Studies on the Riboflavin, Pantothenic Acid, Nicotinic Acid, and Choline Requirements of Young Embden Geese

Studies on the Riboflavin, Pantothenic Acid, Nicotinic Acid, and Choline Requirements of Young Embden Geese J. A. SERAFIN Patuxent Wildlife Research Center, US Fish and Wildlife Service, Laurel, Maryland 2811 (Received for publication November 26, 198) ABSTRACT Four experiments were conducted to examine the riboflavin, pantothenic acid, nicotinic acid, and choline requirements of young Embden geese fed purified diets. Goslings fed diets deficient in either riboflavin, pantothenic acid, nicotinic acid, or choline grew poorly. Feeding a panthothenic acid-deficient diet resulted in 1% mortality. Goslings fed diets containing 53 mg/kg of choline or less developed perosis. Under the conditions of these experiments it was found that: 1) goslings require no more than 3.8 mg/kg of riboflavin and 31.2 mg/kg of nicotinic acid in the diet for rapid growth and normal development, 2) the pantothenic acid requirement of goslings is no more than 12.6 mg/kg of diet, and 3) a dietary choline level of 153 mg/kg is adequate for both the prevention of perosis and rapid growth of goslings. The levels of vitamins found to support normal growth and development of goslings appear to be similar to requirements of other species that have been examined. (Key words: Embden geese, riboflavin, pantothenic acid, nicotinic acid, choline) INTRODUCTION In comparison to the extensive nutritional literature concerning the domestic chick, poult, game birds, and ducklings, little is known about specific nutritional needs of young geese. Several reports (Roberson and Francis, 1963a,b; NRC, 1977; Allen and Storey, 1979) document protein and energy requirements of goslings; and the lysine requirement of White Chinese goslings has been determined (Roberson and Francis, 1966). Briggset al. (1953) demonstrated that choline, folic acid, and nicotinic acid (niacin) are essential in diets for growth and survival of goslings. Battig et al. (1953) showed that nicotinic acid prevents perosis in goslings. Little other specific information is available concerning vitamin requirements of goslings. This report describes a series of experiments using purified diets designed to investigate the riboflavin, nicotinic acid, pantothenic acid, and choline requirements of young Embden geese. MATERIALS AND METHODS General Procedure. Experiments of 3-week duration were conducted with Embden goslings of both sexes, except for Experiment 1, Trial 1, which was a 2-week study. Goslings for each 1 Use of trade names does not imply endorsement of commercial products by the Federal Government. 1981 Poultry Science 6:191-1915 experiment were hatched from eggs obtained at different times from a breeding flock maintained at this laboratory. The average weight of day-old goslings ranged from 99 to 125 g. Goslings were housed in electrically heated, thermostatically controlled battery brooders with raised wire floors when 1-day-old and transferred to wire cages with raised wire floors at 1 days of age. Mortality records were maintained for each experiment. Feed and water were supplied ad libitum, The composition of the basal purified diet and vitamin mixtures used in these studies is shown in Tables 1 and 2. An individual vitamin premix was prepared for each experiment. The pantothenic acid content of the pantothenic acid-deficient basal diet was determined by a method described in the Pharmacopeia of the United States (197) employing Lactobacillis plantarum ATCC 81 (Difco Laboratories, Detroit, MI). 1 Liberation of pantothenic acid from coenzyme A, to permit measuring free and bound forms of the vitamin, was carried out using a modification of the method of Novelli and Schmetz (1951). The niacin content of the niacin-deficient basal diet was determined by the method of the AOAC (197) using L. plantarum ATCC 81. The choline content of the choline-deficient basal diet was determined by the method described by Entenmann et al. (19). USP reference standards were obtained from the United States Pharmacopeial Convention, Inc., Rockville, MD. The total vitamin Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 216 191

VITAMIN REQUIREMENTS OF GOSLINGS 1911 TABLE 1. Composition of the basal purified diet Ingredient Glucose Isolated soybean protein a Soybean oil Cellulose Vitamin mixture Mineral mixture Glycine DL-methionine Protein (N X 6.25) Amount (%) 58.69 27. 3. 3. 1. 6.31.3.7 25.5 RP-1, Ralston-Purina Company, St. Louis, MO. The composition of the vitamin mixtures used is shown in Table 2. c Supplied the following in the diet (g/kg): CaHP 2H 2, 21.6; CaCO a, 18.3; KH 2 PO, 13.9; NaCl, 6.; MgSO, 2.5; (mg/kg) FeS 7H 2, 333.; MnS H 2, 333.; KI, 2.6; CuS 5H 2, 16.7; CoCl 2 6HjO, 1.6; Na 2 MoO 2H 2, 8.3; ZnC 3, 115;andNa 2 Se 3,.22. content of supplemented treatment diets (Tables through 7) was determined by calculation. Because of few instances in which purified diets have been fed to geese, a practical starter ration (Table 3) was fed to groups of goslings in each experiment to evaluate responses from those supplied purified diets. The size and number of replicates per treatment were relatively small because of limited numbers of goslings available and the expense of purified diets. The pen average was considered the experimental unit. Data were tested for significance using the analysis of variance (Steel and Torrie, 196), and differences between treatment means were tested using Duncan's multiple range test (Duncan, 1955). Experiment 1. This experiment was conducted to examine the effect of supplemental riboflavin in a purified diet on the performance of young goslings. A basal diet, containing.3 mg of riboflavin per kilogram by calculation, was supplemented with riboflavin at 1.5, 2.5, 3.5, 5., and 7. mg/kg. These levels were chosen based upon the assumption that the riboflavin requirement of goslings would be similar to known requirements for ducklings and poultry. Each diet was suppled to two replicate groups of 5 goslings in Trial 1, and two pens of 6 goslings each were assigned randomly to each of the experimental diets in Trial 2. Experiment 2. This study was conducted to determine the effect of graded levels of pantothenic acid in a purified diet for young goslings. The basal diet containing.12 mg/kg of pantothenic acid by analysis, was fed alone and supplemented with pantothenic acid at levels of 5., 1., 12.5, 15., and 2. mg/kg. Two replicate pens containing 6 goslings each were assigned randomly to s 1 and 2; all other treatments consisted of four groups of 6 goslings each. Experiment 3. The nicotinic acid requirement of goslings was studied in this experiment by supplementing a purified diet containing 1.2 mg/kg of nicotinic acid by analysis with graded amounts of nicotinic acid ranging up to 75 mg/kg. Relatively high levels of niacin were used in this study since reports (Scott and Heuser, 1952; Battig ef al., 1953) indicated that ducklings and goslings require considerably more nicotinic acid than other avian species for TABLE 2. Vitamin levels in vitamin mixes used in studies of vitamin requirements of young Embden geese Vitamin a Riboflavin, mg Calcium pantothenate, mg Nicotinic acid, mg Choline, mg Thiamine - HCl, mg Inositol, mg Pyridoxine HCl, mg Folic acid, mg Biotin, mg Menadione sodium bisulfite, mg Vitamin B 12, micrograms Ethoxyquin, mg Retinyl acetate, IU Cholecalciferol, IU a-tocopheryl acetate, IU Amount in ' diet (per kg) 15. b 2.C 15. d 3. e 15. 25. 6. 6..6 1.52 6. 125. 5. 5. 11. Water soluble vitamins were obtained from ICN Pharmaceuticals Inc., Cleveland, OH. The vitamin mixture used in Experiment 1 contained no riboflavin. The vitamin mixture used in Experiment 2 contained no calcium pantothenate. The vitamin mixture used in Experiment 3 contained no nicotinic acid. The vitamin mixture used in Experiment contained no choline. The vitamin mixture used in Experiment 1, Trial 1, supplied 19 mg/kg choline in the diet. Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 216

1912 SERAFIN TABLE 3. Composition of the practical starter ration" Ingredient Amount (%) Yellow corn meal.7 Soybean oil meal (9%) 23.75 Wheat standard middlings 13.5 Fish meal (6%) 5. Meat and bone scrap (8%) 5. Corn distillers solubles 3. Alfalfa meal (17%) 3. Brewers dried yeast 2. Dried whey 1.25 Dicalcium phosphate 1. Limestone.5 Soybean oil.5 Salt.25 DL-methionine.8 Vitamin mixture".65 Trace mineral mixture.5 Protein (N X 6.25) 25.7 a The diet was fed in the form of 5/32 in short pellets. Supplied the following in the diet (mg/kg): folic acid,.; riboflavin,.; nicotinic acid, 66; choline chloride, 5; calcium pantothenate, 6.6; biotin,.22; inositol, 138; vitamin B 12,.7; menadione sodium bisulfite,.; ethoxyquin, 125; and (IU/kg): retinyl acetate, 16,55; cholecalciferol, 1653; and a-tocopheryl acetate, 11, premixed in glucose. C Supplied the following in the diet (mg/kg): manganese, 12; zinc, 115; iron, ; copper, 5; iodine, 2.5; and cobalt, 1. preventing bowed legs. Four replicate groups of 6 goslings each were assigned to each of the six experimental diets. Experiment. This experiment was designed to examine the influence of varying amounts of dietary choline on the growth of goslings and the incidence of perosis. The basal diet contained 3 mg of choline per kilogram by analysis. It was fed alone and supplemented with choline at levels of 5, 1, 15, 2, and 25 mg/kg. Two replicate groups containing 5 goslings each were assigned randomly to each of the dietary treatments. RESULTS AND DISCUSSION Experiment 1. The results of feeding goslings a purified diet supplemented with graded amounts of riboflavin in two trials are shown in Table. Goslings fed the diet containing 1.5 mg of added riboflavin per kilogram grew slowly in both trials. Goslings supplied the diet containing 2.5 mg/kg of added riboflavin gained significantly less than those fed diets containing greater a- mounts of riboflavin in Trial 1, but this was not so in Trial 2. Goslings fed diets supplemented with 5. and 7. mg/kg of riboflavin performed no better than those supplied the diet containing 3.5 mg/kg of added riboflavin. Responses in these treatments were similar to those resulting when a practical starter ration was fed. Severely outwardly bowed legs developed in all of the goslings fed diets containing 1.5 and 2.5 mg/kg of added riboflavin in Trial 1. No leg bowing occurred in Trial 2. The onset of this condition was rapid, taking place when the goslings became 1 to 12 days old. Paralysis ensued and goslings lost the ability to stand. Why this condition occurred only in Trial 1 is unclear. The basal purified diet used in this experiment contained.3 mg/kg of riboflavin by calculation. Based on this and the responses obtained when the diet was supplemented with graded amounts of riboflavin, it appears that young goslings require no more riboflavin than 3.8 mg/kg for rapid growth and normal development. This level is similar to those reported for domestic chicks, poults, Bobwhite quail, and ducklings (NRC, 1977). Experiment 2. The pantothenic acid requirement of young goslings was examined in an experiment (Table 5) in which graded levels of calcium pantothenate were added to a purified diet. Only one gosling fed the unsupplemented basal diet survived longer than 2 weeks. Goslings fed a diet supplemented with pantothenic acid at 5. mg/kg grew slowly. All survived for 7 days, but only remained alive at 1 days of age. No mortality occurred among goslings fed diets supplemented with 1. mg/kg of pantothenic acid or more. Goslings fed the diet containing 1. mg/kg of added pantothenic acid gained significantly less than those fed greater amounts of pantothenic acid. Maximal gain appeared to be achieved with the addition of 12.5 mg/kg of pantothenic acid to the diet. Goslings in s, 5, and 6 fed purified diets performed as well as those fed a practical starter ration. The pantothenic acid requirements for the young domestic chick, pheasant, Bobwhite quail, poult, and duckling have been found to fall between 11 and 13 mg/kg of diet (NRC, 1977). Supplementing the basal diet containing.12 mg/kg pantothenic acid with pantothenic acid at 12.5 mg/kg resulted in maximal per- Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 216

VITAMIN REQUIREMENTS OF GOSLINGS 1913 TABLE. Effect of riboflavin supplementation of a purified diet for young Embden geese (Experiment 1) Dietary riboflavin Added Total 3 Trial 1 (1 days) Trial 2 Trial 1 (1 days) Feed/gain Trial 2 Incidei! bowed legs 1 Mortality 2 Trial 1 Trial 2 1.5 1.8 2.5 2.8 3.5 3.8 5. 5.3 7. 7.3 Practical starter 22 a 36 68 c 77 c 67 c 737 c 28 a 116 b 1321 b 1177 b 1288 b 1278 b 1.82 1.62 1.52 1.56 1. 1.3 2.19 a 1.65 b 1.8 b 1.55 b 1.55 b 1.62 b 1 1 (* ) 17 a ' ' c Means within a column not followed by the same letter superscript are significantly different (P<.1). None of the feed/gain values in Trial 1 were significantly different (P>.5). 1 No goslings developed bowed legs in Trial 2. 2 No mortality occurred in Trial 1. 3 Calculated values. formance in this study, indicating that the pantothenic acid requirement of goslings is no greater than 12.62 mg/kg of diet and that the requirement is similar to those of other avian species. Experiment 3. The results of feeding goslings diets supplemented with graded amounts of nicotinic acid are shown in Table 6. Goslings fed the basal diet supplemented with 15 mg/kg nicotinic acid gained significantly less than those fed greater amounts of niacin. Additions of 5 mg/kg of niacin or more did not result in significant gains compared to results obtained when the diet contained 3 mg/kg added nicotinic acid. The performance of goslings fed purified diets containing 3 mg/kg of added niacin or more was comparable to that of goslings supplied a practical starter ration. No bowed legs or perosis developed in goslings in this study. The nicotinic acid requirements for avian species may vary widely since 1) nicotinic acid is synthesized in the body from tryptophane and 2) much of the nicotinic acid present in some feedstuffs is in a bound form that is not readily available. Thus, nicotinic acid requirements depend upon 1) the tryptophane content of the diet, 2) the pyridoxine content of the diet, for this vitamin is required in the synthesis of niacin from tryptophane, and 3) the types of TABLE 5. Effect of pantothenic acid supplementation of a purified diet for young Embden geese (Experiment 2) Dietary pantothenic acid Added Total Feed/gain Mortality Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 216 1 2 3 5 6 7 5. 1. 12.5 15. 2. Practical 1 starter.12 5.12 1.12 12.62 15.12 2.12 229 1 127^ 113 b 17 b 17 b 156 b 2.29' 1.71 1.53 1.66 1.68 1.59 (%) 1 1 a b ' Means within a column not followed by the same letter superscript are significantly different (P<.1). None of the feed/gain values in Trial 1 were significantly different (P>.5). 1 Values for 2 represent data from four goslings at 1 days of age. These values were not included in statistical analysis.

191 SERAFIN TABLE 6. Effect of nicotinic acid supplementation of a purified diet for young Embden geese (Experiment 3) Added Dietary nicotinic acid Total Feec i Mortality 15 16.2 3 31.2 5 6.2 6 61.2 75 76.2 Practical starter 93 a 1332b 132b 138 b 15 b 138 b a b ' Means within a column not followed by the same letter superscript are significantly different (P<.1). None of the feed/gain values were significantly different (P>.5). feedstuffs comprising the ration. A number of reports (Scott and Heuser, 1952; Battig et al., 1953; NRC, 1977) indicate that from 22 to 55 mg/kg of available niacin in the diet may be necessary for maximal growth and normal development of ducklings and goslings. The purified diet used in Experiment 3 contained 27% isolated soybean protein, contributing.19% tryptophane by calculation to the diet, and the vitamin mixture provided 5 mg/kg of supplemental pyridoxine. Analysis of the basal diet indicated a nicotinic acid content of approximately 1.2 mg/kg. Under these conditions, the addition of 15 mg/kg of nicotinic acid to the diet prevented bowed legs and perosis but did not support rapid growth; however, 3 mg/kg of added nicotinic acid in the diet permitted both rapid growth and normal development of goslings. Experiment. The responses of goslings to 1.68 1.73 1.78 1.76 1.7 2. (%) 8 graded levels of choline in a purified diet appear in Table 7. Goslings fed the basal diet grew poorly. Those fed the diet containing 5 mg/kg of added choline grew significantly better than goslings in 1; however, all goslings in both treatments developed perosis by 6 days of age. The condition was characterized by enlarged hocks and bowed legs. Goslings in these treatments exhibited marked nervous excitability and curled toes as well. Paralysis developed in all of the goslings fed the basal diet and may have been a contributing factor to the mortality that occurred. All losses were during the final week of the experiment. Interestingly, this treatment survived rather well considering the low choline content of the diet. Replicate gains in 2, 22 g and 81 g (SD = ± 83 g and 328 g, respectively), were extremely variable, perhaps reflecting the TABLE 7. Effect of choline supplementation of a purified diet for young Embden geese (Experiment ) Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 216 Dietary choline Added Total Feed/gain Incidence of perosis Mortality 1 2 3 5 6 7 3 5 53 1 13 15 153 2 23 25 Practical 253 starter 192^ 616 b 136 c 17C 1c 1389 c 12C 2.56 a 1.8 b 1.8 b 1.68 b 1.66 b 1.73 b 1.6 b 1 1 1 '' c Means within a column not followed by the same letter superscript are significantly different (P<.1).

VITAMIN REQUIREMENTS OF GOSLINGS 1915 perotic condition of the goslings, the small replicate size, and genetic variability in choline needs. Genetic variability alone may explain these results, for responses of chicks to nutrient deficiencies (nicotinic acid and arginine deficiencies, e.g.) are known to show very striking individual variation (Nesheim, 1975). Although the gain by goslings fed the diet containing 1 mg/kg of added choline did not differ significantly from those of goslings fed higher levels of choline when statistical analysis was carried out using data from all treatments (Table 7), 3 gained significantly less (P<.1) than s, 5, and 6 when statistical analysis was performed without 2. Feed utilization was significantly depressed when the basal diet was fed compared to results when diets contained supplemental choline. This study indicated that supplementing a basal diet containing 3 mg/kg of choline by analysis with 1 mg/kg of choline provided an adequate level (13 mg/kg of diet) for preventing perosis in goslings. It appears that a dietary level of 153 mg/kg is sufficient for both the prevention of perosis and rapid growth. REFERENCES Allen, N. K., and M. Storey, 1979. Protein requirements of White Embden geese to 9 weeks of age. Poultry Sci. 58:13. Association of Official Analytical Chemists, 197. Official methods of analysis. 11th ed., Assoc. Offic. Anal. Chem., Washington, DC. Battig, M. J., E. G. Hill, T. H. Canfield, and H. J. Sloan, 1953. Prevention of perosis in goslings by nicotinic acid. Poultry Sci. 32:55-551. Briggs, G. M., E. G. Hill, and T. H. Canfield, 1953. The need for choline, folic acid, and nicotinic acid by goslings fed purified diets. Poultry Sci. 32: 678-68. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1 2. Entenman, C, A. Taurog, and 1. L. Chaikoff, 19. The determination of choline in phospholipids. J. Biol. Chem. 155.13-18. National Research Council, 1977. Nutrient requirements of poultry. 7th rev. ed., Nat. Acad. Sci., Washington, DC. Nesheim, M. C, 1975. Genetic variation in nutritional requirements of poultry. Pages 7 87 in The effect of genetic variance on nutritional requirements of animals. National Research Council, Nat. Acad. Sci., Washington, DC. Novelli, G. D., and F. J. Schmetz, Jr., 1951. An improved method for the determination of pantothenic acid in tissues. J. Biol. Chem. 192: 181-185. Pharmacopeia of the United States, 197. Calcium Pantothenate Assay, 86 866. US Pharmocopeial Convention, Inc. 18 rev. Bethesda, MD. Roberson, R. H., and D. W. Francis, 1963a. The effect of protein level, iodinated casein and supplemental methionine on the performance of White Chinese geese. Poultry Sci. 2:863-867. Roberson, R. H., and D. W. Francis, 1963b. The effect of energy and protein levels of the ration on the performance of White Chinese geese. Poultry Sci. 2:867-871. Roberson, R. H., and D. W. Francis, 1966. The lysine requirement of White Chinese goslings. Poultry Sci. 5:32-329. Scott, M. L., and G. F. Heuser, 1952. Studies in duck nutrition.. Bowed legs in ducks caused by niacin deficiency. Poultry Sci. 31:752 75. Steel, R.G.D., and J. H. Torrie, 196. Principles and procedures of statistics. McGraw-Hill, New York, NY. Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12, 216