WORK which has been reported on

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1 The Stbility of Vitmins A nd D in Mixed Feed Ingredients PART II VITAMIN A F. D. BAIRD, A. T. RINGROSE,* AND M. J. MACMILLAN Nutritionl Lbortory, Ntionl Oil Products Compny, Hrrison, New Jersey WORK which hs been reported on the stbility of vitmin A shows tht there is substntil loss of this fctor in chick rtions nd feed ingredients when these re stored under ordinry conditions for vrious lengths of time. However, there is considerble disgreement s to the mount of destruction or how rpidly such destruction tkes plce. Frps nd Treichler (19) reported losses in vitmin A potency of - percent in to 19 months in lflf lef mel, dried blck-eyed pes, dried green sweet peppers, yellow corn (whole or ground), nd powdered whole milk. Kick nd Bethke (199) reported very little, if ny, destruction of vitmin A in yellow corn (whole, crcked, or finely ground) when stored for one yer. Record, Bethke, nd Wilder (197) stted there ws loss of vitmin A from cod liver oil nd crotene from lflf lef mel when mixed in rtion, but the destruction ws not complete even fter storge period of six months in heted, well lighted room. Frps nd Kemmerer (19) reported tht the vitmin A in cod liver oil when mixed with feed ws entirely destroyed in two weeks when stored t tempertures s low s 7-9 C. Frps nd Kemmerer (197) lso reported tht fish liver oils or concentrtes, when dded to finely divided feeds such s white * Now t the University of Kentucky, Lexington, Kentucky. (Received for publiction Mrch 9, 199) [1] corn mel, white corn mel nd yest, white corn mel nd soy ben oil mel lose their vitmin A potency to the extent of 79 percent to 1 percent when stored t room temperture nd s low s C. for four weeks. It should be noted tht these results by Frps nd Kemmerer (19 nd 197) re bsed upon the spectrogrphic bsorption t millimicrons nd not upon the biologicl efficcy of the vitmin A. The uthors found the white corn mel contined mterils which bsorbed t the sme wvelengths s vitmin A, but did not give the quntity of this "pseudo-vitmin A." Results in our lbortories show tht the bsorption due to interfering mterils in the unsponifible frction from white corn indictes "pseudo-vitmin A" content of bout 1 times the mount of vitmin A dded on the verge by Frps nd Kemmerer to their mels vi fish liver oils or concentrtes. The possibilities of error nd misinterprettion under such conditions re gret nd this my ccount for some of the differences in the findings of Frps nd Kemmerer s ginst the findings of Record et l. (197) nd our findings s reported in this pper. The results reported by Holmes, Pigott, nd Menrd (19) "showed the vitmin A nd D vlue of the oil (cod liver oil) stored in the msh (1 months) to be fully equl to tht of the oil freshly dded to the msh." It is doubtful if these results Downloded from t Penn Stte University (Pterno Lib) on My, 1

2 F. D. BAIRD, A. T. RINGEOSE, AND M. J. MACMILLAN hve ny significnce s to the stbility of the vitmin A in the oil stored in the feed, becuse: (1) the bsl rtion used contined considerbly more pro-vitmin A s crotene from yellow corn nd lflf thn is necessry to dequtely meet chicks' requirements for this fctor, nd () the vrition in growth of the vrious groups of chicks fed the different quntities of cod liver oil ws presumbly due to vritions in the vitmin D intke which ws insufficient in some cses nd dequte in others. EXPERIMENTAL PROCEDURE The N.H. Red chicks used in these experiments were purchsed dy-old from the sme commercil htchery s the chicks for our experiments on the stbility of vitmin D in mixed feed ingredients (Bird, Ringrose, nd McMilln, 199). To the best of our knowledge ll the chicks were htched from eggs produced by one breeding flock consistently fed the sme rtion. The method ws essentilly prophylctic test to eight weeks of ge. The chicks were weighed individully in grms weekly. A record of mortlity nd cuse of deth, if possible, ws mde. BASAL RATION Ground whole white corn Flour whet middlings Whet brn Percent Met scrps ( percent protein). Dried skimmilk Irrdited yest 1. O.S Dried brewers' yest. Pulverized limestone. Slt o.s A source of vitmin D without vitmin A ws supplied in the bsl rtion by O.S percent of irrdited yest which contined,, rt units of vitmin D per grm. Allowing rtio of effectiveness of 1, it ws ssumed tht totl of 1 effective units of vitmin D per 1 grms of rtion ws thus furnished the chicks in the bsl rtion. Additionl vitmin D ws fforded by the fortified cod liver oil used s source of vitmin A which lso contined units, U.S.P. or A.O.A.C., of vitmin D per grm. Therefore ll the chicks received more thn their requirements of vitmin D. Sufficient bsl rtion ws mde up t one time for ll groups in ll experiments. The quntities of fortified cod liver oil were mixed into the bsl rtion t weekly intervls during the running of the first experiment (first weeks), sufficient rtion being prepred in ll cses to be fed in ll subsequent experiments ccording to the desired length of storge. This ment tht the gretest lpse in time over nd bove the stted storge period of the rtions ws one week. In this study this procedure ws thought dvisble. Fortified cod liver oil which contined, units (U.S.P.) of vitmin A per grm ws used s the source of vitmin A. Positive control groups of chicks were run concurrently with ech storge experiment. The fortified cod liver oil ws mixed into the bsl rtion in respective proportionte quntities fresh ech week during the running of ech experiment. Such positive controls were thought necessry s check on the rection or requirements of the different htches of chicks. Twenty chicks were plced in ech group. The groups of chicks in ech experiment nd the unitges of vitmin A incorported per 1 grms of bsl rtion were s follows: Group 1 None negtive control 1 units from fortified cod liver oil ISO units from fortified cod liver oil units from fortified cod liver oil units from fortified cod liver oil 1 units from fortified cod liver oil 7 ISO units from fortified cod liver oil units from fortified cod liver oil 9 units from fortified cod liver oil 1 ISO units from fortified cod liver oil (Positive Control) units from fortified cod liver oil (Positive Control) Downloded from t Penn Stte University (Pterno Lib) on My, 1

3 THE STABILITY OF VITAMINS A AND D IN MIXED FEED INGREDIENTS The stored rtions were kept in burlp scks t indoor summer tempertures in n ordinry feed room t Hrrison, New Jersey, My 1-July 1 to November -December 1, 197. Unitge levels of 1, 1,, nd units of vitmin A per 1 grms of rtion were selected becuse these were believed to cover rnge below nd bove minimum the 1 nd ISO unit groups,,,, nd 7 were eliminted becuse in the two preceding experiments these groups evidenced severe vitmin A deficiency symptoms, low rte of growth, nd high mortlity. Therefore it ws thought unnecessry to test these rtions for ny longer storge period. The sme rtions stored for vrious periods of time were fed in ech ex- Storge Period of Rtions (weeks) Groups Vit. A per 1 gms. rtion 1 units 1 units Averge nd 1 units 7 ISO units Averge nd 7 units units Averge nd units 9 units Averge nd units units TABLE 1. Averge weights of chicks t eight weeks of ge {gms.) (Weighted for sex influence) Positive control groups quntities necessry for optimum results with N.H. Red chicks. Duplicte groups of chicks were fed the sme unitge level of vitmin A. A totl of seven experiments ws conducted covering respectively,,, 1, 1, 1, nd weeks of storge of the rtions. Ech experiment consisted of groups of chicks with the exception of experiments nd. contined nine groups no positive control group being necessry on this experiment where the fortified cod liver oil ws mixed into the bsl rtion fresh ech week. contined only seven groups insmuch s periment with the exception of the positive control groups 1 nd. During the running of ech experiment these positiv.e control groups were fed the sme bsl rtion to which ws dded the respective quntities of the fortified cod liver oil. u i S Rtions stored for weeks 1 1 " 1 " " All the chicks were rered in groups in bttery brooders. Ech group received only its respective rtion nd wter d libitum Downloded from t Penn Stte University (Pterno Lib) on My, 1

4 F. D. BAIRD, A. T. RINGEOSE, AND M. J. MACMILLAN TABLE. Totl percentge mortlity to eight weeks of ge Storge Period of Rtions (weeks) * Groups Vitmin A per 1 gms. rtion units 1 units 1 units 1 units 1 units units units units units 1 units units Positive control groups * Mortlity in experiment, other thn group 1, ws unrelted, occurring during the first two weeks. throughout the eight weeks' experimentl period. On experiment to inclusive post-mortem exmintions were conducted on ech individul, specil cre being tken to observe the presence or bsence of urtes in the kidneys s confirmtory evidence of vitmin A deficiency. For convenience of interprettion urte numbers given in tble were ssigned by multiplying the following rbitrry fctors by the observed percentges of urtes, slight urtes, nd very slight urtes in ech group: urtes 1., slight urtes., nd very slight urtes.1. The sum of these vlues is the urte number. Exmple: Urte number =(u x l)- -(su x.) + (vsu x.1) Where u =% urtes su =% slight urtes vsu =% very slight urtes Observtions were mde during the experimentl period on ny evidences of vitmin A deficiency symptoms: sore eyes (ophthlmi) nd txi DISCUSSION The negtive control group of chicks, which received the bsl rtion only, evidenced severe vitmin A deficiency symptoms t - weeks of ge. However, considerble mortlity occurred in these groups before other symptoms of vitmin A deficiency developed. Atxi (see Tble ) nd sore eyes were common mong these groups, lthough the sore eyes usully developed only in the individuls which lingered beyond the mjority. The txi ws often followed closely by deth. Elvehjem nd Neu (19) reported vitmin A depleted chicks evidenced low rte of growth, high mortlity, nd typicl stggering git (txi), nd Heywng nd Morgn (197) reported txi nd deposit in the burs of Fbricius developed in chicks deficient in vitmin A. The growth rte of our negtive control chicks ws very poor compred with the chicks in ny of the other groups. There ws 1 percent mortlity (Tble ) before the chicks were six weeks of ge. No doubt the bsl rtion contined very little, if ny, vitmin A. Positive control groups 1 nd which received respectively 1 nd units Downloded from t Penn Stte University (Pterno Lib) on My, 1

5 THE STABILITY OF VITAMINS A AND D IN MIXED FEED INGREDIENTS vitmin A per 1 grms rtion ttined good weights (Tble 1) in experiments to inclusive. In experiments,, nd, group 1 showed lower verge weights thn group. The sme ws true for group in. The progressive increse in the number of chicks with observed urtes in the kidneys in experiments,, nd gives further evidence tht there ws some destruction of the vitmin A in the smple of fortified cod liver oil used for the positive control groups. It ws determined by ssy tht the potency of this smple decresed by 1 percent, undoubtedly due to the repeted use of this oil t weekly intervls from smll continer over long period of time (My 1-December 1, 197). The results from groups nd which received 1 units show progressive destruction of the A fctor. Also 1 units vitmin A per 1 grms rtion ws not dequte to meet chicks' requirements. Only prt of the 1 units of vitmin A incorported per 1 grms rtion ws Storge period of rtions (weeks) Groups 1* Vitmin A per 1 gms. rtion units 1 units 1 units 1 units 1 units units units units units 1 units units TABLE --Percentge chicks showing txi (All mortlity previous to two weeks eliminted) 1 destroyed even fter storge period of 1 weeks. Groups nd 7 (1 units) ttined norml growth only in experiment nd ech succeeding experiment showed decline in weight s the vitmin A stored in the rtions ws presumbly undergoing destruction. This result on experiment combined with the results of the positive control groups indictes tht 1 units of vitmin A per 1 grms rtion mixed fresh ech week supplied sufficient vitmin A to meet the requirements of N.H. Red chicks to eight weeks of ge. This checks erlier studies in this lbortory. Our findings lso gree with those of Huge, Crrick, nd Prnge (19), Record, Bethke, nd Wilder (19 nd 197), nd Ringrose nd Norris (19). Others hve reported results showing higher vitmin A requirement for chicks thn our results: Sherwood nd Frps (19 nd 19) reported 1-1 Shermn-Munsell units (17-1 U.S.P. units) vitmin A required per 1 gms. rtion; Wilson, Schroeder, Positive control groups Downloded from t Penn Stte University (Pterno Lib) on My, 1 * Chicks often died so rpidly, mny during the night, tht nottions for txi were impossible to record.

6 F. D. BAIRD, A. T. RINGROSE, AND M. J. MACMILLAN nd Higgins (19) reported chick A requirements to be 1, units per pound or units per 1 grms rtion. Berse nd Miller (197) reported chick requirements for vitmin A to weeks of ge to be 17 Shermn-Munsell units ( U.S.P. units) per 1 grms rtion. These requirements re probbly in line with ours becuse the vitmin A requirements increse with ge during the growing period Storge period of rtions (weeks) Groups Vitmin A per 1 gms. rtion 1 units 1 units 1 units 1 units units units units units 1 units units TABLE. Urtes in the kidneys (Record, Bethke, nd Wilder, 197). Frohring nd Wyeno (19) reported A.D.M.A. units (1 U.S.P. units) nd Biely nd Chlmers (19) reported 7 Interntionl units of (7 U.S.P. units) vitmin A required per chick per dy. We cnnot interpret the results of Frohring nd Wyeno into results to be expected on truly prophylctic tests, nd the results of Frohring nd Wyeno s well s those of Biely nd Chlmers re reported on per dy bsis insted of unitge content of definite quntity of feed. The lck of greement between the reported results on the vitmin A requirements of chicks my, in some cses, be due to one or ll of the following: length of the test period (ge of the chicks), bsl rtions used, source of vitmin A, nd the time intervl of mixing the sources of vitmin A into the rtion. Unpublished work in this lbortory indictes tht 17- units of vitmin A per 1 grms rtion mixed into the diet t the strt of n eight weeks' test period supplied sufficient vitmin A to meet the requirements of N.H. Red chicks. The unit groups nd were nor Positive control groups ml up to nd including experiment rtions stored for eight weeks. When the rtions were stored for more thn eight weeks the growth ttined by the chicks ws stedily downwrd s the storge period dvnced. The symptoms of vitmin A deficiency observed in the chicks becme more pronounced s the length of storge of the rtions incresed. The chicks which received units of vitmin A per 1 grms of rtion from the fortified cod liver oil stored in the rtion up to nd including 1 weeks showed prcticlly norml growth nd no outwrd symptoms of ny vitmin A deficiency, but n increse in the urtes ws observed upon post-mortem exmintion of these groups from experiment (rtions stored Downloded from t Penn Stte University (Pterno Lib) on My, 1

7 THE STABILITY or VITAMINS A AND D IN MIXED FEED INGREDIENTS 7 1 weeks) nd the succeeding experiments. It will be observed tht the growth response of the chicks ws better in experiments nd (Fig. 1). We cnnot ccount for this pprent upswing in the grdully declining verge weights t eight weeks except to sy tht these chicks hd better vitlity presumbly due to groups of chicks decresed nd the observed symptoms of vitmin A deficiency incresed s the storge period of the rtions lengthened. This evidences progressive destruction of the vitmin A. There ws not, however, totl destruction of the vitmin A in ny of the rtions fed, even fter storge period of 1- weeks. *> «7 *> "* = 1 < * Averge *x/^ ^s. 1 N: Ste rge i ^ ^ units of vitmin A ^^^ ^^«w ^V^_go units of vitmin A ^C^**>«^,r>- Averge ^ \ \ ^1 units of viti V-J units of vitnin A Period of Rtions (weeks) I i 1 Fio. 1. Averge weights of the duplicte groups of chicks fed vrious unitge intkes of vitmin A from fortified cod liver oil stored in the rtion up to nd including weeks. lrger storge of the vitmin A fctor from the prents. The mortlity in experiment, other thn group 1, ws unrelted, occurring during the first two weeks. With the bove exception the mortlity ws in generl inversely proportionl to the unitge intke of vitmin A. It incresed in ech group s the storge period of the rtions ws lengthened. Better growth ws ttined by the chicks s the vitmin A unitge intke ws incresed. The verge growth of ll Our findings re in greement with those of Record, Bethke, nd Wilder (197). As previously stted, Frps nd Kemmerer (19 nd 197) reported tht the vitmin A s present in fish liver oils or concentrtes when mixed with feed nd stored t tempertures s low s -9 C. for to weeks ws destroyed to the extent of 79-1 percent, wheres Holmes, Pigott, nd Menrd (19) reported no destruction of the vitmin A nd D vlues of cod liver oil when mixed nd stored in msh for 1 months. The disgreement of our Downloded from t Penn Stte University (Pterno Lib) on My, 1

8 F. D. BAIRD, A. T. RINGROSE, AND M. J. MACMILLAN results with those reported by Frps nd Kemmerer, nd Holmes et l. is probbly due to the experimentl technic used by these workers s we explined in the foreprt of this pper. CONCLUSIONS Vitmin A from fortified cod liver oil, when mixed in the rtion to provide respectively 1, ISO,, nd units of vitmin A per 1 grms rtion, did / not evidence complete destruction even w when the rtions were stored in burlp bgs t summer tempertures for weeks, lthough it underwent progressive destruction s the storge period dvnced. One hundred nd fifty units of vitmin A from fortified cod liver oil when incorported per 1 grms rtion fresk weekly provided sufficient vitmin A to meet chicks' requirements, but units per 1 grms rtion fed fter storge period of eight weeks did not meet chicks' requirements for this fctor. When units of vitmin A from fortified cod liver oil ws incorported per 1 grms rtion nd the rtion stored for 1 weeks t summer tempertures, it provided sufficient vitmin A for prcticlly norml growth nd prevented outwrd symptoms of vitmin A deficiency in chicks to eight weeks of ge, though the mount of urtes in the kidneys incresed t nd fter 1 weeks' storge. REFERENCES Bird, F. D., A. T. Ringrose, nd M. J. McMilln, 199. The stbility of vitmins A nd D in mixed feed ingredients, Prt I Vitmin D. Poul. Sci. 1:-9. Berse, G. E., nd M. Wyne Miller, 197. The vitmin A requirements of White Leghorn pullets during the growing period. Poul. Sci. 1:-. Biely, J., nd Chlmers, W., 19. Vitmin A requirements of growing chicks. I. Preliminry studies with "reference" cod liver oil. Cn. J. Res. 1 :D; C.A. :1; A.F.M.A. Bui. :. Elvehjem, C. A., nd N. F. Neu, 19. Studies in vitmin A vitminosis in the chick. J. Biol. Chem. 97 :7l-. Frps, G. S., nd R. Treichler, 19. Effect of storge on vitmin A in dried foods. Ind. nd Eng. Chem., :., 19. Vitmin A content of foods nd feeds. Texs Agr. Exp. St. Bui. 77. Frps, G. S., nd A. R. Kemmerer, 19. Stbility of crotene nd vitmin A in commercil feeds. Symposium on vitmins, Am. Chem. Soc. An. Meeting., 197. Losses of vitmin A nd crotene from feeds during storge. Texs Agr. Exp. St. Bui. 7. Frohring, W. O., nd J. Wyeno, 19. Crotene nd vitmin A requirements for White Leghorn chicks. J. Nutr. :-77. Huge, S. M., C. W. Crrick, nd R. W. Prnge, 19. Ft-soluble A requirements for growing chicks. Poul. Sci. :1S-1. Heywng, B. W., nd R. B. Morgn, 197. Observtions on some symptoms of vitmin A deficiency in chicks. Poul. Sci. 1:-9. Holmes, A. D., M. G. Pigott, nd D. F. Menrd, 19. The permnency of cod liver oil vitmins in stored feeds. Poul. Sci. 1:7-. Kick, C. H., nd R. M. Bethke, 199. Vitmin A in stored yellow corn. Ohio Agr. Exp. St. Bui. 1:. Record, P. R., R. M. Bethke, nd O. H. M. Wilder, 19. Vitmin A requirements of growing chicks. Poul. Sci. 1:97., 197. Observtions on the stbility of crotene nd vitmin A in mixed rtion. Ohio Agr. Exp. St. Bui. 79:91., 197. The vitmin A requirements of chicks with observtions on the comprtive efficiency of crotene nd vitmin A. Poul. Sci. 1:-. Ringrose, R. C, nd L. C. Norris, 19. A study of the vitmin A requirements of the chick during erly life. Poul. Sci. 1:9-9. Sherwood, R. M., nd G. S. Frps, 19. Vitmin A requirements for growing chicks. Poul. Sci. 1:97., 19. The quntities of vitmin A required by growing chicks. Texs Agr. Exp. St. Bui.. Wilson, W. O., C. H. Schroeder, nd W. A. Higgins, 19. Further studies on the vitmin A requirements of chicks. Poul. Sci. 1 :. Downloded from t Penn Stte University (Pterno Lib) on My, 1