THE RELA'EWE BEGPOTENCY 0F FERMENTATEON BETA- CAROTENE. CRYS?ALL§§¢E EETNC‘AROWNE Afifl WTAMEN A FOR POULTRY Thesis for tha Dug!” of Ph. D. MiG-SEW STATE UNIVERSITY Ca! 1. Hegel $6.5 “4:515 0-169 I LIBRARY Michigan State University This is to certify that the thesis entitled The Relative Biopotency of Fermentation Beta- Carotene, Crystalline Beta-Carotene and Vitamin A for Poultry presented by Cal J. Flegal has been accepted towards fulfillment of the requirements for Ph. D. degree in _EQultr_)L_Science ' K / - F/ ' / . \"//'I:'rfiét(£<" {”{I ’ Mafia: professor v ”w "W. 7 Date _Augu§L_1_Q,_19fi5__ THE RELATIVE BIOPOTENCY 0F FERMENTATION BETA-CAROTENE, CRYSTALLINE BETA-CAROTENE AND VITAMIN A FOR POULTRY BY Cal J. Flegal A Thesis Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Poultry Science I965 ABSTRACT THE RELATIVE BIOPOTENCY 0F FERMENTATION BETA-CAROTENE, CRYSTALLINE BETA-CAROTENE AND VITAMIN A FOR POULTRY By Cal J. Flegal Experiments were designed to determine the utilization of a newly-developed beta-carotene product produced by a fermentation process involving mating of opposite types of the heterothallic mold, Blakeslea trlspora. Comparison was made with the ANRC Vitamin A Reference Standard, vitamin A palmitate and commercially available crystalline and synthetic beta-carotene. Day-old commercial White Rock cockerels were depleted of their vitamin A reserves and then placed on the test diets. Observations were made on growth, feed efficiency, vitamin A plasma concentration, vitamin A liver concentration, and survival on a vitamin A deficient ration after having been on the test diets. Experiments were also conducted to determine the vitamin A reserves of turkeys by placing day-old poults on a vitamin A deficient ration and observing vitamin A plasma and liver concentrations at various intervals. In the first experiment, the levels of the fermentation beta- carotene ranged from Slh to l,#33 IU, crystalline beta-carotene from 548 to l, #53 10 and the ANRC Vitamin A Reference Standard from 539 10 to l,298 10 per pound of diet. In all diets, as the vitamin A activity rose, mean body weights became larger, feed efficiency improved, plasma vitamin A concentration increased and liver vitamin A storage and Cal J. Flegal survival time increased. At any given dietary level, the crystal- line beta-carotene and the fermentation beta-carotene produced similar results for all criteria observed. Neither beta-carotene product. how- ever, performed as well as the ANRC Vitamin A Reference Standard. In the second experiment, the levels of the fermentation beta- carotene ranged from 765 ID to 2,070 IU, crystalline beta-carotene from Bio 10 to 2,050 IU, and the ANRC Vitamin A Reference Standard from l,320 10 to 2,090 IU per pound of diet. The lowest level of the fer- mentation beta-carotene and the lowest three levels of the crystalline beta-carotene did not support the rate of growth obtained with the other diets. There was little difference in the feed efficiency between diets regardless of product or level. With each product, as the vitamin A activity increased in the diet. plasma and liver vitamin A concentrations increased. There were few significant differences in survival time. At any given dietary level of vitamin A activity, the crystalline beta- carotene and the fermentation beta-carotene produced similar results with respect to the criteria used. However, at any given comparable dietary level of vitamin A activity. plasma and liver concentrations of vitamin A were higher in those chicks receiving the ANRC Vitamin A Reference Standard than either beta-carotene product. In Experiment III. the levels of fermentation beta-carotene were 8,950 IU and 2l.350 IU per pound of diet and the levels of the vitamin A palmitate were l0,h00 ID and 2l,700 10 per pound of diet. The liver vitamin A concentration was significantly higher from the vitamin A palmitate than from each comparative level of the fermentation beta- carotene. Cal J. Flegal In these experiments, the beta-carotene products produced similar results for all the criteria used in evaluation, but the ANRC Vitamin A Reference Standard and vitamin A palmitate were more effective than were any of the beta-carotene products in promoting blood plasma and/or liver concentration of vitamin A. Turkey poults were found to have large body reserves of vitamin A. The plasma vitamin A concentration dropped from an initial value of about l#0 micrograms per l00 ml of plasma to about l2 micrograms per l00 ml of plasma; liver vitamin A concentration dropped from an initial value of about #0 micrograms per gram to less than one microgram per gram in ho days on vitamin A deficient rations. ACKNOWLEDGEMENTS The author wishes to express his appreciation to Dr. Philip J. Schaible, Professor of Poultry Science. for his guidance and interest in this study and his direction and many helpful suggestions in the preparation of this manuscript. The author also appreciates the guidance and help provided by Professors H. C. Zindel and T. H. Coleman of the Department of Poultry Science, Dr. D. E. Ullrey of the Department of Animal Husbandry, Dr. E. P. Reineke of the Department of Physiology, and Dr. H. H. Hall, United States Department of Agriculture Northern Utilization Research and Devel0pment Division. Thanks also go to Michigan State University, the Department of Poultry Science and the people of Michigan for providing laboratory and farm facilities used in this study. Finally, the author is indebted to his wife, Mary, for her sacrifice, patience, and encouragement during this strenuous period of study and research. ll ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION REVIEW OF LITERATURE MATERIALS AND METHODS Experiment I -- Experiment II -- Experiment III-- Experiment IV -- RESULTS DISCUSSION CONCLUSIONS LITERATURE CITED TABLE OF CONTENTS Comparison of three dietary levels of fermentation beta-carotene, crystalline beta-carotene, synthetic beta-carotene and the ANRC vitamin A Reference Standard. Comparison of four dietary levels of fermentation beta-carotene, crystalline beta-carotene and the ANRC Vitamin A Reference Standard. Comparison of relatively high dietary levels of fermentation beta-carotene and vitamin A palmitate. The effects of a vitamin A deficient diet on turkey poult plasma and liver vitamin A concentration. iii 14 ill 19 20 2l 25 63 70 72 3-8 3-9 3-l0 3-” 4.1 «.2 11-3 M 4-5 h-6 li-7 h—B “-9 LIST OF TABLES Composition of basal depletion diet used in Experiment I. Composition of basal depletion diet used in Experiment IV Two-week chick weightS. . . . . . . . . . . . . . . . . . Four-week chick weights and feed efficiency . . . . . . . Analysis of variance of two-week chick weights. . . . . . Analysis of variance of four-week chick weights . . . . . Chick plasma vitamin A concentrations . . . . . . . . . . Analysis of variance of two-week chick plasma vitamin A concentrationS. . . . . . . . . . . . . . . . . . . . . . Analysis of variance of four-week chick plasma vitamin A concentrationS. . . . . . . . . . . . . . . . . . . . . . Chick liver vitamin A concentrations. . . . . . . . . . . Analysis of variance of chick liver vitamin A concentrations Survival of chicks placed on the basal ration . . . . . . Analysis of variance of survival. . . . . . . . . . . . . Two-week chick weightS. . . . . . . . . . . . . . . . . . Analysis of variance of two-week chick weights. . . . . . Four-week chick weights . . . . . . . . . . . . . . . . . Analysis of variance of four-week chick weights . . . . . Feed efficiency obtained during the four-week experimental perIOd'Exper'mentIIeeeeeeeeeeeeeeoeee Chick plasma vitamin A concentrations after four weeks on the tOSt diets. O O O. O O O O O O O O O O O O O O O O O O 0 Analysis of variance of chick plasma vitamin A concentrations Chick liver vitamin A concentrations after fOur weeks on the tOSt d‘ets. O O O O O O O O O O O O O O O O O O O O O O 0 Analysis of variance of chick liver vitamin A concentration iv 3h 35 37 38 39 #2 #3 “5 52 53 Table Page h-lO Survival of chicks placed on the basal ration . . . . . . . 56 h—ll Analysis of variance of survival of chicks placed on the basal ration. . . . . . . . . . . . . . . . . . . . . . . . 57 S-l Chick liver vitamin A concentration . . . . . . . . . . . . 59 5-2 Analysis of variance of chick liver vitamin A concentration 60 6-l Poult liver vitamin A concentration . . . . . . . . . . . . 62 LI ST OF FIGURES £12912 I -- Plasma vitamin A concentration (chicks) II -- Mean plasma vitamin A concentration (chicks) III-- Vitamin A liver concentration (chicks) IV -- Plasma vitamin A concentration (poults) vi INTRODUCTION Vitamin A is one of the oldest known vitamins. However, until as recently as l5 years ago, fish liver oils were the only major source of vitamin A. These products differed widely in their potency, stability and purity as well as their supply and price. Therefore, significant dependence was placed on plant carotenes to supply a major portion of poultry and livestock vitamin A requirements. As a result of extensive scientific investigations, today both synthetic vitamin A and synthetic beta-carotene are being commercially produced for feed supplementation. Related carotenoids also are now being used to impart a desirable yellowborange color to such consumable products as eggs, poultry meat, and dairy products. In l957, research workers at the United States Department of Agriculture Northern Utilization Research and Development Division developed a new'method for producing beta-carotene. This new'beta- carotene product is produced by a fermentation process involving mating of opposite types of the heterothailic mold, Blakeslea trlsEora. This beta-carotene product may have an economic advantage over synthetically produced sources of vitamin A. Therefore, if this new product could be utilized successfully by poultry and other livestock, the farmer-producer should realize a lower cost of production. The purposes of the present experiments were: (a) to determine the efficiency of utilization of this newly-developed, beta-carotene product by growing chicks, (b) to investigate some of the relationships of vitamin A and beta-carotene in growing chicks, and (c) to investigate body reserves of vitamin A in the turkey poult. REVIEW OF LITERATURE Vitamin A was first postulated by McCollum and Davis in l9l5. Later, Steenbock and Boutwell (l920) showed that naturally-occurring carotenoids had vitamin A activity. However. it was not until l93l that Karrer, Morph and Schopp. and Karrer and Morph determined the structure of these two products. In l937, Kuhn and Morris produced vitamin A synthetically. Finally, in l950, Karrer and Eugster synthesized beta-carotene. Probably no other nutrient has been so extensively studied as a source of vitamin A for poultry and other domestic animals as has carotene. These studies have produced widely varying results. The relative performance of beta-carotene in Species other than poultry has also been considered. Guilbert gtflgl. (l9h0) reported a carotene to vitamin A ratio. based on weight. of 6:l at levels of intake to provide for freedom from night blindness but little or no storage, and a ratio of l0:l to provide significant liver storage for horses, cattle, sheep and swine. Similar results were obtained by Braude gt 21. (l9hl) for fattening pigs where 300 IU/lb of feed from beta-carotene provided for less liver storage than l00 IU/lb of feed as preformed vitamin A. Gray 25121. (l9h0) compared the U.S.P. reference oil. vitamin A alcohol, the separated esters, and beta-carotene using as a basis of comparison liver storage of vitamin A in rats after feeding a known amount of vitamin A. Greatest recovery was from the U.S.P. reference oil, then separated esters, then alcohol; beta-carotene ran a poor last. Much of the beta-carotene was plainly visible in the feces. Small amounts of beta-carotene were absorbed efficiently but larger amounts were absorbed less efficiently. With fattening lambs, Hoefer and Gallup (l9h7) reported similar results based on blood levels and liver storage of vitamin A after feeding carotene concentrates, alfalfa meal, or fish liver oil. Based on the classic four-week rat curative growth assay. Manuslchlgtflgl. (l96l) found that pure beta-carotene in the form of stabilized beadlets was fully active and that 0.6 mcg. of all-trans beta-carotene equals one IU of vitamin A. However. when carotene levels higher than those necessary for growth were fed to rats for seven weeks in order to induce liver and kidney storage of vitamin A, only one-third to one-fourth as much of the vitamin A from the beta- carotene was stored as when the equivalent levels of vitamin A were fed. In further studies, Marusich and Bauernfeind (l963) found that rats fed h,540 IU beta-carotene per pound of feed had about one-third as much liver storage of vitamin A as did rats fed dry vitamin A at the same level. This indicated an effective carotene to vitamin A ratio of 6:l on a weight basis. At 9,080 IU per pound of feed, liver vitamin A storage from carotene was about one-fifth as high as with the same level of vitamin A. This represents a carotene to vitamin A ratio of l0:l based on weight. Gurcay 95 21. (l950) compared crystalline carotene and crystal- line vitamin A acetate, using both growth rate and liver storage of vitamin A. Turkey poults were placed on a vitamin A deficient diet for two weeks and then on the test diets for six weeks. Based on International Units of vitamin A, crystalline vitamin A acetate was about four times as efficient as corresponding levels of crystalline carotene in supporting growth. At intakes higher than those needed for normal growth, approximately h,000 IU per pound from crystalline vitamin A acetate were equivalent to feeding 30,000 IU/lb from carotene based on liver storage of vitamin A. Poults which received crystalline vitamin A acetate had about twice as much vitamin A in the blood plasma as those which received correSponding levels of crystalline carotene. Chavey'gt‘gl. (l96k) fed turkey poults diets containing stabilized vitamin A and beta-carotene based on vitamin A equivalents. The beta-carotene found in dehydrated alfalfa leaf meal or in a dehydrated flowering aquatic meal blend was not converted to vitamin A to any appreciable extent at either four or eight weeks of age based on liver storage of vitamin A. Levels of vitamin A found in the liver at eight weeks of age were 36h IU for the poults fed 2,000 IU per pound of stabilized vitamin A. At an equivalent level of beta-carotene from either alfalfa leaf meal or the aquatic meal blend. only traces of liver vitamin A were found. Nestler ££_gl. (l9h8) compared crystalline carotene, vitamin A alcohol, vitamin A ester and vitamin A acetate using liver stores of vitamin A as criteria. Vitamin A products were fed from 0 to 10 weeks of age to quail. The crystalline carotene was utilized only one-third to one-seventh as efficiently as vitamin A alcohol, one- half to one-tenth as efficiently as the vitamin A ester and one- fourth to one-seventeenth as efficiently as the vitamin A acetate. Considerable individual variation in vitamin A liver stores were noted in quail on the same diet and with the same parental background. Castano'g£.gl. (l951) compared crystalline carotene and vitamin A acetate and found that crystalline carotene was about one-third to one-fifth as effective as vitamin A acetate, based on vitamin A liver storage in chicks. Harvey‘gt'gl. (l955) found that preformed vitamin A was always more effective than carotene in any given similar preparation used orally, as measured by liver stores and survival time in chicks. Gledhill and Smith (l955) found substantially higher liver stores of vitamin A (about twice as much), significantly greater gains, better feed efficiency and lower mortality in chicks fed dry vitamin A from one day to ten weeks of age, as compared to chicks fed carotene from alfalfa at levels of l,000 IU per pound of feed. Laughland and Phillips (l955) studied the utilization of vitamin A and carotene by normal and deutectomized chicks. They found that liver stores after 8, is, 22, and 29 days were 5 to l0 times higher when vitamin A acetate was fed at l0,000 IU per pound of feed than when 5.6 mg. beta-carotene (9.933 units) was fed. Ely (l959) reported that dry vitamin A was about 2.6 times as effective in promoting chick liver storage as was the provitamin. Carotenes from alfalfa leaf meal and/or corn meal or from purified beta-carotene concentrate were poorly utilized as a source of vitamin A up to 3i days by chicks. 015°“.2£.21- (l959) compared several dry vitamin A sources and vitamin A feeding oil with carotene from cereal grass and obtained similar results. At levels of l00 to l,200 IU per pound of feed no differences were obtained in growth between products. However, based on liver stores of vitamin A and survival time. the cereal grass was greatly inferior. These authors concluded that in short-term experiments, growth may not be a reliable criterion for evaluation of vitamin A availability. Judging by liver storage of vitamin A, feeding oils and dehydrated cereal grass were the poorest sources of vitamin A. Gelatin-coated dry vitamin A preparations were superior to preparations in which the vitamin was coated with wax or fat. or absorbed on vegetable protein. Erasmus _£‘gl. (1959) reported that at correSponding levels, stabilized vitamin A produced vitamin A liver storage about ten times as high as that produced by beta-carotene in chicks which had been artificially infected with coccidiosis. Erasmus'gt‘gl. (l960) found that a level of 800 units of stabilized vitamin A per pound of feed was as effective in maintaining feed consumption and growth rate in chicks artificially infected with coccidiosis. as was a level of 2,h00 units from dry beta-carotene beadlets. Halloran (I960) reported that 2,000 IU of vitamin A per pound of diet from alfalfa leaf meal was insufficient to maintain vitamin A liver levels in 9-month-old laying pullets or l9-month-old laying hens. However, Zimmerman £3 21. (l96l) found that carotene from alfalfa was used satisfactorily as a sole source of vitamin A in laying hens. In broiler-type chicks fed diets containing 2,500 and 5,000 IU t 21. (l961) found that of vitamin A per pound of diet, Marusich vitamin A liver storage was only one-third to one-fourth as large from the feeding of beta-carotene as from the feeding of equivalent levels of dry vitamin A. Similar results were also reported by Marusich gtngl. (l963) in comparing dry beta-carotene beadlets and dry vitamin A beadlets. At levels of 2,500 and 5,000 IU per pound of feed, based on vitamin A liver storage, beta-carotene affords about one-third to one-fourth the storage as true vitamin A at both levels of feeding, revealing an effective beta-carotene to vitamin A ratio of 6 to 8:l, calculated on a weight basis. In this same experiment, when 2,500 and 5,000 IU per pound of feed were fed as dehydrated alfalfa meal or dry vitamin A, vitamin A provided liver stores two to four times higher than beta-carotene based on percent deposition of vitamin A in the liver. This yields an effective beta-carotene to vitamin A ratio of h to 8:l calculated on a weight basis. Utilizing chicks, Olsen gt_gl. (l96h) found that, with growth, livability, and liver storage as criteria, the minimum requirement is at least 600 IU of vitamin A per pound of diet. However, data from chicks fed crystalline beta-carotene indicated that the minimum require- ment was in excess of 350 micrograms or 600 10 per pound; that is, that carotene is a less efficient source of vitamin A activity. As far as growth rate in the normal chick and in the rat is concerned, most investigators have reported that carotene is as efficient as vitamin A (Wilson‘gt‘gl., l936; Record t l., l937; Reynolds t _1., l9h7). It is, therefore, at higher levels of feeding, where liver storage of vitamin A is measurable, that carotene appears to perform less effectively than vitamin A. The relative performance of beta-carotene and vitamin A with re5pect to blood levels of vitamin A has also been reported. Curcay‘gt‘gl. (l950) reported that turkey poults which were fed crystalline vitamin A acetate had about twice as much vitamin A in the blood plasma as those which received corre5ponding levels of crystalline carotene. In comparing crystalline vitamin A acetate and crystalline carotene using chicks, at comparative levels, vitamin A acetate was more effective in causing higher concentrations of the vitamin in the blood plasma (Castano'gt‘gl., l95l). A comparison of various criteria for measuring the biological activity of different sources of vitamin A activity is also of interest. McCoord E£.El- (l93h) observed that the concentration of vitamin A in the blood of rats was no indication of the amount that may be stored in the liver. Krause (i9h9) found that with rats it was difficult to assess the body's need or its reserve of vitamin A on the basis of blood level 23: 33. Lewis 25.21. (l942) observed that growth of rats l0 increased until it reached a maximum when the diet level of vitamin A was increased from 0 to 25 IU daily; it remained here at a maximum up to l,000 IU daily. The average plasma vitamin A concentration also increased with increasing levels of vitamin A intake, reaching an optimum at 50 IU per day and remained at this level with intake up to l,000 IU per day. There was no liver storage at intakes of IO IU or less per day, slight storage at 25 IU per day and increasingly larger storages at higher intakes. In turkeys, Guilbert g£_gl, (l93h) found a direct correlation between liver storage, level of vitamin A in the diet, growth, mortality, and the survival of pen-mates when birds were later placed on the vitamin A deficient ration. Also, large variations in liver storage were found among individuals on the same feed. Record _£.gl. (1937), in observations of growing chicks, found that 50 to 100 micrograms of carotene or 80 to 160 IU of vitamin A from cod liver oil per l00 grams of diet were required for normal growth. However, several times these "minimum" amounts were needed to obtain liver storage. In observations of growing chicks. Johnson‘gt‘gl. (l9h8) found that the critical storage level of vitamin A in the livers of chicks studied appeared to be two or three IU per gram and a high storage of vitamin A in the liver was not required for satisfactory growth. Johnson‘s; 31. (l947) had previously determined that growing chicks grew well when their livers contained from 3 to 10 IU of vitamin A per gram. ll Castano £5 21. (l95l), in experiments with chicks, observed that the vitamin A in the blood was related to vitamin A intake and that some minimum level of vitamin A must be maintained in the blood before measurable liver storage occurred. Further. a high storage of vitamin A in the liver was not essential for satisfactory growth provided sufficient vitamin A was in the daily diet. Therefore, at low intake most of the vitamin A was conceivably employed in biological functions and at higher levels liver storage took place. In this same trial, between 500 and l,000 IU per pound of diet allowed maximum growth while raising the vitamin A in the diet further increased the vitamin A liver storage. In_contrast, Harms 25 21. (l955) observed no difference in average weights of chicks fed dietary levels of vitamin A ranging from 500 to h,000 IU per pound of feed but liver storage was increased as the dietary level of vitamin A increased, however, a depletion period was not utilized prior to the use of the test diets by Harms gt 21. (l955) while Castano gt‘gl. (l95l) placed the chicks on vitamin A depletion diet for lh days prior to the employment of the test diets. In recent years, vitamin A deficiency has been implicated in several disease conditions in poultry, in addition to its long- standing connection with growth, general well-being, and integrity of the epithelial tissue. This has stimulated considerable research in the area of disease and vitamin A. Heiiborn gtflgl. (l9hh) has expressed the opinion that vitamin A deficiencies render the altered epithelium more vulnerable to both l2 bacterial and parasitic attack. Panda and Krishnamurtz (l959) found that insufficient vitamin A favored infestation of chickens with Ascaridia 32111. The latter damages the intestinal mucosa and causes a state of conditioned vitamin A deficiency. Two reports (Bergdoll, 196A; Patterson and McInnis, l96h) have shown that high levels of vitamin A have been beneficial in treating laying hens showing infestation with Capillaria columbae. Erasmus _£“gl. (l959) and Erasmus and Scott (l960) reported that chicks infected with coccidiosis required more vitamin A and had lowered liver levels of vitamin A. Chicks that recovered from coc- cidiosis regained their appetite and grew faster if their diet contained 8,000 IU of vitamin A per pound compared to 800 IU. Scott g_ 21. (196]) also reported that coccidiosis in chicks reduced liver levels of vitamin A and the results strongly indicated that adequate vitamin A nutrition above the minimum levels needed for growth was of primary importance in prevention of the severe lesions and losses from CRO as well as coccidiosis. Panda gt a}. (l96h) reported that the feeding of three to seven times the NRC requirements of vitamin A during the acute phase to chicks infected with coccidiosis resulted in sig- nificantly larger body weight gains than did the feeding of vitamin A at the NRC recommended level. As measured by growth of chicks, Panda gt _a_l_. (l962) reported that in preliminary studies with infectious bronchitis, high levels of vitamin A were beneficial. Squibb and Veros (l96l) showed that l3 vitamin A therapy of chicks with adequate reserves of vitamin A did not improve weight gain or lessen mortality during the period of Newcastle disease involvement. Abbott._t‘_l. (l960), in an experiment with turkeys, found an increased incidence of mycoplasma lesions in embryos from eggs from vitamin A deficient turkeys over those from turkeys fed adequate levels of vitamin A. In chicks, Boyd and Edwards (l962) observed that vitamin A had no influence on the course of mycoplasma or‘E.‘ggll infections and mycoplasma and E, 2211 infections do not have any direct influence on vitamin A absorption or metabolism in the chick. However, Boyd and Edwards (l962) also reported that mortality was greater in mycoplasma infected vitamin A-deficient chicks than in mycoplasma infected chicks receiving adequate vitamin A. MATERIALS AND METHODS Experiment I: Comparison of three dietary levels of fermentation beta-carotene, crystalline beta-carotene, synthetic beta-carotene and the ANRC Vitamin A Reference Standard. This experiment was designed to determine a suitable depletion period for chicks and to establish practical levels of vitamin A. The sources of vitamin A activity in this experiment were: Fermentation beta-carotene -- This product was produced and supplied by the United States Department of Agriculture Northern Regional Laboratory. Peoria, Illinois, as dry fermentation solids. Analysis of the fermentation product at the U. S. Department of Agriculture Northern Regional Research Laboratory indicated that it contained 85 percent all-trans beta-carotene, six percent neo-beta- carotene B and the remainder consisted of unidentifiable material. Synthetic beta-carotene -- This product was supplied by the Hoffman-LaRoche. Incorporation, Nutley, New Jersey as dry beta- carotene beadlets, type 2.h-S. It was a diSpersion of beta-carotene and vegetable oil in a matrix of gelatin and carbohydrate that con- tained #8 percent all-trans beta-carotene, 2i percent neo-beta-carotene B, 20 percent neo-u-carotene and the remainder consisted of unidenti- fiable material. Crystalline beta-carotene -- This product was supplied by General Biochemicals, Chagrin Falls, Ohio as l00 percent all-trans beta- carotene. Vitamin A -- This product was the Animal Nutrition Research Council Reference Standard (Ames, l965). lh l5 All products were prepared for the experiment by diluting the appropriate premix concentration with soybean oil meal containing two per cent of a 2:3 mixture of BHA and BHT. These premixes were chemically analyzed, packaged under nitrogen in sealed glass con- tainers and stored under refrigeration (-l0o F) in the dark until they were used. Commercially obtained one-day-old Cobb's Strain White Rock cockerels were placed into heated, raised-wire chick batteries and placed on the basal depletion diet (Table l). The brooding temperature was initially 90 to 95° F and was decreased 5 degrees F each week until room temperature (about 70° F) was reached. At regular intervals, a few chicks were removed and blood samples taken to determine plasma vitamin A concentration. After l7 days (Figure I), the vitamin A plasma level was very low. The birds were then placed in equalized weight groups, wing-banded, and three replications of ten birds each were randomly distributed in chick batteries. The chicks were placed on the test diets for four weeks. The intended and assayed vitamin A l ._/ activity per pound of the test diets were as follows: ¥ 1/ Vitamin A activity for the beta-carotene products is expressed as IU per pound and was based on the conversion of 0.6 microgram of beta- carotene to one IU of vitamin A activity. l6 Table l. Composition of basal depletion diet used in Experiment I Ingredient Percent Ground white milo 60.0 Saybean oil meal, “0% protein 3&0 Steamed bone meal 2.0 Limestone. ground l.5 Salt. iodized .5 Vitamin premix 2.01 i The premix contained the following per pound of ration: 200 mg riboflavin 500 mg dl-calcium pantothenate l,250 mg niacin 2,000 mg choline chloride 0.5 mg vitamin 012 h,000 I.C.U. vitamin D 200 mg procaine penicillin l gm MnSOu Sufficient soybean oil to make up the two percent Calculated analysis: Unit Protein 2 20.6 Fat % l.98 Fiber % 3-79 Calcium % l.2 Phosphorus ‘% .66 Methionine % .3l Cystine % .3l Lysine % l.20 Prod. energy Cal/lb 922 l7 I.U. Vitamin A Activity/lb. Treatment of ration Intended Assayedi Basal 0 0 Basal + fermentation beta-carotene #00 5l# u + n u n 700 836 n + n u n l,225 1,433 Basal + crystalline beta-carotene #00 5#8 u + u u u 700 876 II + ll H II l,225 1,453 Basal + ANRC Vitamin A Reference Standard #00 539 n + II ll H II ii 700 819 H + n u u H n 1’225 1,298 First weekff Last 3 weeksfi Basal + synthetic beta-carotene #00 3,655 l,3#2 u + H H " 700 7.055 1.916 " + " " " l,225 ll,500 2,320 TAverage of analytical data obtained from feed samples analyzed at the U.S. Department of Agriculture Northern Utilization Research and Development Division. iiThis premix was inadvertently mixed with approximately ten times the requested amount of vitamin A activity. The change between the first week and the last three weeks was made in an attempt to correct the amount of vitamin A activity in the finished feed. The diets were mixed once weekly. They were prepared by adding appropriate amounts of the beta-carotene or vitamin A premixes and the soybean oil meal-antioxidant diluent to the basal depletion diet. Approximately one-half of the weekly needs was fed and the remainder was put in covered metal containers, placed in the dark and refrigerated (-l0° F). After one-half week, the feed not consumed was removed and replaced by the portion which had been kept refrigerated. Each diet l8 was sampled at the time of mixing, at mid-week, and at the end of each week for determination of vitamin A activity. The samples were packaged under nitrogen in sealed containers and kept refrigerated (-l0° F) until analyzed. The chicks were individually weighed at weekly intervals and feed consumption per lot was recorded. Blood samples obtained by heart puncture were taken from each chickZ’at the end of the second and fourth weeks on the experimental diets for plasma vitamin A determinations. After having been on the test diets four weeks, one-half of the birds on each diet were randomly selected, sacrificed, livers removed, wrapped in Saran Wrap and aluminum foil and refrigerated at about -l0° F until assayed for vitamin A. The remaining birds were placed on the basal depletion ration and survival in days was recorded. The plasma vitamin A determinations were made using antimony trichloride according to the method outlined by Yudkin (l9#l). All lobes of each liver were sectioned approximately at the midline and a portion from this area was removed for analysis. Individual liver vitamin A concentrations were determined according to the method des-i cribed by Gallup and Hoefer (l9#6). A Bausch and Lomb Spectronic-20 Spectrophotometer was used. Individual body weights, vitamin A blood plasma concentrations, vitamin A liver concentrations and survival were subjected to analysis of variance (Snedecor, l956); then Duncan's multiple range test (l955) 2 _’ At the end of two weeks, individual blood samples were taken from only one replicate. Pooled samples were obtained from the other replicates. 4'] 19 was used to determine which means were significantly different at the .DI and .05 levels of probability. Experiment 11: Comparison of four dietary levels of fermentation beta- carotene, crystalline beta-carotene and the ANRC Vitamin A Reference Standard. Based on an analysis of the results obtained in EXperiment I, Experiment II was conducted similar to Experiment I with the following exceptions. At one day of age, the chicks were placed on the basal depletion diet for l5 days. Also, 0.2# mg/lb vitamin K, 20 mg/lb zinc and lé'per cent cottonseed oil, at the eXpense of milo, were added to the basal depletion diet shown in Table l. Blood samples for vitamin A determin- ations were taken only after the birds had been on the test diets four weeks. The intended and assayed vitamin A concentrations of the test diets were as follows: I.U. Vitamin A Activity/lb. 122293.21. Ear-1+ Basal 0 0 Basal + fermentation beta-carotene 800 765 u + H U ” l,l20 1:035 u + H " " l,568 19565 II + II n It 2,195 2,070 Basal + crystalline beta-carotene 800 8l0 H + “ " " l,l20 lgogo n + H " " l,568 1.510 II + ll II n 2,195 2,050 Basal + ANRC Vitamin A Reference Standard 800 l,030 u + n n u u n 1.120 L320 n + n u u H H I’568 l,590 u + u n u II H 2.195 2,090 TrjAverage of analytical data from feed samples analyzed at the U. S. Department of Agriculture Northern Utilization Research and Development Division. 20 Experiment III: Comparison of relatively high dietary levels of fermentation beta-carotene and vitamin A palmitate. The purpose of this experiment was to determine the relative ability of the fermentation beta-carotene and a vitamin A product to influence vitamin A liver concentration at relatively high feed levels fed for a one-week period. The sources of vitamin A activity used in this trial were: Fermentation beta-carotene - previously described. Vitamin A palmitate - This material was purchased from Distillation Products Industries, Rochester, New York in the form of PCB-250 Dry Vitamin A Feed Supplement. Commercially obtained day-old Cobb's strain White Rock cockerels were placed into heated, raised-wire chick batteries and placed on the depletion diet as used in Experiment II. After seven days, the birds were placed in equalized weight groups, wing-banded, and four replica- tions of lo birds each were randomly distributed in chick batteries. The chicks were placed on the test diets for one week. The intended and assayed vitamin A activity per pound of the test diets were as follows: Vitamin A activity/lb. of diet 12522222 mi Basal 0 0 " + fermentation beta-carotene loeooo 8:950 n + u u H 25,000 219350 n + PCB-250 dry vitamin A 10.000 '0.“00 u + n u n n n 25,000 2i,700 fAverage of analytical data from feed samples analyzed at the U. S. Department of Agriculture Northern Utilization Research and Devel0pmental Division. 2i After one week on the test diets, all birds were sacrificed, livers removed, wrapped in Saran Wrap and aluminum foil and refrigerated at about -l0° F until assayed for vitamin A. Assay procedures and analyt- ical interpretations were identical to those used in Experiments 1 and II. Experiment IV: The effects of a vitamin A deficient diet on turkey poults' plasma and liver vitamin A concentration. The purpose of this experiment was to examine the effects of a vitamin A deficient diet on the plasma and liver vitamin A concentration of turkey poults. The turkey poults used in this experiment were obtained from eggs that were hatched from the turkey flock maintained at the Michigan State University Poultry Research Farm and consisted of both Beltsville Small White and Broad Breasted Bronze varieties. The breeding flock had been fed the regular turkey breeder diet used at the farm which contained the following: 22 Ingredients Percent of ration Ground yellow corn 50.0 Ground heavy oats l0.0 Soybean oil meal. ##% protein l0.0 Wheat std. middlings 5.0 Wheat bran 5.0 Alfalfa meal, l7% protein 5.0 Fish meal #.0 Meat and bone scraps, 50% 2.0 Dried yeast 2.0 Dried whey l.5 Ground limestone 3.0 Dicalcium phOSphate l.5 Salt, iodized .5 Manganese sulfate, 70% .025 Dry vitamin A, 5,000 IU/gm .l5 Dry vitamin D3, l.500 ICU/gm .l5 Choline chloride, 25% .10 Vitamin 812, 6 mg/lb .06 Vitamin E, 20,000 IU/lb .0# BHT .0l25 Niacin l0 grams Riboflavin 1.0 gram Calculated analysis: Protein % 16.6 PhOSphorus % ,7 Fat % 3.l Vitamin A IU/lb 6,900 Fiber % 5.l Vitamin D ICU/lb' l,020 Calcium % 2.2 23 This experiment consisted of two trials. Trial A -- A blood sample, collected by heart puncture, was obtained from several of the one-day-old turkey poults, before they had consumed any feed, to determine vitamin A plasma concentration. The remainder of the poults were randomly distributed into heated, raised-wire chick batteries and placed on the basal depletion diet shown in Table 2. At regular intervals, several (five or more) poults were removed from the pens and blood samples obtained to determine plasma vitamin A concentration of the pooled samples. Trial B -- This trial was conducted similar to Trial A. However, at certain intervals, the same birds from which blood samples had been taken were sacrificed, livers removed, wrapped in Saran Wrap and aluminum foil and refrigerated at about -lO° F until assayed for vitamin A concentration. A pooled sample of the liver samples was analyzed as previously described. 2# Table 2. Comosition of basal depletion diet used in Experiment IV Ingredient Percent of r9225; Ground white mi lo 38.8 Soybean oil meal, ##% protein 55.2 Steamed bone meal 2.0 Limestone. ground l.5 Salt, iodized .5 Vitamin premix 2.0T 'l’ The premix contained the following per pound of ration: 200 mg riboflavin 500 mg dl-calcium pantothenate l.250 mg niacin 2,000 mg choline chloride 0.5 mg vitamin B]: #,000 I.C.U. vitamin D3 200 mg procaine penicillin l gm MnSOu Sufficient soybean oil meal to make up the two percent. Calculated analysis: Unit Protein % 28.02 Fat % 1.55 Fiber 1 #.78 Calcium 1 1-25 Phosphorus % .73 Methionine % .#l Cystine % .428 Prod. energy Cal/lb 850.3 RESULTS Experiment I The effect on plasma vitamin A concentration when the day-old chicks were placed on the basal depletion diet can be seen in Figure I. The concentration dropped very rapidly and almost in a straight line during the first seven days from an initial value of l9# micrograms per l00 ml to about #3 micrograms per l00 ml of plasma. The decline in vitamin A concentration was less rapid from 7 to l# days when the plasma vitamin A concentration dropped from about #3 micrograms per l00 ml to about five micrograms per l00 ml. Observations were also made on the effects of the basal depletion ration on the appearance of the chicks prior to placement on the test diets. No visible effects were observed during the first four to six days. At about seven days, the chicks appeared to be extremely nervous. At about the same time, mortality at a rate greater than ”normal” appeared and continued at an excessive rate until the birds were placed . on the test diets. At about l2 to l# days, a portion of the chicks exhibited a general weakness, staggering gait, and ruffled plumage. These conditions became more general the longer the birds were kept on the basal ration culminating in death of those chicks not placed on the test diets. The effect of the test diets on weight gain and feed efficiency are shown in Tables 3-l, 3-2, 3-3 and 3-#. Significant differences (Tables 3-l and 3-2) occurred in mean body weights both after two and four weeks on the test diets. 25 26 200 ISO-— [60 - |4O — |20 - 9503 ICC '- .E 00. _ O 8 Lou nos. 60— 4o— 20- I4 l2 IO in days Age 27 Table 3-l. 'Two-week chick weights I.U. Vit. A/lb. Meant body Treatment of basal ration Assayed weights (gms.) Fermentation beta-carotene 5l# 358.7 DFe .. n .. 335 376.5 Dde n n n l.#33 #3#.2 ABCbc Crystalline beta-carotene 5#8 315.9 Ef " " " 876 377.8 CDde " " " l.#53 335.0 CDde Synthetic beta-carotene --II #56.9 ABabc " " " -" “.2 ABebc " " " -- #8#.l Aa ANRC Vitamin A Reference Standard 539 #l6.3 BCDcd ” " " " ” 8l9 “53.l ABabc .. .. " " " l,298 46!.8 ABabc 1 Any two means having the same letter are not significantly different: means not having the same letter are significantly different. Small letters indicate significance at the .05 level; large letters at the .0l level. if Omitted due to incorrect value assigned to premix. complete explanation. See text for i ) 28 Table 3'2. Four-week chick weights and feed efficiency Feed effic. I.U. Vit. A/lb Mean? (body itisdei:.i: :[ngtgggngflnggyllqgtion Jfigggygd weigfl£g_1g!§)r bodv_wt.1 Fermentation beta-carotene 511. 618.2 DEd 3.#l .. n .. 836 666.9 cm 3J0 .. .. .. 1,!»33 771.7 ABab 2.70 Crystalline beta-carotene 5#8 5l7.3 Fe 3.77 .. .. .. 876 608.7 DEd 3.12 .. .. .. 1,853 682.8 BCDcd 2.80 Synthetic beta-carotene "ll 552-3 09‘ 2°93 N u n -- 776.8 “ab 2.57 i. .. .. -- 822.2 Aa 2.#9 ANRC Vitamin A Reference Standard 539 7#2.7 ABCbc 2.71 n n n H " 8i9 770.8 ABab 2.67 .. n .. .. u l,298 801.0 Aab 2.55 i Any two means having the same letter are not significantly different; means not having the same letter are significantly different. Small letters indicate significance at the .05 level; large letters at the 00' I.V.le if Omitted due to incorrect value assigned to premix. See text for complete explanation. “i . S. \a...) 29 Table 3-3. Analysis of variance of two-week chick weights Source Degrees Sum of of of Mean variance freedom squares square F ratio Total 333 2,686,206 Subclass 35 939.077 26,38l #.50** Treatment ll 798.3## 72.577 12.33** Replication 2 28.2l7 l#.l08 2.#l Total int. 22 ll2,5l6 5,ll'+ .87 Error 298 l.7#7.l30 5.363 ** Significant at the .0l level of probability. 30 Table 3-#. Analysis of variance of four-week chick weights Source Degrees Sum of of of Mean variance freedom squares square F ratio TOEOI 292 69198!“66 Subclass 35 2.394.5#0 68.#l5 #.62** Treatment ll 2.03#,9i# l8#.992 l2.50** Replication 2 36.06# l8,032 l.22 Total int. 22 323.563 l#,707 .99 Error 257 3.803.926 l#,80| ** Significant at the .0] level of probability. s e \ e\l\ . ~-‘. I\ 3] It appeared that both the product and the level of vitamin A activity in the diet had an influence on mean body weight. Mean body weights after both two and four weeks on the test diets were larger as the vitamin A activity of the diet was increased for all the products tested. The crystalline beta-carotene fed at 5#8 IU per pound of ration resulted in mean body weights that were significantly smaller than those resulting from all other diets. The synthetic beta-carotene (which was mixed at levels much higher than the other diets) resulted in the largest mean body weights although these were not significantly larger than most of the other mean body weights. The fermentation beta-carotene and the crystalline beta-carotene resulted in mean body weights that were not significantly different at each correSponding level of dietary vitamin A activity with the exception that has been noted above. However, there was a trend toward larger mean body weights at each of the correSponding dietary levels of vitamin A when comparing the fermentation beta-carotene to the crystal- line beta-carotene. At correSponding dietary levels of vitamin A activity, the ANRC Vitamin A Reference Standard resulted in significantly larger mean body weights (after both two and four weeks on the test diets) than the crystalline beta-carotene. This was also true when comparing the fermentation beta-carotene to the ANRC Vitamin A at the two lowest dietary levels. Feed efficiency was closely associated with rate of gain; the faster rate of gain producing the best feed efficiency. The ANRC Vitamin A Reference Standard resulted in a considerably better feed conversion at the two lowest levels than did either the fermentation beta-carotene or the crystalline beta-carotene. 32 The effects of the test diets on vitamin A plasma concentrations are shown in Tables 3-5, 3-6 and 3-7. The product and the level of vitamin A activity had an influence upon mean plasma vitamin A con- centration. In general, as the feed level of vitamin A activity was raised, the mean plasma vitamin A concentration increased. In most instances the plasma vitamin A concentration was higher after four weeks on the test than after two weeks for all products at each level tested. After having been on the test diets two weeks, only the lowest levels of the fermentation beta-carotene and the crystalline beta- carotene failed to raise the plasma vitamin A concentration above the depleted value. However, after four weeks on the test diets, all diets had increased the vitamin A plasma concentrations to a level above the depleted value. Few significant differences in plasma vitamin A con- centrations were observed at two weeks. The ANRC Vitamin A Reference Standard increased the plasma vitamin A concentration more, although not always significantly, at each comparable level than did either the fermentation beta-carotene or the crystalline beta-carotene but not more than did the synthetic beta-carotene. After four weeks on the test diets, at each comparable level of vitamin A activity in the feed, all of the beta-carotene products resulted in similar plasma vitamin A concentrations, except that the highest level of crystalline beta-carotene resulted in significantly smaller (P 0.0l) plasma vitamin A concentration than the other two beta-carotene products at the highest feed level of vitamin A activity. 33 Table 3-5. Chick plasma vitamin A concentrations Means Treatment of basal ration I.U. Vit. A/lb. 'ggaéégngl blOOthl::Ta Fermentation beta-carotene 5l# 6.7 De l6.0 Efg ” " ” 836 l3.5 DEde 18.9 DEefg " " " l.#33 2l.0 BCDbcd 30.5 Bb Crystalline beta-carotene 5#8 7.9 De l5.# E9 " ” ” 876 l5.6 CDcd l8.2 DEefg " " " l,#53 lO.2 CDde 2#.2 CDcd Synthetic beta-carotene --TTT l3.7 CDde l6.# Eef9 " " ” -- 25.8 BCbc 20.5 CDEdef H H " -- 3l.5 ABab 25.8 BCbc ANRC Vitamin A Reference Standard 539 2l.9 BCDbcd 2l.l CDEde H " " ' " 819 28.l ABCb 30.3 Bb H N " "a l,298 #l.l Aa 50.7 Aa 7 Any two means having the same letter are not significantly different; ft m means not having the same letter are significantly different. Small letters indicate significance at the .05 level; large letters at the .0l level. From one replication only. Omitted due to incorrect value assigned to premix. complete explanation. See text for s4“ .i I e - .A e .3 ‘— x _ . C, ,‘ v v ‘i “I . .7) i). l 1 .3 i i.) -v), ‘. "' hfl _ \ e ‘ N l l i ' x. I c O 5 . \ 3# Table 3-6. Analysis of variance of two-week chick plasma vitamin A concentrations Source Degrees Sum of of of Mean variance freedom squares square. F ratio Total 60 8.698 l#5 Treatment li 5.562 506 9.37** Error #9 3,136 6# ** Significant at the .0l level of probability. 35 Table 3-7. Analysis of variance of four-week chick plasma vitamin A concentrations Source Degrees Sum of of of Mean variance freedom squares square F ratio Total 223 28.375.36 Subclass 35 19,#3#.3# 555.27 ll.68** Treatment 11 17,323.35 1,57#.85 33.11** Replication 2 25.50 12.75 .27 Total int. 22 2,085.#9 9#.80 1.99 Error 188 8,9#1.02 #7.56 ** Significant at the .01 level of probability. l I \I\ isle I A l/ 36 The lowest level of the ANRC Vitamin A Reference Standard resulted in a significantly higher (P4: 0.05) plasma vitamin A concentration than did all other products at a correSponding level. At the other two correSponding levels, the ANRC Vitamin A Reference Standard resulted in a significantly higher (P.:.0.01) plasma vitamin A concentration. At 1,298 IU per pound of feed, the plasma vitamin A concentration was significantly higher than with any other diet. In most instances, as the dietary level of vitamin A activity increased, the liver vitamin A concentration also increased, but few significant differences in liver vitamin A concentration occurred (Tables 3-8 and 3-9). There was very little liver vitamin A storage at any of the feed levels from any of the products used in this ex- periment. However, the highest level of the ANRC Vitamin A Reference Standard resulted in significantly higher liver vitamin A concentration than all other treatments. The effects of the test diets on survival are shown in Tables 3-10 and 3-11. There was a trend toward longer survival as the level of the vitamin A activity of the diet increased from each of the products tested. However, few significant differences were observed. 37 Table 3-8. Chick liver vitamin A concentrations Treatment of basal ration Meanl vit. A liver Fermentation beta-carotene Crystalline beta-carotene Synthetic beta-carotene ANRC Vitamin A Reference Standard I.U. Vit. A/lb. concentration Assayed (meg/gm fresh basis) 51# .38 Dd 836 .57 CDd l,#33 l.l# BCbc 5#8 .50 CDd 876 .50 CDd 1.553 .65 CDcd -*l .#o Dd -- .#7 CDd -- 1.3# 80 539 .#7 CDd 819 .86 BCDbcd 1.298 3.32 Aa i Any two means having the same letter are not significantly different; means not having the same letter are significantly different. Small letters indicate significance at the .05 level; large letters at the .01 level. if complete explanation. Omitted due to incorrect value assigned to premix. See text for I ‘ e - . \ ~I|VV ~- x L, Q i "‘ x .5 1 , 38 Table 3-9. Analysis of variance of chick liver vitamin A concentrations Source Degrees Sum of of of Mean variance freedom squares square F ratio Total 138 136.18 Subclass 35 99.91 2.85 8.1#** Treatment 11 95.10 8.61; 2#.68** Replication 2 1.07 .535 1-53 Total int. 22 3.7# .17 .#9 Error 103 36.27 .35 ** Significant at the .01 level of probability. 39 Table 3-10. Survival of chicks placed on the basal ration I.U. Vit. A/lb. Survival Treatment of basal ration Assayed MeanT days until death Fermentation beta-carotene 514 26-25 8355543 .. .. 1. 335 2h.55 BCcde 1. 1. .. 1,#33 29.33 ABCabcd Crystalline beta-carotene 553 21-63 Cc .. n .. 375 23.40 BCde 11 11 .. 1,453 28.9# ABCabcd synthetic beta-carotene --*T 29.23 ABCabcd 1. 11 n: -- 27.83 ABCabcde .. 1. 11 -- 30.83 ABCabc ANRC Vitamin A Reference Standard 539 2#.79 BCcde .. .1 n n 819 32.50 ABab .. .. .. .. 1,298 3#.33 Aa i Any two means having the same letter means not having the same letter are letters indicate significance at the the .01 level. if complete explanation. Omitted due to incorrect value assigned to premix. are not significantly different; significantly different. Small .05 level; large letters at See text for Table 3-11. Analysis of variance of survival SOurce Degrees Sum of of of Mean variance freedom squares square F ratio Total l#2 10.126 Subclass 35 3.838 109.66 1.87** Treatment 11 1,826 166 2.82** Replication 2 672 336 5.72** Total int. 22 l.3#0 60.90 1.0# Error 107 6.288 58.77 ** Significant at the .01 level of probability. #1 Experiment II: Since the procedures in Experiment I were satisfactory, Experiment II was conducted with the changes previously outlined. The plasma vitamin A concentrations followed (now shown) an almost identical pattern to that shown in Figure I and reached a low of about five micrograms per 100 ml of plasma on the l#th day after the chicks had been placed on the basal ration. The effects of the test diets on weight gain and feed efficiency, are shown in Tables #-1 to #-5. The statistical analysis revealed that there were few significant differences in growth reSponses from any of the products or levels tested after two weeks on the test diets (Table #ol). However, the lowest level of the crystalline beta-carotene did result in a somewhat smaller mean body weight although not significantly (P«<..01) smaller than that produced on most of the other diets. After four weeks on the test diets there were significant differ- ences (Pd: .01) in several of the mean body weights (Table #-3). The lowest level of the fermentation beta-carotene and the three lowest levels of the crystalline beta-carotene did not support the rate of weight gain obtained by the diet which supported the greatest weight gain (Basal + ANRC Vitamin A Reference Standard at 1,320 IU vitamin A per 1b.). All of the other diets resulted in mean body weights which were not significantly different (P-¢=.01). Feed efficiency (Table #-5) was quite similar for chicks on all the diets except the lowest level of the crystalline beta-carotene. #2 Table #-1. Two-week chick weights IeUe Vite A/lbe "an? body “so Treatment of basal ration Assayed (gms) Fermentation beta-carotene 765 #27.3 ABabc n u .. 1,035 ##0.3 ABabc .. 11 .. 1,555 1.35.1. ABabc .. .. '1 2,070 ##6.1 ABabc Crystalline beta-carotene 810 #06-3 BC .. .. .. 1,090 1129.6 ABabc .. .. .. 1,510 #22.8 ABab .. .. .. 2,050 ##9.3 ABabc ANRC Vitamin A Reference Standard 1,030 #57.6 Aab .. .. .. .. .. ' 1.320 #58.8 Aab u u u u H 1.590 #6#.3 Aa .. 11 .. .. .. 2,090 1.53.1. ABab T Any two means having the same letter are not significantly different: means not having the same letter are significantly different. Small letters indicate significance at the .05 level; large letters at the eOI IWCIe a I 0 A e 0 lo ‘, L.- ‘ D... a A e 'c \ . \_ . ‘ t 9 ,‘ \ \ . I\ I K \— , Jiii .- .V'Lll CliL;1i(l\i.J :lJ.\\-A Jk--l L). #3 Table #-2. Analysis of variance of two-week chick weights Source Degrees Sum of of of Mean variance freedom squares square F ratio Total 351 l.50#.039 #.285 Subclass 35 190.782 5.#51 1.31 Treatments 11 95.#8l 8.680 2.09* Replication 2 11.275 5.638 1.36 T x R (Int.) 22 811,026 3,819 .92 Error 316 1.313.257 #,156 * Significant at the .05 level of probability. \I ’i l c 'i‘1_:' Table #-3. Four-week chick weights I.U. Vit. A/lb. Meanl body wms. Treatment of basal ration Assayed (gms) Fermentation beta-carotene 765 753-5 CDCd .. .. -. 1,035 788.5 ABCabc .. .. 1' 1,565 807.9 ABCab .. .. .. 2.070 807.6 ABCab Crystalline beta-carotene 810 707.2 Dd .. .1 11 1,090 7#7.u CDcd u n n 1,510 761.6 BCDbcd n u n 2,050 833.2 ABa ANRC Vitamin A Reference Standard 1.030 818-5 ABCab n n n H " 1,320 8#6.0 Aa .. .. .. .. .. 1,590 8111.9 ABCab n n n n " 2,090 835.2 ABa T Any two means having the same letter are not significantly different; means not having the same letter are significantly different. Small letters indicate significance at the .05 level; large letters at the .01 level. Table #-#. Analysis of variance of four-week chick weights #5 Source Degrees Sum of of of Mean variance freedom squares square F ratio Total 3#8 #.135.#32 Subclass 35 859.#7# 2#.556 2.35** Treatments 2 #03.995 201 , 996 19. 36M Level 3 20#.373 68,l2# 6.53** Replication 2 39.799 19.900 1.91 Total int. 28 211.307 75,#67 7.23** Error 31# 3.275.953 10.#33 ** Significant at the .01 level of probability. /\ l‘ r Table #-5. Feed efficiency obtained during the four-week experimental period -- Experiment II I.U. Vit. A/lb. Pounds of feed per pound Treatment of basal ration Assayed of gain in body wt. Fermentation beta-carotene 765 2.58 " ” ” 1.035 2.52 ” " ” 1.565 2.#8 ” ” ” 2.070 2.#9 Crystalline beta-carotene 810 2.70 " " ” 1.090 2.59 " ” ” 1.510 2.52 ” " ” 2.050 2.#6 ANRC Vitamin A Reference Standard 1,030 2.#7 ” " " " 1.320 2.#8 " " " " 1.590 2.52 11 ll 11 11 2,090 2.#9 #7 This diet resulted in a somewhat poorer feed efficiency which was closely associated with the slower rate of weight gain obtained. The effects of the test diets on mean plasma vitamin A concen- tration of chicks that had been on the test diets four weeks are shown in Tables #-6 and #-7 and Figure II. Large variations in blood plasma vitamin A concentration were observed in chicks consuming the same diet. The mean blood plasma vitamin A concentration increased when the feed level of vitamin A activity increased regardless of the product included in the feed. All diets resulted in raising the mean plasma vitamin A concentration well above the depleted concentration value. The assayed value of vitamin A activity for the two beta-carotene products was quite similar at each level tested. At each of the comparable levels tested, the fermentation beta-carotene resulted in a significantly (p.¢..01) higher mean vitamin A plasma concentration than did the crystalline beta-carotene. As the feed level of the vitamin A activity increased, the efficiency of the fermentation beta-carotene was reduced in its ability to raise the blood concentration of vitamin A. This was not true of the crystalline beta-carotene which resulted in a nearly straight line increase in its ability to increase the blood plasma vitamin A concentration in relation to the feed level of vitamin A activity. It was also observed that at all comparable levels, the ANRC Vitamin A Reference Standard resulted in higher, but not always significantly, vitamin A plasma concentrations than did either of the Table #-6. Chick plasma vitamin A concentrations after four weeks on the test diets Mean? plasma I.U. Vit. Allb concentration Treatment of basal ration Assayed (mcg Vit. A/lOO ml) Fermentation beta-carotene 765 28.6 Ggh n n n 1,035 38.1 EFef n u n 1,565 51.2 CDc u u n 2,070 53.9 Cc Crystalline beta-carotene 810 20.9 Hi n n n 1,090 26.# GHh H N H 1.510 33.1 FGfg n u n 2,050 ##.8 DEd ANRC Vitamin A Reference Standard 1,030 #1.3 Ede n n n n H 1,320 52.7 Cc H n H H H 1.590 64.5 Bb u n u H “ 2,090 76.1 Aa 1 Any two means having the same letter are not significantly different; means not having the same letter are significantly different. Small letters indicate significance at the .05 level; large letters at the .01 level. #9 Table #-7. Analysis of variance of chick plasma vitamin A concentrations Source Degrees Sum of of of Mean variance freedom squares square F ratio Total 328 113,950 Subclass 35 83.091» 2.371. 22.61» Treatment 2 #l.#06 20.703 l97.17** Level 3 33.#68 12.823 122.12** Replication 2 197 98.5 .911 Total int. 28 3.023 108 1.02 Error 293 30.355 105 ** Significant at the .01 level of probability. f." . v s- a It /\ 100 m1 plasma Mcg per SO . r1 .. t-“ pinata \ Lan.n concenilat on lenzcks? 80 1+ 70- 60— 50r- 40r- 301- 20— A=ANRC Vitamin A Reference Standard B = Fermentation beta-carotene C = Crystalline beta-carotene l l l l l l 750 1000 1250 1500 1750 2000 IU vitamin A per pound feed 5| beta-carotene products. It was also noted that when the ANRC Vitamin A Reference Standard was increased from l,590 IU to 2,090 IU per pound of feed, the vitamin A blood plasma concentration did not increase correSpondingly. The effects of the test diets on liver vitamin A concentration are shown in Tables h-8 and “-9 and Fig. III. There was a large variation in liver vitamin A concentration in chicks consuming the same diet. Vitamin A liver concentration occurred regardless of product at the lowest level of each product tested. The mean vitamin A liver concentration increased as the level of vitamin A activity in the feed increased stepwise from 800 to 2,l95 IU/lb for all products tested. Large differences resulted between liver concentrations of vitamin A but the differences were not always statistically significant. At the lowest level of vitamin A activity in the feed, there was no significant difference in vitamin A liver concentration. As is evident from Table 4-9, at all of the other levels, the vitamin A liver concentration obtained was significantly higher (.01 level of probability) from the ANRC Vitamin A Reference Standard than from the other products. The next highest level (l.590 IU/lb of diet) produced significantly higher mean vitamin A liver concentration than all diets, except the highest feed level (2,090 IU) of the ANRC Vitamin A Reference Standard, which resulted in a mean liver vitamin A concentration significantly higher than all other diets. At all of the comparable levels of vitamin A activity of the feed, the liver vitamin A concentration obtained was similar for the fermenta- tion beta-carotene and the crystalline beta-carotene. However, at all 52 Table h-8. Chick liver vitamin A concentrations after four weeks on the test diets Mean? Vit.A liver concentration Total Treatment of basal ration I'QRe::;;eA/lb' §::g:g:ifii8) ”T?V::r Fermentation beta-carotene 765 l.22 Efg 2"50 n u n 1,035 1.07 DEfg 31.99 H n n 1,565 3.38 CDde 69.30 n u n 2,070 #.97 Co 9h.50 Crystalline beta-carotene 3'0 .79 £9 l“°56 n n n 1,090 ‘ 1.08 Efg 22.30 H u n 1,510 1.84 DEfg 36.67 H n n 2,050 2.49 DEfg 50.65 Aunt Vitamin A Reference Standard 1.030 2-28 DEefg 50-“0 u n n u n 1,320 n.66 Ccd 101.00 M n u u n 1,590 9.07 80 196.70 n u u n H 2.090 21.32 Aa 471.80 1 Any two means having the same letter are not significantly different; means not having the same letter are significantly different. Small letters indicate significance at the .05 level; large letters at the .0l level. K V 1 ‘. _ 1 1 \ I e A .0 ’ - e 1‘ J e t i a O ‘ ‘ e—LA . I .. x ‘e 1' g I >1, .. e l 'x; e ‘ w... _ i 1 l 1 \C' .4 b s-» 1 \ ‘» ,1 . 1 J ( l a Q.- - ~—‘ f. ' ( l e -' c . l1 l. l k— I .iL L ‘. 1 1 a; - l ‘ 3 JA 1 l ' 1 2 | | i L J L i 5 t ' I l "l. Table hPS. Analysis of variance of chick liver vitamin A concentration Source Degrees Sum of of of Mean variance freedom squares square F ratio Total I75 6,h56.8 Subclass 35 5.963-2 170 “3.57** Treatment 2 2,332.0 1,166 333.lh** Level 3 1,770.8 590 168.57** Replication 2 30.9 15.5 “.43* Total int. 28 1,829.5 65.# l8.68** Error 100 #93.6 3.5 * significant at the .05 level of probability. ** Significant at the .01 level of probability. gm, fresh basis Mcg per 15r- A = ANRC Vitamin A Reference 8 = Fermentation beta-carotene C = Crystalline beta-carotene Standard 1000 1250 1500 IU vitaminA per pound 1750 feed 2000 55 given levels, the fermentation beta-carotene resulted in a slightly higher, but not always significant, mean liver vitamin A concentration than did the crystalline beta-carotene. The lowest level (1,030 IU) of the ANRC Vitamin A resulted in a higher mean liver vitamin A con- centration than did all levels of the crystalline beta-carotene tested and higher than all but the highest level (2,070) of the fermentation beta-carotene. Using liver vitamin A concentration after four weeks on the test diets as the criteria, the relative biopotency of the fermentation beta-carotene and crystalline beta-carotene was different for each of the levels tested when compared with the ANRC Vitamin A Reference Standard. Fermentation beta-carotene (IU) 1,035 1,510 2,070 Total liver vitamin A (% of ANRC) 63.5 35.2 20.0 Liver vitamin A concentration (% of ANRC) 6h.5 35.7 23.3 Crystalline beta-carotene (IU) 1,090 1,590 2,050 Total liver vitamin A (% of ANRC) hh.2 18.6 10.7 Liver vitamin A concentration (% of ANRC) h7.h 19.0 11.7 It is obvious that as the vitamin A activity of the feed increased, the relative efficiency of conversion to liver vitamin A was decreased. Tables h-to and h-ll show the effects of placing the birds on the basal depletion diet after having been on the test diets four weeks. There was a trend toward longer survival as the vitamin A activity of the diet increased from each of the products tested. However, with the exception of the lowest level of the crystalline beta-carotene (810 IU/lb) 56 Table h-lO. Survival of chicks placed on the basal ration 1' I.U. Vit. A/lb. Mean days of Treatment of basal ration Assayed survival Fermentation beta-carotene 765 3h.7 BCc u n n 1,035 39.1 ABbc u H H 1,565 “5.7 ABabc u u n 2,070 ##.0 ABabc Crystalline beta-carotene 810 25-0 Cd n u n 1,090 39.2 ABbc .. .. .. 1,510 34.8 BCc u n n 2,050 #2.1 ABabc ANRC Vitamin A Reference Standard 1,030 “0.3 ABabc 11 n u n H 1,320 l$8.1 ABab 11 11 n .. n 1,590 51.11 Aa .. 11 .. .. .. 2,090 118.0 ABabc i Any two means having the same letter .01 level. are not significantly different; means not having the same letter are significantly different. Small letters indicate significance at the .05 level; large letters at the ‘x , e 5Q. (I ( _1,1 (0 4.; t C «(1 57 Table h-ll. Analysis of variance of survival of chicks placed on the basal ration Source Degrees Sum of of of Hean variance freedom squares square F ratio Total 153 39.977 Subclass 35 13.136 375 2.27** Treatment 2 3.832 1,916 11.61** Level 3 3.393 1.131 5.35** Replication 2 5 2.5 .015 Total int. 28 5.906 211 1.28 Error 129 21,3h1 165 ** Significant at the .01 leveliof probability. 58 which resulted in significantly shorter survival than all other diets (.05 level of probability), few significant differences in survival time were observed. Experiment III: In Tables 5-1 and 5-2 are shown the effects of the test diets on mean liver vitamin A concentration. Significant differences were observed between all diets. Using liver concentration of vitamin A as the criteria for comparison, the relative biopotency of the fermentation beta-carotene was 30.1 per cent (8,950 IU) and 21.7 per cent (21,350 IU) when compared to all the all-trans vitamin A palmitate. Using total liver vitamin A as the criteria, the relative biOpotency of the fermentation beta-carotene was #8.6 per cent (8,950 IU) and h1.0 per cent (21,350 IU) when compared to the all-trans vitamin A palmitate. Experiment IV: The effects of the basal depletion diet on poult vitamin A plasma concentration and liver vitamin A concentration are shown in Fig. IV and Table 6-1. The decrease in plasma vitamin A concentration from approximately 140 mcg per 100 ml plasma was very steep and nearly in a straight line for about 30 days at which time the plasma vitamin A concentration did not drOp any lower than about 12 mcg per 100 ml of plasma. At about ho to #2 days the poults began to show typical signs of vitamin A avitaminosis as described by Moore (1957). No death loss (except those killed by securing blood samples) occurred up to 42 days. Liver vitamin A concentration dropped from a mean of about ho mcg/gm at day one to less than one mcg/gm at #2 days. 59 Table 5-1. Chick liver vitamin A concentration Mean? Vit. A liver conc. Total 1.0. Vit. A/lb. (meg/gm. mcg per Treatment of basal ration Assayed fresh basis) liver Fermentation beta-carotene 8,950 18.09 0d 113.“ ” ” ” 21,350 37.60 Cc 233.2 PGB'ZSO dry vitamin A 10,000 61.00 Bb 390.0 H H H II II 2|.7oo 173.20 M 952.0 Any two means having the same letter are not significantly different; means not having the same letter are significantly different. Small letters indicate significance at the .05 level; large letters at the .01 level. Table 5'2. Analysis of variance of 60 chick liver vitamin A concentration Source Degrees Sum of of of Mean variance freedom squares square F ratio Total 159 712.573.7 Subclass 15 58D,085.6 36,672.h #2.03** Treatments 1 317,980.2 217,980.2 3h5.63** Level 1 170,380.8 170,380.8 185.l9** Replication 3 1,156.3 385.5 .52 Total int. 10 90,568.3 9,056.8 9.8h** Error 10A 132,088.l 920.0 ** Significant at the .01 level of probability. '. 1 I | . . 41 1 1 11‘ i V 1‘ y. .K I I l ‘ ' -‘ I - 1 t 1 -. . “H v a O . e , ‘ A ‘I4‘Va ’ ~ ‘ n4. . . . ‘ ‘0 _. 4 . 1 1 g I 1 v - 1 -. a l I\l\ . , . 1 . T 1 l D ”‘1 . D ‘ O \ I l i I c \a /\ lb . . . ‘ . ' | I ' A . k ' ‘ ' 1\. I 1 . ' ’ - 1 D . 1 . (x l\ 100 m1 plasma Mcg per 160 140 120 100 80 60 40 20 Plasma vitamin A L Trial B CUZiCt‘llL {(10011 Trial 1 l 1 1 IO 20 30 40 Age in days 'pUUEL'S‘ A 62 Table 6-1. Poult liver vitamin A concentration Hean Days of age Hog/gm (fresh basis) 1 39.5 3 30.0 7 18.5 9 10.5 29 2.8 [+2 0 39 DISCUSSION Based upon a review of the literature, there is much controversy concerning the utilization of beta-carotene by poultry and other animals as a source of vitamin A. There also exists doubt as to the need for high levels of vitamin A either in the blood or in the liver. The relationship between dietary, blood and liver concentration is uncertain. Several workers have reported beneficial effects of using therapeutic levels of dietary vitamin A in alleviating certain disease conditions. Much of the information reported relative to the above- mentioned areas of investigation depended upon the criteria employed as measuring devices of the relative performance for vitamin A and/or beta-carotene. In addition, it became obvious that little information was available on the vitamin A reserve in newly-hatched turkey poults. Therefore, it seemed desirable to determine the utilization of a newly deve10ped fermentation beta-carotene product by chicks and to investigate some of the relationships of vitamin A and beta-carotene in growing chicks and to investigate the body reserves of vitamin A in the turkey poult. If this new fermentation beta-carotene product could be in- corporated into poultry rations as a source of vitamin A, it would possibly be economically advantageous to the poultry producer who in recent times has encountered a continuing economic Squeeze between lower prices for his products and rising costs of production. In Experiment I, the initial (day-old) plasma vitamin A concen- tration was higher than that reported by Squibb (1961) and lower than that reported by Castano‘gt‘gl. (1951), while the plasma vitamin A concentration following depletion was about equal to that reported by 63 69 Squibb (1961). These differences are probably due to the difference in the amount of parental carry-over, as shown by Squibb (1961). By depleting the chick of all or nearly all of its vitamin A reserves, a more critical evaluation of the test diets can be made, eSpecially if the test period is of only short duration. In this experiment, the dietary levels of vitamin A selected for all of the products (except the synthetic beta-carotene) were what have been considered below the minimum requirement, approximately the minimum requirement and above the minimum requirement, based on growth. However, at the two lowest levels neither the fermentation beta-carotene nor the crystalline beta-carotene supported growth comparable to the ANRC Vitamin A Reference Standard and only the fermentation beta-carotene supported growth comparable to the ANRC Reference Standard at the highest level. This would lead one to believe that at or below the minimum requirement of vitamin A which is required for growth by chicks, the Vitamin A Reference Standard was utilized more effectively than either the fer- mentation beta-carotene or the crystalline beta-carotene. Although the synthetic beta-carotene was fed at several times the intended levels, differences in growth rate occurred from this product and the highest dietary level produced the largest mean body weights in this trial. It appeared that blood plasma vitamin A concentration was depen- dent on the length of time the chick was on the test diet, level of dietary supplementation, and the dietary source -- whether it was from the Vitamin A Reference Standard or the beta-carotene products. No 65 matter which product was the dietary source, blood pbsma vitamin A concentration was very low unless optimum or near optimum growth was produced. In the case of the synthetic beta-carotene, even though very high levels of vitamin A activity were in the diet and excellent growth was produced, the vitamin A plasma concentrations were similar to the other beta-carotene products. Vitamin A liver concentrations obtained in Experiment I were very low in all cases, except the highest dietary level of the ANRC Vitamin A Reference Standard which resulted in significantly larger vitamin A liver concentration than all other diets. This could very well be the case if one accepts the theory that in all but this high level of the ANRC Reference Standard the chick utilized all of the available vitamin A for metabolic processes. Very few significant differences in survival resulted from any of the diets even though significant differences existed in body reserves of vitamin A. The inability of the synthetic beta-carotene to favorably affect any of the criteria, except growth, even though it was fed at several times the intended levels was probably due to the abundance of isomers of beta-carotene which were not of the all-trans type. The latter have poor biological availability as shown by Moore (1957). The results obtained with the second group of birds fed the basal ration coincided very closely with those of the first experiment, indicating approximately the same parental carry-over. In Experiment II, the dietary levels selected were all above what is generally accepted as the absolute minimum amount of vitamin A 66 activity to produce optimum growth; this accounts for the relatively few significant differences in mean body weights. However, in this experiment, as well as in Experiment I, the lowest level of the beta- carotene products did not support the rate of growth that was obtained in the case of the other diets. This again indicates that the lowest dietary level of beta-carotene used in this eXperiment (regardless of product) was not used as effectively for growth as the comparable dietary level of vitamin A. It was also apparent that the fermentation beta-carotene was at least equal to the crystalline beta-carotene in its ability to support growth of chicks in this eXperiment. In Experiment II, the ANRC Vitamin A Reference Standard increased the vitamin A plasma concentration more than did either of the beta- carotene products at all comparative levels and would, therefore, appear more biologically available for inducing blood concentration of vitamin A. It also appeared that in the case of the fermentation beta-carotene, the highest level used in this test may have been approaching its upper limit in ability to influence vitamin A blood concentration. This did not appear to be true for either of the other products tested. At the lowest dietary level of all products tested, little vitamin A liver concentration was found. However, at each of the other dietary levels, the liver vitamin A concentration was significantly higher from the ANRC Vitamin A Reference Standard than from the other products tested and as the dietary level of the ANRC Reference Standard increased from 1,320 IU to 2,090 IU per pound of diet, a straight line increase in vitamin A liver storage occurred. As the level of both beta-carotene 67 products increased from about 1,000 IU per pound to about 2,050 10 per pound, their ability to produce vitamin A liver storage was reduced. Again, even though large differences existed in the bird's vitamin A reserves from the various diets, few significant differences resulted in survival of the birds placed on the basal ration after having been on the test diets. In these first two experiments, similar reSponses were observed for all of the criteria used to evaluate the utilization of the fermen- tation beta-carotene and the relationships between vitamin A and beta- carotene. In both experiments, when the chicks were fed diets that contained very near or below the requirement of vitamin A needed for optimum growth, the ANRC Vitamin A Reference Standard was utilized more effectively than any of the beta-carotene products in promoting growth. However, when dietary levels were in excess of the known minimum requirement for growth, all products tended to support optimum growth. In both experiments it was obvious that optimum growth could occur without high plasma or liver concentrations of vitamin A. Although optimum growth was obtained without any appreciable vitamin A liver storage, this may not be a practice which is practical for the com- mercial poultryman. The chick can utilize the stored vitamin A and if little was available in the body reserves, the chick could suffer during periods when no dietary vitamin A was available or being ingested. This could be especially important in periods of disease, since the chick usually consumes less feed during sickness and vitamin A has been found to be effective in connection with several disease 68 conditions. It was also observed that even though known differences occurred in the body reserves of vitamin A few significant differences occurred in survival on the basal ration. This could be explained if it were determined that all of the body's reserves of vitamin A were not mobilized or were not available for use by the chick; however, this was not determined. It was also observed that the fermentation beta-carotene and the crystalline beta-carotene influenced the characteristics used for comparison quite similarly; however, both beta-carotene products were inferior to the ANRC Vitamin A Reference Standard in their ability to influence plasma and liver concentrations of vitamin A, while all products influenced survival on the basal ration in about the same manner. In Experiment III, high dietary levels of vitamin A activity were compared for a short period. It appeared that dietary fermentation beta-carotene did not support the liver vitamin A concentrations obtained with comparable levels of dietary vitamin A. However, for liver vitamin A concentration, the fermentation beta-carotene was more effective in this experiment than in previous experiments when compared to vitamin A palmitate. This could have been influenced by the shorter depletion period, large liver size, and product differences. The relative ineffectiveness of the dietary beta-carotene used in these experiments to induce plasma and liver concentrations of vitamin A comparable to that of dietary vitamin A is intriguing. Perhaps there are Specific sites and/or enzymes within the intestine of the chick utilized for conversion of beta-carotene to vitamin A, and there are 69 only slightly more sites and/or enzymes for conversion than are necessary for optimum growth. This could explain the results shown in these experiments. In Experiment IV, turkey poults had a high carry-over of vitamin A in the liver, lived for a much longer period on the vitamin A de- ficient basal ration than chicks, and the vitamin A plasma concentration did not drop as low after #2 days on the basal ration as in the case of chicks after only 1h days on the basal ration. After about 30 days on the basal ration, the poult's plasma reached its lowest concentration in vitamin A. However, vitamin A deficiency symptoms did not appear until 10-12 days later. It is possible that the turkey can live this additional time on the body reserves as there was apparently some liver storage (2.8 mcg per gram) when the plasma vitamin A concentration reached its lowest level. The initial vitamin A concentration in the liver of these turkeys was much higher than the initial vitamin A liver concentration of chicks reported by Squibb (1961). The turkey poult may also have more parental vitamin A carry-over in the yolk due to size of the yolk and the possibility of a higher yolk concentration of vitamin A in turkey eggs. CONCLUSIONS Commercially obtained day-old Cobb's Strain White Rock cockerels were successfully grown at an optimum rate of growth and feed efficiency when fermentation beta-carotene supplied the only source of vitamin A activity. For the criteria established for comparison, fermentation beta- carotene compared favorably with the commercial crystalline beta- carotene; neither beta-carotene product did as well as the ANRC Vitamin A Reference Standard in influencing plasma and liver concentrations of vitamin A. In these experiments, optimum growth of broiler chicks was obtained without high levels of plasma and/or liver vitamin A concentration(s). At levels of vitamin A activity which support optimum chick growth, beta-carotene is fully effective to the extent that .6 mcg of all-trans beta-carotene - l IU of vitamin A. When higher levels are fed to induce liver storage, beta-carotene was about 1/10 to h/lO as effective as vitamin A. Survival time of chicks, which had been on the test diets four weeks and then placed on the basal ration, was at best a poor measure to determine the effectiveness of a product to possess vitamin A activity. In order to accurately discuss the vitamin A requirement of chicks, the requirement must be defined. There was a different requirement for vitamin A for growth, blood concentration, and liver concentration. There was a product biological availability difference. There was a large variability in plasma vitamin A concentration and liver vitamin A concentration in chicks consuming the same diet 7Q 71 Turkey poults hatched from a breeding flock consuming a typical breeder ration lived on a ration devoid in vitamin A for a period in excess of #0 days. LITERATURE CITED Abbott, U. K., D. A. McMartin, H. E. Adler and F. H. Kratzer, 1960. The effect of egg-borne mycoplasma on embryonating turkey eggs. Poultry Sci. 39:315-326. Ames, S. R., 1965. The ANRC Vitamin A Reference Standard, Batch IV. Feedstuffs, Feb. 20, 1965, p. ##. Bergdoll, J. F., l96#. Poor egg production? Try high vitamin A. Everybody's poultry mag., March, p. 11. Boyd, F. M. and H. M. Edwards, Jr., 1962. 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