TEXAS AGRICULTURAL EXPERIMENT STATION A. B. CONNER, DIRECTOR, College Station, Texas BULLETIN NO. 637 ~AUGUST, 1943 THE CAROTENE CONSUMING POWER OF CERTAIN FEEDS G. S. FRAPS, \V. W. MEINKE, RAMOND REISER and R. M. SHERWOOD Division 0f Chemistry and Division of Poultry Husbandry L! ffirlauiterazgp BRARY ,1 Jury: AGRICULTURAL AND MECHANICAL COLLEGE OF TEXAS F. C. BOLTON, Acting President B42 943-4500-L180 SYNOPSIS t: n l: observations indicated that some animal products had of destroying carotene and that such products of high caro- jw: power might injuriously affect chickens if included in __on. Conditions which affect the carotene consuming power were _. Methods for determining it were worked out and are given in _ Samples of meat and bone scraps, meat scraps, tankage, dried ed skim milk and dried butter milk may have a high consuming ‘for carotene. Vegetable feeds “such as corn meal,‘ cottonseed meal, Tgray shorts and wheat bran very rarely if ever have any power to carotene. The carotene consuming power is not constant but _ rease or decrease from time to time. It is removed by autoclav- i_ in as short a period as a week the feed may again have a high ie consuming power. When fed to rats with feed containing A -. quantities of carotene, meat meal with high carotene consum- wer sometimes decreased the vitamin A stored in the liver and es did not. Fed to rats receiving very low quantities of caro- 5- meal with high carotene consuming power slightly decreased _ | of life and the maximum weight attained. Fed to chickens, 6 experiments the high carotene consuming power of meat i’: ; had no injurious effects. In the other two experiments, in one ' 1 the ration contained low quantities of carotene, the by-product w h carotene consuming power appeared to cause vitamin A de- -~ The high carotene consuming power is injurious to chickens ‘Z der exceptional conditions, and when a ration very low in caro- 1 vitamin A is being fed. CONTENTS ‘Page Introduction _______ -_ 5 Experimental 0n method A _ _ _ _ _ _ _ , _ _ _ _ _ . _ _ r . _ _ _ _ _ _ _ _ . _ __ 5 Method of determination ______________ ______________________________________________ _i_- 9 Factors Which affect carotene consuming power ____________________________ 10 Experiments with rats _- ___________________ __ 14 Procedure and results with liver storage 14 Effect on longevity and gain in'weight ______________________________________ -_ 17 Experiments With chickens _______________________________________________ ______________ _. . 17 References _ ___________________________ __ _23 Summary __- __ 22 iCAROTENE CONSUMING POWER OF CERTAIN FEEDS . Maps, W. W. Meinke and Raymond Reiser, Division of Ohem- ’ istry and R. M. Sherwood, Division of Poultry Husbandry. l ionally samples of meat scraps, tankage, and fish meal in the Q chickens have been reported to have a deleterious effect. Kaupp (oted that fish meals with a high free fatty acid content had. an _ e effect, and Te Hennepe (quoted by Schroeder et al) 12 reported eat scraps with a high free fatty acid content apparently reduced production. Schroeder, Redding and Hurber (12) found that fed meat scraps with a high free fatty acid value developed iof vitamin A and D deficiency; the meat scraps after extraction =11 her showed only a partial loss of the ability to produce the vitamin ncies while the extracted fat, when added to wholesome meat I, was not deleterious. Gray and Robinson (4) did not confirm ‘fresults. Sherwood, in the Division of Poultry Science of the Texas ultural Experiment Station, found thatlsome samples of meat and craps produced pronounced vitamin A deficiency. Lease, Lease, t and Steenbock, (7) found that when rancid fat or fatty acid ‘des were fed to rats at the same time with vitamin A or carotene “iwas a marked decrease in the vitamin A stored in the liver. Quack- "* , Cox and Steenbock reported (9) that the presence of alpha-toco- _ or catechol protected the carotene from the rancid fats. Harrell- al (5) reported thatfats which had been heated destroyed the p__ in A potency of other foods fed with them. n meat products are moistened with a solution of carotene in um ether, the petroleum ether allowed to evaporate and the mix- llowed to stand over night, there is sometimes a loss of carotene the presence of the meat product. ‘ .'._ for determining theAcarotene-consuming power of feeds. pfotene oxidase which destroys carotene as reported by Sumner and 5e extracts of legumes, is different from the compounds in meat cts whichpdestroy carotene. A method for the determination of (‘of carotene oxidase in legumes has been reported by Reiser and (10). EXPERIMENTAL ON METHOD g iminary experimental work was necessary in order to develop the 0d for determining carotene consuming power described below. Al- _f leaf meal was not a suitable source of carotene, since destruction Twas both slight and irregular. In the procedure finally adopted a f, n of purified carotene in petroleum ether was added to the, sample, v. conditions which affect this loss have been studied and a method k e (14) and Sumner and Sumner (14) to be present in the water ¢ 6 BULLETIN NO. 637, TEXAS AGRICULTURAL EXPERIMENT STATION the petroleum ether allowed to evaporate and, after a period of time, the carotene that remained was purified and determined by means of a photoelectric colorimeter. The purification was found to be necessary because the petroleum ether usually dissolves colored material from the meat products. An equal amount of the carotene solution treated in the same way but without a sample was used as a standard. Some ma- terials absorb carotene instead of destroying it. Other factors were also found to influence the results and must be carefully controlled in order to secure reliable results. When the carotene solution was added to the meat product in an Erlenmeyer flask, higher losses occurred when the flask was unstoppered than when it was stoppered (Table 1). The vapor of petroleum ether protected the carotene in the stoppered flask. No difference was observed (Table 2) due to the increase in exposed surface when the meat product was spread over the bottom of a 400 ml beaker as compared with a 150 ml beaker. The qhantity of sample affected the results (Table 3), a smaller percentage of carotene being destroyed when 4 or 5 grams of sample was used than when 1 gram was used. The quantity of carotene used had some effect, since a larger percentage was destroyed (Table 4) when 550 micrograms was used with a 5 gram sample than when 116 micrograms was used. Thus, the greater the relative amount of caro- tene present, the greater both the absolute amount and percentage de- Table 1. The efiect of stoppered and. unstoppered flasks on the loss of carotene in contact with feeds. Stoppered feed Unstoppered loss % loss % D-ried skim milk _______________________________________________ __ 17 74 50% protein meat and bone ‘scraps ___________________________ __ 32 ll 50% protein meat and bone scraps ___________________________ __ 1 17 50% protein meat and bone scraps ___________________________ -- 45 68 50% protein feeding tankage with bone ______________________ __ 14 48 50% protein meat and bone scraps ____________________________ -- 1 1 50% protein meat and bone meal- ___________________________ -_ 3 ‘I1 50% protefn meat and bone scraps _________________________ __ 14 +a 50% protein meat and bon-e scraps _______________________ _- 0 2 50% protein meat and bone scraps ___________________________ __ 50 52 60% protein meat meal digester tankage_ ___________________ __ 68 89 50% protein meat and bone scraps ___________________________ -- 4 20 Table 2. The effect of area, of surface exposed. on the Percflltalge of carflené destroyed. ‘ Loss Sample i 5 150 cc beaker 400 cc beaker 1 % % Meat and bone scraps_ _______________________________________ “t 16 22 Meat and bone scraps_ _______________________________________ -- 45 47 Tankag-e _______________________________________________________ "N 45 37 Tankage _______________________________________________________ -- 40 38 Tankage _______________________________________________________ __§ 43 3 Tankagc _______________________________________________________ n‘ 36 THE OAROTENE CONSUMING POWER OF CERTAIN FEEDS 7 he 3. ZEfl-‘ect of the quantity of sample on the percentage of carotene ‘i destroyed. lgm. ~ 4gm. ‘figm. %~ % 15 63 43 45. 24 15 .._ ‘and bone scraps .................................. -- 71 45 ‘ 40 % v ble 4. Effect of amount of carotene on the percentage of carotene destroyed." Lo-ss Grams of 116 aw 550 sample micrograms micrograms micro-grams carotene carotene carotene % % % __________________________________ -_ 1 52 56 56 _____________________________ _- 5 35 52 62 _____________________________ __ 1 50 T2 82 __________________________________ __ 5 39 57 67 lid. The reason for this result is not apparent as one would expect ifyestruction to vary directly with the amount of sample._ ‘I ed skim milk and meat products from which the fat had been ex- 5" have the power of absorbing carotene, holding it from solution Q roleum ether, to the extent- of 16 to 30 per cent of the carotene p. This source of error was avoided by dissolving the milk product rustic potash or treating the meat product with alcohol so as to bring Ibsorbed carotene into solution. s- temperature affects the quantity of carotene destroyed; the data ble 5 shows appreciably greater destruction at 35° C than at 6° C. e 5. '.l.'he efiect of temperature on the loss of the carotene in contact ‘ with feeds. Y Loss l i “rtions of a freshly prepared ‘solution of carotene in petroleum ether ifbe evaporated in a beaker and allowed to stand over night with lit- keno loss of carotene, But, if the solution is stored a few days, especial- gexposed to light, the carotene becomes activated, so that although v.2 is little loss of color from the solution, there may be considerable i‘ n p18 * 8e _______________________________________________________ __ 1B d bone scraps__-- -- - seas sg Q sass s: 8 BULLETIN NO. 637, TEXAS‘ AGRICULTURAL EXPERIMENT" STATION losses of carotene when thesolution is allowed to evaporate and stand over night. It is seen from Table 6 that the loss may‘ be 28 to 70 per cent of the carotene. The loss was less when a brown bottle was used for storage than when a clear glass bottle was used. The activated carotene in an old solution may be partly protected from loss by meat products. As shown by the results in Table 7, there is less loss when such a solution is added to the meat products than when it is placed in a beaker alone and allowed to stand over night. Table 6. Losses of carotene when freshly prepared or when 01d. solutions ' . were used. Carotene in the Loss on exposure to air solution in beaker over night Age of solution in days ............. _- 0 4 i 14 0 1 4 i 14 days days days days i days i days ppm ppm ppm % % ! % Purified carotene No. 1 in \ i brown bottle ______________________ __ 1.30 1.26 . 1.20 2 36 28 Purified carotene No. 1 in l i ' clear bottle- ..... -_- ............... _- 1.26 1.16 1.1.3 o i 45 F as Unpurifled carotene No. 1 in 5 brown bottle ______________________ __ 1.09 1.08 1.03 2 i 38 30 Unpurifieid carotene No. 1 in i clear bottle- ______________________ __ 1.06 1.03 0.96 0 i 49 70 Purified carotene No. 2 in 1 ‘ l brown bottle ______________________ __ 1.28 1.32 1.32 2 i 12 34 Purified carotene No. 2 in _ i clear bottle- ______________________ __ 1.24 1.24 1.24 6 i 45 48 Unpurified carotene No. 2 in brown bottle ______________________ .._ 1.24 1.18 1.17 5 i 20 57 i Unpurifled carotene No. 2 in i i clear bottle _______________________ -_ 1.09 1.28 1.16 5 i 34 56 Table 7. Effect of exposed and old carotene solution on carotene consuming power of animal lay-products. Old solution Fresh solution Exposed Original loss solution solution % used as used as stated stated loss % loss % Meat and bone scraps- --_- 18 0 36 Meat and bone scraps- 71 50 78 Meat and bone scraps 14 0 28 Meat and bone scraps 31 0 38 Meat and bone scraps ~ 76 60 79 Fish meaL 25 0 28 Fish meal- 40 0 42 Sardine” meal ____ __ 40 0 47 Sardine meal 32 0 36 THE OAROTENE CONSUMING POWER OF CERTAIN FEEDS 9 " METHOD OF DETERMINATION er considering the various factors discussed above the method of ' ining the carotene-consuming power of feeds decided upon is as ‘jws: ‘ix-otene solution: Purify 0.1 gm. of SMA carotene by solution in poform and precipitation with methanol (1). Dissolve 50 mg. of Lied carotene in 2 ml of chloroform and make up to 500 ml with flleum ether. Store the solution in a brown bottle in the refrigerator. the solution once a week by pipetting 1 ml into a 150 ml beaker, , ng to stand over night in an incubator at 35° C, adding 100 ml of ileum ether and determining the carotene. If when compared with i of theoriginal solution made up to 100 ml the loss exceeds 5 per "a new carotene solution should be prepared. 1 cedure for tankage, meat and bone scraps and fish meals, weigh 1 f the sample to be tested into a 150 ml beaker and add 1- ml of i==rotene solution, being careful to moisten the entire sample. Place ‘ incubator at 37° C over night. For controls, pipette 1 ml of the gene solution into each of three 150 ml beakers and place“ with the les’. Add 90 ml of petroleum ether to each beaker. . Stir well with a i rod and let stand 5 minutes. Stir again and allow to settle. Make 1 100 ml. Take 50 ml, add 2.5 gm activated magnesium carbonate Shake well and centrifuge. Transfer the solution to another bottle ining about 1 ml of 1 per cent hydrochloric acid to remove any sus- 1.: material, shake, centrifuge and determine the carotene. For a lard, pipette 1 ml of the carotene solution into a 100 ml volumetric and make up to volume. Store in the refrigerator. Calculate the ne destroyed by comparison with the standard. If appreciable loss ‘ = in the controls, a fresh solution of carotene should be prepared and pork repeated. , v ‘cedure for skim milk. Transfer 1 gram to a 150 ml beaker and ml of carotene solution. Allow to stand over night in an incubator 11° C. Add 20 ml of a 2 per cent potassium hydroxide solution. Stir 3a glass rod until completely dissolved. Transfer to a separatory ‘l and wash the beaker with two 1O ml portions of 95 per cent alcohol hree 10 ml portions of ethyl ether. Add the washings to the solu- ‘g- the separatory funnel. Shake. Allow, to settle and add the ether to the original ether extract. Wash the combined ether extracts Q13 portions of 100 ml of water. Dry the ether layer in an Erlenmeyer Tfwith anhydrous sodium sulphate and make up to volume in a‘ 10f lumetric flask. Purify with magnesium carbonate as directed above I7» termine ‘the carotene. ‘ cedure for animal products from which the fat has been extracted. _-1bgm with carotene solution as directed above. In the morning ml of alcohol. Warm for a few minutes on the water bath, cool, ‘dd 95 ml of ethyl ether. Mix well, allow to settle and determine f» otene in the supernatant solution. ' ' . Lulu“... 1O BULLETIN NO. 637, TEXAS AGRICULTURAL EXPERIMENT‘ STATION Table 8. Changes of carotene consuming power during storage. l i Start 2 4 e i s 1 12 Where weeks weeks weeks N weeks 3 weeks stored ‘ a loss % loss % loss % loss % i loss % loss % l 1 Meat and bone scraps _________ Laboratory 9 14 2:» 21 l 17 ; 36 Meat and bone scraps _________ -_, Laboratory 34 7O 67‘ m l £9‘ -- Meat and bone scraps _________ “l Laboratory 5 14 21 1S l 2O 44 Meat and bone scraps _________ __ Laboratory 47 22 18 1 1 7 23 Meat and bone scraps _________ __ Laboratory 821 85' 82 74 1 73 ‘ S4 Fish meal- _____________________ -_ Laboratory 0 16 25 13 . 9 ‘ 26 Laboratory 5 ‘ 6 42 31 g 22 36 _ Laboratory 72 63 67 __ l] __ -_ Sardine meal ___________________ -- Laboratory 3 41 5O 33 a 32 __ Sardine meal ___________________ -- Laboratory 5 34 28 28 * 22 S0 Dried buttermilk _______________ _- Laboratory 0 14 21 -- __ -- Meat and bone scraps _________ __ Incubator 35°C 9 3 11 _- i _ __ Meat and bone scraps _________ __ Incubator 361°C 34 27 11 __ " __ __ Meat and bone scraps _________ __ Incubator 35°C 5 5 8 __ <1 -_ __ Meat and bone scraps _________ __ Incubator 35°C 47 3 5 __ i -- __ Meat and bone scraps _________ _- Incubator 35°C 82' 5-2 35 __ i __ __ Fish meal- _____________________ _- Incubator 35°C 0 14 18 __ 4 __ __ Fish meal_ _____________________ __ Incubator 33°C 5 18 25 _- I __ -_ Dried skim milk ________________ __ Incubator 3L5I°C= 72 29 46 -- 1 -_ __ Sardine meal ___________________ __ Incubator 343°C‘ 3 27 ‘l8 __ l __ __ Sardine meal ___________________ _- Incubator 35°C 5 25 2S __ ‘ __ __ Dried buttermilk _______________ __ Incubator 35°C 0 0 8 -_ __ -_ FACTORS WHICH AFFECT CAROTENE OONSUMING POWER A large number of feeds were tested for carotene consuming power by the methods described. It was found to occur almost entirely with meat products, dried milk products and dried fish. The carotene consuming power is not constant, so that the same sample may give different re- sults from'time to time. Table 8 shows some variations which occurred during storage under laboratory temperature and at 37° C. The caro- tene consuming power of some samples increased and that of others de- creased. Light may increase the carotene consuming power. Samples of meat and bone scraps were divided; one portion of each was placed in clear ‘ glass bottles and another portion wrapped in aluminum foil was placed . in a. similar bottle. After 4 weeks (Table 9), the samples exposed to light had appreciable higher carotene consuming power than those not exposed.‘ As shown in Table 10, portions from the surface of samples in bottles, in some cases, had a higher carotene consuming power than portions taken from below the surface. Autoclaving meat and fish products at 20 pounds pressure for one“ hour decreased the carotene-consuming power of most of the samples} as shown in Table 11. The change was not permanent, as is shown in Table 12. After one week, the autoclavet” mples again had a high carotene-consuming power. When samples which had a high carotene consuming power were ashed, the ash had practically no carotene con- suming power. Table 13. This showed that the ash constituents were not responsible for the consuming power. THE OAROTENE CONSUMING POWER O'F CERTAIN FEEDS 11 U‘! ble 9Q Efiect of light on the sample on destructionof carotene by Ieeds- 1 Start Exposed to Wrapped % hght in foil % % eat and bone scraps __________________________________ -- 5 46 11 eat and bone scraps ................................. __.- 58 69 27 feat and bone scraps __________________________________ -_ 11 33 10 j ‘sh meal _______________________________________________ __ 25 29 25 Tanka ge _ _______________________________________________ - _ 4-8 T5 50' I able 10. Efiect of location of portion used in Sample bottle on destrlwtion " of carotene. 7 Surface Subsurface s sample sample <5 % % _eat and bone scraps- ....... _; _______________________________ -- 41 10 eat and bone scraps ________________________________________ __ 70 , 52 Meat and bone scraps_ _______________________________________ _- 24 10 FFish meal ______________________________________________________ -_ 17 15 l sh meal ______________________________________________________ __ $7 35 Table ll. Eifect of autoclaving upon percentage of earn-tang destroyed. Name 0f Sample Untreated Autoclaved % 60min. % \ eat and bone scraps_ _______________________________________ __ 58 7 ankage with bones ____ -_ 68 7 eat and bone scraps ______________________________ __ 58 14 eat and bone scraps- _____________________________________ __ , 36 3 eat and bone scraps- _______________________________________ __ 45 6 ardine meal- _________________________________________________ __ 16 0 ish meal _____________________________________________________ -.p- 47 1 "- eat and bone scraps- _______________________________________ __ 78 40 linkage _______________________________________________________ __ 94 10 $1 w bone meal ________________________________________________ __ 78 e3 s’ sh meal ______________________________________________________ -_ 27 5 eat and bone scraps ________________________________________ __ 29 3 . eat and bone scraps- ________________________________________ __ 63 7 ankage _______________________________________________________ __ ~ 25 12 ____________________________________________________ __ 46 7 ____________________________________________ __ 42 20 ankage _______________________________________________________ _- 44 20 3 Table 12. Iflifect of storage 1' week after antoclavlng upon percentage of ' carotene destroyed. _ At beginning After 1 week s ~ Name of sample Untreated Autoclaved Untreated Autoclaved, l o % % o __,__.___. - eat and bone scraps ___________ __ 1---- 35 17 -_ 67 ‘eat and bone scraps .......... -_» ‘ b.-- 59 38 78 64 _ ankage __________________________________ -- 50 10 90 63 Meat and bone scraps ___________________ __ 70 35 85 73 _ ankage __________________________________ -_ 63 34 87 79 i ______________________________ __ ' 46 38 __ 65 ff ankage __________________________________ -- 59' 28 86 66 l2 BULLETIN NO. 637, TEXAS AGRICULTURAL EXPERIMENT STATION Table l3. Carotene consuming power of the ash of meat products. Loss of carotene Name of sample Untreated sam- Ash Dle % % Tankage ....................................................... -- 35 5 Meat and bone scraps- _______________________________________ -- 78 5 Tankage _______________________________________________________ -- 45 9 Tankage _______________________________________________________ __ 94 0 Tankage ________________________________________________________ -- 70 0 Meat and bone scraps_ _______________________________________ -- 8 fl Meat and bo-ne scraps- _______________________________________ __ 9 3 Meat and bone scraps ....... -_ --_- 0 0 Meat and bone scraps_ _______________________________________ -- g 8 Meat and bone scraps ---------------------------------------- -_ Table 14 shows that the fat extracted from the feed by petroleum ether consumed carotene in some cases and did not in others While the residue from which the fat had been extracted consumed carotene in all cases. One gram of the feed was extracted six times on a filter paper with 10 cc of 30° to 60° boiling petroleum ether. The extract was col- lected in a 300 cc Erlenmeyer flask provided with a ground glass joint. The ether was removed by reduced pressure and 1 ml of the petroleum ether carotene solution added to the fat and the carotene consumed de- termined. The potency of the residue was determined at the same time. The results are given on Table 14. This experiment showed that most of the substance causing the destruction of carotene remained with the frac- tion insoluble in petroleum ether. Table l4. Percentages of carotene destroyed ‘by fats soluable in petroleum ether and extracted residues. Fats soluble in Extracted Name 0f sample petroleum ether residues loss % Tankage _______________________________________________________ __ 42 5s Meat and bone scraps- _____________________________ __' ________ _- 26 54 Meat and bone scraps---" 40 55 Meat and bone scraps- _______________________________________ -- 20 47 Meat and bone scraps- _______________________________________ __ 2 72 Dried skim milk__ _ 0 , 35 Meat and bone scraps- _______________________________________ _- 0 7s Fish meal ...................................................... _- 0 7s Meat and bone scraps- _______________________________________ __ 0 31 Fish meal ______________________________________________________ __ 5 39 It was thought that the failure to extract the carotene destroying sub- stance with petroleum ether might be due to the presence of fatty acid peroxides which are only, slightly soluble in that solvent, but are soluble in alcohol and ethyl ether. To test this point a series of determinations were- made by boiling 1 gram of the sample with 10 ml of redistilled alcohol for 10 minutes, adding 25 ml of peroxide-free ethyl ether while still warm, and filtering into a 300 ml Erlenmeyer flask provided with a ground glass joint. The extraction was repeated twice with 5 ml of THE CAR-OTENE CONSUMING POWER OF CERTAIN FEEDS >13 Lhol and 15 ml of ether and finally withptwo 15 ml portions of ether V." The alcohol and ether was drawn off by means of reduced pres- i‘. and the carotene consuming power of the fat and of the extracted rues determined. It was found that the carotene is tenaciously ab- ed on the fat-free material and must be_ removed by heating with hol. The procedure was modified to permit this manipulation. Table fishows the results of the determinations. Both the fat and the ex- ted. residue had carotene consuming power which showed that the pptene consuming power was not due entirely to fatty peroxides. e 15. Percentage of carotene conumed by fat free residue and ‘by the a fat extracted from meat products. e Sample Untreated ' Fat-free I Fat * . residue : . and bone scraps __________________________________ __ 10 ' 22 31 a t and bone scraps--- 11 31 18 1t and bone scraps-____ 11 18 23 _ t and bone scraps .................................. -_ 12 25 29 r and bone scraps __________________________________ -- 13 20 7 _t and bone scraps _________________________________ -n._ 38 40 34 t and bone scraps __________________________________ -- 75 36 65 j» age ________________________________________________ __ 70 41 70 ‘t and bone scraps __________________________________ -- 78 42 50 age ________________________________________________ -- 72 38 - 48 w age ________________________________________________ ___ 59 31 54 p- 1129- ----------------------------------------------- --j 33 - 23 40 , ,~ l he effect of autoclaving upon the fat and the fat-free residue was ‘died. Four 1 gram samples of a series of meat products were ex- ted with alcohol and ether as in the method described above. Two j-free and two fatty fractions were autoclaved. The carotene‘ consum- f power of one autoclaved and one untreated fraction of each material determined at once. The corresponding fractions were allowed to jnd one week before their potency’ was determined. The results of ‘e experiment are tabulated on Table 16. Autoclaving destroyed some he potency of both the fat free and fat fractions. The fat free residue not increase in carotene consuming power in a week of storage but '1- carotene consuming power of both the autoclaved and the untreated increased to almost 100 per cent. l 1+- 16. Percentage of carotene consumed by fat and by fat-free residue 01’ meat products before and after autoclaving. Wu"- Immediately After one week Fat-free residue Fat Fat-free residue Fat Auto- Not Auto- Not Auto- Not Aut0- Not * claved auto- claved auto- claved auto- claved auto- claved " cl-aved claved claved and bone scraps ......... -- 8 23 36 " % 11 20 99 98 ~ age _______________________ _- l0 33 35 94 12 32 98 97 ge _______________________ -- 12 29 76 82 13 35 96 95 age _______________________ -_ 11 37 62 84 15 40 9'7 96 ge ____ -_, ________________ -- 12 22 62 a 94 1 13 20 96 97 ' l l4 BULLETIN NO. 6237, TEXAS AGRICULTURAL EXPERIMENT STATION The instability of the active principle, when considered with work of others (7), seemed to indicate that peroxides of the fatty acid are responsible. This simple theory was very attractive, but had to be modified when it was found that repeated extractions with boiling alco- hol and ether (Table 15) did not completely remove the carotene con- suming power. There is the possibility that in the unextracted material only the fatty fraction is active but that after removal of the fat there is a new effect due to surface phenomena. We have seen above how meat scraps protected unstable carotene from oxidation by the air and that this protective power may reside in the fat fraction, as can be seen in Table 15. When meat scraps of low carotene consuming power were extracted, the fat-free fraction had considerably more potency than the unextracted material. In all cases the carotene destroying power of the extracted material plus that of the residue was considerably higher than that of the unextracted sample. Schroeder, Redding, and Huber (12) found that ether extracted meat scraps when fed to chickens produced the same signs of vitamin A and D deficiency as the unextracted ma- terial, indicating that there may be a substance there responsible for the effect rather than just a surface effect. Some premilinary experi- ments have shown that dried blood has a high carotene consuming power but no relation was found between the carotene consuming power of samples of meat scraps and their dried blood content. EXPERIMENTS WITH RATS. It was thought possible that meat by-products of high carotene con- suming power when fed with feed containing carotene might destroy carotene in the digestive tract of animals and so reduce the quantity available for use. Such destruction was found to occur. by Powick (8) and by Lease et al (7) when rancid fats or peroxides of fatty acids were fed to rats, resulting in decreased storage of vitamin A in the livers. A similar ‘method of experiment was applied to meat products. In order to ascertain whether the carotene consuming power was of practical importance, experiments were made to see if it would affect the quantity of vitamin A stored in the livers of rats, or the length of life and weight when fed when only small amounts of carotene were fed. PROCEDURE AND RESULTS WITH LIVER STORAGE. Rats 21 days old were selected from females fed on a diet low in vitamin A potency since the birth of their young, and divided into 2 groups, equalized as to sex and litter. One group received meat meal with a high carotene-consuming power, and the other a similar by- product with a low carotene consuming power. Both groups were fed a diet usually composed in per cent of white corn meal 37, wheat gray shorts 25, casein 12, yeast (irradiated) 3, salt 1, tri-calcium phosphate 1, calcium carbonate 1, meat meal to be tested 20 and alfalfa replacing corn meal to furnish 10 micrograms of carotene per gram, except as THE GAROTENE OONSUMING POWER OF CERTAIN FEEDS 15 eotherwise noted. After 14 days the rats were killed, and the livers re- moved and weighed. The spectro vitamin A was determined by means of 5a Bausch & Lomb medium spectograph by methods already described (3). "i Ten rats per group were used in the first 3 comparisons and 6 per Egroup in the others. The quantity of carotene consumed during each V-experiment was comparatively uniform, being from 1000 to 1100 micro- égrams. Table 17 contains the result of 10 comparisons. In 5 of the 9 com- tiparisons, in which alfalfa was used as the source of carotene the spectro vitamin A was lower in the livers when the high carotene consuming imeat meal was fed than when the meat meal had a low carotene con- fsuming power. With the remaining 4 comparisons, the high carotene consuming meat meal gave equally as good results as the low one. With icod liver oil as a source of vitamin A, in the tenth comparison, the spectro vitamin A in the liver of the rat receiving meat meal of high low consuming power product was used. Apparently the carotene con- suming power of the meat meal interfered with the utilization of caro- tene by rats for liver storage in some cases and did not interfere in jgconsuming power of meat meal does not seem to seriously affect the storage of vitamin_A in the livers of rats. It is to be noted from Table 17 that appreciably less spectro vitamin -A was stored in June and July (35.5 and 48.1 micrograms per liver) Iable 17. Spectro vitamin A in micrograms per liver as affected by 10w and i 7 high carotene consuming- power of meat meals. = Spectro- vitamin A 1- Experiment number and source per liver Percent Month of . of vitamin A potency ---—-——~———-——- high experi- Low High of 10w ment gms. gms. Alfalfa 3114 _____________________________________ __ 116.4- 80.6 69 1940- . November 3114 _____________________________________ __ 83.8 94.5 112 1940 December 3114 _____________________________________ __ 74.0 681.9‘ 9'3 1941 February 3185 ______________________________________ -. 96.8 68.1 70 March 3185 _____________________________________ __ 631.5 57.3 90 May 3229 ______ __; ____________________________ __ 51.6 A 48.1 93 June 3235 _____________________________________ -_ 36.3 35.5 98 July 3418 (no oil) ............................ __ 77-4 77-4 100 1942 ' May g 341s (10% 011) ___________________________ _- 91.5 110.4 121 May God liver oil i e179 ............................ ....... _- 2212.6 190-3 89 March carotene consuming power was 89 per cent of that secured when the ' ‘others. The effect in general was not great, so that the high carotene ' '16 BULLETIN NO. 637, TEXAS AGRICULTURAL EXPERIMENT‘ STATION than in the cooler months, as much as 80.6 in November and 94.5 micro- A grams in December. Substitution of 10% cottonseed oil for 10% corn meal in the ration in another Experiment increased the storage of spectro vitamin A in the liver from 77.4 micrograms without oil to 110.4 micrograms per liver. The effect of some different quantities of meat meal and different quantities of carotene is shown in Table 18. Table 18. Effect of percentage of meat meal in the ration on spectro vitamin A in rat livers. Spectro vitamin A per liver micrograms Carotene 10 parts per million No meat meal ________________________________________________________ _'---. 10% meat meal ---- -- i 20% meat meal _________________________________________________________ _-‘ 30% meat meal --------------------------------------------------------- u: bums»- camel-a» Qeiwu Carotene 5 parts per million No meat meal ---------------------------------------------------------- n; 35-6 101% meat meal _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ . _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _= 311.3 20% meat meal --------------------------------------------------------- - 30% meat meal _________________________________________________________ n} In the experiment described above, the total storage of spectro vita- min A was compared. Treichler, Kemmerer, and Fraps (15) have shown that part of the spectro vitamin A is not vitamin A at all, since it may increase when the rat is fed upon a diet practically free from vitamin A. ‘In one experiment the meat meals were also fed without carotene, with the results given in Table 19. The livers of the rats which did not re- ceive carotene contained 22.7 and 25.1 micrograms of pseudo spectro vitamin A per liver. When correction was made for these quantities, the spectro-vitamin A stored from the meat meal with low carotene con- suming power was 12.4 micrograms per liver, while that stored from the meat meal with high consuming power was 8.7 micrograms per liver. The carotene consuming power of animal products in the diet may zaffect the storage of vitamin A in liver, but not to a great extent. f Table 19. Efiect of meat meal alone and with carotene on spectro vitamin A in livers. Spectro vitamin A Addition and carotene consuming micrograms per liver power of meat meal Total Net* No carotene-low ______________________________________________ __ 22,7 __ ‘Carotene-low _________ __ __ 36,3 12 4 N0 carotene—high _____________________________________________ __ 25.1 __ Qarotene-high _ _ _ _ _ _ . _ _ _ _ _ . _ . _ _ __ 35.1 s 1 *.Corrected for difierence in weight of livers. THE OAROTENE CONSUMIIlG POWER OF CERTAIN FEEDS 17-" Effect of Carotene Consuming Power on Longivity and Gain of White Rats. _= experiments on storage in livers were made on moderate levels tamin A. It was thought that the carotene consuming power might. a greater effect if low levels of carotene were fed. p the time of weaning, litter mates were divided as evenly as pos- ‘lbetween several groups and 4 males and 4 females were placed upon feed containing 0.1 micrograms of carotene per‘ gram, or it's equiva-p . in other forms. The feed in percentage consisted of meat meal 20,. d corn starch 57, cottonseed oil 8, irradiatedyeast 1, non irradiated 9, salt mixture 4 and sodium chloride 1-. It was planned to con- “the experiment until all the rats died, but they lived much longer" v expected, and those alive after 470 to 698 days were killed and the riment discontinued. g: carotene in alfalfa was determined as pure carotene (1), the vita-t A in cod liver oil as spectro vitamin A (3) and reduced to carotene valent, and the pure carotene in yellow corn was calculated to caro- equivalent (2). , » e results of the experiments in which 0.1 micrograms carotene per‘ g“ of feed was used, are given in Table 20. Average length of life 5 average maximum weight were both lower with the rats which re- V, d the meat meals with high carotene consuming power. The dif- ;‘ tees were not great, however, but on the other hand Were compare»- small. Very little carotene was destroyed in the rats by the meat fps with high carotene consuming power. 20. Effect of carotene consuming power of meat meals on IOIIEBViVY and gain 1n weight of white rats. Source of No. N0. Maximum weight of g vitamin A rats rats rats (grams) Aver. of meat meal = potency on living length ‘in ration | (0.1 micro- experi- when Aver. Aver. Aver. of - l grams per ment exp. - 4 4 fe- males life gram of closed males males and fe- days- ration) males ___ i "carotene § 4 F 4 F g '_ H“ ...................... -_ alfalfa I 4 M 1 M 336 212 274 519 3 carotene x f 4 M 2 M g _ _1ng ______________________ _- alfalfa 1 4 F a F m1 192 247 502 ‘ carotene § 4 M 1 M } - _» mg ..................... -__- cod liver oil I 4 F 1 F 3102 204 253 452 ‘carotene A § 4 M _ 1 M I gm ______________________ __ yellow corn ~ ] 4 F 1 F § 250 197 224 478 Experiments With Chickens. gis work was started to attempt to learn the cause of certain chick . f_ = in Texas that appeared to be associated with certain animal pro- ffeeds. When the feeding of the particular animal protein feed was 18 BULLETIN NO. 637, TEXAS’ AGRICULTURAL EXPERIMENT STATION discontinued, the losses usually stopped. It was reported that the chicks seemed to be suffering from vitamin A deficiency. The work of Schroeder (12) indicated that there was a rather wide variation in the free fatty acid content of different samples of meat scrap studied by him, and the work of Schroeder (12) as well as the work of Powick (8) indicated that vitamin A is progressively destroyed or inactivated by free fatty acids. It was thought that this trouble with chicks might be caused by a destruction of all or part of the carotene of vitamin A in the feed. In order to test this hypothesis, and to ascertain th-e importance of such carotene consuming power several experiments were made. Samples of meat scrap were» secured directly from a number of manufacturers. Four samples of 50 per cent protein meat scrap and one sample of 55 per cent protein meat scrap were used in the first experiment. Nine per cent of the different lots of 50 per cent protein meat scrap and 8 per cent of the 55 per cent protein meat scrap were used in the respective rations. These were used in experiment 1, in which 175 micrograms of carotene in oil was used per 100 grams of ration, and in experiment 2 in which 5 pounds of dehydrated alfalfa leaf meal containing from 195 to 142 micrograms of carotene per gram was used per 100 pounds of feed. The rations were made up at two week intervals during the experiment. which continued for 10 weeks. The rations used are given in Table 21. Table 21. Percentage of ingredients of rations for experiment 1 and experiment 2. Experiment 1 Experiment 2 Ground kafir_ _________________________________________________ __ 40* 3 Ground milo ___________________________________________________ __ 6 __ Wheat gray shorts ____________________________________________ __ 20 £0 Ground barley . _ . . _ _ _ _ _ . _ . _ . _ _ _ _ _ _ _ _ . _ _ . _ _ _ _ -_ __ __ Ground whole oats ___________ __ _ 10 l0 Dehydrated alfalfa leaf meal_- _ __ 5 Meat scraps- ____________________ __~ ____________________________ _- 9* 9 41% protein soybean oil meal_ _______________________________ __ 4.5 4.5 43% protein cottonseed meal_ ________________________________ -- 4 5 4.5 Dried whey- ___________________________________________________ _- 8 1 Oyster shell ____________________________________________________ __ 2 2 Salt ____________________________________________________________ __ 1 1 *4T% kafir and 8% meat scraps were used in the rations containing 55% prot-ein meat scraps. According to Table 22, all of the chicks in lot 5 died before the chicks were 8 weeks old. In lot 10, which received the same meat scrap as lot 5, the mortality was 40 per cent even though the chicks received a very liberal supply of carotene from alfalfa leaf meal. With the chickens in both of these lots marked symptoms of vitamin A deficiency were noted. Although the chickens were on Wire floors in battery brooders, a rather large number of the chicks in lot 5 also had coccidiosis. One case was noted in lot 10 and one in lot 4. This indicates the lack of resistance to infections of chicks deficient in vitamin A. The free fatty acid content of the meat scrap which did not cause a loss was 7.8 per cent compared with 2.7 per cent for the sample with THE OAROTENE OONSUMING POWER OF CERTAIN FEEDS 19 Table 22.—Bfi‘ect of meat scraps on _morta.1ity and gains in weight experiments 1 and 2. Experiment 1 g Experiment 2 Lot numbers 1 2 S 4 | 5 6 7 8 9 10 ' (Carotene Tconsuming power ______ __ 0 -- -- -_ 58 0 __ __ __ 58 ;Free fatty acid content jot meat scrap_ _________ __ __ __ _- __ _- 7.8 __ -_ _- 2-7 "Mortality after first two Weeks, percent _________ -_ 8.7 23.1 23.1 32.0 100.0 0' 0 0 0 40-0 "Gains in weight of cock- “ els, gm _______________ -_ 588 568 402 473 _- 599 667 526 510 347 i ains in weight of pul- .ts, gm ________________ __ 487 - 3815 373 417 __ 540 503 ' 397 424 289‘ can gains in ~ eight, gm _____________ __ 538 477 388 445 -- 570 585 462 467 318 rams of feed per -_ am of gain ___________ __ 3.39 3.70 4.28 408' __ 3.58 3.31 3.85 3.79 4.54 hich the mortality was high. The free fatty acids do not account for a vitamin A deficiency. The carotene consuming power of the meat rap that caused the greatest loss was 58 per cent as compared with 0 cent for the meat scrap used in lots 1 and 6 which gave good results. .. On January 2, 1941, Experiment 3 was started with twelve different mples of animal protein feeds ranging in carotene consuming power Kim 0 to 78 per cent. All contained 175 micrograms of carotene from _alfa leaf meal per 100 grams of feed. The chicks were fed ten weeks. he mortality ranged from 0 to 29 per cent, but there was no correla- ‘n between gains in live weight or mortality and carotene consuming iwer as determined by the methods already mentioned. Neither was '_re any marked indications of vitamin A deficiency in any of the fcks. ‘Ti0n May 14, 1941, Experiment 4 was started in duplicate using samples ‘meat scraps with high and with low carotene consuming power; samples fish meal with low carotene consuming power, and a sample of tank- “with high carotene consuming power. No correlation was noted be- Y gains in live weiglgt or the mortality and" the carotene consuming 0f the animal protein feeds in this experiment. Very few birds v symptoms of vitamin A deficiency. These chicks received 175 grams of carotene from alfalfa leaf meal per 100 grams of ration. the spring of 1942, twelve samples of meat scrap were found to “a carotene consuming power ranging from 1.4 to 97.8 per cent. On '_ 24, 1942, the two samples of meat scrap having the highest caro- iconsuming power and the two samples having the lowest consuming were used in rations that were fed in quadruplicate, (Experiment ‘Two series of rations contained 125 micrograms of carotene per 100 of feed and two contained 200 micrograms. On both levels of 20 _ BULLETIN NO. 637. TEXAS AGRICULTURAL EXPERIMENT STATION carotene, one set of chicks received carotene in oil and the other set received carotene from alfalfa leaf meal. In experiment 5 there were few birds showing symptoms of vitamin A deficency, and there was no correlation between the carotene consum- ing power as determined by the chemical method and the gain in live weight or the mortality. In fact, there were no significant differences in mortality. The gains in weight were lowest for ration 3 in all of the dif- ferent series even though this ration was the second lowest in the carotene consuming power. Some factor other than carotene destruction must have caused this poorer gain. In the fall of 1942,\six samples of meat scraps ‘and one sample of blood meal were secured. Experiment 6 was started on January 5, 1943 with rations for each group adjusted to contain the same levels of pro- teins, minerals and vitamins. They contained from 38.9 to 39.65% ground milo, 20% each of ground barley and wheat gray shorts, 4% each of soybean oil meal and cottonseed meal, %% of dehydrated alfalfa leaf meal, 1% distillers’ solubles, from 0.6 to 5.1% of minerals and from 6.25 to 10.0% of the animal protein feed being studied. They also con- Table 23. Relation of carotene consuming power to mortality, gain in weight and vitamin A lesions of chickens. l l l . Lot numbers 1 2. l 3 l 4 l 5 6 7 l l , l ‘ Carotene consuming 1 power, per cent .......... -- 45 54 , 34 1s j 32 3 0 32 Mortality after first l ; l 2 weeks, per cent- _______ -_ 0.0 12.0 I 52.0 4.0 20.0 l 20.0 ~ 25.0 Gain in weight,’ g l F cockelrlels, gm ............. -_ 79v 774 l 623 l 762 l 661 s09 see Gain inwe-ight, . pullets, gm _______________ __ 655 560 \ 471 726 525 1 705 701 Mean gain in - weight, gm _______________ __ 726 667 l 547 744 593 ; 757 C95 Grams of feed per gram of gain ____________ __ 4.63 4.73 l. 6.23 l 4.63 5 79 4.62 5 l3 l . l Per cent having vitamin A l l l \ lesions after 8 weeks on l l l 1 experiment ________________ __ 4.0 ' 40.0 l 50.0 l 12.0 l 17.4 \ 13.0 l 45-0 l tained approximately 100 micrograms of carotene, and 44 units of. vita- min D per 100 grams and 6 grams of manganese sulphate per 100 pounds. As shown in Table 23, decidedly the poorest gains, greatest mortality, greatest percentage of chicks showing vitamin A deficiency at the end of the eighth week of the experiment and the poorest utilization of the feed were found with "the ration containing blood meal fed to lot 3. This blood meal had a carotene consuming power as determined chemically of 34 per cent as compared with 45 and 54 per cent for the feeds used in rations 1 and 2 respectively. The chicks receiving ration 2 showed the third highest percentage of vitamin A deficiency. The gains in live THE OAROTENE CONSUMING; POWER OF CERTAIN FEEDS 21 eight after 12 weeks feeding were greatest and the incidence of vitamin i deficiency was lowest in the case of thechicks in lot 6, ration, 6, but ur chicks died from this pen. This diet contained the meat scrap which pad no carotene consuming power according to chemical determination. he mortality and number of chicks showing vitamin A deficiency at the id of eight weeks of the experiment were low with the chicks in lot 4, ihich received a ration containing a meat scrap having a. 16 per cent rotene consuming power. The weights of these chicks were very satis- '=ct0ry. DISCUSSION OF EXPERIMENTS ON CHICKENS. i Experiment 6 and Experiments 1 and 2 indicate that there is a defi- ‘ite depressing effect by some animal protein feeds on the carotene con- nt of a ration. It appears that there may be some other factor in- _lved other than that represented in the carotene consuming power. his work and the use of this carotene consuming power is further con- fused by the variability of the percentage of destruction by animal pro- in feeds as determined at different times. This is well shown in Tables T,‘ 8 and 24 that feeds that may at first have a low carotene consuming i» but later have somewhat higher consuming power and feeds that ave a very high consuming power may later have a consuming power ' low as 30 per cent. n if It is true that these factors of carotene destruction do not seem to be resent in all animal protein feeds in sufficient amounts to cause trouble i Table 24. Variations in the carotene consuming power of certain feeds. (Per cent.) Consuming power Dates Determined 1-9-41 5-7-41 i‘ 6-3-41 7-2-41 7-29-41 l W0; _________________________________ _.. 0 3 I 9 13 my .... _- _-- 7s 64 t i 50 so ‘f 940 _________________________________ __ es s 11 i s 17 949 ................................. -_ 7e , 24 l 10 60 54 2o 1 32 ' p _ 55 I 1s f 946- -------------------------------- __ 5 1 , ‘Q5 --------------------------------- ' 45 14 I i I ‘I943 ................................. -_ 0 13 I ‘ ________________________________ __ 59 9 -_ 55 a1 Z1 _____________________ _.l ........... -_ 47 22 BULLETIN NO. 637, TEXAS AGRIQULTURAL EXPERIMENT STATION with chicks, but the fact that they do occur in certain feeds of animal origin makes this a real problem for poultrymen who may be feeding rations rather l_ow in carotene. ACKNO\VLEDGMENT Dr. A. R. Kemmerer and other members of the staff made analyses of livers for spectro vitamin A and otherwise helped in the work. J. R. Couch and V. H. Melass of the Division of Poultry Husbandry also assisted in the work. SUMMARY. Certain animal by-products such as meat scraps, tankage, dried fish and dried milk have the power of destroying carotene. Vegetable feeds very seldom had any carotene destroying power. The carotene-consuming power is affected by exposure to air, the quantity of the sample, the temperature and the quantity of carotene in relation to the quantity of the sample. Carotene solutions in petroleum ether became activated in a short time so that carotene is lost when the petroleum ether is allowed to evaporate and the mixture to stand over night. Animal by-products may decrease the loss of carotene in such solutionsf Milk products may absorb carotene as well as destroy it. A method of determining the carotene consuming power of feeds is given in detail. Carotene consuming power may change in storage, so that the same sample may give different results at different times. Samples high in carotene consuming power ‘at first may decrease in this power, and those low in carotene destroying power may increase. Ex- posure to light may increase carotene consuming power. Autoclaving decreased carotene consuming power but the changes were not per- menant, even for as short a period as one week. Both the fat and residue from which the fat had been extracted had carotene consuming power. The carotene consuming power may have been partly due to fatty acid peroxides, but not entirely. Materials subjected to treatment which should have removed fatty acid peroxides still had a high carotene con- suming power. When meat meal having high carotene consuming powers was fed to rats, in 5 of 9 experiments the livers contained less vitamin A than when similar feed having low carotene consuming power was fed. The dif- ferences, however, were comparatively small, and in 4 of the experiments the high carotene consuming power did not decrease the vitamin A stored. Rats fed on a mixture containing 0.1 micrograms of carotene per gram, with meat meal of high carotene consuming power had a slightly shorter average length of life and attained a lower average maximum weight than rats fed corresponding meat meal with low carotene consuming power. The differences were comparatively‘ small. THE OAROTENE GONSUMING POWER OF CERTAIN FEEDS 23 experiments were made to ascertain the effect of carotene con- A‘. power upon gains in weight and mortality on growing chickens. iof. the experiments, the carotene consuming power had no effect. In f the experiments, the high carotene consuming power was detri- “l to the chickens. ds of high-carotene consuming power may sometimes cause injury lflickens through vitamin A deficiency if the ration is low in caro- ior vitamin A potency. Otherwise a high carotene consuming power snot appear to be injurious. . REFERENCES aps, e. s., and Kemmerer, A. R. 1939. Improved method for estimat- '~ ng carotene in feeds. J. A. O. A. C. 22:190-194. .._ ps, G. S., and Kemmerer, A. R. 1941. Determination of carotene and ‘ cryptoxanthin in yellow corn. Ind. Eng. Chem. (Anal. Ed.) 13:306-809. 1 aps, G. S., and Kemmerer, A. R. 1943. Carotene and vitamin A in v-commer-cial butter. Jour. Assoc. Offic. Agr. Chem. 26:158-164. ray, R. E., and Robinson, H. E. 1940. Fr-ee fatty acids and rancidity relation to animal by-product protein concentrates. Poultry Science _ 236-4 . arrellson, R. T., Nelson, P. Lowe, Dyme, H. C., and Nelson V. E. 1939. ‘The effect of treated fat on vitamin A potency. Iowa State College <1 Journal of Science 15:353-364. T upp, B. F. 1931. Fatty acid affects the health of birds. Poultry Cul- _. ture, April, 1931. tease, E. J., Lease, J. G., Weber, J., and Steenbock, H. 1938. Destruction “ of vitamin A by rancid fats. J. Nutrition 16:571. lowick, Wilmer C. 1925. Inactivation of vitamin A by rancid fat. Jour. ‘_ Agr. Res. 3121017-1026. uackenbush, F. W., Cox, R. P., and Steenbock, H. 1941. Stabilization vjof carotene in linoleic ester. Proc. Am. Soc. Biol, Chem. 35:CIV. eiser, Raymond and Fraps, G. S. 1943. Determination of carotene oxidase‘ - inlegume seeds. Jour. Assoc. Offic. Agr. Chem. 26:186-194. Iobinson, Muriel Elaine. 1924. Hemoglobin and methemoglobin as oxi- dative catalysts‘. Biochem. J. 18:255. ‘hroeder, C. H., Redding. G. K., and Huber, L. J. 1936. Some causes and effects of a high free fatty acid content of the meat scraps in poul- try rations. Poultry Science 15:104-114. umner. J. B., and Dounce, A. L. 1.939. A carotene oxidase in beans. Enzymologia 7:130. _ umner. J. B., and Sumner, R. J. 1940. The coupled oxidation of caro- - tene and fat by carotene oxidase. J. Biol. Chem. 1341531. reichler, Ray, Kemmerer, A. R., and Fraps, G. S‘. 1942. The utilization of carotene and vitamin A in the rat. Jour. Nutrition. 24:57-62.