a  weakened condition, the birds made considerable e f f o r t to eat and drink, but they a l l l o s t weight, however, before dying. In a l l r a t i o n s used, a condition was noted i n which feed adhered to the beaks and p a l a t e of the control chicks, and the chicks which received 1/4% of o i l . I t was necessary t o remove t h i s dry, hard accumulation at least two or three times a week.  At four weeks of age, some of the chicks showed a  l a t e r a l displacement of the mandibles, while i n others, the upper and lower mandibles became permanently curvetf.  The  cause of t h i s condition was, no doubt due to'an impairment of the mucous secreting glands of the mouth, induced by a lack of Vitamin A i n the r a t i o n . No t y p i c a l ophthalmic lesions v/ere observed, yet seve r a l chicks shoed s l i g h t s.oreness around the eyes.  These ob-  servations are i n agreement with Elvehj em and Neu (75).  137. A l l chicks that died during the course of the experiment were autopsied and the r e s u l t s or f i n d i n g s recorded (Table 7),  In a number of the c o n t r o l chicks the r e n a l tubules  .were g r e a t l y distended, and the kidneys f i l l e d with accumulations of urates.  Several control c h i c k s , however, exhibited  a t a x i a , without any v i s i b l e accumulation of urates i n the urinary t r a c t .  The chicks s t i l l l i v i n g on the control diets at  the end of the experiment (8 weeks) were k i l l e d and postmortemed.  The two chicks l i v i n g on Ration No. I I , and one on  Ration No. I l l , showed urates In the kidneys and ureters, and t h e i r general appearance and condition indicated that they would have d i e d , probably during the ensuing week. Those on Rations No. I and No. TV shoed s l i g h t accumulations  of urates  i n the kidneys, and no doubt, they would have died i n one or two weeks. The fact that several of the c ontrol chicks l i v e d to the end of the eighth week would i n d i c a t e that the basal r a t i o n s used i n these experiments were not absolutely free from Vitamin A.  Such r e s u l t s are in agreement with the findings of other  i n v e s t i g a t o r s (169)*  Of the four r a t i o n s used, Ration No. 1  (containing 12^% milk and 5% meat scrap) and Ration No. IV (containing 12% commercial casein) apparently had appreciable amounts of Vitamin A.  There were seven and s i x chicks,  r e s p e c t i v e l y , l i v i n g on these c o n t r o l d i e t s at the end of the experiment.  On the other hand, i t would appear that Rations  No. I l and No. I l l (10% and 7 if* of milk r e s p e c t i v e l y ) contained :  the least amount of Vitamin A, since only two chicks on Ration  138. No. I I , and one chick on Ration No. I l l survived t i l l the end of the eighth week.  The s u r v i v a l of only one or two chicks  on these l a t t e r two d i e t s Is most l i k e l y due to the i n d i v i d u a l ? reserves of Vitamin A i n the chick as received from the mother hen. The m o r t a l i t y i n the groups which received the various supplements of Vitamin A during the experiment was less than 22%, demonstrating  that the chicks were brooded under s a t i s -  factory conditions* Up to f o u r weeks of age, the chicks i n a l l groups, except the c o n t r o l s , grew at an almost uniform r a t e . After t h i s , with one exception, the rate of growth of the d i f f e r e n t groups varied d i r e c t l y , according to the percentage of P i l c h a r d O i l fed.  For s ome unaccountable  reason, the group  r e c e i v i n g • the•• \% P i l c h a r d O i l i n Ration No. I grew faster than the 1% group.  This v a r i a t i o n was noticeable at the end of the  second week of the experiment.  As the same sample" of P i l c h a r d  O i l was used to supplement a l l r a t i o n s , the v a r i a t i o n i n t h i s r a t i o n cannot be a t t r i b u t e d to the Vitamin A potency of the o i l used. (See Tables No. 5 and No. 6, and Figures 2 to 5 i n c l u s i v e ) . The growth curves of the chicks which were fed the various basal r a t i o n s , but the same amount of Vitamin A supplement, are shown i n Figures 6, 7, 8, and 9. The growth curves of the chicks which r eceived •%% P i l c h a r d O i l i n Rations No. I , No. I I , No. I l l , and No. IV are shown i n Figure 6.  I t w i l l be seen that Ration No. IV  (12% commercial casein) produced the f a s t e s t growth while  159* Ration No, I I (10% milk) was next i n order.  The r a t e of  growth-cm Rations No. I (lSg-% milk) and No. I l l  micbk) was  p r a c t i c a l l y the same, although considerably behind that produce d by the other two r a t i o n s . In the case of the chicks which received Y/° of P i l c h ard G i l i n the respective rations (Figure 7) i t w i l l be noted that Rations No. I , No. I I and No. IV gave p r a b t i c a l l y the same growth while Ration No. I l l gave the poorest growth . In the case of the chicks which received 1% of P i l c h a r d O i l (Figure 8) i t w i l l be seen that Ration No.  IV  produced the greatest growth while the other rations were i n the f o l l o w i n g order; Ration No. I I ; Ration No. I l l ; and Ration No. I producing  the poorest growth.  The growth curves of the various c o n t r o l groups are found i n Figure 9.  A study of these curves reveals that at the  end of the eight-week experimental period, the chicks on Ration No. I (7 chicks livings) were r a p i d l y l o s i n g weight while those on Ration No. I I (2 chicks l i v i n g ) , showed a marked decrease i n weight, weighing considerably l e s s than the chicks fed Rations No. I; No. I l l ; or No. IV.  The i n d i v i d u a l weekly weights  showed that the c o n t r o l chick, that l i v e d t i l l the end of the experiment on basal Ration No. I l l , grew at a normal r a t e .  The  r a p i d r i s e i n the growth curve between t he seventh and the eighth week of the c o n t r o l c h i c k s which received basal Ration No I I I i s due to the s u r v i v a l of t h i s exceptionally large chick. The chicks fed Ration No. IV (6 chicks l i v i n g ) showed a gradual  140. r i s e i n the growth curve. This, r i s e i n the growth curve may he taken as conclusive evidence that the commercial casein used i n t h i s experiment contained appreciable amounts of Vitamin A. The mean body weights of the d i f f e r e n t l o t s were subjected to s t a t i s t i c a l i n t e r p r e t a t i o n i n Tables No. 8 to No. 14, i n c l u s i v e .  For the r e s u l t s to b e considered s i g n i f i c a n t ,  the d i f f e r e n c e of the two means must be equal to or greater than three times i t s probable e r r o r . In a l l r a t i o n s except Ho, IV, there was a s i g n i f i c a n t difference between the average and the i% and L% groups.  Yfeight  of the \% and  groups  Ration No. IV, however, showed only  a s i g n i f i c a n t difference between the average weight of the and 1% groups.  The fact that i n Ration No. IV the difference  between the average weight of the  and the \% groups was not  s i g n i f i c a n t , i n d i c a t e d further that Vitamin A was presehtrin the commercial casein used i n t h i s experiment. Thus, the superior growth found i n the i% group pf Ration No. IV may be accounted f o r by the assumption that the Vitamin A  -present  in  the casein .supplemented that supplied by the P i l c h a r d O i l . There was no s i g n i f i c a n t difference betweenthe average weights of the  and 1% groups i n any of the r a t i o n s .  This  evidnece demonstrated that -§-% of the P i l c h a r d O i l used i n this experiment supplied s u f f i c i e n t Vitamin A to promote normal growth of chicks up to eight weeks of age.  This further sub-  s t a n t i a t e d the findings of Wood (266), that \% of a good grade of P i l c h a r d O i l meets the Vitamin A requirements of growing chicks to.' eight weeks of age.  Furthermore, these r e s u l t s show  141. to  CO OJ  *  rH -H  "sf<  «  rH 1 1  CO  IO  »  OJ  to  rH  rH  rH  rH  to CO  rH  o>  CD  CO OJ  rH  OJ  a  rH  +.1.  to  02  +  OJ o  «  to  H  1  H-  rH  to to  rH  rH rH  rH rH  to .OJ  CD  CD  «  co  rH  4 i  i i o OJ  <o to  to o  9  +1 TI tO tO »  rH rH  <i  fa o CO  i  O •rH HJ . CO •rl  •  CO  t>  f! o  ©  9  C5V  fH  eis  M  cd T 3  a  +1 CD CD  .  O  «  - t l  +1  o CO  o>  CD  o> CD  &  OJ OJ  CO  •  CO  -{"'!  o OJ  . «  CD  O  +1  o  •  CO  o OJ  IO  9  »  *  to CO  rH  to  CD  CO  CO -p  CO  col co  .1  o |25,  H ipl  N' EH  CD  O CO H CO  • •  rH rH  •H  ©  to  OJ  CO o e CO  rH  rH  rH  «  + 1 +1. OJ tO «  ©  o CO  «  tO CO  fc=J  CO CO  rH  e  a  O  OJ rH  1-1 to  rH  o  rH rH  1  z>CO H rH  + 1 +1 + 1  to  <o  *  »  CD  to o r~  ?>  OJ  OJ  O O  o o  to to  to CO  H  rH  rH •H  «  0  to OJ  o  M EH CO H EH  4-»  EH  ©  <J CO  r-i  © rH. Pi P)  •H  rH rH  rH •H  •H  fH CO  T* fH  •xi  Tj  o  o  rH •H fH  rCl  rH •H rH  O  •CJ fH  co  CO rG  OJ.-.  =5 rH PH  fl o •H  4-»  o  OJ  CO  W  rH •H  Cd r H  tt  rH  O  o  •  OJ  CO  o rH •H fH  rH O fH H-»  S3 O  o  « o  fH CO  o  rH •H rH  O  fH CO  o  rH •rH  PH  o  fH CO  o  rH •H fH  o  •H  +>  cd  «  \  rH  fH •+»  OJ  \  rH  ft  rH O  fl  O  rH  13  142. o  to  a in  0 TH •H ,Q O CO •H . H «H M H CO © >> o i  CO £> 9 O  r-l  HI  rri  - H to  + 1  4 |  tO o>  o  «  f  LO  tl  4-1  to  r-i r-i  CM rH  CM CO 9  CO  to  o CO  CM HI  to  CM • to  CM  CO  LO  A  to LO 9  tO 9  •Hi  LO  CM 9 rH  r-i  r-i 41  a  to r-i  r-i r-i  CM  9 LO  1  4  o  «  to CM  S3 o •ri 4->  CO •ri > ©  • co  «  4l  +1  o  o  •d H CO -d  £> 9 rH  *  s> o  ^'  cn  41 o  t l o  9 CO to  r-  •  •  o>  CO  CO  41  9  to  •41 o  co '9 £> CO  to  a>  to rH 4O I to 9 1  to «  LO  CO  4->  CO  o  4->  43  « I-l r-i  bo  •ri  ©  41 to to  S3 co ©  e CO LO to  o  CM  9 •  CM r-i  41 CM to  oo  00  to  •9 CO  CM HI 41  41' to to  9  to t>  to  Hi •rl  HI ••H  co 9 rH  «  ro  O o  9  o CO r-l  to to to  r-  r-i  + 1  LO 9  to r-i  o> LO  9  to  CM  -tl O  9  o to  O  o  o o  9  CM to  -(->  S3  8  ©  r-i  r-I ft  •ri o  o  ft  -d  •d  CO  CO 43  43  I-l  r-i  H  OJ  r-l  ft S3  o  •ri +5  CO  K  •if S3 o  o  •ri  ft  fH  o  •HI  ft  •ri  CO  P3  HI  HI •  'Hi •ri  O  O  •d  •d  CO 43  co  M  M  I •  o  HI •ri  ft  r-i  O fH H-> S3  o o  N S3  ;  43 o r-i  •ri  ft  o  •ril 4->l col  «  HJ  r-i  r-i  o  o  •H  H  ro  43 o  r-i •ri  •H  •d H  CO 43 o rH •ri  ft ^  ft  CM  ^  r-l  r-l  \  Hi O  U  +> S3 o o  143.  -p  © O  +s -  CO  •H  <H  •rH  •H S3  S3  UO •H CO  W) •H CO  HJ  cd  cd  •H  o  •H  •rH <H  •H S3  •H S3  •H  S3 CO  •rH  CH S3  fH  60  £3 &0  •rH CO  •H  bO •H CO  HJ O  CO  o  S3  o  •rH  S3  HJ  o  cd o •H <H •H  o  <H  cd  S3  CO  o •H  HJ  S3  HJ  •H S3  tao  CO  •H CO  HJ  o  o  © rH O  CO  ©  u ©  ©IrH  CH  CO  CO  o  o-»  o  CO  CO  oj  ©  CO  o 00  • ©  cO  rH rH o PH  CQ  CO  CH  •  o  rH  © o  +  1  CO  S3 ©  rH ©  03  rH  O  o  •CO  02  V5.  ©  en <H •H  £> HI - H  H  ©  o>  CO CO  co w  CO  to  CO  00  CO  <o  to  ©  rH +  H  + 1  1  CO  CO  rH e  » o  CO  to  «  rH + 1  <o  CO -  0  02 ©  H  +1 00 to «  00  rH  CO  n  S3 o  CQ •H  U  CO  f o  o  $3  OJI  Hi  rH  eg  c8  o  ^.  HJ  02  •H cd  rH  or  .:  Ol  \ r-i  H  o  •r-i  +> .  \ rH  rH  02 rH  \ rH  14-4. I  +»  © O  « CO  CO  •H <H •H  CH •ri  •H  •ri  d  o  OJ 02  o  ro  o  •ri  •ri.  «H •ri  «  03  CO  a  a  •ri  •ri  •ri  •ri  •ri  s  CO  CO 4-S  to  HI  CD  »—I  g SI H  CO  CO 4->  o  O IM  o S  HH  •ri  M  M  EH  <H  O  |25  co  o  •ri  «30  -p o  CO  ro o  o  •ri <H  •ri  w •ri  to  •p  c  +>'  fH  ©  o» CM  tO  ©  0  r-i  <H<  r9  £>  o  o  CT>  *  Hi  H  CM;  <H CO  42 O  •ri  P)  M  ft  e  o  525  a  rH  CO  EH  EH!  J=£)  0)  r=3  03  PR Hi  .  ©  o!  PHI  to o  O  rH +i  o  o> to  ©  H © <H  »  •ri  n  • LO e  rH 4 1  o o  .  ©  to o Hi  o CO  to  H  LO  H  •^  rH  £>  HI  co  HI  CO  + 1  +T  + 1  e  9  o  to• CM  z> to  to a>  H  LO  •  0  0  rH  +to 1 LO •  to  o o <5 O H Pq •H Hi  03  o  (12  tal  H CO  t  o o  CM  CM  •ri  o  r-i  O  HI  Pi  o  M  r-i  rH  r-l •  c8  <3  rH  \ rH  o  •ri  CO  \  r-i  r-i  CM \ r-l  •H 4-»  CO  CM  rH  145,  H-> S3 cd o •H <H  © O S3 CO  o •H CH •H  to  CQ  +» O  •H CO  •H  S3 w  S3  tK)  •H  CO  CO  .+> o  +>•  S3  cd o  o •H  •H tH •H  t+H •H  S3  S3  W  •H  •H  CO  CO  CO  a o  J5=l  I  •H  •rH  bO  •H  cd o •H f+H •H S3  S3  CM  S3  •H  4-5  cd o •H fH •H S3  o  . TH  S3 &0  +».  S3  cd  o  rH  13  l ° © fH O f ,  a  in to  ©  OJ  C H P  CO OJ  LO  OJ  to  o  .e CO  CO  cd  CHI  •HI <D  fH PH  CO S3  ©  CO CO «  o  © o S3 © fH  © CM «H M  S3 o  CO •rH  CO  f  r-i  o CO rH  CO OJ  CO CO  rH CO  C-. rH  CO  T1 in  CO rH + 1 CO  CrH + 1 CO  o CO  CO  o OJ rH  CO  03  .1. + 1  02  o  «  ©  a  CO CO  00  Oi  CO  ^  rH  rH  rH  =3 rH  eg  a, «  >  rH rH  ©  rH  -i-  i rH  ^«o  d>  in  •r-i: ' rH.  Oi  CO  5^  rH  rH  rH  CO OJ  <3 03  rH  o o  0  rH  .r-i  146.  +» © o  CD  CD  •H <H •H  HJ  G  O  G  O  •H <H  •rH  •rl  <£»  a  CO  •rH  H->  HJ  HJ  o  O  O S2i  •H  •H  bO  CO  S2i  «H •H  G  CJ •bo •H CO  •H «M  •H <H  •rH  G  W  125  cd  o  •H  <H •H  •rH  G bO  o  G  cd  CO  o  HJ  G  G  cd  o  43  HJ  G  CO  G  o  •rH  G  t>a  •rH  CO  W) •rt CO  550 •H CO  •H CO  HJ  HJ  - p  O  o  &  O  is  '£ r-r  rH  o  ©  .. ©  ©  o  00  o  ©  o  w o  o CM  co  rH  CO «  CO  Oi  ©  . e rH  o  W  CH ,Q  to  PH  CQ  ml  a  rH  CD ,©  o  o ©  ©  fH ©  z>  rH  ~H CO to  o  rH  CO to  CO  CO CO  IO  •CV2  CO  o  .  rH  =*fc  VS. o  CQ 'H CD  rH  Oi  9  rH  CO  H  O CO o  £-  to  iH. ©  CO  rH  O  o r-  rH  1  -t-  +! Oi co o>  CO ©  CO  o  VS.  Vl  0  CO  =*:  ^  ^  \  \  Oi  Oi  rH  rH  rH  H  rH  \rH  rH  rH  «  + 1 • + 1+1 co  CH •H  G  CO e  0  \  > c3  Oi  -CV2  rH  rH  \  \  eg  c8  Oi  to  Oi  Oi  O o  Oi  \ rH  Oi  \ ' rH  \ :rH  H  147. I  © o CO:  o  fa  •rH <H •H  a  •H  CO  4-> S3  4-> S3  •ri  •ri  «H •H  <H •H  •H <H •H  •H  •H «H •H  •H  W) •H  •H  •H  4-» O  4J O £H  4-> O  to o  •<*  O LO  to  rH  O  Oi CO e rH  LO  OJ rH  co  rH  rH  tO rH  + 1  -(- I  + 1  4-> S3 CO  S3  CO  o  O  •PH  <H  S3 bO  S3 &D •ri CQ  •rl  to  4J  ro o  S3 (50  CQ  +»  CQ O  o  CO  o  S3  CO  s  4-J S3  CO  o  •ri  S3 ttO  CQ  4-> S3  CO  o  S3 &0  CQ  r-l  H o  © O H 'S3 © H © 'tHj CO  to  CO  CO e rH  02  • 4  0  nlo  H  PH  CQ S3  ro © o  © o  S3 © M  ©  S3  rH  LO Oi .• LO rH  +1 CO  + 1 OJ  a 00  co  oo o «  to  OJ  o  CM  LO  O O  -H< to  to  ©  O OJ  o>  OJ  «<*  to =fts  o  fH  o o  to to  CO  CQ •H  cO  co rH +.!.  H  rH  «H  c8 rH •  rH  rH  rH  rH  <8  c8  OJ .  rH  CO  ««*•  =*!=  ¥t  ^ .rH  OJ  rH  rH  rH  r-i  148. that no immediate advantage was derived from feeding an excess of Vitamin A.  •  The groups which received the same percentage of •Vitamin A supplement, hut i n d i f f e r e n t r a t i o n s , were compared s t a t i s t i c a l l y i n Tables No. 12 [•£%) , No. 15 (£%), and No.  14  (1%). A study of Table No. 12 ii%)  revealed that the average  weight of the chicks fed Ration No, IV (commercial  casein) was  s i g n i f i c a n t l y higher than the average weight of chicks fed Ration No. I {12%% m i l k ) .  S i m i l a r l y , the average weight of  cbicks fed Ration No.. I I (10% m i l k ) was s i g n i f i c a n t l y higher than the average weight of chicks r e c e i v i n g Ration No. I l l ( 7 i % milk) , and the average weight of chicks fed Ration No. IV was s i g n i f i c a n t l y higher than that of those fed Ration No. I l l These r e s u l t s demonstrated the superiority of Rations No. IV and No. I I over the other r a t i o n s .  Since i t has been shown  above that Ration No. IV contained appreciable amounts of Vitamin A, t h i s r a t i o n cannot be regarded as a suitable Vitamin A-free b a s a l r a t i o n . Table No. 13 ( i % j does not show any  significant  d i f f e r e n c e i n the weights of the. chicks which were fed the various r a t i o n s .  Any trace of Vitamin A that may have been  present i n any of the r a t i o n s d i d not influence the rate of growth of these groups a.s i t . did. i n the case of the•%% groups. Such evidence again points out the fact $ hat when chicks up to eight weeks of age are given i% P i l c h a r d O i l , their Vitamin A requirements f o r normal growth are met.  149. The comparisons of the 1% groups are shown i n Table Ho. 14.  I t w i l l be noted that the only s i g n i f i c a n t difference  was that between the average weight of the chicks fed Ration "Bo-. IV and the weight of those fed Ration Ho. I . The difference between Ration No. I I and R a t i on No. I was nearly significant;. As p r e v i o u s l y stated, there was some unaccountable, reason f o r the slow growth of the group fed Ration No. I with 1% o i l as compared w i t h the group fed ^% o i l .  I t i s doubtful i f any  s i g n i f i c a n c e should be attached to t h i s d i f f e r e n c e . Discussion: I t w i l l be seen from Table No. 7 that, at the end of the eighth week, the group f e d Ration No. I I had two control chicks l i v i n g , and Ration No. I l l , only, one, and that these three chicks showed external symptoms of avitaminosis A. Since several i n v e s t i g a t o r s ( i l l ) have demonstrated chat chicks show marked v a r i a t i o n i n t h e i r i n d i v i d u a l stores of Vitamin A at time of hatching, i t ' may be concluded that the v a r i a t i o n i n l l v a h i l i t y of the chicks r e c e i v i n g the various rations was due not e n t i r e l y to varying traces of Vitamin A i n the r a t i o n s , but to variations- i n i n d i v i d u a l reserves of Vitamin A i n the chicks themselves. Since i t i s apparent that traces, i f any, of Vitamin A i n Rations No. I I and No. I l l were the same, the rate of growth of the groups fed the Vitamin A supplement becomes the most important  fa,ctor f o r consideration. The weights of the  chicks i n the various groups at the end ofthe experiment d e f i n i t e l y show that Ration No. I I i s superior to Ration No.  150.  I l l i h growth promoting powers. The combination of ten pounds of milk and seven and one-half pounds of meat scrap as used i n Ration No. I I can, 'therefore, he d e f i n i t e l y accepted as a. s a t i s f a c t o r y protein s u p p l e m e n t — p r a c t i c a l l y free from Vitamin A — ±  n  place of casein  f o r b i o l o g i c a l t e s t s with chicks. The growth obtained with Rations No. I and No. IV i s decidely greater than that with Ration No. I I , but i t has already been shown that these former r a t i o n s contained an appr e c i a b l e amount of Vitamin A.  Thus i n s p i t e of the increased  growth obtained, these r a t i o n s cannot be recommended f o r use i n Vitamin A determinations with chicks. Besides the cost and time involved i n obtaining Vitamin: A-free casein by e i t h e r a l c o h o l i c extraction or by heating i n an oven, there i s the p o s s i b i l i t y that some of the amino acids may be changed or destroyed i n either or both of these processes.  In the event of any such changes taking  place, i t can be r e a d i l y seen how growing chicks.  detrimental t h i s would be to  On the other hand, when a small quantity of  m i l k , along v/ith a good grade of meat scrap (Ration No. I I ) , i s substituted for treated casein, the cost per pound p r o t e i n i s lowered considerably, while at t h e same time animal p r o t e i n of high b i o l o g i c a l value i s provided. In order to avoid the influence of the i n d i v i d u a l reserve of Vitamin A, i t would be desirable to extend.the length of the Vitamin A experiments from eight to ten weeks.  This  extended period would allow the control chicks more time i n  151. which to exhaust t h e i r store of Vitamin A, thus assuring 100% mortality. I f the supply of Vitamin A fed to the breeding s • stock i s not c o n t r o l l e d , i t would appear desirable to e s t a b l i s h f o r chicks a depletion period s i m i l a r to that used i n the r a t tests.  While no d e f i n i t e time regarding the most s a t i s f a c t o r y  length of t h i s depletion period can be suggested here, the idea would appear to be worthy of i n v e s t i g a t i o n . The depletion periods of chicks hatched from f l o c k s r e c e i v i n g d i f f e r e n t amounts of Vitamin A would ha.ve to be ascertained i n each case. Such a procedure, i f adopted, would lead to the more uniform occurrence of avitaminosis A symptoms,death of control chicks, and more precise evaluation of the potency of Vitamin A carriers. Frohring•and  Wyeno (86) suggested that i n Vitamin A  t e s t s , chicks from only one breeding f l o c k should be used, or i f they are obtained from d i f f e r e n t breeding f l o c k s , that they be d i s t r i b u t e d equally amongst a l l the groups.  The l a t t e r  suggestion e n t a i l s the keeping of extensive records by the person or persons hatching the c h i c k s , end would tend to increase the f a c t o r of v a r i a b i l i t y due to the d i f f e r e n t v i a b i l i t y of the chicks from the d i f f e r e n t f l o c k s .  The idea  of using only one breeding f l o c k as a. source of chicks f o r Vitamin A determinations would appear to be more commendable. At present, the chicks used i n b i o l o g i c a l assays do not vary only from laboratory to laboratory, but they also vary w i t h i n a laboratory, according to the season of the year, s t r a i n of  152. b i r d s , and other conditions.  In Vitamin A assays the i n d i s -  criminate use of chicks from various f l o c k s i s undoubtedly responsible f o r the great v a r i a t i o n noted  i n the rate of  ' g r w t h and f i n a l weight of i n d i v i d u a l c h i c k s . I t probably also accounts f o r some of the v a r i a b i l i t y i n the weight of chicks w i t h i n a group at the end of the test period. To avoid such v a r i a b i l i t y , i t would be necessary to breed and maintain a s t r a i n of breeding stock g e n e t i c a l l y pure f o r rate of growth. Since 'the rate of growth and the f i n a l average weight of chicks at t h e end of the experimental period are the main c r i t e r i a used i n evaluating the pbtency of Vitamin A supplements, i t i s e s s e n t i a l that the chicks i n the various groups should grow at a uniform l e v e l .  To accomplish t h i s , the following  conditions would have to he assured: (a) The s t r a i n of b i r d s should be g e n e t i c a l l y pure for rate of growth. (b) The Vitamin A intake of the breeding stock should be c o n t r o l l e d . (c) The Vitamin A reserve of chicks should be depleted before the actual assay period Is begun. (d) A Vitamin A - f r e e r a t i o n that ensures r a p i d development of avitaminosis A symptoms should be used, (e) The Vitamin A-free r a t i o n i n the presence of s u f f i c i e n t Vitamin A sould promote rapid, normal growth. (f) The conditions of brooding and rearing should be uniform. With a l l of the above conditions under complete experimental c o n t r o l , i t should be p o s s i b l e w i t h i n four to s i x weeks f o l l o w i n g the depletion period to evaluate with great  X 53 e  p r e c i s i o n the potency of Vitamin A c a r r i e r s .  1 5 4 .  Exp e r i me nt No. 5»  The object of t h i s experiment was to compare the h i o l o g i c a l and chemical methods, of assaying B r i t i s h Columbia P i l c h a r d O i l for Vitamin A.  For t h i s purpose, three samples  of P i l c h a r d O i l produced at d i f f e r e n t stages of the f i s h i n g season were used.  Thus, information was also secured on the  e f f e c t of seasonal v a r i a t i o n on the Vitamin A content of P i l c h ard O i l . O i l No. 1 was produced e a r l y i n the season and O i l No 2, i n the middle of the season, and O i l No. 3, l a t e i n the season.  These o i l s were kept i n large volumes i n commercial  storage tanks.  A. sample of ea.ch o i l was taken from the res-  pective tanks and brought t o t he laboratory for b i o l o g i c a l assay. A p o r t i o n of. each o i l was tested by the antimony t r i c h l o r i d e test ( C a r r - P r i c e ) , and the blue unit value recorded. The results' of t h i s t e s t showed that O i l No. 1 contained four blue u n i t s , O i l No. 2, two blue u n i t s , and O i l No. 3, one blue unit. Experimental Methods; Day-old s i n g l e comb White leghorn cockerels were divided into seven groups of f i f t e e n chicks each, and placed i n battery brooders as i n the previous experiments.  They were  fed a basal r a t i o n which consisted of the following ingredients Ground Wheat .  38-g- pounds .  Ground Oats  10  "  Wheat Middlings  20  "  X 5 %3 o Wheat Bran  10 pounds  Skim Milk Powder  10  »  Meat Scrap  7i "  Ground Oyster S h e l l  2  Salt  1 pound  Ix-radiated Yeast  1  Total  "  11  100 pounds  The amount of Vitamin A supplement added to the r a t i o n was e i t h e r 1/8% or 1/4% of each of the three samples of P i l c h ard O i l .  Each o i l was d i l u t e d up to 1/2% by weight w i t h  Y/esson O i l , while the controls received 1/2% of Wesson O i l only.  A l l d e t a i l s of procedure were s i m i l a r to those describ-  ed i n Experiment I I . Results :  : • . :• The average weekly weights of the various groups are  given i n Table No 15.  Growth curves of these groups are shown  i n Figure No. 10. A study of the growth curves shows that the average weight of the groups which received O i l No. 1 are heavier at the end of eight weeks, than the groups which received O i l No. 2 or O i l No. 3 or the c o n t r o l .  S i m i l a r l y the chicks which  were fed G i l No. 2 show higher average weights than the chicks which were fed O i l No. 3 or the ba.sal r a t i o n alone. I t should be noted, however, that the average weight of the chicks which received O i l No. 3 was only s l i g h t l y higher than the average weight of the control chicks.  01  © © S3  « CO  CO  to,  o  © ©  03  © ©  9  to  LO rH o CO to  o Q  o o  9  o Ol  o o  OJ o •SP  to to o rH  CO to  o to  CO CO  o o  LO  *  «  a  OJ to  CO  LO co  LO co  o OJ  to co  o o  OJ to OJ  OJ  to  a>  to  o co  CO co  o  e  9  to CO OJ  to co OJ  to to M  LO OJ  to  CO LO  LO CO  r-i  LO  o  9  9  9  ©  to LO OJ  £>  ©  9  9  to to  to « to  a  LO CO OJ  CO CO OJ  00 to OJ  o> OJ OJ  o o OJ  CO LO  co LO »  CO OI e co r-  CO OJ  o  9  9  to  o to  rH  rH  rH  rH  rH  rH  o o  o OJ  to  O o  LO  o OJ OJ  01  © ©  CO o  rH  CO  HI  z> .rH  to  to  to to  9  «  03  © © to CO  © © OJ  © ©  O  OJ  to  CO «  a  •E-  a>  9  «  OJ  OJ Oi  rH  Oi  OJ OJ  rH  rH  to o  rH  rH  rH  OJ  CO CO  9  4  OJ o>  O o'  ©  9  O  to CO  LO CO  o> 00  co  (>  o CO  H  LO OJ  co CO  OJ to  o to  rto  o  e  OJ  Oi r-i  rH  O o  rH •  ®  CO LO  9  9  CO tO  o> LO  OJ  o  9  co  oo  rLO  t> LO  . e OJ  to  to  rH  r-i  rH  •H  •ri  LO  CO CO 9  CO LO  •d o|  CO] fH rH  rH  =*«  **!  rH  r H •H  •ri  O  o  •d  •d  co  43 o  © ©  iH •H fH  ri  co  43 o  rH •H  ft  o  -d  O  -d H  b 3 43  43  rH  •rH  o  •ri  Pi  cd  o  •ri Pi  0  •H O  -d fH CO  r-i •ri o  •d  U CO  43  .si  rH •H  rH  o  ft  o  •ri  ft  rH  P P a co  o fH  \ rH  9  CO rH to  CO CO  OJ o> OJ  tO Wt  £>  ©  CD..  ©I  LO rH o  LO  OJ  .  156.  o to o E> o to  \ HI.  \ rH  < r-i \  +». S3 o  157  158. , The c o n t r o l chicks showed the e a r l y symptoms of avitaminosis A at three weeks of age.  On post-mortem exami  na t i o n , considerable accumulations of urates i n the kidneys a nd ureters were observed. There were only three chicks l i v i n g i n t h i s group at the end of the experiment. were k i l l e d and examined i n t e r n a l l y .  These three chicks  They showed enlarged  kidneys containing large deposits of urates. K e r a t i n i z a t i o n of the e p i t h e l i a l t i s s u e of the r e s p i r a t o r y t r a c t was observed om several of the c o n t r o l chicks that died during the course of the experiment.  The fact that t h r e e chicks survived to eight  weeks of age on the c o n t r o l diet may be due to l a t e hatching (autumn) and to the large stores of Vitamin A that they could, as a consequence, receive from the parent stock at that time. With the exception of the chicks which were fed O i l No. 3 there were no external symptoms of Vitamin A deficiency i n any other groups.  The chicks which received 1/8% O i l No. 3  showed d i s t i n c t symptoms of avitaminosis A at four weeks of age.  At the termination of the test a t eight weeks, 50% of  these chicks had died.  On post-mortem examination,  accumulat-  ions of urates were found i n the kidneys and ureters of these chicks.  At the end of the experiment, several of the chicks  that were s t i l l l i v i n g i n t h i s group were selected f o r postmortem examination.  They too, showed t o p i c a l avitaminosis A  lesions of the kidneys.  The symptoms of Vitamin A deficiency  i n the group fed 1/4% O i l No. 3 were not so pronounced as those found i n the group which received 1/8% O i l No. 3. The s t a t i s t i c a l a n a l y s i s of the average mean weights  "159. of the d i f f e r e n t l o t s of chicks are given i n Table No. 16,  and  the s i g n i f i c a n c e of the d i f f e r e n c e between these weights i n Tables No. 17 and No. 18. t  A. study of these tables reveals,  t h a t at eight weeks of age the difference between the average weight of the chicks which were fed e i t h e r 1/8% or 1/4%  Pilch-  ard O i l No. 3, and the c o n t r o l c h i c k s , i s not s i g n i f i c a n t . This i n d i c a t e s that P i l c h a r d O i l No* 3 does not supply s u f f i c ient Vitamin A at these l e v e l s to promote normal growth of chicks up to eight weeks of age.  Furthermore, i t w i l l be seen  that the d i f f e r e n c e between the a.veragew eight of the chicks which received 1/8% P i l c h a r d O i l No. 1, end that of the chicks which received either 1/8% or 1/4% P i l c h a r d O i l No. 3 i s statistically significant.  The d i f f e r e n c e i s even more s i g -  n i f i c a n t between the former and the control group.  Similarly  the d i f f e r e n c e between the average weight of the chicks which received the supplement of 1/4% P i l c h a r d O i l No. 1 and any of these three l a t t e r groups, i s s i g n i f i c a n t .  Comparison of the  other groups did not regeal any s i g n i f i c a n t d i f f e r e n c e . Discussion: I t may be r e a d i l y seen from the above r e s u l t s that the sample of P i l c h a r d O i l No. 1 (early season) i s decidedly supe r i o r i n Vitamin A potency to the other two samples of o i l . S i m i l a r l y , the sample of P i l c h a r d O i l No. 2 (middle season) i s superior to the sample of P i l c h a r d O i l No. 3 ( l a t e  season)/  Because these o i l s had been stored immediately upon production, any d e t e r i o r a t i o n that may have taken place during storage would be most pronounced i n sample No. 1, and least i n sample  160. o  CO CO  HJ £ 4-> © -rH  •H rH O -H •H  + 1 .  o  «H' CO  OJ  OJ  + l  -+I  CO  o  r-i  CT>  rH  rH  « LO  r-i  O t>  •IO  4-1  rH  *  r-i  LO OJ o CO  o>  CO  oi CO e  ©3  r-i  CO rH  OJ  LO CO  e  co  co  .+ 1  +1  + 1  00 ^'  j> o  OJ  LO  OJ  CO CO e OJ  LO o  CO  «  «  co  ft  o  CO  OJ  S3  o  •H  4J cd  TJ M  3  O CO •  o rH  o rH  +1' CO  + 1 CO CO  + 1 OJ  o>  CO CO  CO  e>  CO CO  to CO  co «  CO  •H  © <H  LO  o  •  CO +1  co  «  rH  rA r-i  e  D~  s  CO r>  rH t l  rH 00  tt  rH  LO OJ  + 1  + 1  CO tO »  «  e CO  to o rH  HJ  CO  HJ  •H  ©  cd ©  tO  rH  1  + 1  rH  rH  co  tO  £> O  to  CO CO  o  rH  « .rH •  OJ  OJ  CO  =fe  =fe  r-i  rH •H  OJ t l £>  rH  o> 00  © S ©  •H  IO  rH •H  fH  *1  o  Vt-  s  0  o  rH •H ft  IO  O LO  o cd &  rH  + 1 +1 t  0  rH  CO  rH to « to rH  *  rH  4J  rH ft ft  LO LO  co  •<*  •H O X)  CO  o rH •H PH  VI  \  rH  M  co  o rH •H  LO  o •d fH CO  si o  rH •H  ft  ft  VI  VS.  00  \  rH  ft  <o OJ - H  O  to «  'o OJ  o  LO  to  ©  CO CO  "H o  o ©  CO CO  «  CO =te  rH •H O  O  *tf  Xi  fH  rH  •ri  CO  fH CO  o rH •H  rH •H  &  ft  Vi. CO  rH  o to  O  \  i—5  o  ft  V5.  a  r-i O  fH  4-J  \  r-i  a o  o  161. 4-»  G  G  CO  4-»  4->  cO o  cO  G  CO  u  o  «H  • H  <H  «H  •rH  • H  •rH  •rH  u  •H  a  •ri  •r-1  co o •t-i  *<—£  <H  CH  <H  •rH  •rH  G  G  .. bO  to  to  co  bO  <H  <fH  G  G  • H  G  bO  bO  •r-i  •H  to  CO  •p  - P  CO  CO  O  • H  • H  bO  •ri  CO  o  •H  •H  4->  -p o  G  o  •rH  G  • H  P  G co o  o  G  •rH  P  G co  bO  bO  • H  CO  •p  G  • H  •H  to  to o  CO  <H  •rH  • H  CO  •rH  bO  bO  bO  bO  +5  G cO o  co o  G  G  G  HJ  G  o  •r-i  •ri  4->  G  •H  to  o  O  '&  CO  LO  oe  H  00  CO  -4- ! r i  CO  LO  T ! O  a  o  r-H  «  LO LO  r-i  r-i fH P  O  fH - P  G o  O  O  -4- i  CO z>  to  r-i  r-i  r-i  fH P  fH • P  - P  O  O  G o o  CO  =«=  =%=  *tts.  H  r-i  r-i  • H  • H  xs  fH  co  43  o  r—1  •ri Pi  fH  CO  41  o r-i •rH  ft  O  G o  O  J  fH  o  t-i co  43 O  r-i. •ri  ft  CM  CO  r-i •ri  H  HI  • H  O  O  n  -d  fH  fH  CO  CO  o  o ' r-i •ri  43 r H  • H  ft  ft  CM  O  ft  :  HI  cCM  \  r-i  • H  o • d  »  ~f i  CO  *  CM  ~> I + I CM CO  o  a  r-i  CO  «  C0  CO  r-i  H CM  CO  H  CM  o  O  ft  ft  o •  ft  HI  t-i  r-i  «  CO  Us  G o  c«  CM  -Cf  CO  LO CO  * O CM  CM  -1 r-i !  to o  •Z-  «  »  o  fH P  O  O  to  2>  r-i  CO  «  r-i  r-i  o  to to  HI  O  CX> HI j 1! 1 CM  1  Hl  G G o o o . ta  O  I  H  Hi  &  -4- i  CO  LO •  o>  L. 1  «  HI HI  o  O  CO  LO H  e  CD  r-i  o  1 ! T i LO o LO .HI  c8  •rH  r-I  CO  CO  CM  Hi  LO  CM  o  r-i  o  to o  CM  CO  e  CO  LO  CO  CM  CO  «  rH  • ©  -4- 1 j i  CO  »  CO  to  o>  CO  o  ©  <%  °8  HI  H  -o  O  ft CO  *  «  o  *  ft  c8  r-i  l—i  t-i  •O  O  o  ft  ft  ft  ft  .CO  CO  't-i  t-i-  CO:  .G o  r-i • H  ft  *  » «  e  ^. CO  \ r-i  •5° \ r-i  CO  \  r H  \  r-i  \  r-i  r-i  1  \  \  CO  \  1—i  CO  \  r-i  162, -P  S3  co o  CO  c  bO  CO  -p  S3  bO  •rl-  CO  S3 bO  O  3  CO '  tO m  to  in  «  oj  CO CO  CO  o OJ  i-i in  CO a CO  41 o in CO rH  OJ  CO  CO  tO  CO  m co » Oi  in OJ  41CO OJ  CO  OJ  to  CO  i  l> O  OJ OJ  ©  OJ OJ  to  O  +>  OJ o  o  »  o> l  o  rH  to  to  CO  la  OJ  ©  CM OJ  CO  in  rH  + 1  to  OJ o  to  CO  Oi »  ©  in  «  •H  CO  -P  HJ  O  S3 &0  •H  CO  +J  o  •H CH •H  bO  •H  CO  -p  o  •H  bD  o  •H «H •H S3  •rH <rl  •H  CO  -P  o  •rH CH •H S3  S3  cd  cd o  o  o  •H CH •H  bO  •r-i CO  •H  CO  •rH «H •H $3  <H •H S3  cd  cd  o  HJ  S3  S3  o  •r-i  •rl «H •H  -p  CO  a o  G  •H <H •H S3 60 •H  •p  +» S3  OJ OJ  —1- 1  oo  Oi  r 1  o> ©  "T  1  CO CO  to .©  tO OJ  OJ  4!  4 1 .-•  to' Oi  ©  rH  OJ  ©  m OJ  4-1  OJ  Oi  ©  ©  IO  CO  co  rH  to  to  co  CO  OJ  to  to  CO  •H  rH •H  rH •H  rH •H  •r-i  rH  rH •H  to  -.'1=  rH •rH  o fH  CO rG  TJ fH  cd  o  .3  PH  rH •rH  r-i •rH  V^ CO  \  rH  •H  O  o  PH  O  fH  o r-i •H PH  ^' (H  \  <%  <3  °8  rH  H  rH  O  fH  co  ,<3 o  rH •rl PH  rH •H  >  *H  O  O  •X3 h  ta-  CO  o  rH •H PH  iH •H  rH •H  rH •H  c»  PH  rn Cli  r<3 o  rH  •r-i PH  \  \  : H : rH  o  PH  cd  o  PH  cd  <S  rH •H PH  VI ^0  rH  H  rH  OJ  o  O  fH CO  cd  rH •H  o  fH CO  si o  rH •H PH  \  •H  rH •H  o  fH  cd  43  o  PH  rH •H fH  VJ.  VI  S° \  \  rH  •H  c*S  <«  c«  =8  OJ  OJ  OJ  OJ  rH •H  rH tH  rH •H  rH  O  O  •CS fH CO  -cJ  fH CO  o  fH  cd  si  rH •rl  rH •H PH  rH •rH rH  rH •rH  vs.  vs.-  rH  rH  o  o  |  rH  %  .3  Pt  rG  o  rG  r<3 o  fH  rH •H  *fc  VI  V5.  Hi  rH  fH  •d fH  si  =fe  <H •H  O  Si  ¥1  rH  fH. CO  o  cd .£{  si  CO  \  o  Tj fH CO  o  •H rH  O  -  j  JJ j  fH  cd  Si  o  o rH  PH  PH  v?.  VI  VI.  rH  rH  •H  \ H  1  163. No. 3. Therefore, i t may he concluded that P i l c h a r d O i l No. 1 (early season) must have contained more Vitamin A at the t ime of production than the other two o i l s .  This evidence demon-  s t r a t e s that P i l c h a r d O i l - produced early i n the season i s more potent i n Vitamin A than o i l s that are produced at l a t e r stages of the f i s h i n g  season.  I t i s important to note that t he r e s u l t s of the blue unit test were i n agreement w i t h the b i o l o g i c a l assay i n that they showed the differenced i n the Vitamin A content of the three samples of P i l c h a r d O i l .  Consequently the blue unit test  may be used as a preliminary test f o r estimating the Vitamin A potency of P i l c h a r d O i l .  This i s of considerable commercial  importance in;aB much as i t would enable commercial r e f i n e r i e s to s e l e c t t h e i r o i l s on the basis of this t e s t .  Such a p r a c t i c e  would permit: the use of the o i l s with the highest Vitamin A content for medicinal purposes, while the o i l s of low Vitamin A potency could be used for paints and other manufactured products. B a i l e y (6) at Prince Rupert has reported that the blue value of P i l c h a r d O i l i s only a rough estimation of the Vitamin A content o f the o i l . Under such circumstances, t h i s itest may be used only as a rough measure of the r e l a t i g e potency various samples of P i l c h a r d O i l .  of  At the present stage of our  knowledge, the actual Vitamin A a c t i v i t y of P i l c h a r d O i l has s t i l l to be determined by the b i o l o g i c a l assay. The foregoing experiment has d e f i n i t e l y s hown that the Vitamin A content of P i l c h a r d O i l varies with the season of the  164. year, and that o i l produced early i n the season has the highest Vitamin A potency.  I t would he i n t e r e s t i n g to ascertain i f  the vitamin D potency of P i l c h a r d O i l would he p a r a l l e l to the Vitamin A potency at various stages of the f i s h i n g season.  165. • SUMMARY. Experiment No. 1 (1) 140 day-old single comb White Leghorn Cockerels were divided into seven d i f f e r e n t groups. The Vitamin A-free basal r a t i o n used was s i m i l a r t o that of Slvehj em and Neu (75). I t was supplemented with 1/8%, \%, j>-% and 1% P i l c h a r d O i l #1, \% P i l c h a r d G i l #2, and \% Cod L i v e r O i l .  One l o t of chicks  was fed the b a s a l r a t i o n only. (2) When e i t h e r \% P i l c h a r d O i l #1 or \% P i l c h a r d O i l #2 was added to the basal r a t i o n , the rate of growth was as good as when •§-% Cod L i v e r O i l was added to the r a t i o n . (3) The rate of growth of the chicks i n this experiment was compared with the rate of growth of chicks that had been fed graded amounts of Vitamin A as supplied by Reference Cod L i v e r O i l (U.S. Pharmacopoeia, 3000 units of Vitamin A per gram). (4) I t was found that P i l c h a r d O i l #1 contained at l e a s t 300 International Units of Vitamin A per gram and P i l c h a r d O i l #2 at l e a s t 600 International Units per gram. (5) P i l c h a r d O i l #2 would appear to meet' the minimum requirements s p e c i f i e d by the U.S. Pharmacopoeia f o r Cod Liver O i l (600 u n i t s of Vitamin A per gram). Experiment No. 2. (1) 400 day-old single comb White Leghorn Cockerels were divided into 4 groups and each group was subdivided into  4 lots.  Each group was fed a d i f f e r e n t basal r a t i o n .  Ration  No. 1 contained 12-|% powdered skim milk and 5% meat scrap; Ration No.II 10% powdered skim milk and 7f% meat scrap; Ration ' Ho. I l l 7-g-% powdered skim milk and 10% meat scrap; and Ration No. IV l2-g-% commercial casein. (2) The r a t i o n s were s upplemented w i t h 0%, ^%, Y/° and 1% of P i l c h a r d O i l . (3) I t was shovra that Rations No.I and No. IV contained appreciable traces of Vitamin A. (4) Since basal Ration No.II (10% powdered skim milk and 7ir% meat scrap) contained the least traces of Vitamin A of a l l the r a t i o n s used i n t h i s experiment i t was found to be the most s u i t a b l e f o r Vitamin A s t u d i e s . (5) \% P i l c h a r d O i l was found to supply the Vitamin. A requirements  adequately  of grov/ing chicks up to 8 weeks of  age. (6.) At 8 weeks of age the average weights of the d i f f e r e n t l o t s of chicks ( i n grams) were as f o l l o w s : Ration  Control  l A % P.O.  1/2% P.O.  1%  P.O.  #1  279 + 13  667 + 11  774+13  . 736+12  #2  261  705.± 12  766+ 11  785+11  #3,  336  658+11  738±12  7.64+8  #4  324+ 23  718 + 12  759T11  794 it 13  (7) Up to 8 weeks of age, the m o r t a l i t y i n a l l the groups except the controls was l e s s than 2 i % . (8) Several recommendations are suggested  for im-  proving the b i o l o g i c a l Assay of Vitamin A c a r r i e r s . using the chick as the test animal. ' Experiment No. 5. (1) 105 day-old s i n g l e comb White Leghorn chicks were divided  into 7 groups.  The basal r a t i o n used i n t h i s experi-  ment was s i m i l a r to that of Ration Bo. I I i n Experiment No. 2. (2) Three samples of p i l c h a r d O i l that had been produced at d i f f e r e n t stages of the f i s h i n g season were used as Vitamin A supplements. r a t i o n i n amounts of 0%,  These o i l s were added to the basal and  (3) I t was found that the p i l c h a r d O i l produced i n the early stages of the f i s h i n g season was a more potent source of Vitamin A than the o i l s produced l a t e r i n the season. (4) These three samples of p i l c h a r d O i l were tested by the antimony t r i - c h l o r i d e test (Carr-Price  reaction).  (5) The r e s u l t s of the arflmony t r i - c h l o r i d e test p a r a l l e l e d the r e s u l t s obtained w i t h the B i o l o g i c a l Assay. (6) I t i s recommended that o i l s that are produced i n the early stages of the f i s h i n g season should be used for medicinal purposes, because of t h e i r higher Vitamin A content.  168  c  BIBLIOGRAPHY.  1.  Abelin, I . , - N.A. and R., 5: No, 1330, 1935  2.  Ackert, J.E., Mcllvaine, M.iV and Crawford, N. 2., - Am. J . Hygiene, 13: 320, 1931 Ahmad, B., - Biochem, J . , 25: 1195, 1931  3. 4.  Ahmad, B., - J . Soc,. Chem. Ind. , 50: 121, 1931  5.  Ahmad, B,, and Malik, K.S., - Indian. J . Med. Res., 20: 1033,  5a  Anderson, A.C* and Bevine, V.E., - Proc. Soc. Exp. B i o l . Med., 32: 737, 1935  6.  B a i l e y , B.E., - Can. B i o l and f i s h e r i e s 7: No. 31, 1933  7.  Batchelder, E.L., - Amer. J . Of P h y i o l . , 109: No. 3, 1934  8.  Baurnann, 0 .A. and Steenbock, H., - J . B i o l Chem., 101: 561, 1933  9.  Baurnann, C.A. Steenbock, H., Ingraham, M.A. and Fred, E.B., - J . B i o l . Chem., 103: 339, 1933  10.  Baurnann, C.A., R i i s i n g , B.M. and Steenbock, H. , - J . B i o l Chem., 107: 705, 1934  11.  Baurnann, C.A., R i i s i n g , B.M. and Steenbock, H., - N.A. and R., 4: No. 1076,. 1934  12.  Beach, J.R., - C a l i f , agr. Exp. S t s . B u l l 378: 1924  13.  Bearse, G. E. and M i l l e r , M.W., - Poultry S c i . , 16: 39, 1937  14.  Bearse, G.E. and M i l l e r , M.W., - Poultry S c i . , 16: 39, 1937  1.69. 15. 16.  Bethke, H.M., Kennard, D.C. and Sassaman, H.L., - J . B i o l . Chem., 72: 695, 1927 B i e l y J . and Chalmers W., - Can. J . Res., 14: 21, 1936  17.  B i l l s , C.E., - J . B i o l . Chem., 100 Proc. XV, 1933  18.  B i l l s , 0.E., Imboden, M. and Wallenmeyer, E.C., - J . B i o l . Chem, 105: Proc X 1934  19.  B i l l s , C.E., McDonald, E.G., Massengale, D.N., Imboden, M., H a l l , H., Hergert, W.O. and Wallenmeyer, - J.C.J. B i o l . Chem, 109: Proc. V l l , 1935  20.  Bisbey, B. , Appleby, V. , V*eis , A. and Cover, S .U. - M i s s o u r i , Res. Bui. 205, 1934  21.  Brocklesby, H.N. and B a i l e y , 3.E. - P a c i f i c F i s h e r i e s Exp. Sta., Bui. No. 46, 1935  22.  Brockmann, H. and Iecklenbury, M.L., - N.A. and R., 3, No. 4004, 1933  23.  Bruins, H.K., Overhoff, J . and Wolff, I.E., - Biochem J . , 25: 430, 1931  24.  Buchheister, H., - N.A. and R., 5, No. 4415, 1935  24a  Buckner, G.G., Insko, W.M.Jr. and Martin, J.H., - Poultry S c i . 13: 110, 1934  25.  Cady, O.H. and luck, J.M. - J . B i o l . Chem., 86: 743, 1930  26.  C a l i f o r n i a A g r i c . Exp. Sta. Rpt., 79, 1920  27.  Cameron, H.G., - West V i r g i n i a Univ. B u l l . , Ser., No. 15, 1934 Cameron, H.G., - J . Amer. D i e t a t i c Assoc., 11: 189, 1935 Cameron, H.C., - Med. Woman's J . , 42: 266, 1935  28. 29. 30.  Copper, N.S., - Biochem J . , 24: 980, 1930  170. 31. 32.  Copper, N.S., McKibbin, I.M.W. and Prentice, J.H. - Biochem J . , 25: 265, 1931 G h e v a l l i e r , A. and Chabre, P., - Biochem J . , 27: 298, 1933  33.  G h e v a l l i e r , A. and Baert, H., - N.A. and H., 4: No. 2067, 1934  34.  Oook, E. E., - Analyst, 59: 545, 1934  35.  Coward, X.H. and Drummond, J.G., - Biochem J . , 14: 665, 1920  36.  Coward, K.H. and Drummond, J . 0 . , - Biochem J . , 16: 631, 1922  37.  Goward, K.H., - Biochem J . , 17: 134, 1923  38.  Coward, X.H., Biochem J. , 17: 145, 1923  39.  Coward, X.H., - Biochem J . , 19: 500, 1925  40.  Coward, X.H., - J . B i o l . Chem., 72: 781, 1927  41.  Coward, X.H., Key, X.M. and Morgan, B.G.E., - Biochem. J . 27: 873, 1933  42.  Coward, K.H. and Key, K.M., - Biochem J . , 28: 870, 1934  43.  Dann, W.J., - Biochem J . , 26: 666, 1932  44.  Dann, W.J., - Biochem J . , 26: 1072, 1932  45.  Dann, W.J., - Biochem J . , 27: 274, 1933  46.  Dann, W.J., - Biochem J . , 28: 634, 1934  47.  Davies, A.W., - Biochem J . , 27: 1770, 1933  171. 48.  Davies, A.W.. and Moore, T. , - Biochem.J., 28: 288, 1934  49.  Davies, A.W. and Moore, T., - Biochem J . , £9: 147, 1935 Delf, E.M., - Biochem J. , 18: 93, 1924  50. 51. 52.  De Vaney, G.M., Titus, H.W. and lies t i e r , R.B., - J . Agric. Res. 50: 853, 1935 Domayk, G. and Dobeneok, P., - N.A. and R., 3: No. 4017, 1933  53.  Dreyfus, J.R., - N.A. and R., 5: No. 4416, 1935  54.  D r i g a l s k i , W.V., - N.A. and R., 3: No. 187, 1933  55.  " D r i g a l s k i , W.Y.,, - N.A. and R. , 4:  No. 3256, 19.34  56.  D r i g a l s k i , W.V., - N.A. and R., 5: No. 4411, 1935  57.  Drummond, J.O., - Biochem J. , 13: 81., 1919  58.  Drummond, J.C., - Biochem J. , 13: 95, 1919  59.  Drummond, J.G. and Coward, K.H., - Biochem J . , 14: 661, 1920  60.  Drummond, J.G. and Govmrd, X.H., - Biochem J. , 14: 668, 1920  61.  Drummond, J.G. and Coward, K.H., - Biochem J . , 14: 734,.1920'  62.  Drummond, J.G., Golding, J . , Z i l v a , S .S «. and' .Coward, K.H., - Biochem J . , 14: 742, 1920  63.  Drummond, J.G. and Z i l v a , S.S., - Biochem J . , 16: 518, 1922  64.  Drummond, J.G., Channon, H.J. and Coward, X.H., - Biochem J . , 19: 1037, 1925  172. 65.  Drummond, J.G., Coward, E.H. and Hardy, E. - Biochem J . , 19: 1068, 1925  66.  Drummond, J.C. and Morton, S.A., - Biochem J . , 23: 785, 1929 Drummond, J . C , - J . State Med. 42: No. 1, 1933  67. 68.  Drummond, J.C. and Mac Walter, R.J., - Biochem J . , 27: 1342,. 1933.  •69."  Drummond, J . C , G i l d i n g , H.P. and Macwaiter> R.J., - J . P h y s i o l . , 82: 75, 1934  70.  Drummond, J . C and Maewalter, R.J., - J . P h y s i o l . , 83: 236, 1934  71.  Butcher, S . A . . H a r r i s , P.L., H a r t z l e r , E.R. and Guerrant, N.B., - J . N u t r i t . , 8: 269, 1934  72.  Dye, 'M., Medlock, O.G. and C r i s t , J.W., .- - J . B i o l . Chem., 74: 95, 1927  •73.  Dyer, E.J. and Key, K.M., - Biochem j . , 28.:: 8.75, 1934  73a  Eekelen, M.V., Emmerie, A. and Wolff, I.E., - N.A. and R. , 5i No. 130,. ,1934 ..  74.  Elder, C , - J . Amer. Vet.-, Med. Assoc., 87: 22, 1935  75.  Elveh;] em, C.A. and New, V.E.^ - J . B i o l . Chem., 47: 71, 1932  76.  Emmerie, A., EEkelen, M.V. and Wolff, .I.E., - Nature, 128: 495, 1931  77.  Emmett, A.D. and l u r o s , G.O., - J . ^ i o l . Ghem., 38: 441, 1918  78.  Emmett, A.D. and Peacock, G., - J . B i o l . Ghem., 56: 679, 1923  79.  Euler, H.V. and Ahlstrom, 1., . - N.A. and R, , 2: No... 105, 1932  80.  Evans, H.M., . - J . B i o l . Chem., 77: 651, 1928  173. 81.  Fearon, W.R., - Biochem J . , 19:  82.  Finn, D.B., - Can. B i o l , and F i s h . , No. 6, 1931  S3.  Fraps, G.S. and T r e i c h l e r , R. , - Texas, A g r i . Exp. Sta. B u i . No. 477, 1933  84.  Fraps, G.S. Copeland, O.C. and T r e i c h l e r , R., - Texas A g r i . Exp. Sta. B u i . No. 495, 1934  84a  Fraps, G.S., T r e i c h l e r , R. and Kemmerer, A.R., - J . A g r i c , Res.j 53: 713, 1936 F r i d e r i c i a , L.S.j - J . B i o l . Ghem., 62: 471, 1925  85.  888, 1925  86.  Frohring, W.O. and Wyeno, J . - J . Nutr., 8: 463, 1935  87.  Gillam, A.E. and Morton, R.A.,  f  - Biochem J . , 25: 88. 89* 90. 91.  1346, 1931  Gillam, A.E. Biochem J . , 28:  79, 1934  Glanzmann, B., - N.A. and R., 1, No. 2358, 1931 'Goldblatt, H. and Sosmes, K.M., - Biochem J . , 17: 446, 1923 Goode, -G.R. ~ Bui. Basic S c i . Res. 4: 55, 1932  92.  Gordonoff, T. and Ludwig,'F., - N.A. and E.,.5: No. .2514, 1935  93.  Gortzen, J . , ' - N.A.. and E., 5:  94.  Green, H.N., . - Biochem J . , 28: 16, 1934  95.  Green, H.N.j - Biochem J . , 28: 25, 1934  96.  G u i l b e r t , H.R. and Hart, G.H., - 3. N u t r i t , 8:.. 1934  97.  G u i l b e r t , H.R. and Hinshaw, - J . N u t r i t , 8: 1934  No. 2025, 1935  W.R.,  98. : 99. 100.  174.  G u i l b e r t , H.R., - J . N u t r i t , 10: 45, 1935. Gutteridge, H.S., - Dora. Can. Dept. A g r i c . Pub. No. 475, (Tech. B u l l . No. 1), 1935. Gutteridge,. H.S., - Sol." A g r i c , 15: 771, 1935  "101.  Hale, E., - Proc. Amer. Soc. Animal Prod., 126, 1935  101a  Hart, E.B., K l i n e . 0.1., and Keenan, J.A., - Science, 73: 710, 1931  102.  Hart, E.B., Steenbock. H. and Lepkovsky, S., and Hoplin, - J . B i o l . Ghem., 60: 341, 1924 Hathaway, I.L. and Davis, H.P., - Univ. of Neb., Bes. Bui. 69, 1933  103. 104.  Hathaway, I.L. and Davis, H.P., - Univ. of.Neb., Bes. Bui. 79, 1935  105.  Hauge, S.M., Garrick, G.W. and Prauge, R.W., - P o u l t r y S c i . , 6: 135, 1927  106.  Hauge, S.M., - J . B i o l . Ghem., 108: 331, 1935  107.  Hayaski, S., , - N.A. and R. , 6: No. 1603, 1936  108.  Heilbron, I .M., Kamm, E..D. and Morton, .B.A., - Biochem. J . , 21: 78, 1927  109.  Heilbron, I .M., Gillam, A.E. and Morton, B.A., - Biochem J . , 25: 1352, 1931  110.  Heilbron, I.M., Gillam, A.E. and Morton, B .A., - Biochem J . , 25: 1352, 1931  111.  Heilbron, I..M. and Phipers,. R.E,, -Biochem J . , 29: 1369, 1935  112.  Heilbron, I.M.. Parry, E.G. and Phipers, R.F., - Biochem J . , 29; 1376, 1935 H e l l e r , Y.6. and St. J u l i a n , R.R., - J . N u t r i t , 4:. 227, 1931 .  113.  114.  175. Hinshaw, W.R. ana Lloyd, W.E.H., H i l g a r d l a , - 8. .. • .Ho. 9:, 281, 1934  .115.  Holmes, A.D., D o o l i t t l e , A.W.. and Moore, W.B., - J . Am. Pharm. Assoc. 16: 1927  116.  Holmes, A.D., Tripp, If. and Campbell, P.A., - Poultry S c i . , 15: 71, 1936  117.  Holmes, A.D., Tripp, :F.. and Campbell, P.A., - J . N u t r i t . , 11: 119, 1936  118.  Holmes, H.N., Cassidy, H., Manly, R.S. and H a r t z l e r , E.R., - J . Amer, Chem. Soc. , 57: 1990, 1935  119.  Holmes, H.N. and Bromund W.H., - J . B i o l . Chem., 112: 437, 1936  120.  Holmes, H.N., Corbet, R.E. and H a r t z l e r , E.R., - Indust. Eng. Chem., 28: 133, 1936  120a ' Hoist, W.E. and Newlon, W.E.., - Univ. of C a l i f . Bui.. 417, 1935 121. 122.  Honeywell, H.E., Dutcher, R.A. and Cly, J.0., - J . N u t r i t , 3 : . 491, 1931 Hopkins, E.G., * Bioohem J . , 14: 725, 1920  123.  Horn, Z. and Sandor, S t . , •- N.A. and R., 4: No. 2074, 1934  124.  Hughes, E.H.. ' - J . A g r i c . Res. , 47: 487,1933  125.  Jameson, H.L., Drummond, J.G., and Coward, K.H., . - Bioohem. J . , 16: 482, 1922  126.  J a v i l l l e r , M. and Emerique, L., ... - N.A. and R., 1: No. 2349, 1931  126a  J e f f r e y , E.P., - New Jersey Agric. Exp. Sta. 23: 3, 1936  127.  Jones, W.S.'and Christiansen, W.J., - J , Amer. Pharm. Assoc., 24: .465, 1935  127a  J u l l , M.A., . n "Poultry Husbandry", 1930  176. 128.  Karrer, P., Buler, H.V. and Hellstrom, H„, - N.A. and H., 1: No. 1488, 1931  129.  Karrer, P., Mori', E. and Schopp, K., - N.A. and R., 3: No. 2354, 1933  130.  Karrer, P., Morf, R. and Schopp, K., - N.A, ana R., 3: No, 1522, 1933  131.  Kennedy, G and Butcher, R.A., - J . B i o l . Ghem., 50: . 339, 1922  132.  K l i n e , O.L., Schultze, M.O., and Hart, E. B., - J . B i o l . Ghem., 97: 83, 1932  133.  Koenig. M.C., Kramer, M.M. and Payne, L.E., P o u l t r y S c i . , 14., 178, 1935  134.  Kuhn, R., Brockmann, H., Scheunert, A., and S c h i e b l i c k , M., -N.A. and R., 3: No. 4003, 1933  135.  Dauber, H.J. and Rocholl, H., - N.A. and R., 5: No. 2512, 1935  136.  Lewis, J.T. and R e t i , L., - N.A. and R., 5: No. 160, 1935 l i i c f c e , H., - N.A. and R., 5: No. 4413, 1935  137. 138.  lindholm, H.R.V., - N.A. and R., 6: No. 198, 1936  139.  Lovern, J.A., Edishury, J.R. and Morton, R.A. - Biochem. J . , 27: 1461, 1933  140.  Lovern, J.A. and Sharp, J.G., - Biochem J . , 27: 1470, 1933  141.  Luce, E. and Maclean, S, - Biochem. J . 19-1925  142.  McGollum, E.V. and Davis, M. , - J.. B i o l . Ghem., 15: 167, 1913  143.  McGollum, E.V., Simmonds, N. and Steenbock, H - J . B i o l . Ghem., 29: Proc. XXVI 1917  144.  McGollum, E.V., Simmonds, N. and Beeker, J.E. - J . B i o l . Ghem., 64: 161, 1925  145. 146.  177. MoCoord, A.B. and Luce-Clausen, 3.M., - J., N u t r i t , 7: 557, 1954 McDonald, E.G., - J . B i o l . Chem., 100: Proc. LXIX, 1933  147.  McDonald, E.G., - J . B i o l . Chem., 103: 455, 1933  148.  McEarlane, W.D. and Rudolph, 1. , - S c i . A g r i c . , 16: 398, 1936  149.  Macwalter, R.J. and Drummond, J.G., - Biochem J . , 27: 1415, 1933 Macwalter, R.J., - Biochem, J . , 28: 472, 1934  150. 151.  Mallon, M.G. and Clark, M., - B i o l . Chem., 54: 763, 1922  152.  Malraberg, M. , - N.A. and R., 5: No. 3755, 1935  153.  Marcus, J. K., - J . B i o l . Chem. 90: 507, 1931  154.  Medical Research Council Special Report, S e r i a l No. 202, 1935  155.  M i l l e r , H.G., - O i l and Soap, 12: 51, 1935  155a  M i l l e r , • M.W. and Bearse, G.E., - Wash. A g r i c . Exp. Sta., B u i . 292, 1934  156.  Milne, H.I., - 28th Annual Meeting of Poul. S c i . , 1936  157.  Mohler, J.R., - U.S. Debt. Agr., Bur. Anim. Indus:, 1934  158.  Monaghan, B.R. and Schmitt, E.G., - J . B i o l . Chem., 96: 378, 1932  159.  Moore, T., -Biochem J . , 21: 870, 1927  160.  Moore, T., - Biochem J . , 22: 1097, 1928  161.  Moore, T., - Biochem J . , 25: 275, 1931  177. Moore, T., - Biochem J . , 25: 2131, 1931 Moore, T. , - Biochem J . , 26: 1, 1932 Moore, T., - Biochem J . , 27: 888, 1933 Morton, B.A. and H l i l b r o n , I.M., - Biochem J . , 22: 987, 1928 Morton, R.A., and H l i l b r o n , I.M. and Thomson - Biochem J . , 25: 20, 1931 Nelson, E.M., Walker, R. and Jones, D.B., - J . B i o l . Ghem. 92: Proc. VI, 1931 N o r r i s , R. J . , - B u l l . Basic S c i . Res., 3: 89, 1931 N o r r i s , H. J . , - B u l l . Basic S c i . Res., 5: 23, 1933 Notevarp, 0., - Biochem J . , 29: 1227, 1935 Osborne, T.B. and Mendel, L.B., J.B., - J . B i o l . Chem., 15: 311, 1913 Osborne , T.B. and Mendel, L.B., - J . B i o l . Ghem., 17: 402, 1914 Osborne, T.B. and Mendel, I.B., - J . B i o l . Chem., 37: 187, 1919 Palmer, I.S. and Kennedy, G., - J . B i o l . Ghem., 46: 559, 1921 Parkhurst, R.T., - Poultry Craftsman and The P a c i f i c Poultryman, 51: 3, 1936 Polak, A. and Stokvis, J.A., - N.A. and R., 2 No. 98, 1931 Potter, M. T. - Science 76: 195, 1932 Przezdziecka, A., N.A. and R., 5: No. 2498, 1935  178*  178.  Querner, E.R.V., - N.A. and R., 5: No. 2490, 1935  179.  Randoin, L and Netter, R., - N.A. and R., 3: No. 2813, 1933  180.  Rea, J.L. and Drummond, J.G., - N.A. and R. , 2: No. JJ304, 1932  181.  Record, P.R., Bethke, R.M. and Wilder, O.H.H., - P o u l t r y S c i . , 14: 297, 1935  182.  Record, P.R., Bethke, R.M. and Wilder, O.H.M., - P o u l t r y S c i . , 16: 297, 1935  183.  Report of the Conference on Vitamin Standards, 1931  184.  Reti, I . , - N.A. and R., 5: No. 3748, 1935  185.  Ringrose, R.G. and N o r r i s , L.C., - P o u l t r y S c i . , 15: 390, 1936  186.  Rosenheim, B. and Drummond, J.G., B Biochem J . , 19: 753, 1925  187.  Rosenthal, E. and E r d e l y i , J . , - Biochem.. J. , 28: 41, 1954  188.  Rosenthal, E. and E r d e l y i , I . , - Biochem J . , 28: 41, 1934  189.  Rosenthal, E. and E r d e l y i , J . , - N.A. and R., 4: No. 1085, 1934  190.  Rosenthal, E. and Weltner, M., - Biochem J . , 29: 1036, 1935  191.  Rowntree, J . I . , - J . N u t r i t , 3:  192.  R u s s e l l , W.G. and Webex, A.L., - Proc. Soc. Exp. B i o l , and Med., 29: 297, 1931  193.  R u s s e l l , W.G., - New Jersey A g r i c . , 16: 6, 1934  194.  R u s s e l l , W.G. and Taylor, M.W., - J . N u t r i t , 10: 613, 1935  195.  R u s s e l l , W.G. and Taylor, M.W., Chichester, D.E., and Wilson L . I . , - New Jersey A g r i c . Exp. Sta. B u i . No. 592, 1935.  345, 1931  1.79. Sampson, M.M. and Korenchevsky, V., - Biochem J . , 26: 1322, 1932 Scheunert, A. and S c h i e b l i c k , M., - N.A. and R., 3: No. 2814, 1933 Scheunert, A. and S c h i e b l i c k , M., - N.A. and R., 3: No. 2815, 1933 •Schlutz and Morse, - Amer. J . B i s . C h i l d . , 30: 199, 1925 Schlutz and Z e i g l e r , - Biochem J . , 69: 415, 1926 Schneider, E. and Windmann, E., - N.A. and R., 4: No. 3251, 1934 Schroeder, C.H., Higgins, W.A. and Wilson, W.O., - 27th Annual Meeting of Poul. S c i . 1935 Seifried, 0 . , . - J . Exp. Med. 52: • No. 4, 519, 1930 Seifried, 0 . , - J . Exp. Med. 52: No. 4, 533, 1930 Seifried, 0 . , - N.A. and R., 5: No. 2513, 1935 Sherman, H.C. and Munsell, H.E., - J . Amer. Chem. Soc. , 47: 1639, 1925 Sherman, H.0. and Cammack, M.I., - J . B i o l . Chem. 68: 69, 1926 Sherman, H.C. and Batchelder, E. L., - J . B i o l . Chem., 91: 505, 1931 Sherman, H.C. and Todhunter, E.N., - J . N u t r i t , 8: 347, 1934 Shrum, G.M. and How, T.G., - Can. J . Res., 13: 93, 1935 Sherwood, R.M. and Eraps, G.S., - Texas Agric. Expt. Sta. Bui. No. 468, 1932 Sherwood, R.M. and Eraps, G.S., - Roma Sept. 1933-XI No. 41  180. Sherwood, R.M. and Fraps, G.S., . - Texas A g r i c . Exp. Sta. B u i . No. 493, 1934 Sherwood, R.M. and' fraps, G.S ., - .Poultry S c i . 14: 297, 1935 Sherwood, R.M. and Fraps, G .S., - Texas Agr. Exp. Sta. B u i . No. 514, 1935 Sherwood, L.C. and luckner, W.G., . - J . N u t r i t . , 9: 123, 1935 Siraonnet, H., Busson, A. and A s s e l i n , L., - 0.R. Soc. B i o l . , 109: 182, 1932 .* Simonnet, H., Busson, A. and A s s e l i n , 1., - G.R. Soc. B i o l . , 109: 358, 1932 Sj orslev, N., - J . B i o l . Chem., 62: 487, 1925 Skarzynski, B., .-N.A. and R. 3: No. 4005, 1933 Smith, E.L. and Hazley, 'V. , .. - Biochem J. , 24: 1942, 1930 Smith, J • H«C •, • - J . B i o l . Ghem., 90: 597, 1931 Smith, L.W. and Morgan, A.F., - J . B i o l . Chem. 101: .43, 1933 Smith, L.W.j - Poultry S c i . , 12: 331, 1933 S t e e l , J.P., Lancet, - 229: 290, 1935 Steenbock, H., Kent, H.E. and Gross, E.G., - J . B i o l . Ghem. 35: 61, 1918 Steenbock, H. , Boutwell, P.W. and Kent, H.E., - J . B i o l . Ghem., 35: 517, 1918 Steenbock, H. and Gross, E.G., - J . B i o l . Chem., 40: 501, 1919 Steenbock. H. and Boutwell, ".W., - J . B i o l . Ghem., 41: 81, 1920 •SteenbocK, H. and Gross, E.G., - J . B i o l . Ghem., 41: 149, 1920  181. Steenbock. H. and Boutwell, P.W., - J . b i o l . Ghem., 41: 163, 19E0 Steenbock. H. and Boutwell. ".¥«,, - J . B i o l . Ghem., 42: 131, 1920 Steenbock. H., S e l l , M.T. and B u e l l , M.V., - J . B i o l . Ghem., 47: 89, 1921 Steenbock. H. , S e l l , M.T, and Boutwell, v.VI., - J . B i o l . Ghem., 47: 303, 1921 Steenbock. H., S e l l , M.T. and Nelson, E.M., - J . B i o l . Ghem., 56: 327, 1923 Steenbock. H., Nelson, M.T. and Black, A., - J . B i o l . Ghem. 62: 275, 1925 Steenbock. H., S e l l , M.T. and Nelson, E.M., - J . B i o l . Chem., 56: 327, 1923 Stephenson, M. , '. ",, - Biochem J . , 14: 715, 1920 Strauss, K . , - N.A. and R  . ,  5: No. 2513, 1935  Suquira, K . and Benedict, S . R . , - J . B i o l . Ghem., 55: 33, 1923 Sure, B., X i k , M.C., Walker, K.J. and Smith, M. - Univ. of Arkansas, Bui. No. 286 Sure, B. and Smith, M.E., - Proc. Soc." Exp. B i o l , and Med.., 28: 439, 1930-31 Sutton, T.3., S u t t e r f i e l d , H.E. and Xrauss, W.E - Univ. of Ohio, B u i . 545 Tornblom, N., - N.A. and R  . ,  5: No. 157, 1935  Truesdail, R.W. and Boynten, 1.0., - Indust. Eng. Ghem,, 23: 1136, 1931 Turner, R . G . and Loew, E.R., - J , of I n f . Diseases, 49: 1931 Thatcher, H.S. and Sure, B., - N.A. and R., 2: No. 932, 1932  18 2 a Turner, R.G. and Loew, E.R., - J . of N u t r i t , 5: No. 1. 1932 Turner, R.G. and Loew, E.R., - J . Inf. Diseases, 52: 102, 1933 Tyson, M.D. and Smith, A.E., - Am. J . Path, 5: 57, 1929 U n i v e r s i t y of Idaho, Annual Report, B u i . 205, 1933 United States Pharmacopoeia, 1937 V a i l , 0.E., Tobiska, J.W. and Douglass, E., - Oolarado State College Bui. No. 18, 1936 Wendt, H., and Schrolder, H., - N.A. and R., 5: No. 2520, 1935 Wendt, H., .- N.A. and R., 6: No. 220, 1936 Wilber, J.W., H i l t o n , J.H. and Hauge, S.M., - J . of Dairy Science, 16: No. 2., 1933 " i l l i m o t t , S.G., Moore, T and Wokes, E., - Biochem J . , 20: 1292, 1926 Willirnott, S.G. and Moore, T. , - Biochem J . , 20: 1292, 1926 Willimot, S.G. and Wokes, E., - Biochem J . , 21: 887, 1827 W i l l i m o t , S.G., - Biochem J . , 22: 67, 1928 Wilson, W.H., - J . B i o l . Ghem., 51: 455, 1922 Wilson, W.H., - Biochem J . , 21: 1054, 1927 Wilson, W.O., Schroeder, G.H. and Higgins, W.A., - 28th Annual Meeting of Poul. S c i . 1936 Wokes, E. and Willimot, S.G., - Biochem J . , 21: 419, 1927  1.8 3 « 264.  Wokes, F. , • - Bioohem J . , 22: 997, 1928  265.  Wolfe, J.M. and S a l t e r , H. J r . , - J . N u t r i t . , 4: 185, 1931 Wood, G.W., - Thesis, U.B.G. 1936  266.  .  266a  Woods, E. , Atkenson, E.W., l e l l h o u s e n , H. and Johnson, R.P., - J . Dairy S o i . , 19: 581, 1936  267.  Z i l v a , S.S., .. - Biochem J , , 13: 164, 1919  268.  Z i l v a , S .S ., Drummond, J.G. and Graham, }&, - Biochem J . , 18: 178, 1924 Zoltan, I.A., - N.A. and R., 5: No. 866, 1935  269.  U.S.P.X Interim Revision Announcement No. 2  A 1934 REVISION OF THE TEXT AND ASSAYS FOR COD LIVER OIL OF THE  PHARMACOPOEIA OF THE UNITED STATES TENTH DECENNIAL  REVISION  This announcement by interim revision of new standards and assays for medicinal Cod Liver Oil is in recognition of the notable advancement in scientific knowledge concerning vitamins, and for the purpose of officially establishing in this country the new International Units for Vitamins A and D. The Pharmacopoeia has accepted these International Vitamin A and Vitamin D Units as the basis for its new standards, and the new " U.S.P. X 1934 Vitamin A Unit" and " U.S.P. X1934 Vitamin D Unit" are identical with the corresponding International Units. It is hoped that this action by the Pharmacopoeia will largely overcome the present confusion in label and literature statements concerning the Vitamin A and Vitamin D potency of many products. This confusion in the past has been due to the use of unofficial units of varying vitamin value.* Following the established policy of pharmacopceial revision the Committee has had the advice and cooperation of many experts in the preparation of this new text. Two conferences, with more than thirty recognized authorities in vitamin work,[present at each, laid the foundations for the assays. There has since been established what is known as the "U.S.P. Vitamin Advisory Board." This Board has completed the assay texts and supervised the preparation of an official "Reference Cod Liver Oil" of known Vitamin A and Vitamin D potency, expressed in International Units. This is now being distributed by the Pharmacopoeia Board of Trustees as a basis of comparison in assays. The new assays require the use of this official "Reference Cod Liver Oil" as a basis of comparison. This  "Reference Cod Liver Oil" was independently assayed, in com-  * A statement of the approximate relationship of various unofficial yitamin units, now employed i n the study and labeling of products containing Vitamin A and Vitamin D , has been issued by the U.S.P. Vitamin Advisory Board. Information concerning this can be obtained by addressing the Chairman of the U.S.P. Committee of Revision. .1  parison with the international standards, by fifteen different laboratories who reported their results to the Vitamin Board. The members of this Board compiled and evaluated the data submitted and determined from these figures the Vitamin A and Vitamin D potency of the "Reference Oil." The Board has also arranged for the periodic recheck of the potency of this "Reference Oil" so that it may serve as a satisfactory standard when used by manufacturers of products containing Vitamin A and/or Vitamin D . The "Reference Cod Liver Oil" is obtainable through the office of the Chairman of the Committee of Revision. This Revision for the first time establishes an official vitamin standard for Cod Liver Oil. Heretofore, the Pharmacopceial requirement for vitamin content and assay has been optional and reflected the situation when the TJ.S.P. X became official some years ago. At that time there was little or no quantitative information upon which to base the proper minimum Vitamin A potency for a medicinal Cod Liver Oil and the requirement that, when assayed Cod Liver Oil should contain at least 50 Vitamin A units per gram, was primarily to insure the •exclusion of manipulated oils in which all vitamins had heen destroyed. A t that time "Vitamin D " was not even known by that designation but was referred to as "an antirachitic factor." The new official standards insure a Cod Liver Oil of excellent quality, corresponding in potency to what is now largely being sold in this country as "U.S.P. Oil." In addition to the introduction of the vitamin standards and methods of assay, the following physical or chemical standards have been introduced or amended: A standard for permissible color intensity for the official oil has been added. The limit of free acid and the saponification number take into consideration the occasional use of carbon dioxideas a preservative for the Oil. The directions for the determination of unsaponifiable matter have been so modified as to eliminate possible sources of error. A chill-test has been introduced to insure the absence of excessive amounts of stearin. The new official vitamin standards and assays and the chemical and physical standards as prepared by the Sub-Committee on Organic Chemicals of the Committee of Revision, have been officially adopted by the General Committee of Revision and the Board of Trustees. By action of the Board of Trustees the new standards for Cod Liver Oil will become official on January 1, 1935. E. F U L L E R T O N C O O K  May  1, 1934  43rd St. and "Woodland Ave.. Philadelphia, Pa. Chairman oj the Committee oj Revision ojthe U.S. Phartnacopatia.  "SALT MIXTURES" For  Diets i n Biological  Assays  In r e v i e w i n g the s a l t m i x t u r e s d u r i n g t h e r e v i s i o n of the Pharmacopoeia i t was found t h a t the f o r m u l a s p u b l i s h e d i n the U,S,p„ I n t e r i m R e v i s i o n Announcement No,. 2 were not s t r i c t l y i n o c n f o r m i t y w i t h the fovirulas as o r i g i n a l l y published.!, The formulas 3?.cw l-.apc: bee.n oa.i.oul.vred t«» r e p r e sent t h e SGHW content of s a l t s and a c i d s as i n the c r i g l n a . , b u t i n terms of o f f i c i a l p r o d u c t s . T h i s w i l l enable those u s i n g these formul a s t o secure more e a s i l y the i n g r e d i e n t s and prepare the m i x t u r e s . 1  The formulas as t h e y w i l l appear  i n t h e U.S.P.XI are as f o l l o w s :  S a l t Mixtures For p r e p a r i n g the s a l t mixtures the a v a i l a b l e form o f each chemical i s t a k e n to f u r n i s h the s t i p u l a t e d e q u i v a l e n t o f each c h e m i c a l . S a l t M i x t u r e No.  1  • Calcium Carbonate (U.S.p.)..., Magnesium Carbonate (U.S.P., 40 p e r c e n t MgO)....... Sodium Carbonate, anhydrous (U.-.S.P, Reagent)......... «.... Potassium Carbonate ( U S P „ , d r i e d a t 180°C)„ Phosphoric A c i d (U..S,P,, 86.5 per cent),,.... H y d r o c h l o r i c A c i d (U.S.P,, 36 p e r c e n t ) . . S u l f u r i c A e i d (U.S,P., 96 per c e n t ) . , C i t r i c A c i d (u.S.p..). .. F e r r i c C i t r a t e (U.S.P, Reagent, 17.5 per cent Fe) Potassium Iodide (U.S.p.) „„ Manganese S u l f a t e (U.S.P. Reagent, MnS04.4H20) ,... Sodium F l u o r i d e (U.S.P. Reagent) Potassium Alum (U.S.P.) , , t  w  134.8 Gm. 28.9 Gm. 34,2 Gm. 141.3 Gm. 119.3 Gm. 148.3 Gm. 9.6 Gm. 111.1 Gm, 7.44 Gm. 0.020 Gm. 0.117 Gm. 0.062 Gm. 0.044 Gm.  D i s s o l v e the c i t r i c a c i d i n a s u f f i c i e n t q u a n t i t y o f hot d i s t i l l e d v/ater and add the s o l u t i o n to the mixed carbonates. Then add the p o t a s sium i o d i d e , manganese s u l f a t e , sodium f l u o r i d e and potassium alum, p r e v i o u s l y d i s s o l v e d i n d i s t i l l e d v/ater.. Then add the f e r r i c c i t r a t e d i s s o l v e d i n the h y d r o c h l o r i c a c i d . D i l u t e the s u l f u r i c a c i d with d i s t i l l e d water; add t h e phosphoric a c i d and add t h i s a c i d mixture t o t h e mixture p r e v i o u s l y prepared and s t i r u n t i l e f f e r v e s c e n c e ceases. Evaporate the f i n a l mixture to dryness, i n a c u r r e n t of a i r a t from 90° t o 100°C, and reduce t h e r e s u l t i n g p r o d u c t t o a f i n e powder.  S a l t M i x t u r e Mo. 2 Sodium C h l o r i d e (U.S.p.) ....... Magnesium S u l f a t e (U.S.P. ) Sodium Biphosphate (U.S.P,) Potassium Phosphate ( K 2 H P O 4 ) . . . . .* Calcium Biphosphate Ferric Citrate  1.73 5.45 j , . . 3.47 9.54  [CaH4(PO4)2.H2O].,..i.  5.40  (U.S.p. Reagent, 17.5 p e r cent P e ) . . , , . . ,  C a l c i u m L a c t a t e (U.S.P.) j Mix the f i n e l y powdered s a l t s u n i f o r m l y .  Respectfully  E. HJLLERTON COOK, Chairman  Gm.  1.18 Gm» 13  submitted,  Gm. Gm. Gm.. Gm.  Gm.  OLEUM MORRHILE Cod Liver Oil Ol. Morrh.  The partially destearinated fixed oil obtained from fresh livers of Gadus Morrhua Linne" and other species of the Family Gadidm. Cod Liver Oil may be flavored by the addition of not more than 1 per cent of any one or any mixture offlavoringsubstances recognized in this Pharmacopoeia. Co Liver Oil contains in each Gm. at least 600 U.S.P. Units of Vitamin A and at least 85 U.S.P. Units of Vitamin D . * The Vitamin A potency and Vitamin D potency of Cod Liver Oil when designated shall be expressed in "United States Pharmacopoeia Units" per gram of oil and may be referred to as " U . S . P . Units" per gram of oil. To indicate the adoption of the new standards the statement " U . S . P . X —Revised 1934" may be used. Description and physical properties—A thin oily liquid, having a peculiar, slightly fishy, but not a rancid odor, and a fishy taste. Cod Liver Oil is slightly soluble in alcohol, but is freely soluble in ether, chloroform, carbon disulphide, and in ethyl acetate. Tests for identity and purity—Specific gravity 0.918 to 0.927 at 2 5 ° C . A solution of 1 drop of Cod Liver Oil in 1 cc. of chloroform, when shaken with 1 drop of sulphuric acid, acquires a violet-red tint, gradually changing to reddish-brown. When viewed transversely in a tall, cylindrical, standard oil-sample bottle of colorless glass of about 120 cc. capacity, the color of Cod Liver Oil shall not be more intense than that of a mixture of 11 cc. of colorimetric cobalt, T.S.,t 76 cc. of colorimetric ferric T.S.f, and 33 cc. of distilled water, in a similar bottle of the same internal diameter. * One "United States Pharmacopoeia Unit of Vitamin A " is equal, in growth promoting and antiophthalmic activities for the rat, to one International Unit of Vitamin A as defined and adopted by the Conference of Vitamin Standards of the Permanent Commission on Biological Standardisation of the League of Nations in June of 1931; one "United States Pharmacopoeia Unit of Vitamin D " is equal, in antirachitic potency for the rat, to one International Unit of Vitamin D as defined and adopted by the Conference of Vitamin Standards of the Permanent Commission on Biological Standardisation of the League of Nations in June of 1931. t Reagents for Color Determination: Colorimetric Cobalt T.S.—Containing 59.496 Gm . of cobaltous chloride (CoCl . 6H 0) in 1000 cc. of solution. Dissolve about 65 Gm. of cobaltous chloride in enough of a fluid made by mixing 25 cc. of hydrochloric acid with 975 cc. of distilled water to make a volume of 1000 cc. Measure 5 cc. of this solution into a 250 cc. flask, add 15 cc. of a solution of sodium hydroxide (1 in 4) and 5 cc. of solution of hydrogen dioxide. Boil the mixture gently for ten minutes, cool, add 2 Gm. of potassium iodide and 20 cc. of sulphuric acid (1 in 4). When the precipitate has dissolved titrate the liberated iodine with tenth-normal sodium thiosulphate, using starch T.S. as the indicator. One cc. of tenth-normal sodium thiosulphate corresponds to 0.023799 Gm. of C0CI2.6H2O. Adjust the volume of the cobaltous chloride solution so that 1 cc. contains 0.059496 G m . of CoCl .6H 0. Preserve the solution in a bottle with a well-fitting glass stopper. 2  2  2  2  Colorimetric Ferric T.S.—Containing 45.053 Gm. of ferric chloride (FeCl .6H 0) in 1000 cc. of solution. Dissolve about 55 Gm. of ferric chloride in enough of a fluid made by mixing 25 cc. of hydrochloric acid with 975 cc. of distilled water to make a volume of 1000 cc. Assay the solution as directed under Liquor Ferri Chloridi, U.S.P. X , using 10 cc. of the solution, accurately measured. Adjust the volume of the solution so that 1 cc. contains 0.045053 Gm. of F e C l . 6 H 0 . Preserve the solution in a bottle of amber glass having a well-fitting glass stopper. 3  3  2  2  Dissolve 2 Gm. of Cod Liver Oil, accurately weighed, in 30 cc. of a mixture of equal volumes of alcohol and ether, the mixture having been previously neutralized with tenth-normal sodium hydroxide, using 5 drops of phenolphthalein T.S. as- the indicator, and boil the oil solution gently under a reflux condenser for ten minutes. Cool and titrate the mixture with tenthnormal sodium hydroxide to the production of a i pink color which persists after shaking for thirty seconds. Not more than 1 cc. of tenth-normal sodium hydroxide is required (free acid). ' Weigh 5 Gm. of Cod Liver Oil into a 250 cc. flask, add a solution of 2 Gm. of potassium hydroxide in 40 cc. of alcohol and heat under a reflux condenser for two hours, keeping the alcohol gently boiling. Evaporate the alcohol on a water bath, dissolve the residue in 50 cc. of hot distilled water, and transfer the solution to a separatory funnel, rinsing the flask with' two 25-cc. portions of distilled water which are added to the solution in the separator. Cool the mixture to from 15° to 20° C , and extract with two successive portions of 50 cc. each of ether, adding a few drops of alcohol. Combine the ether extracts in another separator, and wash the ether solution, first with 20 cc. of tenthnormal potassium hydroxide, then with 20 cc. of fifth-normal potassium hydroxide, and finally with successive 15-cc. portions of distilled water until the washings are not reddened by the addition of 2 drops of phenolphthalein T.S. Transfer the ether solution to a tared beaker, rinse the separator with 10 cc. of ether, and add the rinsings to the beaker. Evaporate the ether just to dryness on a water bath and dry the residue for thirty minutes at 100° G. *Cool the beaker in the desiccator for thirty minutes and weigh. The residue shall not exceed 1.30 per cent of the weight of Oil taken for the assay (unsaponifiable matter). Fill a tall, cylindrical, standard oil-sample bottle of about 120 cc. capacity with Cod Liver Oil, at a temperature between 23° and 28° C , stopper, and immerse the bottle in a mixture of ice and distilled water for five hours. The oil remains fluid and does not deposit stearin (undestearinated cod liver oil). Saponification value: not less than 180 and not more than 192. When carbon dioxide has been used as a preservative, the oil must be exposed in a shallow dish in a vacuum desiccator for twenty-four hours before weighing . the sample for determination of the saponification value. Iodine value: not less than 145 and not more than 180. P r e s e r v e in a cool place, in well-closed containers which have been thoroughly dried before filling. Cod Liver Oil may be bottled or packaged in a vacuum or in the presence of an inert gas. Assay—Proceed as directed under Assays for Vitamins A, and D in Cod Liver Oil, ' pages 4 to 11. Preparation—Emulsum Olei Morrhua?. A V E R A G E DOSE—(Administer three times daily)—Infants: Metric,  4 cc.—Apothecaries, 1 fluidrachm. Adults: Metric, 8 cc.— Apothecaries, 2 fluidrachms.  Assays for Vitamins A and D in Cod Liver Oil (To replace the Vitamin Assay on page 489 in the U.S.P. X) Definitions—As used herein, unless the | context otherwise indicates, the term assayer means the individual immediately responsible for the interpretation of the assay; the term assay group means a group of rats to'which the assay oil shall be administered during the assay period; the term assay oil means a God Liver Oil under examination for its vitamin potency; the term assay period for the Vitamin A assay means the interval in the life of a rat between the last day of the depletion period and the twenty-ninth day thereafter or between the last day of the depletion period and the death of the rat; the term-assay period for the yitamin D assay means the interval in the life of a rat between the, last day of the depletion period and the eleventh day thereafter; the term assemble means the procedure by which rats are selected and assigned to groups for the purpose of feeding, care, and observation; the 4  term control group means a group of rats receiving no Cod Liver Oil during the assay period; the term daily, for the Vitamin A assay, means six days of each week of the assay period; the term daily, for the Vitamin D assay, means each of the first eight days of the assay period; the term declining weight means a condition of a rat when the, body weight of the rat on any given day is equal to or less than the body weight of the rat on the seventh day prior to the given day; the term depletion period means the interval i n the life of a rat between the last day of the preliminary period and the first day of the assay period; the term dose means the quantity of the Reference Oil or of the assay oil to be fed daily to a rat during the assay period; the term fed means made readily available to the rat or administered to the rat by mouth; the term ground gluten means the clean, sound product made from wheat flour by the almost complete removal of starch, and contains not more than 10 per cent of moisture, and, calculated on the water-free basis, not less than 14.2 per cent of nitrogen, not less than 15 per cent of nitrogen-free extract (using the protein factor 5.7), and not more than 5.5 per cent of starch (as determined by the diastase method); the term group for the Vitamin A assays means six or more rats maintained on the same required dietary regimen during the assay period; the term group for (he Vitamin D . assay means seven or more rats maintained on the same required dietary regimen during the assay period; the term ophthalmia means a pathological state of the eye and/or the conjuctiva and/or the tissues anatomically related to the eye, readily discernible macroscopically and usually associated with Vitamin A deficiency; the term preliminary period means the interval in the life of a rat between the seventh day after birth and the first day of the depletion period; the term rachitogenie diet means a uniform mixture of the food materials, and in the proportions named, in either of the following formulas: Rachitogenic Diet No. 1 Whole Yellow M a k e , ground. Whole Wheats ground. Ground Gluten : ". Gelatin Calcium Carbonate (CaCO ) Sodium Chloride (NaCl) Rachitogenic Diet No. 2 Whole Yellow Maize, ground. Ground Gluten Calcium Carbonate (CaC0 ) Sodium Chloride (NaCl) s  3  33 per 33 per 15 per 15 per . . 3 per 1 per  cent cent cent cent cent cent  76 per 20 per 3 per 1 per  cent cent cent cent  The term Vitamin A test diet means a food material consisting of the following proportions of the named ingredients of the quality specified: Vitamin A Test Diet casein.,•••••••/ 18 per cent Salt Mixture (see page 6) : 4 per cent Yeast, dried , 8 per cent Starch..... ..65 per cent Vegetable Oil 5 per cent yitamin D , a sufficient amount Not less than 3. U.S.P. Units of Vitamin D shall be provided in each gram of diet and this vitamin shall be carried by the yeast or the vegetable oil. The ingredients of the Vitamin A test diet shall be free from Vitamin A or shall have been treated so as to reduce the Vitamin A content to such a degree that when the Vitamin A test diet is fed to the control group two-thirds or more of the rats shall manifest, prior 5  to the eleventh day of the assay period, symptoms of Vitamin A deficiency characterized by both declining weight and opthalmia. The dried yeast shall carry the Vitamin B complex in such concentration that a daily dose of 0.15 G m . shall permit an average gain i n weight of at least 3 Gm. per week in rats during an interval of four weeks between the twenty-fifth and sixtieth days of age, when the rats are provided ad libitum with a ration which is adequate for optimal growth, except that the ration shall be devoid of the Vitamin B complex. Salt Mixture No. 1 Calcium Carbonate (CaC0 ) Magnesium Carbonate (Mg.COs) Sodium Carbonate ( N a C 0 ) Potassium Carbonate ( K C O ) . . . Phosphoric Acid (100 per cent) Hydrochloric Acid (100 per cent) Sulphuric Acid (100 per cent) Citric;Acid ( 1 H 0 ) . . Ferric Citrate ( 1 V H 0 ) Potassium Iodide ( K I ) . . . . . . Manganese Sulphate ( M n S 0 ) Sodium Fluoride (NaF) Potash Alum [ K J A I ^ S C M ) * ]  134.8 G m . . 24.2 Gm. 34.2 G m , 141.3 G m . 119.3 G m . ....166.9 Gm. 9.8 G m . 111.1 G m . 5.7 G m . 0.020 G m . 0.079 G m . 0.248 G m . 0.0245 G m .  3  2  3  2  s  2  2  2  4  The available form of each chernical substance is taken i n sufficient quantity to furnish the stipulated equivalent quantity of each chemical. The mixed carbonates and ferric citrate are added to the mixed acids. The specified quantities of K I , . MnS04, N a F , and K2A1 (S04)4 are added as.solutions of known concentrations and the resulting mixture is evaporated to dryness i n a current of air at from 90 ° to 100 ° C. and ground to a fine powder. 2  Salt Mixture N o . 2 Sodium Chloride (NaCl) 0.173 G m . Anhydrous Magnesium S u l p h a t e . ( M g S 0 ) . . . . . . . . 0.266 G m . Sodium Phosphate ( N a H P 0 . H 0 ) 0.347 G m . Potassium Phosphate ( K H P 0 ) 0.954 G m . Calcium Acid Phosphate [ C a H ( P 0 ) . H 0 ] 0.540 G m . Ferric Citrate ( i y H 0 ) 0.118 G m . Calcium Lactate 1.300 G m . Uniformly mix the finely powdered salts. The assay of Cod Liver O i l for Vitamin A and Vitamin D potency shall be by comparison with the U.S.P. "Reference Cod Liver O i l , " by assay procedures conforming i n all respects to the following specifications: 4  2  2  4  2  4  4  2  4  2  2  2  Method of Assay for Vitamin A The Vitamin A assay, comprising the recording of observations of groups of,rats throughout specified periods of their lives, while being maintained on specified dietary regimens, and the interpretation of such data, is as follows: Preliminary period—Throughout the preliminary period each rat shall be raised under the immediate supervision of or according to directions specified by the assayer. Throughout the preliminary period the rats shall be maintained on a dietary re'gimen which shall provide for normal development in all respects, except that the supply of Vitamin A , or precursors of Vitamin A , shall be limited to' such a degree that rats weighing between 40 and 50 G m . and not: exceeding twenty-eight days of age and  •6  subsisting on a suitable V i t a m i n A deficient ration a n d water for a n i n t e r v a l not exceeding forty-five days shall manifest symptoms characteristic of V i t a m i n A deficiency. Depletion period—A r a t shall be suitable for the depletion period when the age of the rat does not exceed twenty-eight days, a n d if the b o d y weight of the rat shall exceed 39 G m . , a n d does not exceed 50 G m . , and if the a n i m a l manifests no evidence of injury, or disease, or anatomical abnormality w h i c h might hinder growth a n d development. Throughout the depletion period each rat shall be provided w i t h the V i t a m i n A test diet and distilled water a d l i b i t u m , and during this period no other dietary supplement shall be available to the animal. Assembling rats into groups for the assay period-—Rats w h i c h are suitable for the .. assay period shall be assembled into groups. F o r each assay o i l there shall be one or more assay groups. I n the assay of one assay o i l there shall be provided at least one control group a n d a t least one reference group, b u t one control group a n d one reference group m a y be used for the concurrent assay of more than one assay oil. T h e interval of assembling rats into groups shall not exceed sixty days. O n any one day during the i n t e r v a l of assembling rats into groups, the t o t a l number of rats that shall have been assigned to make up any one group shall not exceed b y more than two the number of rats t h a t shall have been assigned to make up any other group. W h e n the assembling of a l l groups s h a l l have been completed the total number of rats i n each group shall be the same, and the number of rats of one sex i n each group shall be the same. N o t more t h a n three rats from one l i t t e r shall be assigned to one group. W h e n the assembling of a l l groups shall have been completed, the average weight of the rats i n any one group on the day beginning the assay period shall not exceed b y more than 10 G m . the average weight of the rats i n any other group on the day beginning the assay period. Assay period—A rat shall be suitable for the assay period, provided that the depletion period shall have exceeded twenty-four days and shall not have exceeded forty-five days, a n d provided t h a t a r a t shall manifest evidence of V i t a m i n A deficiency characterized %y declining weight a n d / o r ophthalmia. T h r o u g h o u t the assay period each r a t of the control, reference, and assay groups shall be kept i n an i n d i v i d u a l cage a n d shall be p r o v i d e d w i t h t h e V i t a m i n A test diet a n d d i s t i l l e d water, a d l i b i t u m . T h r o u g h o u t the assay period each r a t i n any assay group shall be fed daily a dose of the assay oil, and throughout the assay period each rat i n any one reference group shall be fed d a i l y a dose of the reference o i l . T h e reference o i l a n d / o r the assay o i l m a y be diluted before feeding w i t h a n edible vegetable o i l free f r o m V i t a m i n A . D i l u t e d o i l shall be stored i n the dark at a temperature not exceeding 50° F . T h e period of storage shall n o t exceed seven days. N o t more t h a n 0.1 cc. of the diluted o i l shall be fed as a daily dose. D u r i n g the assay period a l l conditions of environment shall be maintained as u n i f o r m l y as possible w i t h respect t o t h e assay, reference, a n d control groups. Recording of data—-On the day beginning the depletion period a n d at intervals of not more t h a n seven days for the first twenty-one days of that period there shall be a record made of the b o d y weight of each rat. F r o m the twenty-first day of depletion period u n t i l the end of the assay period a record shall be made of the b o d y weight a n d eye condition of each r a t at intervals not exceeding five days. T h e eye condition shall be. designated as n o r m a l , watery, sensitive t o light, swollen, bloody exudate, purulent, opacity of cornea, or any combination of these terms.. A record shall be made of the failure of a rat to consume the prescribed daily dose of reference or assay o i l .  Vitamin A potency of the assay oilr—-In determining the V i t a m i n A potency of the assay oil the performance of the rats of the assay a n d reference groups shall be c a l c u lated for each group on the basis of the difference between the average weight of t h e 7  s u r v i v i n g rats arid the average weight of the same rats on the d a y beginning the assay ..period. T h e d a t a f r o m the reference group s h a l l be considered v a l i d for establishing the V i t a m i n A potency of t h e assay o i l o n l y w h e n t w o - t h i r d s o r more of t h e t o t a l n u m b e r of animals c o m p r i s i n g a reference group shall have made i n d i v i d u a l l y b e tween t h e beginning d a y of t h e assay period a n d t h e t w e n t y - e i g h t h d a y thereafter a n increase i n b o d y weight w h i c h shall equal or exceed 12 ' G m . a n d shall not exceed 60 G m . , a n d the d a t a f r o m a n assay o r reference group s h a l l be considered v a l i d for establishing the V i t a m i n A potency of the assay o i l o n l y when t w o - t h i r d s or'more, b u t not less t h a n six, of t h e rats of a n assay o r reference group have s u r v i v e d t w e n t y eight d a y s of the assay period. T h e d a t a f r o m a n assay group s h a l l b e considered v a l i d f o r establishing t h a t a n assay o i l conforms w i t h t h e U . S . Pharmacopoeia s t a n d a r d f o r V i t a m i n A i n c o d l i v e r o i l only when t w o - t h i r d s o r more b u t riot less t h a n six rats s h a l l have made i n d i v i d u a l l y between t h e beginning d a y of the assay period a n d the t w e n t y - e i g h t h day thereafter a n increase i n b o d y weight w h i c h shall equal or exceed 12 grams. T h e d a t a f r o m a r a t shall be considered v a l i d for establishing t h e average performance of a reference o r assay group o n l y o n t h e conditiont h a t the r a t has consumed the prescribed dose of o i l for at least t w e n t y - t w o days of the assay p e r i o d . A V i t a m i n A assay shall n o t b e considered v a l i d unless t w o - t h i r d s o r more of the t o t a l n u m b e r of animals comprising the control group shall, prior t o the eleventh d a y of the assay period, manifest s y m p t o m s of V i t a m i n A deficiency characterized b y b o t h d e c l i n i n g weight a n d o p h t h a l m i a . T h e V i t a m i n A p o t e n c y of t h e assay o i l is then calculated according t o the following procedure: L e t " R " equal the d a i l y dose i n - m i l l i g r a m s of the reference o i l necessary t o p r o duce i n a reference group an average gain i n weight, " G , " of not less t h a n 12 G m . a n d not more t h a n 60 G m . L e t " A " equal t h e d a i l y dose i n milligrams of t h e assay o i l t h a t w i l l produce in a n assay group a n average gain i n weight equal t o o r greater t h a n " G . " If the p r o d u c t of  —  X [units p e r gram of V i t a m i n A contained i n the refer-  ence oil] is equal t o or greater t h a n 600, then the assay o i l contains 600 or more u n i t s of V i t a m i n A. per gram of o i l a n d complies w i t h the U . S . Pharmacopoeial requirement for V i t a m i n A potency.  ;  Method of Assay for Vitamin D  T h e V i t a m i n D assay, c o m p r i s i n g the recording of observations of groups of rats, t h r o u g h o u t specified periods of their lives, w h i l e being m a i n t a i n e d o n specified dietary regimens, a n d the interpretation of s u c h d a t a , i s as follows: Preliminary period—Throughout the p r e l i m i n a r y period each r a t shall be raised under t h e i m m e d i a t e supervision of or according t o directions specified b y t h e a s sayer. T h r o u g h o u t the p r e l i m i n a r y period the rats s h a l l be m a i n t a i n e d o n a dietary regimen w h i c h s h a l l p r o v i d e f o r n o r m a l development i n a l l respects, except t h a t the s u p p l y of V i t a m i n D s h a l l be l i m i t e d t o such.a degree t h a t rats, weighing between 40 a n d 60 G m . a t a n age of t w e n t y - o n e t o t w e n t y - e i g h t days, a n d subsisting for ari i n t e r v a l of three weeks o n a suitable rachitogenic diet, shall manifest evidence of severe rickets. Depletion period—-A r a t s h a l l b e suitable f o r the depletion period when the age of the r a t does not exceed t h i r t y days, a n d i f the b o d y weight of the r a t s h a l l exceed 44 G m . , a n d does n o t exceed 60 G m . , a n d i f t h e a n i m a l manifests n o evidence of i n j u r y , o r disease, o r a n a t o m i c a l a b n o r m a l i t y w h i c h m i g h t hinder g r o w t h a n d d e velopment. T h r o u g h o u t the depletion period each r a t shall be p r o v i d e d w i t h t i e  .8'  rachitogenic diet and distilled water a d l i b i t u m , a n d during this period no other dietary supplement shall be available t o the animal.  Assembling rats into groups for (he assay period—Rats w h i c h are suitable for the  assay period shall be assembled into groups. F o r each assay o i l there s h a l l be one o r more assay groups. I n the assay of one assay o i l there shall be p r o v i d e d at least one reference group, b u t one reference group m a y be used for the concurrent assay of more thdn one assay o i l . T h e i n t e r v a l of assembling rats into groups shall not exceed sixty days. O n any one day during the i n t e r v a l of assembling rats i n t o groups, the t o t a l n u m b e r of rats t h a t s h a l l have been assigned to make up a n y one group shall n o t exceed b y more t h a n t w o the n u m b e r of rats t h a t shall have been, assigned to make u p a n y other group. W h e n t h e assembling of a l l groups shall h a v e been completed the t o t a l n u m b e r of rats i n each group shall be the same, a n d the number of rats of: one sex i n each group shall be the same. N o t more than three rats from one l i t t e r shall be assigned to one group. W h e n the assembling of a l l groups shall have been completed the average weight of the rats i n any one group on the day beginning the assay period s h a l l not exceed b y more t h a n 8 G m . the average weight of the r a t s i n a n y other group on the day beginning the assay period.  Assay period—A r a t shall b e suitable for t h e assay period, p r o v i d e d t h a t the depletion period shall have exceeded eighteen days a n d shall n o t h a v e exceeded t w e n t y five days, a n d provided t h a t a r a t shall manifest evidence of rickets characterized b y a distinctive, wabbly, rachitic gait and b y enlarged joints. T h e presence of rickets m a y also be established b y examination of a leg bone of one member of a l i t t e r b y the " l i n e t e s t " described below. E a c h r a t shall be kept i n an i n d i v i d u a l cage a n d shall be p r o v i d e d w i t h the rachitogenic diet a n d distilled water, a d l i b i t u m . O n a n y calendar d a y of the assay period the assay a n d reference groups s h a l l receive a rachitogenic diet compounded f r o m t h e same lots of ingredients. T h r o u g h o u t the first eight days of the assay period each r a t i n a n y one assay group shall be fed d a i l y a dose of the assay oil, a n d throughout the first eight days of the assay period each r a t i n any one reference group shall be fed daily a dose of the reference o i l , except t h a t the following deviation f r o m the d a i l y feeding shall be permissible: t h a t the d a i l y dose m a y be doubled on the day preceding a one-day h o l i d a y falling w i t h i n t h e first eight days of t h e assay period. D u r i n g the remainder of the assay period (i. e., the n i n t h d a y a n d t e n t h d a y ) neither t h e assay o i l n o r t h e reference oil shall be fed. O n the eleventh d a y of the assay period each r a t s h a l l be k i l l e d and one or more leg bones examined for healing of the rachitic metaphysis according to the "line t e s t " described below. T h e reference o i l a n d / o r the assay o i l m a y be d i l u t e d before feeding w i t h a n edible vegetable o i l free f r o m V i t a m i n s A and D. T h e d i l u t e d o i l s h a l l be stored i n the d a r k at a temperature not exceeding 50° F . a n d t h e duration of t h i s storage s h a l l n o t exceed t h i r t y days. N o t more t h a n 0.1 cc. of t h e d i l u t e d o i l shall b e f e d as a daily dose. D u r i n g t h e assay period a l l c o n d i tions of environment (particularly w i t h reference t o physiologically active radiations) shall be maintained as uniformly as possible w i t h respect t o the assay a n d reference groups! Line test—The line test shall be made on the p r o x i m a l end of a t i b i a or distal end of a radius or ulna. T h e end of the desired bone is removed from the a n i m a l and cleaned of adhering tissue. A longitudinal median section shall be made through the end of the bone w i t h a clean, sharp blade to expose a plane surface through the j u n c t i o n of t h e epiphysis a n d diaphysis. I n a n y one assay the same bone of a l l the animals must be used a n d sectioned through t h e same plane. B o t h sections of the bone shall be rinsed i n distilled water and shall then immediately be immersed i n a 2 per cent aqueous solution of silver nitrate for one minute. T h e sections s h a l l then be rinsed i n distilled water and the sectioned surfaces of the bone shall be exposed 9  i n w a t e r t o d a y l i g h t or other source of actinic l i g h t u n t i l the calcined areas have developed a clearly defined s t a i n w i t h o u t m a r k e d discoloration of the uncalcified areas. • Records' shall be made i m m e d i a t e l y of the extent a n d degree of calcification of the rachitic metaphysis of every section. I t shall be permissible t o use modifications of the described procedure for staining, p r o v i d e d t h a t s u c h modified procedures c l e a r l y differentiate between calcified a n d uncalcified areas. Recording of data—On the day beginning the assay period a n d on the t e n t h day thereafter a record shall be made of the b o d y weight of each rat. A record.shall be made of the q u a n t i t y of rachitogenic diet consumed per r a t per diem during the assay period. N u m e r i c a l values shall be assigned to the extent a n d degree of calcification of the r a c h i t i c metaphysis of the bones examined b y the line test so t h a t i t w i l l be possible t o average the performance of each group. Vitamin D potency of the assay oil—In determining the V i t a m i n L> potency of the assay o i l t h e average performance of groups w i t h respect t o healing of the r a c h i t i c metaphysis s h a l l be considered, p r o v i d e d t h a t the average performance of a reference group w i t h respect to calcification of the r a c h i t i c metaphysis shall be determined b y the d a t a f r o m rats w h i c h i n d i v i d u a l l y show a n extent a n d degree of calcification i n the r a c h i t i c metaphysis as determined b y the line test equal t o or greater t h a n a c o n d i t i o n described as a positive macroscopic evidence of calcification, b u t less :than a n extent a n d degree of calcification described as complete healing. T h e d a t a f r o m a reference group s h a l l be considered V a l i d f o r establishing t h e V i t a m i n D p o t e n c y of the assay o i l o n l y w h e n t w o - t h i r d s or more, b u t n o t less t h a n seven rats, show i n d i v i d u a l l y a n extent a n d degree of calcification of the r a c h i t i c metaphysis equal to or greater t h a n a c o n d i t i o n describ8d=as positive macroscopic evidence of calcification, b u t less t h a n a n extent a n d degree of calcification described as complete healing. T h e d a t a f r o m a n assay group shall be considered v a l i d for establishing t h a t a n assay o i l conforms w i t h the U . S . Pharmacopoeia s t a n d a r d for V i t a m i n D i n c o d l i v e r o i l o n l y w h e n t w o - t h i r d s or more, b u t n o t less t h a n seven rats, show i n d i v i d u a l l y a n extent a n d degree of calcification of t h e r a c h i t i c metaphysis equal t o or greater t h a n a c o n d i t i o n described as positive macroscopic evidence of calcification. T h e d a t a f r o m a r a t shall be considered v a l i d for establishing the average performance of a group o n l y o n the condition t h a t the weight of the rat o n the eleventh d a y of the assay period s h a l l equal or exceed the weight of t h e r a t on t h e beginning d a y of the assay period; a n d t h a t t h e r a t has consumed 2 o r m o r e G m . of the rachitogenic diet o n each d a y of t h e assay p e r i o d a n d 40 G m . or more of the rachitogenic diet d u r i n g t h e assay p e r i o d a n d o n the c o n d i t i o n t h a t the r a t has consumed each prescribed dose of c o d l i v e r o i l w i t h i n t w e n t y - f o u r hours f r o m the t i m e it. was fed. T h e V i t a m i n D potency of the assay o i l i s then calculated according to the following procedure. L e t " R " equal t h e d a i l y dose i n m i l l i g r a m s of the reference o i l necessary t o p r o duce i n a reference group a n average extent a n d degree of calcification " C " n o t less than a c o n d i t i o n described as a narrow continuous line of calcification b u t less t h a n a n extent a n d degree of calcification described as complete healing. L e t "A"  equal the d a i l y dose i n m i l l i g r a m s of the assay o i l t h a t w i l l produce i n  a n assay group a n average extent a n d degree of calcification equal to or greater than " C . " If t h e p r o d u c t of J^^  J *  [units p e r gram of V i t a m i n D , contained i n t h e refer-  ence oil] is equal to or greater t h a n 85, then the assay o i l contains i n . each gram 85 or more u n i t s of V i t a m i n D a n d complies w i t h the U . S . Pharmacopoeial requirem e n t for V i t a m i n D potency. 10  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0105547/manifest

Comment

Related Items