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The availability of Lysine autoclaved in the presence of carbohydrate [and] The animal protein factor… Stevens, Joan Millicent 1950

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THE AVAILABILITY OP LYSINE AUTOCLAVED IN THE PRESENCE OF CARBOHYDRATE. A Thesis submitted i n P a r t i a l Fulfilment of The Requirements f o r the Degree of MASTER OF SCIENCE IN AGRICULTURE In the Department of POULTRY HUSBANDRY The University of B r i t i s h Columbia May, 1950. Joan M. Stevens ABSTRACT (a) The A v a i l a b i l i t y of L y s i n e A u t o c l a v e d i n the Presence of a C a r b o h y d r a t e . Heat treatment has l o n g been known to improve the v a l u e of soybean o i l m e a l f o r c h i c k growth . However, more r e c e n t l y i t has been demonstrated t h a t too much heat or too l o n g a h e a t i n g p e r i o d lowers the a v a i l a b i l i t y of c e r t a i n of the amino a c i d s p r e s e n t . T h i s lowered a v a i l a b i l i t y was a c -companied by a brown c o l o r a t i o n . I n v e s t i g a t o r s had shown t h a t such a brown c o l o r a t i o n occurs i n other m a t e r i a l s as a r e s u l t o f a r e a c t i o n between r e d u c i n g sugars and amino a c i d s . As a r e s u l t t h i s s tudy was undertaken to determine whether a s i m i l a r r e a c t i o n was r e s p o n s i b l e f o r the d a r k e n i n g i n c o l o u r of the soybean d u r i n g h e a t i n g and whether or not such a r e a c t i o n a f f e c t e d the n u t r i t i v e va lue of the mea l . The r e s u l t s of the s tudy showed t h a t a decrease i n n u t r i t i v e v a l u e d i d accompany the browridsoj* o f soybean o i l -m e a l . I t was a l s o shown t h a t t h i s decrease was m a i n l y due to a l o w e r i n g o f the a v a i l a b l e m e t h i o n i n e . As the l y s i n e gave the g r e a t e s t growth r e s p o n s e , the s tudy was c o n t i n u e d u s i n g c r y s t a l l i n e l y s i n e and c e r e l o s e . I t was shown the r e a c t i o n was a f f e c t e d by the time of h e a t i n g , the r a t i o of l y s i n e to c a r b o h y d r a t e p r e s e n t , and the presence o f w a t e r . Assays were c a r r i e d out both b i o l o g i c a l l y and m i c r o -b i o l o g i c a l l y . ABSTRACT (b) The Animal P r o t e i n F a c t o r i n R e l a t i o n t o the N u t r i t i o n o f the C h i c k . A s e r i e s of t h r e e exper iments were c a r r i e d out to d e t -ermine , i f p o s s i b l e , the v a l u e of Animal P r o t e i n F a c t o r C o n c e n t r a t e s i n p r o a c t i c a l c h i c k r a t i o n s when f e d to c h i c k s which were not d e p l e t e d of the f a c t o r ( s ) . Experiment I was set up u s i n g a b a s a l r a t i o n which would c o r r e s p o n d to a p r a c t i c a l s t a r t e r r a t i o n u s i n g wheat and a mixture o f coarse g r a i n s as the c e r e a l s . The r a t i o n was f e d wi th and wi thout a source o f a n i m a l p r o t e i n and was supplemented at v a r i o u s l e v e l s wi th the A . P . F . produced by-Merck and Company. Experiment I I was set up u s i n g a h i g h energy type of d i e t c o n t a i n i n g corn and soybean such as i s used f o r b r o i l e r p r o d u c t i o n . Males and females were f e d s e p a r a t e l y to determine i f t h e r e was a s eparate response o f c o c k e r e l s and p u l l e t s t o the A . P . F . S u p p l e m e n t a t i o n . Experiment I I I was set up a g a i n u s i n g a c o r n soybean r a t i o n . The purpose of t h i s experiment was to compare the Animal P r o t e i n F a c t o r Concentra te s produced by Merck and L e d e r l e r e s p e c t i v e l y . From the r e s u l t s i t appears t h a t supp lementa t ion w i t h A . P . F . c o n c e n t r a t e s i s p r a c t i c a l but that the r e s u l t s v a r y w i t h the l e v e l and the type o f d i e t u s e d . A p p a r e n t l y t h e r e i s an optimum l e v e l above which f u r t h e r supp lementa t ion e x e r t s a d e p r e s s i n g e f f e c t upon c h i c k growth . T h i s i s a l s o t r u e where f i s h m e a l i s i n c l u d e d i n the d i e t . L e d e r l e gave a g r e a t e r response than d i d the M e r c k ; p r o d u c t both i n growth and f e e d e f f i c i e n c y . ACKNOWLEDGMENTS The writer wishes to express appreciation and gratitude to Professor Jacob B i e l y , of the Department of Poultry Husbandry, f o r h i s kindly guidance i n the conduct of t h i s study* Thanks are also due to Professor E.A.Lloyd and Mrs* 8. March of the Department of Poultry Husbandry, f o r t h e i r constructive c r i t i c i s m i n the pre-paration of the manuscript. TABLE OF CONTENTS* ESS© INTRODUCTION 1 REVIEW OF LITERATURE... • 3 EXPERIMENT l a . Materials and Methods • 6 Results and Discussion...............V. 8 Summary .'• 10 EXPERIMENT l b . Materials and Methods. . V . . V . . . . . . » » . . ^ f . 11 Results and Discussion................. 13 Summary. 14 EXPERIMENT II Review of Literature 15 Materials and Methods.................. 16 Results and Discussion................. 18 Summary........... 20 GENERAL SUMMARY... 21 BIBLIOGRAPHY 23 (1) INTRODUCTION On account of the short supply and high p r i c e s of animal proteins i n recent years, there has been an increasing demand for concentrates of plant o r i g i n . Among the d i f f e r e n t plant proteins, soybean oilmeal has proved to be the most s a t i s f a c t o r y f o r feeding poultry. However, because of i t s l e s s e r n u t r i t i v e value i n i t s raw state and the discovery of improvement through heating, much a t t e n t i o n has been given to the processing of soybean i n the production of soybean oilmeal. I t has been demonstrated that the n u t r i t i v e value of soybean oilmeal i s improved to a c e r t a i n extent by heat treatment. However, i f the heat treatment i s unduly prolonged, i t has been demon-strated that the n u t r i t i v e value decreases.(7) The l o s s i n n u t r i t i v e value has been shown to be due to either destruction or a lowered a v a i l a b i l i t y of the amino acids and vitamins.(9,10) The increase i n brown coloration accompanying the l o s s of n u t r i t i v e value suggests some connection with the M a i l l a r d or "Browning Re-action.' 1 The l a t t e r has been found to take place i n mixtures containing reducing sugars and proteins and was f i r s t reported by M a i l l a r d i n (2) 1912. (1) Investigators (2,3,4,) found i t to possess p r a c t i c a l s i g n i f i c a n c e i n the prooesses of drying milk and eggs, and the storage of molasses. In studies connected with these products, i t was established that the re-action was not b a c t e r i a l i n nature, but that i t was affected by the i n i t i a l pH, the presence of moisture and oxygen. However, at that time l i t t l e was known concerning the effeot of the re-action on the n u t r i t i v e value of the protein and carbohydrate involved. More recently with the i d e n t i f i c a t i o n and i s o l a t i o n of the amino acids, a closer study of the re-aotion has become possible. I f the brown coloration of soybean oilmeal on processing could be shown to be due to the M a i l l a r d re-aotlon, I t would seem to be desirable to learn the ef f e c t of the re-aotion on the protein supplied by the soybean oilmeal. O) REVIEW OF LITERATURE/ EXPERIMENT Ia & l b . The deleterious e f f e c t of autoclaving too long or at too high a temperature has been demonstrated by numerous investigators. (5-8) Evans and MoGrinnis (7) attributed the decrease i n n u t r i t i v e value from overheating to the lowered a v a i l a b i l i t y of methionine. Riesen et a l . (9) i n work with raw, heated and over-heated soybean oilmeal showed that for the amino acids studied the amount l i b e r a t e d by aoid hydrolysis was unaffected by heat treatment except In the case of l y s i n e , argenine, and tryptophane, and that the l i b e r a t i o n of these three was decreased by prolonged heating. Clandinin et a l . (10) supplemented a p r a c t i c a l type of d i e t containing overheated soybean with vitamins, methionine, and l y s i n e and produced growth i n chicks comparable with that secured from properly oooked soybean oilmeal. Thus overheated soybean oilmeal i s apparently d e f i c i e n t i n avai l a b l e l y s i n e as well as i n methionine. The l y s i n e i n proteins other than soybean oilmeal has been shown to be affected by heat treatment. Harris and M a t t i l l (11) i n work with kidney and l i v e r t i s s u e found that hot alcohol extraction produced no l o s s i n nitrogen from the proteins but caused a U) decrease in the amino nitrogen. To explain this, they suggested the combination of the free amino groups with other re-active groups, forming enzyme resistant linkages. They farther-suggested that i f the free amino groups in protein are for the most part lysine, slightly more than half the epsilon-amino groups of lysine disappear during hot alcohol extraction, possibly due to linkage with available oarboxyl, kh&inb^  or hydroxyl groups. Greaves et a l . (12) using rats, showed that casein heated for JO minutes at 140° was deficient in lysine. However, Block et a l . (15) showed that the same amount of lysine could be isolated chemically, from raw oasein, or casein that had been heated for 65 minutes at 150°. Z i t t l e and Bldred (14) confirmed the work of Block et a l . (15) finding that lysine was chemically present but not released by enzymes. More recently by the use of microbiological techniques i t has been demonstrated that a part of the lysine in heated soybean oilmeal was destroyed or was unavailable to micro-organisms. (10) A re-action has recently been demonstrated between amino acids and reducing sugars in potato chips. This re-aotion might explain the loss of nutritionally available lysine following heat treatment. Patton and Pyke (1^) working with potato chips, (5) found that the presence of amino acids and reducing sugars produced the browning re-action on heating. Glyoine and glucose produced such a re-action, but so did other combinations of amino acids and reducing sugars. In no case did any sugar or amino acid produce browning when heated alone. There may be some s i m i l a r i t y between t h i s re-aotion and the re-action involving l y s i n e i n overheated soybean oilmeal or casein. Lysine, as one of the essential amino acids, i s the one most usually d e f i c i e n t i n animal d i e t s . I t was f o r t h i s reason i t was chosen i n preference to methionine as a basis of t h i s study. The following experiments were undertaken to determine the e f f e c t of heat treatment on l y s i n e i n the presence and absence of carbohydrate,with a view to es t a b l i s h i n g a basis for the lowering of the a v a i l a b i l i t y of the amino acids, p a r t i c u l a r l y l y s i n e , by the prolonged heat treatment of soybean oilmeal. (6) EXPERIMENT Ia - MATERIALS AND METHODS A study was undertaken to determine whether pure l y s i n e autoclaved alone and i n the presenoe of a oarbohydrate could be u t i l i z e d by the chick as a supplement to overheated soybean oilmeal. A diet composed of natural ingredients was fed, i t s composition i s given i n Table I. The soybean oilmeal included i n the d i e t s fed,received one of two treatments. Some diets contained meal of a high n u t r i t i v e value which had been autoclaved f o r | an hour at 100° C. This w i l l be referred to as properly cooked meal. The others contained meal of a low n u t r i t i v e value which had been autoclaved for 4 hours at 120° C. and w i l l be referred to as over-heated soybean oilmeal. This l a t t e r meal would be of low n u t r i t i v e value and would be d e f i c i e n t i n methionine, l y s i n e , and vitamins.(10) The colour of the overheated soybean oilmeal became dark brown, whereas there was only a s l i g h t ohange i n the color of the meal that was autoclaved & an hour at 100° 0. New Hampshire chicks, not sorted as to sea, were used i n t h i s experiment. From the time of hatching u n t i l one week of age they were kept i n a battery brooder and fed a complete s t a r t i n g r a t i o n . A t the end of t h i s time the birds were divided at random TABLE I . Composition of Basal Diet. 66 Ingredients. Ground corn Ground wheat....... Ground barley Dehydrated a l f a l f a B~Y feed I250 gammas r i b o f l a v i n per gm) Ground oyster s h e l l . . . . . . . . . . . . . • Dicalcium phosphate......... Salt Soybean oilmeal L i v e r paste. Choline chloride Vitamin D. supplement x MnS04 # per cent 22 26 10 5 0 2 1 0 31 .0 0 .0 .4 .0 .0 .5 .0 .2 .1 x 100 A.O.A.C. mnits activated animal sterols were added to each 100 gm of diet, # Added at a l e v e l of 4 ounces per ton of d i e t . (7) i n t o sixteen groups. They were wing-banded, weighed and maintained for the following three weeks on the experimental d i e t s . Individual weight records were made weekly. The DL form of l y s i n e * and methionine was added to the d i e t s . In the d i e t s i n which l y s i n e was added as a supplement, i t was added at a l e v e l of 0.6%. When the Methionine was used i t was added at a l e v e l of 0.3%. These amounts i n addition to that oontained i n the basal d i e t , were s u f f i c i e n t to meet chick requirements, as sp e c i f i e d by Almquist and Grau.(l6) The l y s i n e added to di e t 7 was weighed out into a beaker and autoclaved f o r 4 hours at 120° C. During the heating i t changed to a s l i g h t l y brown colour, shrank, and formed a soft cake i n the bottom of the beaker. This cake was ground with a mortar and pestle before being added to the d i e t . The l y s i n e added to d i e t 8 was weighed and then mixed i n an open tray with cerelose. (1 part l y s i n e to 10 parts oerelose) The mixture was autoclaved for 4 hours at 120°. The lysine-oerelose mixture formed a black spongy mass which was mixed with some of the basal d i e t and ground i n a Wiley m i l l . 3E The l y s i n e used was i n the form of l y s i n e monohydrochloride xs Manufactured by Corn Products Refining Co., A r g o , I l l . RESULTS AND DISCUSSION -EXPERIMENT l a . Table II gives the d i e t s fed, number of chicks per group, and the average weight at twenty-eight days of age. There was no mortality throughout the experiment. The growth r e s u l t s show that the chicks fed the d i e t containing soy-bean oilmeal autoclaved t h i r t y minutes at 100° grew much better than those fed the d i e t containing soybean oilmeal autoclaved four hours at 120° • Methionine alone had l i t t l e supplementary value to the overheated soybean oilmeal, but when both methionine and l y s i n e were added, the overoooked meal produced growth nearly equal to that of the chicks fed the soybean oilmeal autoclaved f o r t h r i t y minutes at 100° •Methionine or both methionine and l y s i n e added to the basal d i e t containing properly heated soybean oilmeal gave only a s l i g h t increase i n growth. Autoclaving pure l y s i n e f o r four hours at 120° had no e f f e c t on i t s value as a supplement to overheated soybean oilmeal. On the other hand, autoclaving l y s i n e i n the presence of oerelose e i t h e r destroyed i t or made i t unavailable. These r e s u l t s indicate that the l y s i n e re-acts with some group present i n oerelose. Such a r e - a c t i o n might be analogous to the browning re-action of amino acids and reducing sugars suggested by Patton and Pyke (15), as cerelose i s mainly composed of glucose. I t i s i n t e r e s t i n g to note the s i m i l a r darkening i n colour TABLE I I . E f f e c t of Autoclaving Lysine i n Presence of Carbohydrates on I t s Supplementary Value for Overheated Soybean Oilmeal. Autoclaving of Supplements to d i e t I n i t i a l Time Tempera-ture Ho.chicks per l o t . 0 hrs. C 1. ™ 100 Hone 12 0.30 12 2. 100 DL-me t h i onine 12 12 3. i 100 0.3$ w n 8 0.6$ DL - Lysine 8 4« 4 - 120 Hone 12 12 5- 4 120 DL- Methionine 12 12 6. 4 120 0.30 II it 8 0.60 DL - Lysine 8 7- 4 120 0.30 DL / Methionine 8 0.60 treated DL-lysine # 8 8. 4 120 0.30 DL- Methionine 8 0.60 treated DL-lysine ## 8 Average weight at 28 days. gm gm. 238 222 230 M 246 246 246 229 252 241 155 156 156 164 173 169 222 223 223 215 234 225 140 I48 A Average of duplicates. # Lysine was separately autoclaved for 4 hours at 13O 0 0 ##Lysine was separately autoclaved as a 1:10 mixture with oerelose,4 hours at 120 of lysine heated with oerelose and the darkening of soybean oilmeal* In both cases the darkening may have been due to a re-action between carbohydrate and the amino acids present. It seems probable that when lysine becomes unavailable on the heating of casein, i t i s due to some re-action other than one with a reducing sugar, since there i s very l i t t l e carbohydrate i n casein. However, i t may be possible that some group common to both casein and carbohydrate re-acts with the lysine. Assuming that there are two types of re-actions, i t i s possible that they both oocur i n overheated soybean oilmeal. Even i f i t i s assumed that lysine becomes unavailable on heat treatment due to the formation of an enzymatic-resistant linkage with some other substance present, i t s failure to support the growth of micro-organisms after acid hydrolysis i s d i f f i c u l t to explain. Evans and McGlnnis after autoclaving soybean oilmeal one hour at 130° 0. found that only 10% of the lysine was available on acid hydrolysis for the growth of STREPTOCOCCUS FAECALIS R. Riesen et a l . (9) found 48# of the lysine available to LEUCONOSTOC MESENTEROIDES P-60 after acid hydrolysis of soybean oilmeal that had been autoclaved for-four hours at 15 pounds pressure* Therefore, i t seems that the explanation of this i s that the lysine becomes unavailable to the bacteria Involved as the lysine i s apparently stable at the temperature used i n both the above cases. - 1 0 -SUMMARY. o 1 . Lysine autoclaved alone f o r four hours at 1 2 0 supplemented overheated soybean oilmeal f o r chick growth as e f f e c t i v e l y as unheated l y s i n e * 2* Lysine autoclaved with oerelose f o r four hours at o 1 2 0 C. was eithe r destroyed or rendered unavailable f o r chick growth* 3* Supplementary methionine and l y s i n e d i d not Improve the growth of chicks fed a d i e t containing soybean oilmeal o which has been autoclaved at 1 0 0 C. f o r t h i r t y minutea* 4 . The decreased n u t r i t i v e value of overheated soybean oilmeal was almost f u l l y corrected by supplementary methionine and lysine* 5* Methionine alone had l i t t l e supplementary value to overheated soybean oilmeal* -11-KATEI1IALS A1TD 1IETI-I0DS EXT5RIIT11TT l b From the f i r s t "biological experiment i t was apparent the cerelose Then heated with lysine at a r a t i o of 10:1 either destroyed i t or made i t unavailable to the chick. An experi-ment was undertaken to determine the effect of d i f f e r e n t r a t i o s of cerelose to lysine i n autoclaving upon the a v a i l -a b i l i t y of the amino acid. Diets were also included to deter mine the effect of autoclaving lysine i n the presence of a protein free of carbohydrate. The protein used was soybean protein. In experiment lb the s?me procedure was followed as i n experiment Ia. Unsexed Hew Hampshire chicks were fed a complete st a r t i n g r a t i o n for a week and then wing-banded and placed on the experimental r a t i o n for the following three weeks, \7eight records were made weekly. The experiment was set up using duplicate groups of ei^ht chicks each. The basal diet was the same as was used i n experiment Ia (table I ) . IJine d i f f e r e n t diets were fed, s i x of them containing the over-heated soybean o i l meal, so that they were de f i c i e n t i n methionine and l y s i n e . The lysine and methionine supplements were added at the same lev e l s as i n the previous experiment. The negative control was supplemented with methionine alone and the p o s i t i v e control with lysine as w e l l as methionine. Lysine.-cerelose mixtures at r a t i o s of 1:1, 1:S, 1:5, and 1:10 were autoclaved for 4 hours at 120^. These cons-t i t u t e d the supplements to the other four diets containing -12-overheated soybean o i l meal. The other three diets contained soybean protein instead of the soybean o i l meal. One of them contained raw soybean protein supplemented with methionine. The other two con-tained soybean protein autoclaved f o r four hours at 1 2 0 ° . One of these was supplemented with Methionine and the other with methionine and l y s i n e . On autoclaving the 1 : 5 & 1 : 1 0 mixtures* the same spongy masses were formed as i n the previous experiment. These two mixtures were therefore mixed with some basal diet and ground i n a Wiley m i l l . The 1 : 1 and 1 : 2 lysine-cerelose mixtures formed a thick black syrup. As a r e s u l t i t was taken into solution i n water and the solution mixed into the feed wMch. was then sieved. -13-PJSSULTS AKD DISCUSSION- EXPERIMENT Tb Table I I I gives the diets fed, chicks per group, and the average weekly weights. I t was d i f f i c u l t to explain why the weights at four weeks were only approximately 175 grams instead of about 100 grams higher. However recent experi-mental evidence has shown that soybean o i l meal i s a source of an unknown factor required for chick growth. Since some soybean o i l meals are d e f i c i e n t i n t h i s factor (and i f that was the case with the one used i n t h i s experiment,) i t might have caused a general lowering of the weights i n t h i s case. In any event, the r e s u l t s show s i g n i f i c a n t differences between.the experimental treatments, moreover,; such a deficiency would af f e c t a l l groups a l i k e . As would be expected from the r e s u l t s i n Experiment Ia, the lysine and methionine had a good supplementary effect on the overheated soybean o i l meal, whereas the methionine alone had l i t t l e supplementary value. In both a r a t i o of 1:5 and 1:10 the cerelose apparently makes the lysine completely unavailable to the chick following autoclaving of the mixtures. The weights i n both diet 3 and diet 4 were the same as the weights of the nega-ti v e control, so that the lysine apparently had no supplement-ary value. In the 1:2 and the 1:1 mixtures, only a portion of the lysine was unavailable. However, even i n the 1:1 mixture a portion of the lysine was rendered unavailable to the chick. A l l the diets containing soybean protein gave as good Table I I I - The E f f e c t of Varying the l e v e l o f Carbohydrate mixedrwith lysine during Autoclaving on the Supplementary Value of the Lysine for Overheated Soybean Oilmeal. Diet No. Vegetable Protein. He-at Treatment Supplement to Heat Treatment Wt at of Veg.Protein. the Diet. of l y s i n e . 28 days. 1. 1 Soybean Oilmeal i 4 hrs® 120° 0.30 Methionine Hone 88 2. IT n i n ti 0.30 Methionine 0.60 DL Lysine None lbO 3. I I I I I I II 0.30 DL.Methionine 0.60 DL- Lysine 4 hrs & 120° © 1:10 r a t i o with 85 glucose 4. n II II n 0.3> DL .Methionine 0.60 DL Lysine 4 hrs & 120% 1:5 r a t i o with glucose 87 b. n i II n w 0.30 DL Methionine 0.6$ DL Lysine 4 hrs & 120° & 1:2 r a t i o with, glucose 106 6. n I I I I u 0.60 Dlt Methionine 0.6% DL Lysine 4 hrs 120% 1:1 r a t i o with glucose 124 v. Soybean Protein Unheated 0.30 DL Methionine 177 0. n II 4 hrs at 120° 0.3% DL Methionine 170 9. H II n ti 0.30 DL Methionine 0.60 DL Lysine None 172 -14-growtii as the p o s i t i v e control. Apparently from these r e s u l t s the amino acids, p a r t i c u l a r l y lysineare s t i l l present at high enough lev e l s even after autoclaving for four hours, at 120°C. This i s to "be expected since there i s not reducing sugar present to re-act with the protein. Summary 1. Lysine when i t i s mixed with cerelose at a r a t i o of 1:5 or 1:10 and autoclaved f o r four hours at 120°, "becomes n u t r i t i o n a l l y unavailable for the chick, 2. Lysine when mixed with cerelose at a r a t i o of 1:2 or 1:1 becomes only p a r t i a l l y unavailable upon autoclaving f o r four hours at 120°. 3. Heat treatment of soybean protein does not improve i t s n u t r i t i o n a l value for the chick. 4. Autoclaving soybean Protein for four hours at 120° did not a f f e c t i t s n u t r i t i v e value for chicks. 5. The addition of Methionine and Lysine to overheated soybean protein had no supplementary effect on chick growth. -15-Experiment I I - Review of - Literature Studies on the formation of humin i n protein hydro-lysates have demonstrated that heating causes a re-action to occur "between carbohydrates and certain amino acids (1,17,18). Since that time t h i s re-action, often referred to as the M a i l l a i r d reaction has "been important where the development of a "brown colour during processing i s detrimental i n foods. More recently i t has been demonstrated that t h i s re-action between reducing sugars and amino acids causes the l a t t e r to become n u t r i t i o n a l l y : unavailable.Patton and H i l l (19) using the microbiological assay found that on autoclaving there was a loss off tryptophane, l y s i n e , methionine and the amino vitamins of the B Complex. They found that i t was possible to minimize t h i s loss by the use of sucrose, which i s not a reducing sugar, instead of glucose i n the media. In work with soybean o i l meal, Patton et a l . (20) showed that heat damage was due to a s i m i l a r re-action between amino acids 3,nd reducing sugars. Riesen et a l . (9) also working with soybean o i l meal found that the l i b e r a t i o n of lysine arginine and methionine was decreased by prolonged heating. vTaisman and Elvehjam (21) demonstrated that the n u t r i t i v e value of edestin was lowered following autoclaving, and that i t could be restored by the addition of l y s i n e . The work of Riesen et a l . (9) on soybean o i l meal and further the work of Clandinin (22) on fishmeals has shown that the l i b e r a t i o n of lysine from heat damaged protein materials by enzymatic hydrolysis i s much lower thaxi by acid hydrolysis. - 1 6 -H a t e r i a l s and Methods A series (series 1), consisting of nine samples i n quadruplicate of 50 mg. of L-Lysine monohydrochloride mono-hydrate (equivalent to 36.5 mg. L-Lysine) and nine samples i n quadruplicate of 50 mg. of L-Lysine monohydrochloride • monohydrate and 250 mg. glucose^, were placed i n 125 ml. orlonmyer f l a s k s and heated i n an autoclave at f i f t e e n pounds steam pressure for 0, 4, '7-\, 15 and 30 minutes and f o r 1, 2, 4 and 8 hours respectively. A second series (Series 2), i d e n t i c a l to these except that 2 ml. of d i s t i l l e d water was added to each f l a s k , was set up and heated i n an autoclave i n the manner referred to at>ove. Following heat treatment two samples of each quadru-p l i c a t e i n "both series were subjected to acid hydrolysis and two to enzymatic hydrolysis and the amount of L-Lyaine present -was determined m i c r o t i o l o g i c a l l y . The methods of hydolysis and microbiological procedure employed were similar to those employed by Reisen et a l (8). A tabulation of the samples, treatments and r e s u l t s i s given i n Tables IV and V^ TABLE IV. Libe r a t i o n of L-Lysine. Series 1 - Heated Without Water. Sample Treatment L-Lysine Liberated. Acid Ensymatic Ho. Description Water. Autoclave. Hydrolysis. Hydrolysis. 1 . 3 6 . 5 mg. L-Lysine* None No Heat 3 7 . 9 » » 37-01 2 . " ti M 4 min.® 15# 3 6 . 1 3 7 . 8 i i ii 7 1 / 2 Mln .@15# 3 5 . 9 3 3 . 0 4 . .» ii a 15 min.© 15# 3 6 . 4 3 6 . 7 5 . " ii ii 3 0 min.® 15# 3 6 . 5 3 6 . 5 6 . " it ii 1 hr.® 15# 3 5 . 8 3 6 . 5 7 . * ii « 2 hr. ® 15# 3 4 . 8 3 4 . 4 8 . " ii ii 4 hr. @ 15# 3 3 . 9 3 5 . 8 9 . " ii ii 8 hr. @ 15# 3 5 . 3 3 4 . 5 1 0 . 3 6 . 5 mg.L-Lysine* 250 mg.Glucose ii No heat 3 9 . 0 3 6 . 2 1 1 . " II it 4 min. @ 15# 2 8 . 5 2 2 o 3 1 2 . " a H 7 1 / 2 min @ 15# 2 3 . 2 1 7 . 1 1 3 . > it ii 1 5 min @ 15# 1 7 . 9 1 3 . 9 1 4 . , v it ii 30 min.® 15# 1 5 . 0 8 . 3 1 5 . > ii ii 1 hr. @ 15# 7 . 4 3 . 4 1 6 . » II ii:: 2 hr. ® 15# 5 . 6 2*2 1 7 . 5 ii ^ i i 4 hr. @ 15# 5 . 3 1 . 3 1 8 . " ii ii 8 hr. @ 15# 4 . 8 1 . 0 50 mg.L-Lysine monohydrochloride monohydrate was ac t u a l l y used. This i s equivalent to 3 6 . 5 mg. L-Lysine. ** Average of duplicates. Table V. Liberation of L-Lysine Series 2-Heated with Water Sample Treatment L-Lysine Liberated* No. Description Water Acid Autoclave. Hydrolysis. Enzymatic Hydrolysis. i . 36.5mg.L-Lysine * 2 ml •No heat 37.9*» 37.0*» 2. H « tt 4 min.® 15# 36.6 37.2 3. it ii ii 71/2 min.@15# 34.1 37.6 4. ti ii it 15 min.® 15# 35.8 37.7 5. it ti it 30 min.® 15# 33.7 36.2 6. ft ii ii 1 hr. @ 15# 35.3 34.5 7. —i It ii ii 2 hr. @ 15# 35.4 34.0 8. it ii ii 4 hr. @ 15# 36.1 33.4 9. ii ii ii 8 hr. @ 15# 37.0 33.8 10. 36*5 mg.L»Lysine 250 mg. Glucose ii No heat 39.0 36.2 11. tt it tt 4 min. @15# 36.5 30.1 12. ii if ii 7 1/2 mln.@15# 36.0 30.8 13. it ii ii 15 min.@15# 35«0 30.6 14. it ii it 30 min.®15# 35.6 33.3 15. 11 ii ii 1 hr. @ 15# 34.2 31»3 16. It it it 2 hr. ® 15# 32.7 28.7 17. It ii ii 4 hr. ® 15# 30.1 26.4 18. ii ii ii 8 hr. ® 15# 28.4 22.6 * 50 mg. L-Lysine monohydroohloride monohydrate was actually used. This i s equivalent to 36.5 mg-Lysine. ** Average of Duplicates. These workers suggest that enzymatic hydrolysis values give a more ' e l i a b l e index of the n u t r i t i v e value of heat treated protein materials than do acid hydrolysis values. Experiment 2 wa,s undertaken to determine whether or not the heating of a lysine - glucose': mixture i n an autoclave for d i f f e r e n t periods of time affects the l i b e r a t i o n of lysine by acid and enzymatic hydolysis i n a manner s i m i l i a r to that reported by the l a t t e r workers (9, 22) i n soybean o i l meal and fishmeals. -18-Resuits and Discussion The heating of L-Lysine dry or i n the presence of water produced no v i s i b l e browning of the lysine or lysine-water solution. On the other hand the heating of a mixture of L-Lysine and glucise-; dry or i n the presence of water resulted i n v i s i b l e browning of the mixture. The degree of browning was more pronounced i n the dry series than i n the water series. Furthermore the degree of browning increased as the time of heating increased i n much the same way as noted i n the soybean o i l meal series of Clandinin et_ a l (6). Indeed, heating the lysine-glucQseo mixture dry not only CB.used various degrees of browning but also was responsible for a. decrease i n the water s o l u b i l i t y of the residues to such an extent that microbiological assays on the residues without acid or enzymatic hydrolysis was considered inadvisable. The r e s u l t s of the microbiological assay work, shown graphically i n Figures 1 and 2, indicate that heating L-Lysine alone i n an autocalve at f i f t e e n pounds steam press-ure for up to eight hours has no appreciable effect on i t s -a v a i l a b i l i t y following acid or enzymatic hydro3.ysis. On the other hand i t i s apparent from the graphs that heating L-Lysine i n the presence of glucose-! has a pronounced effect on i t s a v a i l a b i l i t y subsequent to acid or enzymatic hydro-l y s i s . Further the reduction i n a v a i l a b i l i t y of lysine i n the heated lysine-glucoses samples i s much more pronounced when heated dry than when heated i n the presence of water. .It w i l l also be noted from the figures that the greatest -19-decrease i n the a v a i l a b i l i t y of lysine i n these heated mixtures i s effected during the f i r s t hour of hea/ting. This i s p a r t i c u l a r l y true i n the case of the lysine-glucloaeo mixture heated dry. I t i s of interest to point out here the p a r a l l e l between these r e s u l t s and the marked decrea.se i n a v a i l a b i l i t y of lysine reported by Clandinin ( 9 ) as occuring i n f i s h meal overheated f o r a short time during flame drying. The date show that i n both series enzjanatic hydrolysis released considerably less lysine from the heated l y s i n e -glucose samples than acid hydrolysis. This f i n d i n g suggests the formation on heating of an enzyma.tic r e s i s t a n t linkage between lysine and glucose such as the one postulated by Evans and Butts ( 1 0 ) , I f such i s the case the reaction between lysine and carbohydrate might consist f i r s t of the formation of an enzyma.tic res i s t a n t but acid non-resistant linkage followed by the formation of an enzymatic and a,cid r e s i s t a n t linkage or disintegration of the lysine molecule. . -SO-Summar^ ; 1. The l i b e r a t i o n of lysine by acid and enzymatic hydro-l y s i s from a lysine-glucose mixture heated i n an autoclave at 15 pounds pressure f o r varying periods of time was greatly reduced by the heat treatment. 2. The decrease i n l i b e r a t i o n of lysine was greater when the mixture was heated dry than when heated i n the presence of Y/ater, 3. The l i b e r a t i o n of lysine by enzymatic hydolysis from a heated lysine-glucose mixture was less than the l i b e r a t i o n of lysine by acid hydrolysis, suggesting the formation on heating of an enzymatic r e s i s t a n t linkage between glucose and l y s i n e . -21-General Summary* The foregoing experiments have demonstrated that soybean o oilmeal autoclaved f o r 30 minutes at 100 C. i s of high n u t r i t i o n a l value whereas soybean oilmeal autoclaved f o r 4 o hours at 120 C. was of low n u t r i t i o n a l value (overheated). The overheated meal when supplemented with l y s i n e and methionine was equal i n n u t r i t i o n a l value f o r the chick to the properly heated meal. Lysine autoclaved In the presence of glucose i s e i t h e r destroyed or becomes n u t r i t i o n a l l y unavailable to chicks and bacteria. The re-action of l y s i n e with glucose i s affected by the r a t i o of ly s i n e to glucose present as measured by b i o l o g i c a l t e s t i n g with the chick. Lysine autoclaved alone f o r four hours at 120° remained avail a b l e to the chick as well as to bac t e r i a . Soybean protein, a product free of carbohydrates, was unaffected by autoclaving f o r four hours at 120°C, and waB not improved by supplementing with l y s i n e and methionine. Lysine,autoclaved i n solut i o n with glucose, was more available to bacteria than l y s i n e autoclaved i n a dry mixture. o The l i b e r a t i o n of lysine-glucose mixture heated at 120 C, varies inversely with the length of the heat treatment. Acid hydrolysis following heat treatment releases more l y s i n e f o r b a c t e r i a l growth than does enzymatic hydrolysis. This -28 -would suggest the formation of an enzymatic r e s i s t a n t linkage. From the above observations, i t seems quite l i k e l y that the decrease i n the n u t r i t i v e value of soybean i s due to a r e - a c t i o n between the amino acids and reducing sugars present. This re-action would be s i m i l a r to the M a i l l a r d or "Browning re-action", which was demonstrated i n p a r t i c u l a r with l y s i n e as t h i s amino a c i d i s e s s e n t i a l and i s the one exerting the most l i m i t i n g e f f e c t i n soybean oilmeal as a p r o t e i n source. The others are present i n l a r g e r amounts and should a portion of them become unavailable there i s s t i l l enough i n the free form to f u l f i l l the n u t r i t i o n a l requirements of the chick. As the re-action i s not as yet to date thoroughly understood, and i s affected by the conditions of heat treatment, care should be taken, In the processing of soybeans and other protein concentrates to maintain high n u t r i t i v e q u a l i t y . -23-BIBLIOGRAPBY. 1. Maillard, L.C. Compt. rend.54:66 (1912). 3. Homer, A*, BiochenuJ. 7:101 (1913)!. 3. Roxas, M.L., J.Biol.Chem. 27:71 (1916), 4. Patton, A.R. and Hill.E.G., Science 107:68 (1948). 5. Patton.A.R., Hill,E.G.,& Foreman E.M. , Poultry So. 27:680 (1948)-. 6. Riesen.W.H. ,' Olandinin D.R. ,Elvehjem,C.A., and Cravens,W.W., J.Biol. Ohem. ,167:143 (1947). 7. Waisman,H.A. ,and ElvehJem,C.A. ,J.Nutr. 16:103 (1938). 8. Stevens,J.M. and MoGinnis J.Biol.Chem. ,171:431(1947.) 9. Clandinin.D.R., Poultry So., 28:128 (1949). 10. Evans R.J. , and Butts, H.A., J. Biol.Chem. 7 175:15 (1948). 11. Greaves ,E. 0. ,Morgan A. F., and Love en M.K. , J. Nutr. 16:115 (1938 P. 12. Block,R.J., Jones,D.B., and Gersdoff, C.E.F., J.Biol. Chem. 105;667 (1934). THE ANIMAL PROTEIN FACTOR IN RELATION TO THE NUTRITION OF THE CHECK. Joan M* Stevens A Thesis submitted i n P a r t i a l Fulfilment of The Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE In the Department of POULTRY HUSBANDRY The University of B r i t i s h Columbia May," 1950* TABLE OF CONTENTS. Page INTRODUCTION. 1 REVIEW OF L I T E R A T U R E . 2 EXPERIMENT I Methods and Materials..................... 38 Results and Discussion.••••••••••••••••••• 40 EXPERIMENT II Methods and Materials •. • • 47 Results and Discussion................... 49 EXPERIMENT III Metho ds and Materials................... 55 Results and Discussion 57 GENERAL SUMMARY. 62 BIBLIOGRAPHY.................................... 69 AOKNCTOBDGMBNTS. The writer wishes to express appreciation and gratitude to Professor Jacob Biely, of the Department of Poultry Husbandry, for his kindly guidance i n the conduct of this study. Thanks are also due to Professor E.A.Lloyd and Mrs. B. March of the Department of Poultry Husbandry, for their constructive c r i t i c i s m i n the preparation o f the manuscript. IMRODUCTION. Th© recent discovery of the animal protein factor(s) and the identification of vitamin B l g as one of i t s principal constituents i s one of the most important contributions made to the recent knowledge of poultry nutrition. Through greater knowledge of the use of Animal Protein Factor Concentrates, the elimination of, at least, part of the animal protein i n poultry rations becomes a p o s s i b i l i t y . Interest i n the use and nutritive value of such products has increased with the general shortage and high price of animal proteins. At present many studies are being conducted on the animal protein factor complex, and u n t i l many of these are completed and evaluated, i t w i l l be d i f f i c u l t to estimate the true value o f the oomp lea: i n poultry nutrition. I t i s possible that the use of commercial preparations w i l l make i t possible to formulate poultry rations without animal protein. Review of Literature The existence of a factor or factors associated with animal  proteins and required f o r chick growth established. The value of feeds of animal o r i g i n i n the feeding of poultry and non-ruminant animals has long been recognized. However, more recently, i t became apparent that the benefit was not due to any of the known amino acids or vitamins i n t h e i r composition. The f i r s t d i r e c t evidence of the e x i s t -ence of such a factor(s) came from the work of Byerly, T i t u s , and E l l i s (15). In t h e i r Investigation study of the e f f e c t of the d i e t of hens on the composition of the egg, they i n -cluded a study of embryo mortality of eggs from hens on various rations. In t h e i r observations, they noted a high incidence of mortality i n the second week of Incubation with eggs from hens on a r a t i o n i n which a l l the protein came from a vegetable source. In future work, these investigators (65) reported that t h i s factor was d i s t i n c t from r i b o f l a v i n and that i t was present i n dried grass, dried pork l i v e r , and a combination of meat meal, fishmeal and dried buttermilk. About the same time, Van de Hoorn, Branion, and Graham (106) demonstrated the presence of a chick growth factor associated with casein. However, much of the significance of these early studies was overlooked because of the emphasis that was being placed on amino acids and other u n i d e n t i f i e d factors . 5 -and the d i f f i c u l t i e s e ntailed at that time i n providing f o r same. Reports continued to appear, however, which demonstrated the need f o r animal protein. Christiansen, Deobold, Halpin, and Hart (21) showed that d i e t s containing soybean oilmeal at l e v e l s to provide adequate protein would not support optimal chick growth, although Almquist (1) had shown soybean protein adequate to supply the amino a c i d requirements of the chick, Christiansen et a l . (21) found that a r a t i o n deriving i t s pro-t e i n from soybean o i l meal gave excellent growth when supple-mented with 3-4$ sardine or menhaden f i s h meal. Vegetables supplements such as linseed o i l meal or corn gluten meal gave no added growth response. Meat meal gave some improvement and the e f f e c t was enhanced by the addition of skim milk. However, even both milk and meat meal did not give a growth response equal to that produced by fishmeal. These r e s u l t s were confirmed by Carver and Evans (18) who noted that supplementing such diets with animal products containing only small amounts of protein, produced a marked improve-ment i n growth. Johnson et a l . (46) reported an unknown growth f a c t o r present i n casein, l i v e r , and a small amount i n dried skim milk. I t was not i n evidence i n a l f a l f a , ground yellow corn, wheat, or soybean o i l meal. This f a c t o r was soluble i n ether and thermostable. Schumacher and Heuser (91) - .4-reported a f a c t o r which was e s s e n t i a l f o r growth, maintenance, ©gg production and h a t c h a b i l i t y . It was water soluble, heat stable, dlalyzable and p r e c i p i t a t e d by 90% alcohol. Berry et a l . (4) produced chick of a greater average weight by supplementing a r a t i o n with whey solubles. Heuser and Norris (37) showed that to obtain optimum growth and feed e f f i c i e n c y up to eight weeks of age, rations containing large amounts of soybean oilmeal should also contain 2-3% of animal protein concentrates. This was confirmed by Hammond and T i t u s (36) who obtained more rapid growth by including 2% sardine meal In a soybean r a t i o n and Wilgus and Zander (110) who reported that soybean was not s a t i s f a c t o r y as the sole source of pro-t e i n . They found that as l i t t l e as 1.25% meat and bone scrap s u f f i c e d to promote growth but that 2.5% was the minimum amount that would produce optimum growth. Thus by 1945, i t was established animal proteins contained a factor or factors necessary f o r optimum chick growth, H i l l , Scott, Norris and Heuser (40) showed the factor(s) to be of p r a c t i c a l importance by demonstrating "that with combinations of commonly used feedstuffs, p r a c t i c a l chick rations can be formulated which are not rendered e n t i r e l y complete by supplementing with r i b o f l a v i n alone but require inaddition an u n i d e n t i f i e d growth factor or factors which makes them more s a t i s f a c t o r y f o r feeding." As a r e s u l t , reports began to appear regarding the effectiveness of various proteins as supplements to rations f o r chicks deriving t h e i r vegetable from protein sources* Berry et a l . (5) found that the water soluble portion of f i s h press water was an excellent source of the f a c t o r although i t contained almost no protein. They, therefore, postulated the unknown substance to be a vitamin. Cravens, McGibbon, and Halpln (24) confirmed these r e s u l t s using f i s h press water or ground f i s h v i s c e r a and demonstrated that the factor was not r i b o f l a v i n . Heuser et a l . (38) found that a basal r a t i o n contain-ing expeller processed soybean o i l meal supported better growth than a diet containing solvent processed soybean oilmeal. They found l i v e r paste, livermeal, f i s h solubles and fiBhmeal a l l possible sources of the chick growth fa c t o r . Similar sources of the f a c t o r were noted by Mlshler et a l . (62) and Essary et a l . (28). In 1946, Patton et a l . (7?) supplemented a p u r i f i e d d i e t with fishmeal and methionine but could demonstrate no growth response, although they did get a growth response when f i s h products were added to a corn-soybean r a t i o n . They, therefore, suggested that the requirement f o r the f a c t o r supplied by fishmeal was p e c u l i a r to a corn-soybean r a t i o n . However, McGlnnin and Carver (55) e f f e c t i v e l y demonstrated that the requirement f o r the factor(s) i n f i s h and l i v e r was not affected by the soybean oilmeal - 6 -or the cereals present In the d i e t . Robblee et a l . (80) confirmed the work of McGinn!s and Carver by demonstrating a growth response on a p u r i f i e d r a t i o n . Combs, Heuser, and Morris (22) using microbiological assay made a very complete study and demonstrated that 2% fishmeal would produce an increase i n growth and feed e f f i c i e n c y when added to a r a t i o n complete i n amino acids and known vitamins. As early as 1942, Hammond (35) had noted the favourable e f f e c t of cow manure on chick growth, and i n 1945 Whitson et a l . (108) showed that 8% cow manure was as e f f e c t i v e as 3% sardine meal i n improving the growth of chicks r e c e i v i n g a soybean oilmeal basal. Rubin and B i r d (83) d i f f e r e n t i a t e d the growth factor i n cow manure from any factors previously described. Nichol et a l . (70) found that cow manure, l i v e r extract and f i s h solubles a l l gave good growth when used to supplement a soybean d i e t . Therefore, i t seems l i k e l y that they a l l contained the same growth factor(s) associated with animal proteins. The same growth stimulating e f f e c t was noted by Stephenson et a l . (102) i n s o i l . An a l l vegetable d i e t was e f f e c t i v e l y supplemented f o r chick growth, by the addition of 1% s o i l which wastaken from a f i e l d which had not been manured f o r three years. Kennard and Chamberlain (48) found that deep l i t t e r on the f l o o r served as a source of - 7 -the u n i d e n t i f i e d factor(s) f o r chick growth. The Requirement of Breeding Hens f o r an unknown  ha t c h a b i l i t y factor associated with Animal Proteins  and i t * s i d e n t i f i c a t i o n with the Chick Growth Factor. When, by 1945, the requirement of chicks f o r a f a c t o r or factors associated with animal protein became generally accepted, attention was focused on breeding hens to determine whether or not t h e i r requirement f o r animal proteins was due to the same factor required f o r chick growth. Byerly and associates (15) had noted the lowered h a t c h a b i l i t y on a d i e t where the protein came from a vegetable source. They had also (16) noted that on such a r a t i o n the hat c h a b i l i t y was lower i n the Winter than i n the Spring and Summer months. This was not due to d i r e c t sunlight as a r t i f i c i a l l i g h t produced a s i m i l a r e f f e c t . However, there was no seasonal v a r i a t i o n when the d i e t contained a high q a l i t y beef which was 10$ livermeal. G i l l i s , Heuser, and Norris (30) noted a fac t o r i n l i v e r extract which was d i s t i n c t f o r pantothenic a c i d and was necessary f o r h a t c h a b i l i t y . I t was heat stable and only active i n the presence of s u f f i c i e n t pantothenic acid. McGinnis, Heuser, and Norris (56) concluded fromtheir experiment work, that there i s an un i d e n t i f i e d factor(s) required for reproduction not supplied in a diet where soybean oilmeal i s the sole source of protein. This factor i s present i n meat scrap and l i v e r paste and i s distinct from choline, but i s not found i n dried brewer's yeast.. Cravens, Halpin, and McGlbbon (23) found that i t was necessary to Include f i s h solubles or livermeal i n add-i t i o n to riboflavin i n order to maintain high hatchability in birds kept i n batteries. 2.5% milk in the diet did not improve hatchability and, therefore, apparently lacked some factor present In fis h solubles and livermeal. Whitson, Titus, and Bird (108) found that 6% cow manure dried at 80° to destroy i t s androgenic properties and added to a hen's diet containing no animal protein produced a significant Improvement in hatchability. They also noted a seasonal effect with hatchability being higher i n the Spring and Summer months. These same workers found that hens fed 30% soybean as the sole source of protein produced eggs of lowhatchability. During a 43 week period, only 66% of the f e r t i l e eggs hatched from hens on the soybean basal whereas 84$ of the f e r t i l e eggs from hens on a diet contain-ing sardine meal hatehed. The deficiency in the soybean was corrected by the inclusion of 5% cow manure, 10% sardine meal, or 10% milk. The deficiency of the basal was further revealed by the low v i a b i l i t y of the chicks hatched. Chicks from hens on the soybean basal had a mortality of 16-38$ i n - 9 -the f i r s t week, whereas mortality i n chicks from hens r e -ceiving the supplemented d i e t was 1-11J&. Mortality was as high i n the birds receiving, a d i e t containing J>% eardine meal. However, i t i s l i k e l y that t h i s l e v e l of sardine meal was not sufficient to supply the needs of d e f i c i e n t chicks. From these r e s u l t s , again i t seems l i k e l y that the cow manure factor(s) i s s i m i l a r to the factor(s) associated with animal proteins* Bethke et a l . (7, 8) established that the h a t c h a b i l i t y f a c t o r associated with animal proteins was not choline, methionine, r i b o f l a v i n , pantothenic a c i d , n i a c i n , pyridoxins, f o l i c a c i d or b i o t i n . McGinnis and Carver (54) found that chicks from hens fed on a corn soybean r a t i o n grew poorly on a plant protein r a t i o n containing the f a t soluble vitamins. Mortality was high even i f the d i e t was complete i n a l l I d e n t i f i e d factors. I f fishmeal was added to the d i e t , growth improved and mortality stopped. I f the breeding hens received a r a t i o n containing 1.7j£ fishmeal, t h e i r o f f -spring grew better than those from the hens rec e i v i n g no fishmeal, and i f h% fishmeal was added even better gains were made. Therefore, i t was concluded that the growth response must be due to some fa c t o r i n the fishmeal which was passed to the chick through the egg. I f the breeding d i e t contained 5% fishmeal,or 3% f i s h solubles, or 0.5$ of an - 10 -alcohol f r a c t i o n of l i v e r , the eggs produced contained s u f f i c i e n t of the factor(s) to supply the chick f o r the f i r s t four weeks of l i f e . The findings of McGinn!s and Carver (54) were con-firmed by Robblee and associates (80) who found that chicks from hens on a d i e t containing f i s h solubles d i d better on a soybean r a t i o n than d i d chicks from hens re-ceiving no animal protein. Bethke et a l . (9) obtained s i m i l a r r e s u l t s using fishmeal instead of f i s h solubles. The chicks grew better i f they were hatched from hens re-ceiving fishmeal as well as soybean oilmeal than i f they were receiving soybean oilmeal alone. I f , however, the d i e t contained k% fishmeal, the chicks grew well regard-l e s s of the hen's d i e t . Rubin and Bird (85) working with an a c i d p r e c i p i t a t e from a water soluble extract of cow manure, which was known to produce optimum chick growth when added to a soybean basal, found that i t also supported high h a t c h a b i l i t y i n hens raised on a d i e t free of animal protein. Storage of the f a c t o r i n the hen was shown. When hens were placed on a d i e t of a l l vegetable protein, they continued to produce eggs of high h a t c h a b i l i t y f o r 12-15 weeks following the change from the d i e t containing either fishmeal or the a c i d p r e c i p i t a t e from the cow manure extract. - 11 Rubin, Bird and Rothchild (86) demonstrated that the chick growth and h a t c h a b i l i t y factor(s) i n cow manure was also present inheh feces. As i t was not present i n the d i e t of these hens, i t was evident that synthesis must have occured. The urine free feces from hens i n which an a r t i -f i c i a l anus had been produced, contained a larger amount of the factor(s) than did the normally voided mixture of feces and urine. However, McGinnis, Stevens, and Groves (58) showed that feces which had been frozen a f t e r voiding con-tained very l i t t l e of the growth factor(s) whereas feces c o l l e c t e d under the same conditions and incubated f o r 72 hours at 3G°C contained a considerable amount of the animal protein f a c t o r ( s ) . Therefore, i t became apparent that the synthesis took place a f t e r the feces had been voided. The re s u l t s of these workers seemed to explain, l o g i c a l l y , the seasonal v a r i a t i o n noted by Byerly et a l . (16) and whltson and co-workers (108) Groscke, Rubin and B i r d (33 & 34) found the h a t c h a b i l i t y i n birds on a corn-soybean r a t i o n i n open pens improved i n the Spring and Summer months but i f they were kept i n batter-ie s there was no improvement. They att r i b u t e d t h i s to coprophagy. They overcame the low h a t c h a b i l i t y by adding cow manure to the d i e t . Petersen, Wiese, and Lampman (75) confirm-ed the work of Groscke et a l . (33 & 34) noting that 2% meat scrap i n the d i e t gave s i g n i f i c a n t l y better h a t c h a b i l i t y when the birds were kept on the f l o o r than when they were kept i n ba t t e r i e s . B i r d , Rubin, and Groschke (12) found that i n d i v i d u a l s within a floc k varied greatly i n t h e i r a b i l i t y to reproduce i n the absence of the h a t c h a b i l i t y f a c t o r . Over a ten month period c e r t a i n birds on a d e f i c i e n t d i e t continued to l a y eggs that hatched over 70$. Under the conditions of the experiment, t h i s could not be explained by coprophagy or storage of the factor. For t h i s reason, they found i t necessary to divide t h e i r f l o c k i n two portions i n order to obtain depleted chicks f o r experimental purposes. Kennard, Bethke, and Chamberlain (47) investigated the l i t t e r where deep l i t t e r was used as a source of dietary factors e s s e n t i a l f o r h a t c h a b i l i t y . Birds on b u i l t up l i t t e r had a ha t c h a b i l i t y of 80$ compared with 32$ i n birds on fresh l i t t e r receiving the same d i e t . By including 2.5$ whey i n the d i e t , the h a t c h a b i l i t y was improved to 56$, and i f 2$ meat scrap was included the h a t c h a b i l i t y rose to 67$. If both whey and meat scrap were Included, the h a t c h a b i l i t y of the birds on fresh l i t t e r equalled that of birds on deep l i t t e r . These same investigators experimented with the deep l i t t e r as a source of dietary factors f o r chicks, and found i t to be' a potent source of c e r t a i n f a c t o r s , but i t did not support maximum chick growth i f the d i e t lacked a l f a l f a and - 13 -animal proteins. Thus, by 1948, the unknown growth and hatchability factor(a) associated with animal proteins was definitely established to be necessary i n rations for poultry. As a result, investigators began to look for satisfactory and efficient sources. Wiese, Petersen, and Lampman (109) compared various supplements i n a breeding ration and found that 5*3% fishmeal was sufficient to give good growth in the chicks hatched u n t i l they reached four weeks of age when maintained on a soybean basal. Meat meals were a less potent source of the factor(s) and 7.7% rendering plant or 9»2% packing plant meat meal did not give maximum growth to four weeks. Mortality i n chicks from hens fed a peameal, or peameal and meat scraps supplemented diet was high, regardless of the diet fed the chicks. However, chicks from hens on a soybean basal gave maximum growth when fed a diet containing 6% fishmeal, 5% dried skim milk, and 5*5% meat meal. Lindstrom and associates (53) found that when hens had been depleted and were changed to a ration containing f i s h -meal, the hatchability improved within 12-18 days. On the other hand, hens which were depleted and then fed a complete ration for six weeks had a carry-over for 10 weeks after being changed back to the incomplete ration. In open pens there was sufficient coprophagy to supply the unknown factor ( B ) indefinitely. - 1 4 As the e f f e c t of the hens' di e t on the chicks hatched became d e f i n i t e l y understood, the importance of using depleted chicks f o r experimental purposes became evident. This was done by maintaining the hens on fresh l i t t e r and feeding a vegetable protein d i e t . This was a very import-ant step as i t made a more or l e s s quantitative assay f o r the factor(s) possible and explained many of the discrep-ancies i n previous tests. I t was because of the lack of a proper assay gi v i n g reproduceable r e s u l t s that work on the factor(s) proceeded slowly f o r such a period previous to t h i s . Properties of the Growth and Hatchability Factor  associated with Animal Proteins as determined through  B i o l o g i c a l Testing with Chicks. Because of I t s close association with animal protein, the unknown factors became commonly referred to as the "Animal Protein Factor" which i n turn was shortened to A.P.F. The need f o r A.P.F. i n the d i e t of chicks from hens on vegetable rations was now established making the assay f o r the factor reasonably accurate. As a r e s i t , much work was undertaken to determine the chemical and physical properties of A.P.F. and i t s e f f e c t upon the metabolism of 15 the chick. Rubin and Bird (83) having demonstrated that the growth factor i n cow manure was different from any factors previously described and that i t was precipitated from water solution by acid (85) and related to, i f not identical with the factors in fishmeal and l i v e r , did further work (84) on the factor(s). A concentrate was obtained from cow manure of which 3*75 to 7.5 mg. per 100 gm. of diet supported maximum growth in depleted chicks. The active factor in the concentrate was soluble i n water, 50$ ethyl alcohol, 95$ ethyl alcohol but insoluble in chloroform and ether. It was stable i n the dry state at 100° for one hour and was not destroyed by autoclaving in solution for fifteen minutes at 120° at 15 pounds pressure. It did not dialyse through a cellophane membrane. McGinnls, Stevens, and Groves (59) made a study of the unidentified factor i n l i v e r paste which had supplementary values similar to the cow manure factor. They found that the factor was destroyed by oxidation. It was soluble In water at pH 2,3,4, or 5« It was insoluble in acetone and dialyable through a cellophane membrane. A concentrate was prepared that would support maximum growth i n depleted chicks when fed at a level of 3 mgms per 100 gms. of diet, methionine did not replace the concentrate in.the vegetable basal. The properties of the concentrate taken from l i v e r 16 ..-paste corresponded to those of the concentrate taken from cow manure except that the cow manure fac t o r was not dialysable through a cellophane membrane. Bird, Rubin, and Groschke (11) did further work on the concentrate from cow manure and found the fa c t o r to be water soluble at pH 3 i f the protein was f i r s t removed. They also noted i t soluble i n 8 0 % acetone though insoluble i n 100$ acetone. It was stable to autoclavlng f o r 2 hours at a neutral pH but was destroyed on autoclavlng 1 hour with a c i d or on standing i n an al k a l i n e solution. Robblee and associates (79) worked on a si m i l a r growth promoting concentrate prepared from f i s h solubles. The growth promoting fa c t o r i n t h i s concentrate was soluble i n water, i n 7 0 % methanol, and 7 0 % ethanol; i t was somewhat soluble i n absolute methanol and very s l i g h t l y i n 95% ethanol. I t was insoluble i n ether and acetone and dialyzed through a c e l l o -phane membrane. It was heat stable f o r 2 hours at 100°over a range of pH 3 to pH 9. There was no lo s s i n a c t i v i t y following enzymatic digestion of the solution. The properties of t h i s concentrate corresponded to those of the l i v e r paste concentrate of McGinnls et a l . I t was also s i m i l a r to the cow manure concentrate except that i t dialysed a cellophane membrane and was not r e a d i l y soluble i n 9 5 % ethanol. Fensack, Bethke, and Kennard (73) found sardine.meal e f f e c t i v e l y supplemented a d i e t d e f i c i e n t i n the h a t c h a b i l i t y - 17 -and chick growth factor(s). They found that the active principle could be extracted from the sardine meal by 80$ hot ethanol. It was adsorbed by Dareo G-60 at pH 3 from a water solution of an ethanol extract of fishmeal but was not adsorbed at pH 3 from an 80$ ethanol solution of a similar extract. These investigators did similar work (74) on f i s h solubles. They found the factor from both products to be soluble i n 75$ acetone and 80$ ethanol. It was Insoluble on Darco G 60 and adsorbed on Darco G 60 at pH 3 from a water solution. It could be eluted with 10$ ammonium hydroxide i n 95$ ethanol with a loss In activity of about 20$. The factor(s) were colorless In solution. From results obtained in the preceding reports i t seemed lik e l y that the factor was a water soluble vitamin related to the B Complex. From the slight variations among reports i t appeared that there might be more than one factor concerned or that the same factor might exist i n more than one form depending on the source. Using f i s h solubles as a source of the animal protein factor, Mishler (63 & 64) noted a difference i n require-ments according to the sex of the birds. 1.5$ f i s h solubles supported maximum growth in males whereas 0.5$ supported maximum growth i n females. 18 McGinn!s, Hsu, and Graham (57) reported that the animal protein factor as supplied by l i v e r paste was required f o r protein metabolism. They found that i f vitamin free casein or alpha protein from soybean was added to a d i e t containing no animal protein, that the non-protein nitrogen of the blood was greatly elevated. However, i f the alcohol soluble f r a c t i o n of l i v e r paste was added with the protein the non-protein nitrogen of the blood remained normal i n d i c a t i n g that normal p r o t e i n metabolism had taken place. Hobblee and co-worker (81) established a re l a t i o n s h i p between induced hyperthyroidism and the requirement f o r an uni d e n t i f i e d chick growth fa c t o r . Hyperthyroidism was induced by feeding d i s s i c a t e d thyroid or iodinated casein and counteracted by l i v e r extract. When t h i s was done, i t was possible to get a greater response i n chick growth when assaying f o r the animal protein f a c t o r . I s o l a t i o n of Vitamin B^2 i t s r e l a t i o n s h i p to Pernicious Anemia; In spite of the large amount of work done on the animal protein factor(s) i n r e l a t i o n to i t ' s growth promoting e f f e c t , i s o l a t i o n of the f i r s t member of the animal protein f a c t o r complex came about e n t i r e l y independently of studies i n that - 19 regard. Vitamin B 1 2 was Isolated through attempts to p u r i f y the fa c t o r responsible f o r the anti-pernicious anemia e f f e c t of l i v e r extracts. The presence of such a fa c t o r i n l i v e r acting as a s p e c i f i c agent i n the dietary treatment of pernicious anemia had been described as early as 1926 (6). Then i n 1944, Castle and associates (19) had postulated the e x i s t -ence of an e x t r i n s i c f a c t o r e f f e c t i v e against Addison's pernicious anemia i n beef muscle, milk, and eggs that was an u n i d e n t i f i e d , thermostable component of the vitamin B. complex. Subsequently, many investigations were carr i e d out i n anattempt to puri f y t h i s postulated f r a c t i o n . The work proceeded slowly, mainly due to the lack of a s a t i s f a c t o r y assay as a guide i n the f r a c t i o n a t i o n work, and a shortage of patients suffering from pernicious anemia. However, i n 1948, Smith (100) i n England and Rickes and co-worker (77) i n the United States working independently, announced, almost simultaneously, the i s o l a t i o n of a c r y s t a l l i n e compound from l i v e r which i n microgram quantities produced p o s i t i v e hemacological responses In patients with Addisonian pernicious anemia. Rickes and co-workers (77) c a l l e d t h e i r new compound vitamin B^* The reason f o r designating t h i s name was - 2 Q ~ -merely that i t had not been used i n the B. series and would connote n u t r i t i o n a l s i gnificance u n t i l such time as the b i o l o g i c a l r o l e or chemical structure of the compound was learned. Smith and Co-workers (100) i s o l a t e d almost 1 gram of material from four tons of ox live??. They noted two red pigments both highly active against pernicious anemia, but i t appeared that one arose from the other by p r o t e o l y s i s . The compounds were p u r i f i e d by repeated chromatography and i t became apparent that the colour and a c t i v i t y were inseparable. The compounds active against pernicious anemia as i s o l a t e d by Smith (100) were amorphous s o l i d s about the colour of cobalt s a l t s . This colour changed gradually from red to orange on exposure to l i g h t . The molecular weight was about 3,000. The product was exceedingly soluble i n water and nearly anhydrous alcohol. I t was Insoluble i n ether and chloroform. I t i s i n t e r e s t i n g to note the s i m i l a r i t y of these s o l u b i l i t i e s with the s o l u b i l i t i e s of the chick growth factor. Chemically, the compound was found to contain nitrogen, calcium, and sodium. From h i s study, Smith f e l t the follow-ing conclusion warranted. "True pernicious anemia and i t ' s associated neurological disturbances do not require a n u l t l p l i c i t y of factors but respond to a single, f a c t o r , with an e f f e c t i v e dose equivalent to some 20 gamma d a i l y . This i s — 21 •» therefore, one of the moat potent of known p h y s i o l o g i c a l l y active substances." In a l a t e r report Smith (101) reported the presence of 4$ cobalt or 1 atom of cobalt and 3 atoms of phosphorous. The presence of cobalt was confirmed by Rickes et a l . (78) af t e r a spectrographic analysis. Following the I s o l a t i o n of vitamin B 1 2 » West (107) car r i e d out a number of c l i n i c a l t r i a l s and demonstrated p o s i t i v e hematological responses i n three patients. In one patient a f t e r a single dose of 150 micrograms administered Intramuscularly, the r e t i c u l o c y t e count rose from 0.5$ to 27$ i n f i v e days, and the red blood c e l l count rose from 1.5 to 3.4 (x 10^) i n 23 days. The other two patients received 6 and 3 micrograms of vitamin B^g respectively and showed good re t i c u l o c y t e responses and p o s i t i v e red blood c e l l counts and hemoglobin r i s e s i n the f i f t e e n day period of the t e s t . Berk and co-workers (3) also report the effectiveness of B^ 2 In the treatment of pernicious anemia. In a single case of the neurological complication of perniclousanemla, combined system disease, the patient had become sensatized to l i v e r extracts and they found that, although f o l i c a cid produced an i n i t i a l hematological response, the patient suffered a relapse and simultaneouslydisplayed the neuro-l o g i c a l symptoms. Following B 1 2 therapy, both hematological and neurological responses were noted. When therapy was - 22 — discontinued f o r a week, neurological worsening was noted but abated when the therapy was r e - i n s t i t u t e d . Thus, i t would seem that the c r y s t a l l i n e vitamin B^2 was responsible f o r the neurological improvement observed. In connection with t h i s report, i t i s i n t e r e s t i n g to note that the patient, although sensative to l i v e r , was neither sensative to f o l i c a c i d nor to vitamin B^2» The study also would seem to indicate that B^2 ^B more e f f e c t i v e than f o l i c a c i d i n the treatment of pernicious anemia. A frequent manifestation of pernicious anemia i s changes i n the mucosa of the tongue. The usual treatment with ex-t r a c t s of hog l i v e r or hog stomach produces a prompt regression of the symptom. Shieve and Rundles ( 9 0 ) made a study of the r e l a t i v e response of the l i n g u a l manifestations of pernicious anemia to treatment with pteroglutamic acid and vitamin B ^ . In several cases, complete r e s t o r a t i o n of normal l i n g u a l mucosa was obtained through the administra-t i o n of pteroglutamic acid. In other patients some e f f e c t was noted following therapy with f o l i c a c i d , but an even greater e f f e c t was noted i f l i v e r extract was also admini-stered. In s t i l l other patients, no response to pteroglutamic acid was obtained. In these patients, also, pteroglutamic had no e f f e c t upon the hematologic and neural manifestations of the disease. Where vitamin B^2 was used, no f a i l u r e In treatment occurred and the authors found the vitamin adequate i n replacing l i v e r extracts as determined by hematologic, neurologic, and l i n g u a l responses to therapy. Bethell (6) investigated the e f f e c t of vitamin B 1 2 o n macrocytic anemias other than pernicious anemia. He found that vitamin B±2 therapy l e d to s a t i s f a c t o r y remissions i n a l l pernicious anemia patients but In other macrocytic anemia i t produced various r e s u l t s . Apparently, B^2 cannot replace f o l i c a c i d i n states where there e x i s t s a n u t r i -t i o n a l deficiency of that vitamin. The o r a l administrations of B 2 2 was i n e f f e c t i v e . Hog duodenal mucosa was e f f e c t i v e i n that i t f a c i l i t a t e s the absorption and u t i l i z a t i o n of dietary B^2* The Development of the Microbiological Assay f o r vitamin Bjg In 1947, Shorb (95) noted that Lactobacillus l a c t i s Dorner required,in addition to a l l the amino acids and vitamins, two unknown factors* One of these was a heat stable f a c t o r supplied by l i v e r extract and was referred to as T. J . In the assay of various feedstuffs f o r the presence of these f a c t o r s , Shorb noted that good agreement with the mouse assay of Wooley was obtained* Further work (96) demonstrated that the concentration of L.L.D. i n l i v e r extract almost p a r a l l e l e d the effectiveness of the 24 * extracts i n the treatment of pernicious anemia suggesting that the L.L.D. factor was the therapeutically active p r i n c i p l e i n l i v e r extracts. Thus, with the i d e n t i f i c a t i o n of vitamin B 1 2 i n 1 9 4 8 , Shorb ( 9 7 ) tested the compound and found i t extremely active i n the microbiological t e s t and that comparison with a standard l i v e r extract Indicated that the fac t o r i s eithe r wholly or p a r t i a l l y responsible f o r the L.L.D. a c t i v i t y of the extract. However, d i f f i -c u l t i e s were encountered i n the method of assay due to the many variables. Shorb and Briggs ( 9 8 ) found that Lactobacillus l a c t i s Dorner dissociated on c e r t a i n media to produce variants which grew without any supplementation other than tomato Juice. They obtained good r e s u l t s with the assay where a stable culture of the organism was used together with p u r i f i e d l i v e r extracts or c r y s t a l l i n e B^2* E r r a t i c r e-sults were obtained, however, with a d i s s o c i a t i n g culture or with some crude materials due to the presence of i n h i b i -tory substances. This i n h i b i t i o n was found to be due i n part to high concentrations of f o l i c a c i d , serine, p -aminobenzolc acid, zanthine, MnSC-4, NaCl, and FeSO^. In 1 9 4 8 , Wright ( 1 1 3 ) announced a method of assay f o r B 1 2 using Lactobacillus leichmannii. The media i n general was the same as f o r other B vitamins with the addition of . * 25 -t r y p s i n i z e d vitamin free casein. One d i f f i c u l t y was to release the f a c t o r i n microbiologically available form. To do t h i s , t r y p s i n was found to be the best agent. Wright suggested t h i s assay was more reproducible, contain-ing fewer sources of v a r i a t i o n . Capps, Hobbs, and Fox (17) also demonstrated more reproducible r e s u l t s using l a c t o -b a c i l l u s lelchmannii which had l i t t l e tendency to di s s o c i a t e . They found the organism to respond within the range of 0.01 to 0.10 gamma per tube. Shive and co-workers (93 ) reported that thymidine replaced B 1 2 i n the n u t r i t i o n of Lactobacillus l a c t i s Dorner, and i n further work ( 94 ) found i t also replaced B^2 l n n u t r i t i o n of Lactobacillus lelchmannii. These r e s u l t s indicated that B-^ i s involved i n the biosynthesis of purines and thymidine but d i d not preclude the p o s s i b i l i t y that these substances i n turn might also be involved i n the biosynthesis of vitamin Bi2» La c t o b a c i l l u s lelchmannii was found to require f o l i c a cid i n addition to thymidine but a f t e r a l a g phase thymine was found to replace the f o l i c acid. Skegges et a l . (99 ) reported that by using Lacto-b a c i l l u s lelchmannii they were able to obtain highly s a t i s f a c t o r y r e s u l t s which showed excellent c o r r e l a t i o n with the mouse growth assay f o r the animal growth f a c t o r . The e f f e c t s of ascorbic a c i d and a i r as noted by Shive et 26 a l (94) f o r Lactobacillus l a c t i s Dorner were reported f o r Lactobacillus leichmannii by these workers. They found these e f f e c t s could be minimmzed by autoclavlng the t e s t s f o r f i f t e e n minutes at 120°, but that the growth stimulation by thymidine must be taken into account. Hoffman et a l . (42) noted that f o l i c acid and paramino-benzolc a c i d were necessary f o r Lactobacillus leichmannii but that PABA gave no growth stimulation i n the absence of f o l i c acid. Wright (114) i n a further study of the a b i l i t y of thymidine to replace vitamin B 1 2 found that although the above proved the case with Lactobacillus leichmannii, that i n the n u t r i t i o n of Leuconostoc citrovorum and Strepto-coccus faecales there i s not the same thymidine - vitamin B 1 2 r e l a t i o n s h i p . Of i n t e r e s t , also, i n r e l a t i o n to the microbiological assay wasthe discovery by Winsten and Elgen (111) of the Joint occurrence of thymidine and B.^ 2 i n natural products. They found, using chromatography that i n the microbiological assay using Lactobacillus leichmannii 313, no l e s s than six e n t i t i e s were capable of supporting growth i n a B 1 2 d e f i c i e n t media. This was confirmed by Winsten & Eigen (112) i n further work, who noted two forms of B 1 2 . and f i v e other growth factors including thymidine i n natural materials. Their r e s u l t s suggested a desoxyriboside - 27 nature of the more potent growth factors. In work with thermobacterium, Hoff-Jorgensen (41) noted that guanine desoryriboside has the same a c t i v i t y as thymidine i n replacing vitamin B 1 2 . He also noted a s t r a i n of Thermobacterium where thymidine could not be replaced by vitamin B12. This i s of i n t e r e s t i n r e l a t i o n to the observations of Winsten & Eigen. Eonditschek and associates (49) made a study of the n u t r i t i o n of Lactobacillus l a c t i s Dorner and found the following to be some of the reasons f o r f a i l u r e to obtain reproducible r e s u l t s i n B^ 2 assay where t h i s bacteria was used as the t e s t organism. Lactobacillus l a c t i s w i l l not grown i n the absence of C02» even when B12 i s eliminated by factors producing anaerobic conditions such as the addi-t i o n of reducing substances, the removal of oxygen or the lowering of the oxidation- reduction p o t e n t i a l . Aeration or oxidizing substances i n h i b i t Lactobacillus l a c t i s but t h i s i s overcome by B^* The requirement f o r B12 under anaerobic conditions i s related to the peroxide content of the cultures. Caswell et a l (20) confirmed these r e s u l t s and showed that the B3.2 requirement decreased as the volume increased. They also showed that ascorbic acid eliminated the requirement f o r B^2* They do, however, present a procedure giving good r e s u l t s and i n which the tomato juice - 28 -i s replaced by a mixture of fumarlc acid and sodium ethyl oxalacetate. Hoffman et a l . (43) studied the assay using Lactobacillus leichmannii with a view to standarifing the method to obtain reproducible r e s u l t s . I t was found that the additional reducing agent such as t h i o g l y c o l l c acid stimulates the growth of organisms and protects vitamin B 1 2 from destruction during the autoclavlng of the samples. They suggest the formation of a growth factor from glucose i n the media during autoclavlng. When sucrose was included i n the media there was no growth as the factor was not formed. I t was necessary to measure the e f f e c t of desoxyribosides by assaying the sample before and a f t e r a l k a l i n e gydrolysls which destroyed the vitamin B^2. Roberts et a l . (82) studied the incorporation of radio-active Phosphorous by Lactobacillus leichmannii growing i n media with and without B^2. The t o t a l phosphate uptake was measured and the uptake was greater i n the media containing vitamin B 1 2. This tends to substantiate the concept that B 1 2 i s involved i n nucleic acid synthesis. More recently two other methods of assay for the anti-pernicious anemia f a c t o r , Hutner et a l (45) found i t possible to assay f o r the factor using a species of Euglena - 29 -which has a quantitative requirement* This has the advan-tage that the growth i s unaffected by thymidine but as the Euglena required about 0.01 gammas f o r " h a l f maximum growth w a l e v e l approximately one-tenth that required by Lactobacillus lelchmannii, the t e s t i s very d e l i c a t e * A cup plate assay was described by Bacharach and Cuthbertson (2,25) and Foster et a l (29) THE MULTIPLE NATURE OF THE ANIMAL PROTEIN FACTOR. With the Isolation of vitamin B^g and the subsequent development of relatively accurate methods of assay, It became possible to carry out more exacting experimental work on the sources and characteristics of the vitamin. As a result, i t soon became evident to the investigators that they were dealing with more than a single factor i n connection with animal proteins. This had already been postulated by some of the workers prior to the Isolation of vitamin B 1 2» although when the vitamin was f i r s t isolated, i t appeared in some investi-gations to entirely replace animal proteins. In 1947, Scott, Norris, and Hueser (92) demonstrated a growth response to whey be chicks receiving a diet containing 10$ fishmeal, sufficient to supply the A.P.F. They further demonstrated that this response was due to Streptogenin which was present as an Impurity i n the whey. This whey factor referred to as factor S. was also present in f i s h and l i v e r . After the discovery of B-^ 2 Ott et a l . (71) demonstrated that with an a l l vegetable diet containing 40 or 70$ sdybeah deficient chicks showed growth responses to as l i t t l e as 6 gamma per kil o of diet and produced optimum growth using less than 30 gamma per k i l o . From their results, they - 31. conclude B^ 2 i s either i d e n t i c a l with or cl o s e l y r e l a t e d to the Animal Protein Factor. Novak & Hauge (69) i s o l a t e d a fa c t o r from r i c e polishing which was also present i n l i v e r extract. They showed t h i s f a c t o r which was required f o r chick and rat growth to be d i s t i n c t from the other known vitamins and postulated factors including the cow manure factor; i t i s t e n t a t i v e l y c a l l e d vitamin B-^j. L i l l i e , Denton & Bird (51) found c r y s t a l l i n e B 1 2 equivalent to l i v e r extract and the cow manure fa c t o r i n producing growth when injected into the breast muscles or given o r a l l y . These i n j e c t i o n experiments served to demonstrated that the B^g had a d i r e c t a c t i o n on the chick not mediated through the i n t e s t i n a l f l o r a * Similar experimental work was carr i e d out by Nichol et a l . (66) who showed that pure vitamin B^ 2 c a n replace the animal protein f a c t o r a c t i v i t y of condensed f i s h soluble and injectable l i v e r preparations. A f t e r a two week t e s t period, chicks receiving 1.5 gamma of vitamin B- 2^ per one hundred grams of r a t i o n , averaged eighty-eight grams and those receiving 3% condensed f i s h solubles averaged eighty-six grams. Birds injected with 0.1 gamma of B 1 2 per b i r d per day averaged seventy-two grams H i l l (39) carried out experimental work which confirm-. - 3 2 -ed the work of Scott et a l . (92). He noted the supple--mentary e f f e c t of whey upon soybean but found that fishmeal was s t i l l required f o r optimum growth. He, therefore, postulated a multiple deficiency. Bird and co-workers (10) d i d further work with the unknown growth factor. They found that with both chickens and turkeys the requirement f o r the f a c t o r was l e s s during l a t e r stages of growth. They found that when chicks are eight weeks o l d i t i s possible to replace the f i s h (4%) with soybean o i l meal e n t i r e l y without impairing growth. With turkeys they replaced the fishmeal (6%) with meat meal and obtained as good r e s u l t s . This was confirmed by L i l l i e et a l . (52) who found i t possible to replace the fishmeal i n poult d i e t s with meat meal at eight weeks of age. With chickens they found soybean o i l meal could be used to replace the fishmeal at eight weeks of age. Nichol and co-workers (68) i n work with l i v e r extracts noted that some l i v e r extracts which were active against pernicious anemia were i n a c t i v e f o r chick growth. They also noted that crude l i v e r f r a c t i o n s discarded i n the commercial preparation of A.P.F. extracts were highly active i n promoting chick growth. Daniel and co-workers (26) demonstrated two factors - 33 -required f o r growth by Lactobacillus easel. One of these was the l i v e r paste fa c t o r and the other they whey fac t o r of H i l l ( 3 9 ) . They suggest that i t may be the whey fa c t o r responsible f o r l i m i t i n g the a c t i v i t y of l i v e r paste. They also found soybean a variable source of the whey factor. ... Stokstad & Jukes (105) found that-a mash containing a supplement from Strephtomyces aureofaciens produced a growth response separate from vitamin B^ 2. The fac t o r g i v i n g t h i s response was not i n yeast, a l f a l f a , d i s t i l l e r s solubles or f i s h solubles. They, therefore, postulated the n u l t l p l e nature of the animal protein factor. Nichol and co-workers ( 6 7 ) found that i t was possible to produce a f o l i c a cid deficiency i n the presence of vitamin B 1 2 . The presence of vitamin B^2 i n a f o l i c d e f i c i e n t d i e t increased the growth rate of the chicks, but the birds remained anemic, poorly feathered and paralysed. The addition of f o l i c corrected these symptoms. In further work these same investigators ( 2 7 ) established a relationship between B 1 2 , F o l i c acid and vitamin C i n chick growth. They noted that both vitamin C and B^ 2 stimulated chick growth when the birds were fed semi-p u r i f i e d r a t i o n with or without f o l i c acid. The addition of both these vitamins gave a greater response than eith e r 34-alone. They also observed greater f o l i c storage i n the l i v e r when the two vitamins were present i n the d i e t . Conversely f o l i c a cid i n the d i e t stimulated the synthesis of vitamin Bi2« Menge, Combs and Shorb (60) demonstrated growth responses to l i v e r and whey as supplements to a d i e t con-ta i n i n g vitamin B-j^. The die t contained extracted soybean protein as the protein source. Therefore, apparently, l i v e r and whey contain f a c t o r s not present i n the soybean protein and d i s t i n c t from vitamin B^g. Schaefer et a l . (87, 88, 89) noted a re l a t i o n s h i p of vitamin B 1 2 and choline i n chicks and r a t s . Renal damage i n r a t s was l e s s when a diet low i n choline and methionine wassupplemented with vitamin B 1 2 . Th i r t y micrograms of vitamin B^ 2 was s u f f i c i e n t to replace h a l f of the choline or methionine. They also noted a s i g n i f i c a n t sparing e f f e c t of B^g on choline f o r chicks. At eight weeks of age, the weight gain i n chicks receiving only 0.2% choline plus vitamin B 1 2 i n a soybean basal was equal to that of chicks receiving 0.6$ choline and no vitamin B 1 2 i n the same basal. G i l l l s & Norris (31) confirmed these findings. They noted more transmethylation to take place i n the presence of adequate Animal Protein Factor(s). I t seemed that a p a r t i a l deficiency of the Animal Protein Factor(s) creates 55-or stimulates metabolic processes requiring excess methyl groups. Briggs et a l . (14) supplemented a corn soybean d i e t with vitamin B^g. They found that one mgm of vitamin B^g per one hundred pounds of feed produced heavier chicks and greater feed e f f i c i e n c y than d i d two mgm per one hundred pounds of basal. Graham and co-workers (3?) showed that the n u t r i t i o n value of the soybean had an e f f e c t upon the requirement f o r animal protein. Mushett & Gtt (63) noted that twenty-five gamma of vitamin B-j^ prevented gizzard lesions almost completely i n chicks from hens on a commercial breeders r a t i o n . Ten gamma per k i l o was required by chicks from hens on a depleted r a t i o n to obtain the same r e s u l t s . Stevens, B i e l y , and March (103) found that although the addition of a commercial Animal Protein Factor Supplement produced no e f f e c t on growth of undepleted chicks during the f i r s t four weeks. Feed e f f i c i e n c y was greatly increased. This was true even i n a d i e t containing fishmeal, meat meal, and dried whey. Thus, apparently, a l e v e l of the animal protein factor(s) s u f f i c i e n t to produce maximum growth was not necessarily s u f f i c i e n t f o r maximum feed e f f i c i e n c y . -Richardson and Blaylock (76) found that amino acids and vitamin B 1 2 as supplements to soybean and cottonseed meal produced as good growth as 5% fishmeal. From t h i s the p r o b a b i l i t y appeared that a part of the added response to animal protein over c r y s t a l i n e vitamin B 1 2 was due to the amino acids present i n the supplements used. Stockstad and co-workers (104) have recently demonstrated the added growth they obtained using a supplement from Streptomyces aureojfia-clHns was due to the aureomycin present. This gave growth e n t i r e l y seperate from any response to that given by fishmeal as the a n t i -b i o t i c does not exi s t i n natural protein concentrates. They also noted that there i s no carry-over of t h i s f a c t o r from the maternal d i e t * I t i s apparent that further work should be done to establlshthe true value of the animal protein factor&s) i n r e l a t i o n to the n u t r i t i o n of the chick. Much of the work to date has been done on chicks hatched from depleted hens and fed p u r i f i e d or semi-purified d i e t s . Such experiments are of value i n determining the requirement and the functions o f the factor(s) f o r growth and metabolism. However, the findings of such experiments do not indicate the possible e f f e c t of the Animal Protein •» -3B7 •»' Factor f o r chicks on p r a c t i c a l d i e t s and hatched from hens receiving breeders rations containing s u f f i c i e n t animal protein. This study was undertaken with a view to establishing the value of the Animal Protein Factor as supplied by available commercial preparations i n p r a c t i c a l d i e t s for chicks. The f i r s t experiment was set up using New Hampshire chicks from hens receiving a p r a c t i c a l breeding d i e t containing s u f f i c i e n t animal protein as supplied by fishmeal. The purpose of the experiment was to determine whether such undepleted chicks required a d i e t containing animal protein or whether there Has s u f f i c i e n t carry over from the hen to support maximum growth. Treatments were also included to determine whether the Animal Protein Factor Concentrate prepared by Merck and Coi was capable of supporting maximum growth i n undepleted chicks when added to a l l vegetable d i e t s . This supplement was aldo included at various l e v e l s i n order to determine i f possible the optimum l e v e l of supplementation. -38-E X P E R I M E H T I - M E T H O D S A M ) M A T E R I A L S . Hew Hampshire cockerels were wing banded and divided at random into 36 groups of approximately f i f t e e n chicks each. Eighteen d i f f e r e n t d i e t s were fed i n duplicate to the chicks which were placed i n battery brooders. The composition of the basal diets fed i s given i n Table 1. The diets contained 16, 18, or 20 per cent protein either with or without a source of animal p r o t e i n . The d i e t s without animal protein contained soybean oilmeal as the vegetable protein source and were supplemented with •75, 1.5, and 2,25 milligrams of vitamin B^ 2 Per 100 pounds of d i e t . The vitamin B j ^ w a s supplied i n the form of a commercial animal protein f a c t o r concentrate, The l e v e l s of vitamin B 1 2 included were based on the work of Ott et a l . (71) who showed that, using depleted chicks, 6 gamma of the vitamin per k i l o of d i e t was s u f f i c i e n t to give a growth response, but that 30 gamma per k i l o were required to produce maximum growth. Control groups were kept at each protein l e v e l of the a l l vegetable d i e t with no supplementation with the animal protein factor concentrate. Fishmeal was the animal protein concentrate used and was included at a l e v e l of 3 per cent. The protein l e v e l was varie d by adjusting the amount of soybean oilmeal A* The commercial product used i n the study was Merck*s Animal P r o t e i n Supplement. This preparation i s a by-product of a n t i - b i o t i c fermentation and contains 12,5 milligrams per pound of vitamin B l 2 as determined by b i o l o g i c a l and microbiological assay TABLE I BASAL BrSgS/EXPSBIMMT I INGREDIENTS 210 iQf0 180 Soybean Soybean Soybean F i s h m e a l F i s h m e a l F i s h m e a l B a s a l B a s a l B a s a l B a s a l B a s a l B a s a l Wheat Germ Wheatmeal A C h o l i n e C h l o r i d e P r e - m i x P u l v e r i z e d Oats P i n h e a d Oatmeal N i a c i n P r e - m i x AA D r y B P r e - m i x AAA Co rnme a l B a r l e y m e a l C e r e a l Grass B r a n Soybean O i l m e a l F i s h m e a l S a l t MnSO, p r -mix -AAAA Bonemeal L imestone 2.5 2.5 2.5 15-5 23.0 28.0 2-0 2.0 2.0 10.0 10.0 10.0 10.0 10.0 10.0 0.15 O . l f 0.15 0.15 0.15 O . l f 10.0 10.0 10.0 10.0 10.0 10.0 5»o 5.0 5.0 5.0 5.0 5.0 25.0 . 17.5 12.5 1.0 1.0 1.0 0.2 0.2 0.2 3.0 3.0 3 - 5 0.5 0.5 2.5 2.5 2.5 ig.o 25.0 30.5 2.0 2.0 2.0 10.0 10.0 10.0 10.0 10.0 10.0 0.15 0.15 0.15 0.15 0.15 0.15 10.0 10.0 10.0 10.0 10.0 10.0 5.0 5.0 5.0 5.0 5.0 5.0 19.0 13.0 3.0 3.0 3.0 1.0 1.0 1.0 0.2 0.2 0.2 _ — 2.5 2.5 1.0 0.5 0.5 A A p r e - m i x o f C h o l i n e i n a 1:9 r a t i o w i t h s h o r t s . AA A p r e - m i x c o n t a i n i n g N i a c i n , P a n t o t h e n i c A c i d and R i b o f l a v i n . AAA A p r e - m i x c o n t a i n i n g D e l s t e r o l AAAA A p r e - m i x c o n t a i n i n g 1 ounze p e r pound o f Manganese S u l f a t e . -39-added. The calcium and phosphorus l e v e l s were maintained through adjusting the amount of bonemeal and limestone added* Again a group- was fed each protein l e v e l with and without animal protein factor concentrate. Where the dfet was supplemented with the A.P.F., i t was added at a l e v e l of 2.25 milligrams of vitamin B^2 P®r 100 pounds of d i e t . Thus i n a l l there were s i x groups i n duplicate maintained on diets containing fishmeal, three of them contained fishmeal and three of them contained f A.P.F. as w e l l as fishmeal. The chicks were weighed weekly for the f i r s t four weeks and again at the end of the s i x t h week. Feed consumption records were taken at the end of the fourth and s i x t h weeks. EXPERIMENT I - RESULTS AND DISCUSSION. T a b l e I I g i v e s the d i e t s f e d , and the l e v e l of i s u p p l e m e n t a t i o n w i t h A . P . P . as w e l l as the week ly w e i g h t s o f each g r o u p , and the average we ight s o f d u p l i c a t e g r o u p s . G r a p h I and G r a p h I I i n d i c a t e the r e l a t i v e w e i g h t s o f tbsr-c h i c k s i n the d i f f e r e n t l o t s a t f o u r and s i x weeks o f age r e s p e c t i v e l y . A t b o t h f o u r and s i x weeks, t h e r e a r e s i g n i f i c a n t d i f f e r e n c e s i n the w e i g h t s o f the b i r d s r e c e i v i n g the t h r e e d i f f e r e n t l e v e l s o f p r o t e i n . T h e r e a r e a l s o s i g n i f i c -ant d i f f e r e n c e s between the group r e c e i v i n g the soybean b a s a l and the groups where the soybean b a s a l i s supplemented w i t h ' ' e i t h e r f i s h m e a l or the a n i m a l p r o t e i n f a c t o r . A t the 20% p r o t e i n l e v e l , the 3 p e r c e n t f i s h m e a l b a s a l produces c h i c k s w h i c h average 291.6 grams i n we ight a t f o u r weeks o f age , whereas the a l l soybean b a s a l produces c h i c k s w h i c h average 272.1 grams i n ' w e i g h t . T h i s i s a d i f f e r e n c e o f 20 grams or 7 p e r c e n t in , body w e i g h t . A t 18^ p r o t e i n i n the d i e t , t h e r e i s a d i f f e r e n c e o f 9 p e r c e n t i n body weight i n f a v o u r o f the groups r e c e i v i n g the f i s h m e a l . A t the 16^ p r o t e i n l e v e l , the d i f f e r e n c e i s o n l y 0 o i n f a v o u r of the l o t s r e c e i v i n g f i s h m e a l i n the b a s a l . T h i s may be due e i t h e r to the f a c t t h a t a t t h i s l e v e l t h e r e i s a b o r d e r - l i n e amino a c i d d e f i c i e n c y or t h a t t h e r e i s a l owered r e q u i r e m e n t f o r the f a c t o r s s u p p l i e d Table II Treatment 0 weeks 1 week 2 weeks 3 weeks 4 weeks 6 weeks, 1.20$ Soybean- No.A.P.F. 34.7 70.57 124.8 199.1 261.0 497.1 34.9 71.57 131.5 192.7 283.2 527.0 *34.'8 *71.1 *128.2 *195.9 *272.1 *512.5 2. " n .75 A.P.F. 35.4 71.28 125.6 202.5 276.75 515.5 34.4 69.6 126.9 196.6 288.1 536.5 *34.9 *70.44 *126.25 *199.5 *282.42 *526.0 3. " n 1.5 ». 34.8 71.49 134.57 218.7 310.46 548.8 34.5 71.61 133.23 217.8 295.9 536.8 *34.7 *71.55 "=133.9 *218.25 *302.77 *542.8 4. " n 2.25 34.9 71.1 130.21 195.8 281.07 504.2 34.8 70.3 136.3 190.3 305.7 544.4 *34.9 *70.7 *133.2 *193.0 *293.7 *524.3 5.18# «. -No.A.P.F. 34.9 64.5 110*0 175.5 251.5 483.3 34.4 65.3 115.8 184.2 265.3 503.2 *34.7 *64.9 *112.9 *178.8 *258.4 *493,25 6. B ff -.75 A.P.F. 35.0 68.0 126.23 203.2 288.6 559.0 34.5 66.3 116.15 188.1 276.4 526.8 *34.8 *67.15 *121.2 *195.6 *282.5 *542.9 7. » "-1.5 " 35.4 69.3 125.38 206.0 277.8 540.5 34.4 64.1 120.4 199.8 283.08 514.2 *34.9 *66.7 *122.9 *202.9 *280.8 *527.35 8. " "-2.25 • 35.1 70.21 124.9 200.9 264.1 535.8 33.8 69.8 123.07 202.7 298.2 534.0 *34.5 *70.6 *123.9 *201.8 *281.15 *534.9 T 9.16$ Soybean - No.A.P.F. 35.2 63.14 103.28 162.57 649.01 484.0 34.5 64.3 108.6 170.0 253.8 481.1 *34.8 *63.7 *105.9 *166.2 *251.25 *482.55 10. " « -.75 A.P.F. 35.0 66.3 112.38 173.23 257.6 508,2 35.9 64.5 114.07 185.4 273.2 526.8 *35.4 *65.4 *113.2 *179.3 *265.4 *516.0 11. " " -1.5 • 34.8 62.36 110.2 172.0 248.6 485.0 34.9 59.35 109.2 183.5 252.0 517.8 *34.85 *60,85 *109.6 *177.7 *250.3 *501,4 12. * » -2.25 " 35.1 64.0 113.07 180.0 269.2 531.3 34.9 63.3 110.7 174.7 257.0 504.5 *35.0 *63.6 *111.8 *177.3 *263.1 *517.9 13.20$ Fishmeal-No A.P.F. 34.64 72.5 131.6 205.4 284.7 553.2 35.4 70.57 132.2 216.07 298.6 563.7 *35.02 *71.2 *131.9 *210.7 *291.65 *558.45 14. " » -2.25 » 34.9 70.64 128.61 189.8 287.5 516.1 34.6 74.42 134.7 206.3 300.5 578.4 *34.7 *72.5 *131.6 *198.0 *294.0 *547.25 15.18$ » . No.A.P.F. 35.7 75.6 134.3 213.1 295.6 564.9 34.6 70.7 122.86 199.46 276.8 532.3 *35.1 *73,6 *128.5 *206.28 *286.2 *548.6 16 " » -2.25 A.P.F. 34.9 69.3 126.16 187.7 281.5 547.7 34.4 71.23 126.5 209.2 278.9 517.8 •34.6 *70.2 *126.3 *198.4 *280.2 *532.75 17.16$ " No A.P.F. 35.8 67.0 116.07 181.8 272.1 517.2 34.7 59.0 103.09 188.0 253.9 510.4 *35.2 *63.0 *109.5 *195.5 *263.0 *513.8 18. » « -2.25 A.P.F. 34.9 60.13 102.5 168.3 254.8 497.1 33.9 61.9 104.0 173.8 241.9 478.2 *34.4 *60.9 *103.2 *171.0 *248.35 *487.65 G R A P H ^ o * CU o in o (P M i 0 o CD 01 V / / ' / / / 7 / / / 7 \ , / , , 7/ / / y / / ' / / / 75 M a m f+vr/xot? / / / / R T? FT / l O O / / / / ; / 7 7 7 ^ , v \ W \ ? 2-5" r-u- UPP/too* \ X X \ \ C \ v \ \ \ i B R S ^ I L M O - W » K !22J M9r~. »-«»Vfec>*\: / / ' / / / / / / / / / / \ . 2zs Mr.'»-f>P/ioo1 No. ^ I 3 4 6 7 8 9 l o 11 I2 13 I4 15 16 /7 !8 G P A P » H 3C N \ \ \ W N \ W \ \ \ \ V \ \ 'too* 14 15 1 6 1 7 18. -41-by animal protein at this level. This w i l l be dealt with i n greater detail later. By the end of six weeks the ohioks whioh were fed the fishmeal basal rations containing 18 and 20$ protein were 11$ heavier than those fed the soybean basals at the same protein levels. The supplementation of the 20$ soybean basal with the animal protein supplement was most effective at 1^5 mgm/100 pounds of diet at both four and six weeks of age. At four weeks the addition of the 1%5 mgms of B 1 2 (Merck's A.P.F supplement) gave greater growth response than the 3$ fishmeal. The addition of 2v25 mgms of B 1 2 (Merck1 s A.P.F. supplement) per one hundred pounds of soybean basal gave lower average weights than 1.5 mgms^ but s t i l l equal to and slightly greater than the 3$ fishmeal basal. 2.25 mgm B L G (Merck's A.P.F. supplement) added to the fishmeal basal gave a slight lnorease i n growth at four weeks indicating that 3$ fishmeal might not supply quite sufficient of the animal faotor(s). At six weeks the 3$ fishmeal supplementation gave greater growth than the soybean basal with Animal Protein Factor supplementation. The addition of 2.25 mgms of the B . (Merok's A.P.F. supplement) to the fishmeal 12 basal deoreased the growth but the average weight was s t i l l above that of the lots reoeiving the soybean basal regardless of the addition of animal protein factor -42-supplement. With the soybean basals containing 16 and 18$ protein,' 0,75 mgm of B 1 2 (Merck's A.P.F, Supplement) was apparently sufficient to meet the A,P,F, requirement. In the groups receiving 16 and 18$ protein, 1 the soybean basals supplemented with A,P,F, more nearly equals the growth of the groups receiving diets containing 3$ fishmeal than at the 20$ protein l e v e l . In fact the 18$ diet containing ,75 mgm of A.P.F, at six weeks produced birds averaging the same as the birds receiving the 20$ soybean diet containing 1,5 mgm of B, (Merok's A,P.F. Supplement) and almost equal to the 18$ protein containing fishmeal. At 16$ however, the soybean basal containing ?.75 mgm of A.P.F. (Merck*s A.P.F. Supplement) although i t produced chicks equal i n weight to the 16$ fishmeal basal produces chicks weighing signifioantly less than the 18$ diets irrespective of supplementation. From Inspection of Graph II It i s noted that at six weeks of age 2,25 mgms B^g (Merck*s A.P.F.Supplement) i n addition to 3$ fishmeal depressed growth at every protein levels At 20$ protein,' growth was depressed 2.0$, at 18$ the growth was depressed 2.9$ of the body weight and at 16$ the depression amounted to 5.09$ of the body weight. While the depression was very small, i t was, nevertheless, significant and It i s readily seen that i t increased as the protein level of the diet dropped. This would suggest one of two things, 1 either -43 the requirements for A.P.F • i s higher at the higher protein level or the tolerance of i t s presence Is greater at the higher protein l e v e l . Inspection of the groups receiving the soybean basal plus the B^g concentrate tended to support the fact that there may be a higher requirement at the higher protein l e v e l . I t w i l l be remembered that maximum growth was obtained on the S0$ soybean basal with 1.5 mgm B l g (Merck1 s A.P.F. Supplement) per one hundred pounds of feed of the A.P.F. supplement, whereas at 16 and 18$ .75 mgm B l g (Merck's A.P.F. Supplement) per one hundred pounds of feed supported maximum growth. Also i n the groups receiving the soybean basal supplemented with .75 mgm of vitamin B l g (Merck's A.P.F.Supplement) produced as efficient growth as the diet containing 3$ fishmeal. With the diets containing 18$ protein, the greatest feed efficiency at four weeks was again found i n the group receiving 3$ fishmeal. At this protein level at six weeks the same diet again produoed the greatest feed efficiency. Of the l&/o soybean basals supplemented with the B-L2 concentrate, .75 mgm gives the greatest effioiency of feed u t i l i z a t i o n . The soybean basal at 16§$ protein supplementation and with .75 mgms of the vitamin B-^ g concentrate gave Table I I I . l FEED EFFICIENCY EXPRESSED AS POUNDS FEED PER POUND OF flfflre.WRTffflT'', DIET* 4 Wks. 6 Wks:. r 20$ Soybean basal - No,A,P,F. 2.11 2; 56 n ft » -,75 mgm/10C# 2.07 2.49 n n " - 1.5 mgm/100# 2.04 2.72 " - 2.25 mgm/100# 1.99 2.55 18$ Soybean basal - No,A,P.F. 2.17 2.62 tt tt " -.75 mgm/100# 2 .12 2.57 n n " - 1.5 mgm/100# 2.19 2.75 n n " -2.25 mgm/100# 2.21 2.58 16$ Soybean basal- No. A.P.F. 2 ? . l l 2.92 tt « » J^75 mgm A.P.F. 2.12 2.55 tt n w - 31.5 mgm A.P.F, 2!.21 2f,63 n tt " - 2.25 mgm.A.P.Cr. 2.15 2.61 20$ Fishmeal basal- No. A.P.F. 1.95 2.54 tt tt " - 2.25 mgm A.P.F. 2; 12 2. 63 18$ Fishmeal basal - No.A.P.F, 2.01 2.45 n » M -2.25 mgm A.P.F. 2.05 2,51 16$ Fishmeal basal - No.A.P.F. 2|17 2.69 " -tf.25 A.P.F. 2.22 2,87 G R A P H ^ l£>0-(0 0 0 i 9> CO I It 4,8 to z It St E jc to Z05 "RatAK-oS F E E D Tfew. T^CWAUP O F S O W  V^/gi CM T  4.15 £Z5 Z.3S e+5 Z8S 295 the greatest feed effioienoy at four weeks. However, by six weeks, the soybean diet without supplementation gave the poorest feed effioienoy while the soybean basal supplemented with .75 mgm of the A.P.F. concentrate gave the greatest feed efficiency, A l i k e l y explanation of this i s that since the ohioks were not from depleted stock, there was sufficient carry-over from the hen diet to permit optimum growth rate to four weeks. Comparing the diets containing 3$ fishmeal at a l l three protein levels, i t i s apparent at both four and six weeks that the feed efficiency was decreased by the addition of 2,25 mgms of vitamin B 1 2 (Merok's A.P.F, Supplement.) This i s i n agreement with the growth results where $.25 mgms of B^g depressed growth at each protein level. In the groups reoeiving the three soybean basals, greatest feed efficiency was given by the addition o f .75 mgm of B 1 2 concentrate per one hundred pounds of basal diet. In every oase where the soybean basals were fed except the 20$ has a l at four weeks, the poorest feed efficiency was given when the soybean basal was supplemented by 1,5 mgm of vitamin B l g (Merck's concentrate). At four weeks with the 20$ basal 2.25 mgm of B l g (Merok's concentrate) gave the p oorest efficiency. The results obtained are i n agreement with the - 45-results of the experimental work done by Briggs et a l * (14). In feed effioienoy t r i a l s they noted that diets containing 1 mgm of orystallin© vitamin B l g per one hundred pounds of diet gave greater feed effioienoy than diets containing 2 mgm of vitamin B l g per one hundred pounds. There seems no obvious explanation of this unless there i s some re-arrangement of metabolism when there i s a higher level of vitamin B^ 2 present i n the diet. Altogether i t seems that the faotors supplied by the Animal Protein Factor Supplement are definitely related to feed effioienoy ani ohiok growth. From the results i t also appears that the requirements are very exaoting i n relation to the diet fed and the protein level of the diet. Indications are that the requirement i s greater at a higher level of protein i n the diet. Much of the work done previously by other investigators had been i n relation to corn-soybean basals. For this reason and from the experimental results obtained here, i t was f e l t worth-while to run a second experiment using a basal containing corn-meal as the main cereal! As a result Experiment II was set up i n much the same manner as Experiment I, exoept that the soybean basals were not supplemented with 1,5 mgm of and the diets containing fishmeal were omitted. Again the -46-B l g was supplied through the use of the concentrate of Merck and Company. As i t had been shown by Mishler et al.( ) that pullets and oookerels responded differently to the growth factor(s) as supplied by f i s h solubles, i t was f e l t worth-while to determine i f there was a similar difference i n response to supplementation with the Animal Protein Factor Conoentrate. -47-EXPERIMENT II . MATERIALS AND METHODS* Leghorn cockerels were wing-banded and divided at random into nine lots of approximately twenty chicks each* Leghorn pullets were similarly wing-banded and divided into nine lots of about twenty chicks each* Nine different diets were fed; one to each l o t of cockerels and one to each lot of pullets* The birds were kept for five weeks In battery brooders* Table IV gives the composition of the diets fed. The main difference between these diets and those fed In Experiment I i s the use of corn as the main cereal rather than a mixture of corn, wheat, bar ley and oats. This was done In order to give a ration comparable to the high-energy low-fibre,type of diet. As i n Experiment I the diets contained three levels of protein. They were calculated to contain sixteen, eighteen and twenty-one percent protein. Soybean was used as the source of vegetable protein and the diets were supplemented at each protein level with ,75 and 2,25 of B l g per one hundred pounds of diet, as supplied by the same commercial A,P,F* supplement used In Experiment I, Control groups were also fed at each protein level with supplementation, with the vitamin B._ concentrate, X Supplied by Merck & Co, TABLE IV. Baaal Dieta - Experiment II. 16$ 18$ 21$ Soybean BasalSoybean Basal.Soybean Basal. Wheat Germ 2 . 5 2.5 2 . 5 Ground Wheat 15.0 15.0 15.0 Plnhead Oatmeal 10.0 10.0 10.0 Bran 5.0 5.0 5.Q Dehyd.Cereal Grass 2.5 2.5 2.5 Iodized Salt 1.0 1.0 1.0 Limestone 1.0 1.0 1.0 Bonemeal 2.0 2.0 2.0 Cornmeal 43.8 37»6 29.5 Soybean Oilmeal 16.8 24.0 31.2 MnS04 6 gm 6 gm 6 gm Niacin 200 mgm 200 mgm 200 mgm Pantothenic Acid 200 mgm 200 mgm 200 mgm Riboflavin 160 mgm 160 mgm 160 mgm Dry D, 3 0,065 l b . 0.065 lb 1,065 l b . Choline Chloride 0.2 l b . 0.2 lb 0.2 l b . The ohioks were weighed and feed consumption records taken at the end of the second, third,fourth and f i f t h weeks*. -49-RESULTS AND DISCUSSION- EXPERIMENT II.. Table V gives the sex of eaoh l o t , the diets fed, the level of A.P.F. supplementation, and the weekly weights of eaoh group. Graph IV gives the average weights of the duplicate male and female groups on similar diets presented graphically and Graph V gives the weights of eaoh group separately. Both Graphs present the weights at the end of the f i f t h week. The basis of testing the separate response of the pullets and cockerels to the different diets was the work of Mishler et a l . (63). In work with f i s h solubles they noted a higher requirement for maximum growth i n males then i n females. Table VA gives the increase over the weight of the unsupplemented basals produced by supplementation at eaoh protein l e v e l . At 16 and 18% i t w i l l be noted that ,75 mgm of B^ 2 (Merck's Concentrate) gave about twioe the response i n females that i t did i n males. 2.25 mgm of B^g produoed about equal gains i n both males and females. At 21% the results obtained were very irregular, for some reason growth was retarded i n one of the groups of females. This was f e l t due to the extremely cold weather encountered at the time of the test. The results obtained i n the 16 and 18$ diets Diet Sex 1,16$ Soybean - No A.P.F. Male Fema le 2. -.75 A.P.F. Male Female 3. -2.25 A.P.F. Male Female 4.18$ Soybean - No A.P.F. Male Female 5. -.75 A.P.F, Male Female 6. -2.25 A.P.F. Male Female 7.21$ Soybean - No A.P.F. Male Female 8. -75 A.P.F. Male Female 9. -2,25 A.P.F. Male Female TABLE 7. 2 weeks 3 weeks 4 weeks 5 weeks 129.3 184,8 249,7 274.6 123.5 173.2 209,5 242.4 •126.4 *179.0 *222.95 *258.5 129.0 180.4 244.6 282,3 128.0 181,1 279,3 252.7 *128.5 *180.8 •232.0 *267.5 130.4 182.3 255.9 306.2 123.7 168.8 228.9 267.6 •127.0 *175.6 *242,4 *286.9 132.6 190.9 257,1 296,0 123.8 177.2 230.0 267.8 *127.0 *184,1 *243.6 *281.9 136.3 201.2 269.6 301.1 134.4 189.8 254.5 274.2 *135.4 *195.5 •262.1 •287.6 128.2 185.3 257.8 316.6 133.5 184.8 241.1 285.1 *130.9 *185.1 •249.5 •300.3 130.4 192.2 266.9 316.5 132,8 187.2 240.5 290.4 •131.6 •189.7 *Z5Z.7 ^303.4 125.6 194.1 261.9 337.8 137.2 187.7 250.3 292.6 •130.4 *191.9 *256.1 *Z15.Z 132.0 208.4 271.4 343.2 141.4 202.9 273.2 332,0 •136.7 *2Q$.& *Z12.Z •SS?^ MrtLgS I9N D FeiM(tt.e.s f=\~r 5 W E E K S . 360 -4 3oo 0; 4' i 3 0 I E u. I E CO 1 I 4 a ' E -9: 5: 8 G R A P H 3T M a l e s cxt 5" weeks FemoJes cxt 5 weeks 4 0 0 -350. 300-Z40-u; a* < i Z a rl u: < ui.' E « ^ • It f « Z a-.16 °7<> Soy bea n ba $a I 2.1 % 1 8 % Table VA. PERCENT IN BODY WEIGHT OVER BASAL AT 5 WEEKS DDE T O A . P . E . S U P P L E M E N T A T I O N . Diet Vitamin B _ _ Female Male (Merck) ± d > 16$ Soybean .75 4.25 2.98 2.25 10|39 l i f S l iS$ Soybean ,75 2^39 1.69 2.25 6.46 6.96 21$ Soybean f75 0.75 6..73' 2,25 14.32 8.43 50-however tended to substantiate the work of Mishler et al.(63) as i t appears pullets are more a ffeoted by lower levels of vitamin B l g. As Mishler used crystalline vitamin B^g i t apparently was this vitamin which produces the results and not some other component used i n the A.P.F .Supplement used i n this experiment. In the groups reoeiving the Soybean basals there was a significant difference i n the weights of the birds reoeiving the three different protein levels In their diets. There i s also a significant difference at each protein level when the soybean basal was supplemented with the animal protein factor. From Table V or Graph IV It i s seen that i n the soybean basal containing 16$ protein the addition of .75 mgms of vitamin B l g conoentrate p roduced an Increase of 9.0 gms or 3.5$ i n body weight over the basal. The addition of 2.25 mgm of B l g concentrate produced an increase of 11$ i n body weight or 28.4 grams. In the 18$ Soybean basal the diet containing no supplementation produced ohicks weighing 281.9 grams whioh was 23.4 or 9$ heavier than the ohicks receiving the 16$ soybean basal without supplementation. Therefore the 16$ soybean basal with 2.25 mgms of the vitamin B._ concentrate added per hundred pounds produced heavier chicks than the 18$ Soybean basal without supplementation. The addition of .75 mgms of the B 1 2 concentrate per one hundred pounds of the 18$ Soybean basal pro duced an Increase of 5.7 gms or 2,02% i n body weight of the chicks. 2.25 mgms of A . P.F. supplement added per one hundred pounds of 18$ Soybean basal, p roduced an increase of 18.4 gms or 6.3$ i n body-weight. This i s i n support of the evidence i n Experiment I that there i s a greater requirement for the factor(s) supplied by the animal protein factor supplement at a higher level of protein i n the diet 5. The groups receiving the 21$ soybean basal are 7.6$ heavier than those receiving the 18$ soybean basal, but only 1.0$ heavier than the groups receiving the 18$ soybean basal plus 2.25 mgms of the B l g concentrate. The addition of .75 mgms per one hundred pounds of A . P.F. to the 21$ soybean basal gives an increase of 15.2 gms or 3.9$ i n body weight. The addition of 2.25 mgms of A . P.F. produced an increase of 34.2 gms cr 11.2$ of the body weight. In this experiment there was no depression of growth at any protein level when the A . P.F. supplement was added at a level of 2.25 mgms per one hundred pounds of diet. This Is not i n agreement with the results of -52-Experiment I nor i n agreement with the work of Briggs et a l . (14). In both oases a depression was noted above one mgm of B^g per one hundred pounds of diet. As Briggs was using crystalline vitamin B 1 2 this depression must be due to some re-action with the diet used and not due to the presence of some fa otor other than the Vitamin B^g i n the A.P.F. supplement used i n Experiment 3?. There i s also the pos s i b i l i t y that there was a greater oarry-over from the diet of the breeding hens i n Experiment I. When the hatching eggs were collected for Experiment II the weather was extremely cold and there would be no opportunity for the hens to obtain additional A.P.F. through coprophagy. I f such were the case then a higher level of supplementation with B^g would be required to bring about a depression similar to that i n Experiment I. There i s also a pos s i b i l i t y this effect i s due to the particular soybean used i n the diet as Graham et a l . (32) showed a different response to souroes of the Animal Protein Factor dependent on the Soybean oilmeal i n the diet. The feed efficiency results are presented i n Table VI and shown graphically i n Graph VI. In the lots receiving 16 and 18$ protein,at five weeks feed efficiency was greatest where the soybean basal i s supplemented with .75 mgms B l 2 supplement per one hundred pounds of diet*. Where the chicks received the Table 71. FEED EFFICIENCY EXPRESSED AS POUNDS OF FEES/ POUND OF BODY* WEIGHT. Diet. 2 weeks. 3 weeks 4 weeks. 5 weeks 16$ Soybean basal- No A.P.F. 1.5 2.27 2.5 3.4 n n. n -.75 Mgm A.P.F. 1.5 2.14 2.4 £.98 " n « _2.25 » » 1.5 2.19 2.29 2.99 18$ Soybean basal- No .A.P.F. 1.57 2.12 2.35 3.12 " n » -.75 Mgm A.P.F. 1.48 1.77 2.04 2.93 » » " -2.25 " » 1.46 1.9 B.13 2.95 21$ Soybean basal - No .A.P.F. 1.30 2.14 2.45 3.08 " n n _ - 7 5 Mgm A.P.F. 1.50 1.88 2.27 3.16 " " •»: - 2.25 Mgm A.P.F. 1,42 1.99 2.42 2.85 G T C A P H 1ZT < in < 3 - + Q z < u ti > o ;.3.S--. CL U , CU u: • ; J « z m o: c»i Fe«o EFcicieNCV E X P R E S S E D A > P Q W O J Q F F E C P / P O U M O O F t 3 O D y Oei&ri 21% soybean basal, feed was greatest where the birds were fed 2,25 mgms of B^g of A.P.F. Supplement per one hundred pounds of feed. Again this i s In support of the greater requirement for the factors supplied by the Animal Protein Factor Supplement i n a ration con-taining a higher level of protein, The second experiment also shows the relation of the Animal Protein Factor containing vitamin B 1 2 to both growth and feed efficiency i n ohicks from undepleted hens. Evidence i s presented to show that the results obtained.depend on the ration to which the supplement was added. It further substantiates the evidence that the requirement for the animal protein factor i s higher where the diet i s higher i n protein. It also shows through the feed efficiency results that the best level for supplementation with vitamin B^g seems to be at a level below two mgms per one hundred pounds of diet, I'his i s not evidenced by the growth results i n regard to this particular diet. In both Experiment I and Experiment II the source of B^g was the commercial product prepared by kerek and Company, Due to the probable presence of other factors i n the concentrate besides vitamin B 1 2, i t was thought advisable to compare different animal protein factor concentrates as supplements to practical chick rations. Therefore Experiment III was set up using -54-bo th the Lederle and Merck A .P.F, supplements us undepleted ohicks. The Lederle produot was also fed i n a diet containing animal protein! EXPERIMENT I I I - MATERIAL AND METHODS. Leghorn cookerels were wing-handed, and divided at random into twenty-eight groups of twenty chicks each. Fourteen different diets were fed i n dup l i c a t e , to the chicks which were placed i n battery brooders under experimental conditions^ The composition of the basal diets fed i s given i n Table V I I . Twelve of the fourteen diets fed contained soybean oilmeal as the only protein concentrate and were adjusted to contain 17 or 21$ protein. The other two diets contained 11,5$ fishmeal as the protein source and were adjusted to contain 17$ soybeari. The 17 and 21$ soybean basals were both supplemented with 0.3$ methionine; 1.5 or 2.25 mgms per one hundred pounds of feed from Merck's Animal Protein Factor Supplement; x or 0.3 or 0.5$ of A K Lederle Animal Protein Factor Supplement. The methionine was added as i t was f e l t that a diet dependent on Soybean oilmeal as i t s source of protein might be deficient i n that amino aoid. One oft the t wo fishmeal basals was supplemented with 0.5$ of A A fermentation product containing 12.5 mgm of vitamin B^g per pound. AA A souroe of A.P.F.which carries no vitamin B 1 2 guarantee §pecial Gmeliat a b y g r o w i n g a mold(S,auresfaoiens) i n Table VII, Baaal Diets- Experiment I I I , rrfo m 17$ Soybean Basal, Soybean Basal. Fishmeal Basal. Wheat Germ 2.5 2.5 2.5 Ground Wheat 15.0 15.0 15.0 Pulverized Oats 10.0 10.0 10.0 Bran 5.0 5.0 5.0 Feeding O i l 0.5 0.5 0.5 Iodized S a l t 1.0 1.0 1.0 Choline 0.2 0.2 0.2 BY 1.0 1.0 1.0 Niacin 100 mg. 100 mg. 100 mg. Pantothenic Acid 100 mg. 100 mg 100 mg. MnS0 4 6 gms 6 gms 6 gms Cornmeal 42.0 31.0 51.0 Soybean 19.8 30.8 Bonemeal 2.0 2.0 1.0 Limestone 1.0 1.0 1.0 Fishmeal ~« 11.5 -56-Loderle»s A,P,F«, Supplement and fed to duplicate groups of chicks. The other group was fed i n duplicate as a control. The chicks were weighed at the end of the second, third and fourth weeks, Feed consumption records were made at each weighing. - 57-RESULTS AND DISCUSSION - EXPERIMENT III. Table vTXT gives the diets fed, the source of A.P.F. and the level of supplementation as well as the weekly weights of each group. Graph 711 gives the four week weights, using the averages of duplicates. There was a significant difference between the birds receiving the different levels of protein i n their diet. There was also a significant difference between the 17$ basals with and without fishmeal. Supplementation of the Soybean basals with either Merck's A.P.F. supplement, or Lederle's A.P.F. supplement produced a significant difference i n body weight. The Lederle product, however, produced a growth response significantly greater than did the Merck p roduct^. Supplementing the 17$ fishmeal basal with the Lederle product also produced significantly greater weights than did the fishmeal basal alone. The 17$ Soybean basal produced chicks which averaged 22 5.6 grams at the end of four weeks. Supplementation of this Soybean basal with 0.3$ d l methionine produced no increase i n body weight Indicating there was no deficiency of methionine. The addition of 1.5 mgm vitamin B 1 P (Merck's Table 7 I H Group 2 weeks. 3 weeks 4 weeks 1. 17$ Soybean Basal 2. " 2. » 4. » 5. " 6. » B 0.3$ d l ^ Methionine 12 " 0. 104.4 110.4 •107.4 1.06.7 105.1 *105.9 1.5 mgm/lOO# Meroks B 105.3 Supplement #3. 1 2 107.1 *106.2 2.25 MerokB B Supplement 106.1 104.9 •105.5 " 0.3$ lederle A.P.F. Supplement 120.1 117.3 *118.7 122.7 114.5 *118.6 156.6 164.7 •160.6 164.6 154.2 •159.4 159.4 162.8 •161.1 164.4 158.2 •161.3 188.7 180.5 •184.6 191.8 181.5 •186.6 217.6 233.6 •225.6 219.3 219.6 •219.5 225.4 231.8 •228.4 234.4 229.4 •231.9 274.6 261.9 •268.2 273.7 264 .5 •269.1 7.21$ Soybean Basal 8." 9." 10" 11" 12" * " 0.3$ d l methionine " 2.25 " 112.1 118.9 •115.5 118.8 •119.3 " 1.5 mg/l00# Meroks B.„ A.P.F. 120.7 12 118.7 •119.7 123.1 122.7 •122.9 " 0.3$ Lederle A.P.F. Supplement 131.8 132.9 •132.3 « 0. 13.17$ Fishmeal Basal 14.» n & Lederle A.P.F. O.J 136.3 129.9 •133.1 125.3 111.2 •118.2 128.4 136.9 •132.6 175.9 196.8 •186.3 184.8 199.5 •192.1 192.0 187.7 •189.8 180.0 188.0 •184.0 218.5 205.3 •211.9 212.2 208.9 •210.5 200.6 174.7 •187.6 204.5 217.8 •211.1 T 248.9 285.9 •267.4 265.0 273.1 •269.0 270.63 273.3 •271.9 280.6 267.5 •274.1 308.4 300.5 •304.4 304.6 304.5 •304.5 288.45 243.0 . •26f . r 298.7 ; 313.2 •305.9 3 0 0 -•ZSO— — u; I o 2 J 4 VI 4 CO I < ui Q » i HI r i 0 UI 3 J v. tl) I i c: d £ 1 Ui £ U a-<£ 1 tt I ' = » ' i • :?*r I u: u J of d; 1 1 a ui j or UI 0 !P 0 I zj -1 < « r « ID »r 0 I ui Z 0, I H ui s 19 3 ft* ui u; 23 ul E » u <• 111 4 0 ui in 6 1 05 ui J a! ui a o cc 0 z 1 _ a a CC j ul £ 1 D: Ui _i a; ul D ui - i 1/1 6 -58-A.P.F. Conoentrate) per one hundred pounds of feed to the 17% soybean basal produced a 1 4 increase i n body weight over the basal alone at the end of the fourth week. The addition of 2.25 mgm B_0 of the Merck Concentrate to the same basal produced a 2.9$ increase In body weight. Neither of these increases are ver y marked, but this i s l i k e l y due to the carry-over to the chicks of the animal p rotein faotor(s) stored from the diet of the breeding hens. Thus had the birds been kept on the experimental diets for another week the differences would probably have been greater and more comparable to Experiment I I . The addition of 0.3$ Lederle A.P.F. Supplement to the 17% Soybean basal produced birds which averaged 269.1 grams at the end of the fourth week as compared with 225.6 for the birds on the straight 17$ Soybean basal. This is an increase of 18.9% i n body weight. The addition of 0.5% A.P.F.Supplement gave an increase of 18% In body weight over the basal. This i s not a signifioantly greater average body weight than the 0.3% produced, but there was no depression of growth. Apparently 0.3$ i s sufficient to produce maximum growth and there i s no advantage to be gained through the use of larger amounts. The Zl$b Soybean basal produced chicks with an -59-average weight of 267.4 gms. The 17% Soybean basal p lus 0,3% and 0,5% of the Lederle A.P.F. Supplement produced chicks averaging 268.2 and 269.1 gms respect-ively. Thus the addition of the supplement to the plant basal produced growth responses equal to 4% protein i n the absence of animal protein. The 17% basal containing fishmeal produced chicks averaging 288.45 grams at four weeks. This was also equivalent to the 17% Soybean basal plus the Lederle supplement'. In other words the 17% Soybean basal plus the A.P.F. supplement produces as good growth as does the 17% fishmeal basal. The supplementation of the 17% fishmeal basal, supposedly complete i n A.P.F. produces a further growth response. This appears as a complicating factor because i f the fishmeal basal gave the growth response over the Soybean basal due to the factors present i n the Animal Protein Factor concentrate, then there should have been no further response when the Animal Protein Factor Concentrate was added to the fishmeal basal. With the 21% Soybean basal, the addition of the Merck's A.P.F. Concentrate produces slight Increases i n growth and exhibits no depression of growth even when used at a level of 2.25 mgm per one hundred pounds of feed. The addition of Lederle*s Concentrate produced an increase of 13.7$ i n body weight at both the 0.3$ and the 0.5$ levels i n the diet. This response to the 21$ Soybean basal p i us the A.P.F. Concentrate produced chioks weighing 304.5 and the 17$ fishmeal basal plus 0.5$ of the A.P.F. supplement p roduced chicks averaging 305.9. This may be due to a better amino aoid balance i n the fishmeal bub there s t i l l remains the possibility of the presence of some other factor, particularly since investigators have shown Soybean to be balanced i n amino acids for chick growth. As i n Experiment II there i s no depression i n growth at higher levels of the A.P.F. Supplement. This i s not i n agreement with Experiment I nor with the work of Briggs et a l . (14). Again the only explanation seems to be through some connection with the diet. The feed effioienoy results are presented i n Table VIIIAand shown graphically i n Graph VIII. Methionine gave no significant responses i n either growth or feed efficiency. In the 17$ Soybean basal .75 mgm of B l g of the Merck Supplement produced the greatest feed effioienoy of the 17$ soybean basals. When 2.25 B mgms per Group fable T i l l A 1. 17$ Soybean Basal 2. * n "0.3 01 Methionine 3. » " " 1,5 mgm/100# Meroks B Supplement #3. 1 2 4. " n "2.25 Meroks B Supplement 12 5. " " "0.3$ Lederle A.P.F.Supplement 6. " n n 0.5$ Lederle A.P.F.Supplement 7. 21$ Soybean Basal 8. « » " 0.3$ d l Methionine 9. » n "1.5 mg/lO0# Meroks B l 2Aiif?F* ; 10. » " 2.25 mgm/100# " " «* 11. " n 0.3$ Lederle A.P.F.Supplement 12. " * 0.5$ " •» ' » 13. 17$ Fishmeal Basal 14. 17$ Fishmeal & Lederle A.P.F. 0.5$ 2 weeks 3 weeks 4 weeks 1.33 1.5 1.29 1.42 1.36 1.38 1.41 1.32 1.26 1.37 1,37 1.28 1.3 1.28 1,82 1.78 1.69 1*87 1,81 1.80 1.76 1.69 1.77 1.62 1.52 1.64 1.63 1.53 2.13 2.06 2.03 2.2 2.22 2.08 2,02 1.98 2.08 2,07 1.88 1.94 2.16 1.52 G ' R A P H S H E ii a <£ o J 4 o T CO z ui 0 9" 0 6? u z c HI z 10 i ! i I - J * IT i r 5 u' <£ a! Ul Ul J c« IP d i tt. « ri Ul I Z o D ! vl K ui E «; a-< it: u * « £ ui ui LI o ul c * ° 5 ° Hi -1 .„<> </> N el w a 6* r? 0 u.' a: 6 N <r --61-one hundred pounds was added the efficiency was lower than that of the unsupplemented basal. This i s i n agreement with Experiment I and the work of Briggs et a l . 0.3$ of the Lederle concentrate produced no improvement In feed effioienoy and 0.5$ p reduced only a slight improvement. In the 23$ basal neither level of supplementation with the Merck product produced any improvement i n feed efficiency. 0.3$ of the Lederle product produced the best results regarding efficiency at this protein level*. 0.5$ of the Lederle Supplement was sl i g h t l y less efficient than 0.3$ but s t i l l more efficient than the basal. Most striking was the effeot of the addition of 0.5$ of the Lederle supplement to the 17$ Soybean basal. This produoed an increase of 29$ i n feed efficiency over the basal. This i s In agreement with the work o f Stevens, Biely and March (103) who showed a 21.9$ increase i n feed efficiency on adding the Merck Product to a diet containing soybean and fishmeal. -62-GrEMERAL SUMMARY. A series of three biological experiments was carried out to determine, i f possible, the effect of vitamin B , as supplied by oommerioal animal 12 protein factor preparations, on ohiok growth. The chicks used i n the experiment were from hens receiving a complete breeding diet, and therefore had a carry-over of the factor from the maternal diet. The basal diets were patterned as closely as possible after commercial rations. In this way the results would be applicable to practical rations. In Experiment I the basal diet used oontained a mixture of cereals, mainly wheat. The basal used i n Experiment II and III contained oorn as the main cereal and the ration was of the high efficiency type reoently used i n the production of broiler meat. In Experiment I and II the animal protein factor oonoentrate used was that produced by Merck and Company containing 12.5 mgm of vitamin B _ per pound. 1 5 2 In Experiment III both Merck* s preparation and the animal protein supplement produced by Lederle were used. The Lederle product carries no guarantee of vitamin B^g content but i s known to contain aureomyoin which has additional growth promoting properties (104). Experiment I was set up to determine whether -63-or not A.P.F. supplementation produced as good chick growth as did the addition of fishmeal to a soybean basal. Treatments were also included to determine at what level A.P.F, supplementation was most effective and whether i t was advantageous to supply A.P.F. i n addition to fishmeal. Experiment II was set up to determine what effect the A.P.F. supplementation had on a corn-soybean diet. Males and females were fed separately i n order to determine whether or not there was a difference i n response as demonstrated by Mishler et a l . (63) • Experiment III was planned to compare the commercial A.P.F. preparations produced by Merck and Lederle. Treatments were included to determine whether or not the Lederle A.P.F. supplement gave any growth response i n addition to that produced by fishmeal. From the experimental work oarried out, i t was shown practical to supplement chick diets with animal protein factor ^concentrates. However, these concentrates must be used judioiously i n relation to the oparticular ration i n which they are to be fed. In the use of the product manufactured by Merck,' indications were that the requirement was high when the diet contained a higher level of protein. This i s logical as the chick growth factor has been shown to be related to protein metabolism (57). In 64-Ixperiment I when this supplement was added to a practical diet containing wheat as the main cereal and no animal protein, optimum growth was produced by 1.5 mgm of B^g per one hundred pounds of diet at the 16 and 18% protein levels and by 2,25% at the 20% protein level. In this same experiment when 2.25 mgm of the A.P.F. supplement was added to a similar diets oontaining fishmeal at correspond-ing protein levels, there was a depression i n both growth and feed consumption. Possibly had the level of A.P.F. added to the fishmeal basals been lower i t would have proven advantageous. In every case the greatest feed effioienoy given by the soybean basals and A.P.F. was almost the same as that given by the fishmeal basal without supplementation. The use of the A.P.F. i n the soybean rations i n Experiment I produced growth almost equal to the addition of fishmeal to the basal, but i n no oase produced any additional growth response. I t gave the best response i n the 18% protein diet. Feed efficiency i n Experiment I on the soybean basal oontaining 20% protein was greatest when the diet contained 2.25 mgm of vitamin B l g. When the diet contained 16 and 18% protein, greatest feed efficiency was given by the addition of '.75 mgm of A.P.F. to the soybean basal. At every protein level the addition o f 1.5 mgm gave the poorest feed efficiency. At the 18 and 20% protein levels the addition of Ifi5 mgm gave even poorer feed efficiency than the basal alone. In Experiment II the separate response of the pullets and cockerels to the supplementation of the 16 and 18% soybean basal diets with MerokVs A.P.F. concentrate, was i n agreement with the work of previous investigators. In both oases the pullets gave twice the response of the males to .75 mgm of B i n the 12 diet. Bothv males and females gave a similar response to the addition of 2[.25 mgm of vitamin B i n the diet. 12 Merck's A.P.F. supplement when added to the basal diet used i n Experiment II gave increases i n average weight at four weeks at both the 1.5 mgm of B^g per one hundred pounds level and the 2.25 mgm level. There was no depression i n growth at the higher level of the A.P.F. supplementation at any protein level. This i s not i n agreement i n Experiment I but might be explained by a greater carry-over of vitamin B. from the maternal diet as the hatching eggs for Experiment I were oolleoted i n hot weather when faeoal synthesis of the A.P.F. i s more l i k e l y to occur. There i s also the po s s i b i l i t y i t i s due to the soybean used i n the diet as i t was demonstrated by Graham (32) that different soybean oilmeals produced different responses to the addition of a,souroe of A.P.F. 66-Feed ef f i o i e n o y i n Experiment I I was greatest at the 2.25 mgm of B^ g l e v e l of supplementation i n the 21$ protein d i e t . I n the 16 and 18$ protein d i e t s apparently .75 mgm of B^ g per one hundred pounds was s u f f i c i e n t to support maximum feed e f f i c i e n c i e s . I n Experiment H I again there was no depression i n growth produced by the Merck's product. The addition of methionine did not improve growth or feed e f f i c i e n c y i n d i c a t i n g that the die t contained s u f f i c i e n t of t h i s amino a c i d . The exceptional part of t h i s experiment was the growth response produced by the addition of Lederle»s A.P.F. supplement. From the results,* 0.3$ gave as good a response as 0.5$y although there was no depression at the high l e v e l of supplementation. In t h i s experiment, the addition of Merck's A.P.F. to the soybean basal did not produce growth equal to the 17$ fishmeal basal as i t did i n Experiment I . Again, t h i s might be due to the oarry-over of the fac t o r or to the soybean used i n the d i e t . There was just as great an increase when the 17$ fishmeal basal was supplemented with the A.P.F. I n f a c t the 17$ soybean basal when supplemented with the Lederle A.P.F. gave as good growth as the 21$ unsupplemented soybean basal and the 17$ fishmeal basal. I t was obvious that the Lederle product contained some fa c t o r neither i n the fishmeal nor i n the Merck A.P.F. supplement• Subsequent to the experimental work Stookstad (104) demonstrated that i t was aureomyoin responsible for this additional growth response i n connection with this diet. I t i s not understood whether the action of the antibiotic i s direct or whether i t i s mediated through the effect on the Intestinal f l o r a . From the experimental work presented the following conclusions seem warranted. (1) Diets containing soybean as the only source of protein need supplementation with an Animal Protein Factor concentrate even when the chicks are from hens receiving a practical breeding ration for most efficient growth and feed u t i l i z a t i o n . (2) Under the experimental oonditions i n this study, there was a greater response by females to a; lower level of A.P.F. supplementation. (3) Supplementation with the A.P.F. ooncentrates i s definitely advantageous as far as feed efficiency i s concerned but apparently the effect Is relative to the diet fed and the level of supple-mentation. (4) The Lederle supplement added to a diet already containing fishmeal produced exceptional improvement both i n connection with feed efficiency and growth responses. . (5)-It also seems that the B 1 p requirement was higher at the higher 1^ levels of protein i n the diet ;. (6) From Experiment I i t seems there i s -68-an optimum l e v e l of supplementation beyond which there i s a depression of growth and feed e f f i c i e n c y In a soybean basal. The same effe c t was obtained when fishmeal i s supplemented with Heroic's A.P.F, supplement. I n corn-soybean rations there was no depression at high l e v e l s of supplementation with the Merck product." This may be due to eit h e r storage of the factor i n the chick or the dietary ingredients. Vitamin B, 2 alone i s not responsible f o r the growth responses obtained from the animal protein i n the chick d i e t . Evidence suggests a m u l t i p l i c i t y of the factor beyond vitamin B,„ and aureomyeih. -69-BIBLIOGRAPHY (1) Almquist; H.J. ; Mecchi, E ; Kratzer, F . H . ; and Grau, C.RE. Soybean Protein as a Source of Amino Acids for the Chick. J . N u t r i t i o n 1942, 24:385. 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