Available Biotin Content of Barley. R. K. WAGSTAFF, D. C. DOBSON AND J. O. ANDERSON Poultry Department, Utah State University, Logan, Utah

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1 SR 9, C B AND BA 133 STUDIES WITH HENS 3 Three different methods of administration of the isotopes to the hens were tried and the advantages and disadvantages of each discussed, as related to obtaining information about specific biological processes. ACKNOWLEDGEENTS The authors gratefully acknowledge the thoughtful suggestions and interest given by Dr. J. C. Driggers and Dr. N. S. Hall of the University of Georgia and the University of Tennessee-Atomic Energy Commission Agricultural Research Laboratory, respectively. Assistance given by R. C. cllhenny in solving some of the counting problems encountered is appreciated. The mathematical treatment of the data by Dr. J. L. Carmon and r. J. C. ortson of the Statistics Institute, University of Georgia, is sincerely appreciated. REERENCES Brassuer, H., 197. Isomorphisme du phosphate tristroncique hydrate et de l'hydroxylapatite 3 Sr 3 P 2 Ba(OH)2. Bull. Soc. ranc. iner, Crist. : Comar, C. L., R. H. Wasserman and.. Nold, 196. Strontium-calcium discrimination factors in the rat. Proc. Soc. Exptl. Biol. ed. 92: Driggers, J. C, and C. L. Comar, 199. The secre- tion of radioactive calcium (Ca-) in the hen's egg. Poultry Sci. 2: 2-2. Driggers, J. C, R. L. Shirley, G. K. Davis and. R. ehrhof, 191. The transference of radioactive calcium and phosphorus from hen to chick. Poultry Sci. 3: Hashizume, T., H. Nozaki, K. Hiroe, S. Horii, Y. Takei, H. orimoto and Y. Kaishio, 192. Calcium metabolism in the laying hen. Bull. Nat. Inst. Agri. Sci. Chiba-Shi. Japan Series G. No. : Lagergren, C, and O. Carlstrom, 197. Crystallographic studies of calcium and strontium hydroxyapatites. Acta Chem. Scand. 11: -. Lange, N. A Handbook of Chemistry, p Handbook Publishers, Inc., Sandusky, Ohio. Likins, R. C, A. S. Posner,. L. Kunde and D. L. Craben, 199. Comparative metabolism of calcium and strontium in the rat. Arch. Biochem. Biophys. 3:72-1. Nozaki, H., S. Horii and Y. Takei, 19. Utilization of shell Ca by chick embryo. Bull, of Nat. Inst. Agri. Sci., Series G. No. 9, 9-9. Partington, J. R., 19. A Textbook of Inorganic Chemistry, p. 76, acillan and Co., Ltd., London. Romanoff, A. L., and A. J. Romanoff, 199. The Avian Egg, p John Wiley and Sons, Inc., New York. Shirley, R. L., J. C. Driggers, J. ccall, G. K. Davis and N. R. ehrhof, 191. Excretion and retention of P-32 and Ca- by laying hens. Poultry Sci. 3: Wasserman, R. H., C. L. Comar and.. Nold, 196. The influence of amino acids and the other organic compounds on the gastrointestinal absorption of calcium- and strontium-9 in the rat. J. Nutrition, 9: Available Biotin Content of Barley R. K. WAGSTA, D. C. DOBSON AND J. O. ANDERSON Poultry Department, Utah State University, Logan, Utah Downloaded from at Penn State University (Paterno Lib) on ay, 216 OST ingredients used in poultry feeds are considered to contain enough biotin so that biotin supplementation of most practical rations is not needed. The National Research Council's Publication (19) gives the biotin (Received for publication June ) content of barley as. milligrams per pound or.1 micrograms per gram. This would mean that a ration containing % or more of barley should contain adequate biotin to meet the chick's requirement of. milligrams per pound (National

2 R. K. WAGSTA, D. C. DOBSON AND J.. ANDERSON Research Council, 19). In some of the experiments conducted at this station to study the value of certain enzyme preparations in rations with a new variety of hulless barley, a semi-purified type of diet was fed which contained 7% hulless barley and no added crystalline biotin. Chicks fed this diet developed typical biotin deficiency symptoms, similar to those described by Hegsted et al. (19), within three weeks. The symptoms did not appear when 1 micrograms of biotin were added per kilogram of diet. This suggested that either the hulless barley contained less biotin than varieties of barley with a hull (termed barley in this paper) or that the biotin was not available to the chick. This paper presents results of experiments conducted to determine the biotin content of hulless barley by chick assay, to compare several grains as a source of biotin for the chick, and to determine the value of adding biotin to simplified practical rations containing a high level of hulless barley. PROCEDURE The composition of three of the basic rations used in these experiments is given in Table 1. The composition of ration 11 is not given; it contained the same amount of purified casein, gelatin, corn oil, DLmethionine, and vitamins as ration 1. A complete mineral mixture (see paper by luckiger and Anderson, 199, for the composition of the complete mineral mixture), 9 grams of vitamin-free casein, and grams of arginine HC1 were included in each kilogram of ration 11. Cerelose made up the remainder of this ration. The protein content of ration 11 is the same as ration 1. The purified and vitaminfree caseins were used as they were obtained from the Nutritional Biochemicals Corporation, Cleveland, Ohio. Each of the grains used in these experi- TABLE 1.Composition of the basic rations ' in the experiments Ingredients Hulless barley oil oil meal (% prot.) DL-methionine Casein, purified Gelatin Alfalfa, dehy. (17% prot.) Limestone flour Calcium carbonate Calcium phosphate Dicalcium phosphate, feed grade (1.% P.) Trace mineral mix* Salt Dibasic potassium phosphate Vitamin mixf Crude bacterial amylase Ration * Contains 6% manganese, 2% iron,.2% copper,.12% iodine,.2% cobalt,.6% zinc, and 26.% calcium. t The vitamin mixture used in ration 1 contained the same vitamins as rations used by luckiger and Anderson (199) except that the biotin was omitted. The vitamin mixture used in rations 16 and 111 supplied the following per kilogram of ration: 1, I.U. of vitamin A, 2, I.C.U. of vitamin Dj 1. mg. menadione sodium bisulfite, 13.3 mg. alpha-tocopherol acetate, 1. mg. of riboflavin, 3.6 mg. of pantothenic acid,.2 mg. of niacin,.2 mg. of vitamin B12, mg. of choline and 22 mg. of chlortetracycline. ments, except the hulless barley, was obtained from commercial grain dealers in this area. Samples of the barley and hulless barley used in these experiments were sent to the Wisconsin Alumni Research oundation where the biotin content of each was determined microbiologically by the method of Wright and Skeggs (19). The barley was found to contain.13 micrograms of biotin per gram and the hulless barley.1 micrograms per gram by this method. The enzyme supplement used in some of the experiments was a crude bacterial amylase obtained from Dawe's Laboratories Inc., Chicago, Illinois. Where it was used in the rations, it was added at a level of grams per kilogram of ration and replaced the grain. Straight-run Vantress X Hubbard 3 chicks were used throughout these experiments. They were progeny of hens fed a high barley breeder ration. Chicks used in experiments conducted to determine the available biotin content of hulless barley were fed ration 1 for one week and then Downloaded from at Penn State University (Paterno Lib) on ay, 216

3 BIOTIN IN BARLEY SOS fed the experimental rations for two weeks. In the remainder of the experiment the chicks were started on the experimental rations when they were taken from the incubator, and unless otherwise indicated the experiments were continued for four weeks. The chicks were distributed to comparable weight groups of 7 or birds at the time the experimental rations were started. They were reared in electrically heated battery brooders for the first four weeks and then moved to unheated batteries if the experiment was continued longer. eed and water were offered freechoice. our experiments have been conducted which involved the addition of biotin to practical chick starting rations in which hulless barley was the only grain. In three of the experiments the chicks were carried beyond four weeks of age. Ration 16 was fed for the first four weeks in each of the experiments; the basic ration fed when birds were continued beyond this age was ration 111. Composition of both of these rations is given in Table 1. Ration 16 contained more soybean oil meal and less grain than ration 111 but was otherwise the same. Protein content (NX6.2) of ration 16 was 23.%; ration 111 contained 2.1%. In each experiment a cornsoybean oil meal ration was fed. replaced hulless barley in ration 16 to make the ration fed during the first four weeks to chicks on this treatment; that fed after this period contained 3% more soybean oil meal than ration 111 and the grain was corn. Other modifications of rations 16 and 111 were fed in one experiment. In one of these modifications the hulless barley was replaced with barley, in another 1% of fish meal replaced part of the soybean oil meal in ration 16 to produce the ration fed during the first four weeks of the experiment and 7.% was used in the ration fed during the remainder of the test. The same levels of meat and bone meal were used in another modification. Protein, calcium, and phosphorus levels were adjusted to be approximately equal to the levels found in hulless barleysoy diets. RESULTS AND DISCUSSION our experiments were conducted to determine how much of the biotin in hulless barley was available to the chick. Levels of, 2,, 7, 1, and 2 micrograms of biotin were added to each kilogram of ration 11, and to ration 1 both with and without the addition of grams of crude bacterial amylase per kilogram. Six groups of chicks were fed each of the 1 rations. A summary of the average growth rates obtained in these experiments is found in Table 2. The growth rate of chicks fed rations with suboptimum levels of biotin was variable. This made it difficult to determine the biotin requirement exactly. About 9 micrograms of added biotin were required per kilogram of ration 11 to allow optimum growth. About 6 micrograms of added biotin were required with ration 1. This requirement was not influenced by the addition of the crude bacterial amylase to the diet, although growth rate was increased by this addi- TABLE 2.Biotin requirement of chicks fed rations 1 and 11 Level of biotin added 7/kg Average 2 week gain/chick* Enzyme Ration 1 Enzyme ' Six groups of eight chicks per treatment. Ration Downloaded from at Penn State University (Paterno Lib) on ay, 216

4 6 R. K. WAGSTA, D. C. DOBSON AND J. O. ANDERSON TABLE 3.Effect of biotin addition to ration 1 with various grains Grain used Avg. weight- wk. % t- snowing m ration -Biotin Biotinf dermatitis* Wheat Hulless barley Barley ilo Oats * Data given only for groups of chicks fed rations without added biotin. our groups of eight chicks per treatment. f Biotin added at level of 2 micrograms per kilogram of ration. tion. Chicks fed ration 1 developed biotin deficiency symptoms with about the same frequency and severity as chicks fed ration 11 with 2 micrograms of biotin added per kilogram. Thus, it would appear that the chicks were obtaining about 2 to 3 micrograms of biotin from the hulless barley added to one kilogram of ration 1. Couch et al. (19) found that the type of carbohydrate in the diet affected the amount of biotin synthesized in the intestines of the mature fowl. Differences in microbiological synthesis and use of biotin in the digestive tract probably affected the estimate of the available biotin in hulless barley in these chick tests. It is felt that the estimate obtained should be a good estimate of the effective quantity available to the chick. Since ration 1 contained 7.% hulless barley, it was estimated that hulless barley contained only.3 micrograms of biotin per gram which was available to the chick or only about one-third of that found by microbiological assay. Experiments were conducted in which grains other than hulless barley have been used in ration 1; the rations were fed both with and without the addition of 2 micrograms of biotin per kilogram of ration. The results of two experiments are summarized in Table 3. The data have been analyzed statistically by the analysis of variance. The differences produced by the addition of biotin, the use of the different grains, and the interaction between these two are all highly significant, (P<.1). Slow growth and a high incidence of dermatitis were noted among chicks fed ration 1 with hulless barley, barley, or wheat. The final average weights were increased about % by the addition of biotin to these diets. These grains must provide about the same amount of available biotin to the chick; this quantity is less than that provided by the other three grains. Less than one-third of the biotin found in barley by microbiological assay appeared to be available to the chick. Oats appeared to be the best source of biotin, although most of the hulls had been removed mechanically from the oats used in these experiments and the estimate may not hold for whole oats. ry et al. (197) found that water treatment of barley improved its nutritional value for the chick. Jensen et al. (197) showed that the addition of a fungal enzyme preparation to a high barley diet improved its value. It seemed possible that part of this response might be the result of making more biotin available to the chick from the barley. The results of one factorial type of experiment involving soaking hulless barley in water and addition of biotin and crude bacterial amylase to ration 1 are presented in Table. The soaking procedure was the same as used by ry et al. (197). Three groups of eight chicks were fed each of the diets given in the table. ore experiments of this type have been conducted and the results have been similar to those given here. Addition of enzyme supplement to the ration or use of water soaked hulless barley did not affect the amount of response obtained by the addition of biotin. Both the water Downloaded from at Penn State University (Paterno Lib) on ay, 216

5 BIOTIN IN BARLEY 7 TABLE.Effect of water soaking hulless barley and addition of enzyme supplement on the availability of biotin Treatment of hulless barley or addition to ration 1 Soaked Enzyme* Biotin* Average -week weight Gain eed % % ortality showing dermatitis 7S 1 * Where enzyme was added a crude bacterial amylase was added at a level of grams per kilogram; where biotin was added it was added at a level of 2 micrograms per kilogram. soaking and enzyme addition increased the growth rate of the chicks. No mortality occurred among the chicks fed ration 1 in this experiment, and there has been little mortality among chicks fed this ration in any experiment. When water soaked hulless barley was used in ration 1, or an enzyme supplement was added to the ration which increased the growth rate of the chicks, nearly one-third of the chicks died before the end of the fourweek experiment. Chicks fed these diets seemed to develop biotin deficiency symptoms faster than those fed the ration without added enzymes or soaked hulless barley. No evidence was obtained which would indicate that soaking the hulless barley or addition of enzyme to the ration increased the availability of biotin to the chick. Generally it is felt that the practical rations fed chicks and turkey poults contain adequate biotin to meet the needs of the birds. Patrick et al. (192), cginnis and Carver (197), Robblee and Clandinin (193), and Slinger and Pepper (19) have reported results of experiments which indicated some benefit was obtained by the addition of biotin to practical type rations fed to turkey poults. The 7 rations used in these experiments did not contain as much of the ingredients usually considered to be good sources of biotin as most turkey rations fed today. The experiments reported in this paper with ration 1 indicated that the wheat, barley, and hulless barley used did not provide much biotin to the chicks, and suggest that rations containing high levels of these grains may benefit more from the addition of biotin than rations based on corn, milo, or oats. A summary of the results of the four experiments involving the addition of biotin to practical rations containing a high level of hulless barley is found in Table. Three groups of chicks were started on each of the rations in experiments 1, 2, and 3. Twenty-four birds of each sex were fed each of the rations in the fourth experiment. The average body weight at four weeks was increased slightly by the addition of biotin to ration 16, but the difference was not statistically significant. The average gain/feed ratio for chicks fed ration 16 was.1 both with and without the addition of biotin. It was. for chicks fed the corn-soy ration. The differences in weight gain produced by the addition of biotin, tended to become greater after the birds were fed the lower protein rations. The average unweighted difference in 7 or week weight produced by the addition of biotin to the barley and hulless barley rations in the experiments summarized in Table, is 36 grams. This difference was found to be statistically significant when the differences were analyzed by a t-test, (P<.2). This average difference includes the differences from the rations containing fish meal; the fish meal appeared to provide more biotin than the soybean oil meal that it replaced. The average final weights of the birds fed the hulless barley-soy diet with added biotin Downloaded from at Penn State University (Paterno Lib) on ay, 216

6 R. K. WAGSTA, D. C. DOBSON AND J.. ANDERSON TABLE.Value of biotin addition to chick rations containing a high level of hulless barley Grain in ration Protein suppl. in ration Age in weeks Sex Average body weight -Biotin Biotin* Exp. 1 Exp. 2 Exp. 3 Barley Exp. f & fish, meat & bone ,6 1,1 2 1, 1,17 1 1,392 1, ,331 1, ,1 1,29 1,36 1, ,7 1, ,3 1,16 3 1,6 1,22 2 1,31 1, ,73 1,23 1, 1,191 1,36 1,2 * Biotin added at a level of 1 micrograms per kilogram of ration. f All birds on the hulless barley-soy rations in this experiment were fed ration 16 for the first four weeks. The average weights of the two groups were the same at four weeks of age. in these experiments were nearer those of birds fed a corn-soy diet than in previous tests in which biotin was not added to the rations. The gain/feed ratio was not increased by the addition of biotin to the hulless barley rations. The average ratio for birds fed to eight weeks was.39 with the hulless barley-soy rations and.2 with the corn-soy rations. While the improvement in performance produced by the addition of biotin was not great, the data indicate that it would be advisable to include some biotin rich ingredient(s) in chick rations containing high levels of hulless barley. Such ingredients would probably be needed in rations containing high levels of barley or wheat or mixtures of the three grains. SUARY Biotin content of hulless barley was determined by chick and microbiological Downloaded from at Penn State University (Paterno Lib) on ay, 216

7 BIOTIN IN BARLEY 9 assay. The chick assay indicated that it contained.3 micrograms of biotin per gram, while.1 micrograms per gram were found by microbiological assay. Barley appeared to contain about the same amount of biotin available to the chick as the hulless barley, although the microbiological assay indicated that it contained.13 micrograms per gram. The availability of the biotin in hulless barley was not increased by soaking the grain in water or by the addition of a crude bacterial amylase to rations containing this grain. The hulless barley, barley, and wheat used contained less biotin that was available to the chick than the corn, milo, or oats used. None of these grains supplied enough biotin to the chick to meet its requirement completely when fed at a level of about 7% of the diet. The addition of biotin to simplified chick rations based on hulless barley, tended to increase the growth rate of the chick. ACKNOWLEDGENTS These experiments were supported in part by a grant from Kennecott Copper Corporation. Some of the vitamins used in these experiments were supplied by Lederle Laboratories Division, American Cyanamid Company, Pearl River, N. Y.; erck and Company, Rah way, N. J.; and Nopco Chemical Company, Harrison, N. J. The enzyme supplement was supplied by Dawe's Laboratories, Inc., Chicago, 111.; and the Delamix by Limestone Products Corporation of America, Newton, N. J. REERENCES Couch, J. R., W. W. Cravens, C. A. Elvehjem and J. G. Halpin, 19. Relation of carbohydrate to intestinal synthesis of biotin and hatchability in mature fowl. J. Nutrition, 3: luckiger, H. B., and J. O. Anderson, 199. Amino acid requirements of the chick. 1. Effect of thyroxine and kind of protein on the arginine, methionine and glycine requirements. Poultry Sci. 3: ry, R. E., J. B. Allred, L. S. Jensen and J. c- Ginnis, 197. Influence of water treatment on the nutritional value of barley. Proc. Soc. Exp. Biol. ed. 9: Hegsted, D.., J. J. Oleson, R. C. ills, C. A. Elvehjem and E. B. Hart, 19. Studies on a dermatitis in chicks distinct from pantothenic acid deficiency. J. Nutrition, 2: Jensen, L. S., R. E. ry, J. B. Allred and J. c- Ginnis, 197. Improvement in the nutritional value of barley for chicks by enzyme supplementation. Poultry Sci. 36: cginnis, J., and J. S. Carver, 197. The effect of riboflavin and biotin in the prevention of dermatitis and perosis in turkey poults. Poultry Sci. 26: National Research Council, 19. Nutrient requirements for domestic animals. No. 1. Nutrient requirements for poultry. National Research Council, 19. Composition of cereal grains and forages. Publ.. Patrick, H., R. V. Boucher, R. A. Dutcher and H. C. Knandel, 192. The nutritional significance of biotin in chick and poult nutrition. Poultry Sci. 21:76. Robblee, A. R., and D. R. Clandinin, 193. The use of calcium pantothenate and biotin in practical poult starters. Poultry Sci. 32: Slinger, S. J., and W. E. Pepper, 19. The effect of antibiotics on the turkey poults' need for supplementary biotin and pantothenic acid. Poultry Sci. 33: Wright, L. D. and H. R. Skeggs, 19. Determination of biotin with Lactobacillus arabinosus. Proc. Soc. Exp. Biol. ed. 6: 9-9. Downloaded from at Penn State University (Paterno Lib) on ay, 216 NEWS AND NOTES {Continued from page 7) N.T.. NOTES irst Vice-President. G. Smith, Ellicott City, At the 1961 National Turkey ederation conven- aryland; Second Vice-PresidentR.. cphertion held in Chicago, the following officers were rin, Sunnymead, California; Third Vice-President elected: PresidentS. Stout, Richland, issouri; R. Thompson, Ellsworth, Iowa; and ourth Vice- {Continued on page 36)